Pioneers In Microbiology: The Human Side Of Science

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b2530 International Strategic Relations and China’s National Security: World at the Crossroads

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World Scientific Publishing Co. Pte. Ltd.5 Toh Tuck Link, Singapore 596224USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

Library of Congress Cataloging-in-Publication DataNames: Chung, King-Thom, 1943– author. | Liu, Jong-Kang, author.Title: Pioneers in microbiology : the human side of science / King-Thom Chung, Jong-Kang Liu.Description: New Jersey : World Scientific, 2017. | Includes bibliographical references and index.Identifiers: LCCN 2016040514| ISBN 9789813202948 (hardback : alk. paper) | ISBN 9789813200364 (pbk. : alk. paper)Subjects: | MESH: Microbiology | BiographyClassification: LCC QR41.2 | NLM WZ 112.5.B3 | DDC 579--dc23 LC record available at https://lccn.loc.gov/2016040514

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b2806 Pioneers in Microbiology: The Human Side of Science

Preface

Microbiology is a major building block of modern science, and in the past has often been taught as a small section of botany or another discipline in universities. The microbiology discipline can reveal different facets of life science. A large university may place the microbiology department in different colleges or schools. Medical colleges certainly will embrace micro biology because microbiology is an important part of medical training. Engineering schools will place microbiology in the department of civil engineering, biochemical engineering, or other areas because microorganisms are related to many engineering processes. Microbiology is also an important discipline in the college of agriculture, because microorganisms can be involved in soil science, agronomy, horticulture, water conservation, food science, fermentation, enology, environmental conservation, and so on. Nowadays, biotechnology, recombinant DNA technology, molecular biology, genetic engineering, bioinformatics, and so on, are emerging as important; all of them deal with microorganisms in one way or another. Microbiology becomes a basic science.

Microbes are masters of biosphere. They are the progenitors of all life on earth. It is estimated that the biomass of 5 × 1031 microbial cells with a weight of 50 quadrillion metric tons is heavier than all plant or animal cells combined. Microbes carry out more photosynthesis than green plants. They are the providers of food and beverages, including cheese, beer, wine,

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other forms of alcohol, pickles, sauerkraut, solvents, organic acids, amino acids, vitamins, flavor nucleotides, pigments, anti-biotics, antitumor agents, enzymes, plant insecticides, coccidio-stats, gums, alkaloids, plant growth factors, and animal growth promotants, along with others.

We can recall that the human life span was only 47 years in the year 1900, when microbial infections (germ theory) were not widely known. Disease control was often based on folk beliefs or somewhat pseudoscientific fictions. Human lives were rather miserable until germ cell was discovered. In the year 2015, the life span in United States was 76.9 years for males and 81.6 years for females. We have learned to fight many diseases with the knowledge of microbiology. There are more than 23,000 bioactive secondary metabolites made by many microbes; many of these products are used in medicine and agriculture. In 1995, a prominent microbiologist Arnold Demain (b. 1927) determined that glutamate was used for food flavoring as monosodium glutamate at 1.5 million tons per year, lactic acid at 2,500,000 tons per year, and penicillin at 31 million kg per year. More recently, there we have statins, immunosuppresants, antiparasitic agents, and other biopharmaceuticals, such as monoclonal antibodies, recombinant human insulin, which are widely used in medicine. Another major area where microbes are involved is in the production of biofuels, such as hydrogen, methane, bioethanol, and biobutanol. As stated by Louis Pasteur back in the 19th century, Arnold said: “The microbe will have the last word”.

Microbes are ubiquitous. They are present in the areas of boiling mud with huge gas bubbling, the bottom of oceans where heat is being released from earth’s interior, and in the vents of chimneys. They are also important inhabitants of the human body. Approximately 1014 microbial cells inhabit our body, primarily in the gastrointestinal tract. We are basically symbiotic with microbes. Microbes participate in the biogeochemical cycles, including carbon cycle, nitrogen cycle, and sulfur cycle. Microbes can be producers, consumers,

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or decomposers in the ecosystem. They are essential to the biosphere, the region of earth inhabited by living organisms, including humans. Microbes also carry on fundamental biochemical processes.

Besides, microbes are the best paradigms for experimental study. With the rapid growth rate and a large population easily obtained in the laboratory, they have been a favorable subject in experimental biology. Therefore, the fundamental biochemical principles are chiefly obtained by the study of microorganisms. Many Nobel Laureates were either microbiologists or used microorganisms as their experimental subjects.

As stated by Selman Waksman (1888–1973), “The microbe has come of age … there is no field of human endeavor, whether it be in industry or in agriculture, whether it be in the preparation of foodstuffs or in connection with shelter and clothing, whether it be in the conservation of human and animal health and the combating of disease, where the microbes does not play an important and often a dominant part”.

Microbiology is a vital branch of life science. However, compared to mathematical and physical sciences, it was not significantly developed until recent history. About 2600 years ago, Siddhartha Gautama (Buddha), the creator of Buddhism, first stated that there were 84,000 lives (microbes) in a drop of water. In the 1st century, the Roman scholar and writer Varro proposed that tiny invisible animals entered the body through the mouth and nose to cause diseases; Lucretius, a philosophical poet, cited “seeds” of disease in his De Reum Natura (on the Nature of Things). Humans did not recognize the existence of microbes until Robert Hooke who in 1665 illustrated the microfungi, and around 1677–1678, when Antonie van Leeuwenhoek (1632–1723) described the animalcules (microbes). Leeuwenhoek observed all the major kinds of microorganisms which includes protozoa, algae, yeast, fungi, and bacteria in spherical, rod, and spiral forms. The word virus, the other important microbe, was first coined by Martinus W. Beijerinck (1851–1931) who also demonstrated many aspects of the

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application of microbiology. The presence of microbes was not generally accepted. The microbial role of causing diseases was later proved by Louis Pasteur (1822–1895) and Robert Koch (1843–1910). Microbiology began to be noticed. The importance of microbes in soil such as nitrogen, carbon, and sulfur cycles were first introduced by Sergei N. Winogradsky (1856–1953). Microbes play an important role in the biogeochemical cycle. The discovery of the microbial role in human disease or ecology is a gigantic step in human history. For example, historical roots, such as Bubonic plague, in the mid-14th century (1347–1351) wiped out 25 million people — one-fourth of the population of Europe and neighboring regions in just five years.

Pasteur, Koch, Beijerinck, and Winogradsky were generally recognized as major pioneers of microbiology. They were chiefly responsible for the establishment of microbiology as an independent scientific discipline. But microbiology was not taught as an independent course in the United States until 1931 at Stanford University by C. B. Van Niel (1897–1985). And now, microbiology is a big enterprise both in academia and industry.

”Pioneering” means breaking new ground, moving out into the unknown. Pioneers are not limited to a few people, such as Pasteur, Koch, Beijerinck, or Winogradsdky. There were many people who made significant contributions to the knowledge of microbiology. Yet, those pioneers were not known to the general public including those who are immersed in modern microbiology research. Their stories or simply their names are not included in general textbooks and are often neglected subjects in literature and history books. Their stories should be told. The process of making this wonderful microbiological knowledge known is equally important as wars, politics, or the arts.

The authors call the stories of the life experiences of pioneer microbiologists the “human side of microbiology”. To read the story of pioneer microbiologists is like reading literature or poems. Human values and creative thinking brought about

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such genius and marvelous results. Their contributions to the progress of civilization cannot be ignored. It is a human legacy. The stories of these pioneers will inspire all who read them, especially young people. The same challenges that stimulated Robert Koch, Louis Pasteur, Martinus Beijerinck, or Sergei Winogradsky can inspire young minds to creativity and further contribute to human civilization.

There were numerous pioneers, and every year in history has had its share of pioneering. In this book, the authors explore the life experience of those who were considered major pioneers in microbiology. Our civilization was built by fellow human beings. Every step of progress is accomplished by human efforts. We are appreciative of those human efforts, especially those pioneers of our field — microbiology.

After we read thorough the life stories of those pioneer microbiologists, we may find there is something in common among them. Those common qualities of pioneer microbiologist are worth studying. First of all, they seemed to have prepared minds. The prepared mind is important for a successful life. The prepared mind is a dedication to a goal, which is something worthwhile to pursue in life. Prepared minds set their sails for the endless sea of the unknown. It is not an ambition. An ambition may lead to greediness and selfishness. But, the prepared mind is to do something good that nurtures the opportunities in life, but only a few people grasp the opportunity when it arises and executes to its destiny.

Second, most pioneer microbiologists seemed to have a strong compassion toward life and human beings. They seemed to have lived to relieve human suffering, which drove them to work tirelessly and persistently. They possessed strong love for their families, friends, and nations. They are great human beings.

Third, they were interested in finding the truth. They were determined to find the truth and stood for the truth they found. After all, science is an exercise of finding truth.

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There are many other qualities, such as dedication, persistence, and hardworking spirits that are also common to those microbiologists. None of them was strongly tempted by political power or wealth. None of them was simply looking for wealth by studying microbiology. They had set high goals and worked hard to achieve their goals. They were content in a small laboratory or office and worked diligently.

Despite the fast growing knowledge of microbiology and its influence on human civilization, there is every reason to believe that there are huge unknown areas of microbiology that are awaiting discovery. Human maladies, such as cancer and AIDs are still taking their tolls and are awaiting microbiologists to provide solutions. Likewise, environmental pollutions, accumulation of nonbiodegradable substances, are awaiting microbiologists to solve these problems. These are just few examples. There are ample areas of microbiology that need to be studied. There are many opportunities for present and future microbiologists to continue to discover new findings. The past sheds the light for the future. The future of microbiology is just as bright as in the past. There is ample room for young minds to get in and start their trailblazing. Civilization will continue to progress only as microbiologists are tirelessly continuing their search for truth.

The choice of pioneers to be written about in this book was entirely arbitrary. It may be because their life stories were easier for the authors to find than in the case of others. Or it may be that some of their stories were most appealing to the authors. Although some stories were available fragmentary in different sources, a collective issue is necessary. The authors did not exclude the female microbiologists. Most female pioneer microbiologists were also doing medical research. The stories of female microbiologists were published in another book Women Pioneers in Medical Research by one of the authors of this book (K. T. Chung). This book covers the biographical stories that were primarily of men. This author’s endeavor started in 1989,

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when web searches were not as convenient as today. Due to the limitation of time, only 57 pioneers were covered in this book.

King-Thom ChungThe University of Memphis, United States

Jong-Kang LiuNational Sun Yat-sen University, Taiwan

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Acknowledgments

We are greatly indebted to many senior microbiologists and colleagues in the completion of this book. We were determined to write this book when we began teaching microbiology over 30 years ago, because no matter how fascinating the science is, knowledge is still discovered and fueled by the innovation of human beings. Without their endeavors, this knowledge would not be revealed, and the impact of microbiology to our world would not be recognized. The first author, King-Thom Chung, was inspired by microbiologists like Cornelis B. Van Niel (1897–1985) and Robert E. Hungate (1906–2004) who introduced numerous stories of pioneers such as Louis Pasteur (1822–1895), Robert Koch (1843–1910), Martinus W. Beijerinck (1851–1931), and Albeit J. Kluvyer (1888–1956) to the author because of their personal experiences with them. Their stories impressed this author profoundly. Later, many equally important pioneer microbiologists such as Joshua Lederberg (1925–2006), Hubert A. Lechevalier (1926–2015), H. Boyed Woodruff (1917–2017), Deam H. Ferris (1912–1993), Herman Phaff (1913–2001), Arnold Demain (1927–), Ralph W. Wolf (1921–), Marvin P Bryant (1925–2000), Bruce Ames (1928–), Richard L. Crowell (1930–), Peter C. Doherty (1940–), etc., encouraged the authors to write the stories of pioneers and/or include their own stories. They also verified the accuracy of the manuscripts and edited some of the manuscripts. Most importantly are the pioneers themselves

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who recounted their life experiences, allowing us to commemorate their stories. The choice of pioneers covered in this volume was entirely arbitrary, relying on the opportunity of gathering their information, names of sequences appearing in textbooks, journal articles or other literature describing them. There are many more pioneers not covered in this volume because time is limited and the writers are aging. There are many female pioneers not covered because many of them have been described in the first author’s book Women Pioneers of Medical Research: Biographies of 25 Outstanding Scientists (Mcfarland & Company, Inc., Publishers, Jefferson, North Carolina, and London, ISBN 978-0-7864-2927-1).

King-Thom Chung and Jong-Kang LiuJune 2017

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About the Authors

Dr. King-Thom Chung is a professor emeri-tus of Microbiology and Molecular Toxicology at the University of Memphis. He pioneered research on azo-induced cancer. His work on the genotoxicology of aromatic amines which metabolize from azo dyes led to the abate-ment of several azo dyes and their metabolic products, aromatic amines, to be used in industries in the European Union. He is the

author of more than 100 papers and the book Women Pioneers of Medical Research (McFarland & Company, Inc., 2009, ISBN:0786429275). He has also written six biographical books on bioscientists in Chinese.

Dr. Jong-Kang Liu is a professor emeritus of Biological Sciences at the National Sun Yat-sen University, Kaohsiung, Taiwan. His main research interest is environmental microbiol-ogy, and he has developed many bacterial strains that can degrade various industrial pollutants such as cyanide, dioxin, trichloro-ethylene, and pentachlorophenol. He has published more than 80 papers and has five

patents in the area of bioremediation. Besides that, he has also written 75 popular science articles, as well as five popular science books in Chinese.

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Contents

Preface v

Acknowledgments xiii

About the Authors xv

Chapter 1 Antonie van Leeuwenhoek (1632–1723): The First Microbiologist 1

Chapter 2 Robert Hooke (1635–1703): The First to Observe the Existence of Microorganisms 10

Chapter 3 Lazzaro Spallanzani (1729–1799): Fighting Against the Odds 14

Chapter 4 Edward Jenner (1749–1823): The First and Greatest Success of Immunization 20

Chapter 5 Agostino Maria Bassi (1773–1856): Pioneer of Studying Contagious Disease 31

Chapter 6 Ignaz Philipp Semmelweis (1818–1865): Savior of Mothers 36

Chapter 7 Louis Pasteur (1822–1895): The Master of Microbiology 49

Chapter 8 Ferdinand Julius Cohn (1828–1898): Pioneer of Bacteriology 69

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Chapter 9 Joseph Lister (1827–1912): Pioneer of Antisepsis 75

Chapter 10 Heinrich Anton de Bary (1831–1888): Pioneer of Mycology 84

Chapter 11 Thomas Jonathan Burrill (1839–1916): Pioneer of Microbe and Plant Diseases 90

Chapter 12 Gerhard Henrik Armauer Hansen (1841–1912): Pioneer of Leprosy Studies 97

Chapter 13 Robert Koch (1843–1910): The Great Medical Microbiologist 105

Chapter 14 Élie Metchnikoff (1845–1916): Phagocytosis and Immunology 121

Chapter 15 Charles Louis Alphonse Laveran (1845–1922): Discoverer of the Malaria Parasite 133

Chapter 16 Martinus Willem Beijerinck (1851–1931): Pioneer of General Microbiology 143

Chapter 17 Walter Reed (1851–1902): Yellow Fever Fighter 154

Chapter 18 Emile Roux (1853–1933): Diseases Fighter 167

Chapter 19 Emil von Behring (1854–1917): Pioneer of Serology 174

Chapter 20 Erwin F. Smith (1854–1927): Father of Plant Pathology 182

Chapter 21 David Bruce (1855–1931): Pioneer of Veterinary Microbiology 188

Chapter 22 Sergei N. Winogradsky (1856–1953): Founder of Soil and General Microbiology 195

Chapter 23 Kitasato Shibasaburo (1853–1931): First to Isolate Clostridium tetani and a Pioneer of Serology 214

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Chapter 24 Theobald Smith (1859–1934): The Captain of American Microbe Hunters 224

Chapter 25 Alexandre Yersin (1863–1943): Pioneer of Plague Fighter 237

Chapter 26 Albert Leon Charles Calmette (1863–1933): Antituberculousis and BCG Vaccination 242

Chapter 27 Charles J. H. Nicolle (1866–1936): Pioneer of Typhus Studies 260

Chapter 28 Howard Taylor Ricketts (1871–1910): Pioneer of Rickettsial Diseases Studies 265

Chapter 29 Chaim A. Weizmann (1874–1952): Pioneer of Industrial Microbiology and First President of Israel 271

Chapter 30 Oswald Theodore Avery (1877–1955): Microbiological Genetic Transmission and DNA 281

Chapter 31 Frederick Griffith (1879–1941): Discovery of Transformation 289

Chapter 32 Alexander Fleming (1881–1955): The Discovery of Penicillin 296

Chapter 33 Albert Jan Kluyver (1888–1956): Unity of Biochemistry and Pioneer of General Microbiology 309

Chapter 34 Gerhardt J. Domagk (1895–1964): Pioneer of Sulfur Drug Chemotherapy 315

Chapter 35 Paul Henry de Kruif (1890–1971): Gas Gangrene Research and Historian of Microbiology 322

Chapter 36 William C. Frazier (1894–1991): Pioneer of Dairy and Food Microbiologist 329

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Chapter 37 Ira Lawrence Baldwin (1895–1999): Pioneer of Agricultural Microbiology and Education 339

Chapter 38 Cornelis B. van Niel (1897–1985): Educator and Pioneer of Bacterial Photosynthesis and General Microbiology 349

Chapter 39 Max Theiler (1899–1972): Yellow Fever Vaccine Developer 358

Chapter 40 René Jules Dubos (1901–1982): Pioneer of Bacterial Antibiotics and Environmental Microbiology 369

Chapter 41 Barbara McClintock (1902–1992): Pioneer of Microcellular Directed Genetics 373

Chapter 42 George W. Beadle (1903–1989): Pioneer of Biochemical Genetics 381

Chapter 43 Edward Lawrie Tatum (1909–1975): Pioneer of Molecular Genetics 397

Chapter 44 Horace A. Barker (1907–2000): Pioneer of Anaerobic Metabolism 408

Chapter 45 Deam Hunter Ferris (1912–1993): Pioneer of Epizoonotic Studies 419

Chapter 46 Herman J. Phaff (1913–2001): Pioneer of Yeast Biology 430

Chapter 47 Harold Boyd Woodruff (1917–2017): Antibiotics Hunter and Distinguished Soil Microbiologist 441

Chapter 48 Ralph S. Wolfe (1921 to Present): Pioneer of Biochemistry of Methanogenesis 464

Chapter 49 Esther Miriam (Zimmer) Lederberg (1922–2006): Transduction and Replica Plating 472

Chapter 50 Marvin P. Bryant (1925–2000): Bacteria in Methanogenic Ecosystems 477

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Chapter 51 Joshua Lederberg (1925–2008): Pioneer of Microbial Genetics 485

Chapter 52 Hubert A. Lechevalier (1926–2015): Antibiotics Hunter and Actinomycetologist 493

Chapter 53 Thomas D. Brock (1926 to Present): A Successful Modern Microbes Hunter 504

Chapter 54 Arnold L. Demain (1927 to Present): A Giant of Industrial Microbiology 516

Chapter 55 Bruce N. Ames (1928 to Present): A Pioneer of Genetic Toxicology and Molecular Mutagenesis 527

Chapter 56 Richard L. Crowell (1930 to Present): Cellular Receptors and Viral Infection 538

Chapter 57 Peter Charles Doherty (1940 to Present): Pioneer of Immunology 550

Name Index 561

Subject Index 573

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Chapter 1

Antonie van Leeuwenhoek (1632–1723): The First Microbiologist

“ . . . from a craving after knowledge, which I notice resides in me more than other men. . . I have thought in my duty to put down my discovery on paper, so that all ingenious people might be informed thereof. . . my work springs only from an inclination I have to inquire into the beginnings of created things. . . .”

— Antonie van Leeuwenhoek

Source: https://en.wikipedia.org/wiki/Antonie_van_Leeuwenhoek(US Public Domain image)

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Introduction

Pliny the Elder (23–79 AD) wrote that the ancients of his time used tiny glass globules filled with water as a lens. (If you wish to get an idea of what they saw, take a bacteriological plati-num wire loop and dip it in clear water. You will find that it is a perfect lens with considerable magnification.) But the ancients including Archimedes (287–212 BC) and Claudius Ptolemy (100–170 AD) who had some knowledge about light refraction at the surface of glass or water, did not have any knowledge of the action of lenses. It was not until the time of Alhazen (965–1039 AD) that the action of lens was described. In his Optics (Opticase Thesaurus Alhazeni Arabius Basil, Lib. Vii, 1572, pp. 44–45), he says: “If an object is placed in a dense spherical medium of which the curved surface is turned towards the eye and between the eye and the centre of sphere, the object will appear magnified”. Vitello also wrote a book on optics in about 1270, but it is doubtful that Vitello had any real knowledge of optics. Contemporary with Vitellio was Roger Bacon (1219–1292), who described experiments with lenses. From his writing, he seemed to know the real value of lenses. Spectacles were invented probably by that time perhaps by Bacon, but it was stated on the grave of Salvino d’Armato (or spelled Salvino d’Armati (1258–1317)), a nobleman of Florence that he invented spectacles, but kept the secret of their manu-facture. It is Alessandro della Spina (–1313) of Pisa, who learnt how to make spectacles (probably from Armati) and made the method known to the public. Giordano da Rivalta (1260–1311), a fellow monk of Spina, said that the art of making spectacles was one of the most useful skills. It was only twenty years since its invention, and he had known the inventor. The use of spec-tacles and the method of making them began to spread over Europe and led to the invention of the microscope and telescope.

It was certain that the telescope was invented first. Jean Hendrick van Swinden (1746–1823) found evidence that Jacobus

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Metius (1571–1631), a native of Alkmaar in the Netherlands, applied to the State General for exclusive rights of selling tele-scopes that he had invented in 1609. Anton Maria Schyrleus de Rheita (1597–1660) in his Oculus Enoch et Eliae (published in 1645, p. 337) ascribed the invention was by Hans Lippershey (1570–1619) in 1608. However, Pierre Borel (1620–1671), physi-cian to the French king, in his De Vero TelescopiiInventore (On the True Inventor of the Telescope, published in 1655) claimed that the invention was Zacharias Janssen (1580–1638), son of Hans Janssen, a spectacle maker of Middleburg, Holland in 1590.

When the telescope was invented, it had immediate practi-cal value. It was useful in war and at sea. Galileo Galilei (1564–1642) also made a telescope in 1609. Galileo and Nicolaus Copernicus (1473–1543) were well known in the battle between “science and religion”, as they turned the telescope on the moon, the planets, and the cosmos. For the first time, Galileo observed the satellites of Jupiter and the phases on Venus.

Exploration of the world’s surface required vast outlays of capital, ships, and personnel. Even astronomy was dependent on costly astronomical structures and required coordinated observations and communications between scientists at many locations. The adventures with the microscope usually required only one individual and his trusty microscope! A single person, anywhere, could venture out into a world as unknown as that explored by Christopher Columbus (1451–1506) with his three ships and crews.

The microscope opened up vistas as great as any seen by Francis Drake (1540–1596), Ferdinand Magellan (1480–1521), and the other navigators, even as overwhelming as the cosmic space revealed by the telescope and astronomy. But microscopic revelations came slowly to the general public and even to sci-entists — and in a far different manner.

It is also believed that Zacharias Janssen might have made the first microscope. Historian believe that the microscope was an “accidental” discovery, as there was no reason for its use at the time, and the major stimulus to use was probably curiosity.

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The name “microscope” was given by a physician, Giovanni Faber (or Johann Faber) (1574–1629), who called the new opti-cal tube, a microscope because it allowed the viewer to see minute objects. But there was strong medieval distrust of opti-cal devices as being perhaps a work of evil standing between man and his “God-given” faculties. The cruse and fuzzy images of these microscopes probably reinforced this opinion!

Galileo used his telescope as a microscope and reported see-ing “flies which looked as big as lambs and were covered with hair”. He was not able to perfect lenses suitable for satisfactory microscopes. People first questioned what could be seen through the telescope and considered Galileo’s descriptions of moons and planets as “optical illusions”. The mass of evidence finally out weighted these suspicions, but people seriously doubted the first description of microscopic life.

Antonie van Leeuwenhoek (1632–1723): The First Microbiologist

Antonie van Leeuwenhoek (1632–1723), a merchant in the city of Delft, Holland, typifies the lone microscopist who is able to amaze the world and perplex his contemporaries with descrip-tions in words and pictures (micrographs) of the microbial uni-verse — not only the species of organisms, but also their environment. He also typifies what we will observe again of microscopists — they may be found in undistinguished circum-stances or in great universities.

Leeuwenhoek’s observations were made using simple micro-scopes, which were simply magnifying lenses. The magnifying lens had been used for research as noted in the preface to Mouffets’s Insectorum sivi Minimorum Animalium Theatrum, a manuscript now a British Museum, written about 1590. It was republished by Sir Théodore Turquet de Mayerne (1573–1655) in 1634; it is there stated that de Mayerne was accustomed to observing small insects with a magnifying glass.

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Antonie van Leeuwenhoek was born on 24 October 1632 in Delft, The Netherlands. His father died when Antonie was five years old; his mother sent him away to school when she remar-ried in 1640. In school, he learned basic mathematics and physi-cal sciences. It seems Leeuwenhoek was not preparing for one of the learned careers of the day or for the university since he never studied Latin. He left school in 1648 and apprenticed in a dry good store in Amsterdam. In 1653, he returned to Delft, married and sets up his own haberdashery. He was widowed twice and had six children: only one, Maria, lived to adulthood. She took care of him until his death at 91. Before he was 40 years old, he was appointed chamberlain to the sheriff of Delft. His duties were to open, clean, and lock the town’s law offices. He earned his living with haberdasher and chamberlain duties but his hobby of playing with magnifying lenses appeared to take most of his time.

This unknown and uneducated merchant/janitor developed an idiotic love for grinding lenses, which were to examine cloth for quality. He learned to grind and polish tiny lenses, which he mounted in metal plates. He made at least 550 of these in his lifetime. He grounded the best microscope lenses in the world in his day. He also turned his lenses onto everything he could place under them including muscle fiber of a whale, scales of his own skin, rainwater, human saliva, animals’ feces, fly brains, beetle eyes, spider spinnerets, frog skin, fish scales, and so on.

He was a very precise and mulish man. He looked through those lenses again and again. He was laughed at and ridiculed by his neighbors because of his strong love of lenses. Between 1657 and 1677, the newly formed Royal Society of London actively solicited works that “promote natural knowledge”. A corresponding member of the society who lived in Delft, Regnier de Graaf (1641–1673) did not laugh at him. On the contrary, he was amazed at what Leeuwenhoek could see through these lenses. Regnier de Graaf introduced him to the Royal Society of London in 1673. The Royal Society requested

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Leeuwenhoek to write and tell of his discoveries through these lenses. Leeuwenhoek knew only his native Dutch and wrote in that language in a small and neat handwriting. Leeuwenhoek was fortunate, or we are fortunate that Leeuwenhoek had a friend, Hoogvliet, who helped to translate Leeuwenhoek’s let-ters into Latin. In the first letter, he described how microbes multiplied in grounded pepper in water, until there were 2,700,000 microbes in a drop of water! Most of the members of Royal Society scoffed, but not all. Robert Hooke (1635–1703) and Nehemiah Grew (1641–1712) were commissioned to con-struct better microscopes (the compound microscope in England would not reveal the bacteria that Leeuwenhoek could see with a single lens!) and see if they could verify Leeuwenhoek’s report. Leeuwenhoek was disturbed by the Royal Society doubters and sent them affidavits from prominent citizens of Delft as well as his calculations in meticulous arithmetic (nearly as accurate as those done by modern microscopists with elabo-rate equipment). On 15 November 1677, Robert Hooke appeared at a meeting of the Society with his new microscope and ani-malcules from the ground pepper, which Leeuwenhoek had described. Every one crowded around and peered through the microscope. Leeuwenhoek’s first letter and letters communi-cated later were published in Philosophical Transactions.

Leeuwenhoek was right. Not long after that, on 8 February 1680, Leeuwenhoek was unanimously elected as a fellow of the Royal Society and was sent a handsome coat of arms of the Society in a silver case. This was a big day for Leeuwenhoek and he wrote that he would serve the society the rest of his life, a promise he kept; he never attended a meeting of the Society for the rest of his life. On his deathbed van Leeuwenhoek asked John Hoogvliet to translate two more letters into Latin as his parting gift to the Society. The Royal Society of London sent a doctor, Samuel Molyneux (1689–1728), to visit him and report on his microscopes. Today, we know that the best of his microscopes, although they were simple lenses, were far supe-rior to the compound microscopes in wide use at the time.

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They had a resolution of about a millionth of a meter. We believe that Leeuwenhoek had an excellent eyesight.

Leeuwenhoek observed, described, and made micrographs of the major types of bacteria and protozoa. In his 39th letter (1683), Leeuwenhoek described rods, cocci, and spirochetes in scraping from his teeth. He described that there were more animalcules living in the scum on the teeth in a man’s mouth than there are men in the whole kingdom especially in those who didn’t clean their teeth. He computed detailed measure-ments of the areas, volumes, and numbers of organisms he observed. During his whole life, Leeuwenhoek communicated a total of 375 letters in Dutch to the Royal Society; many of were of great length. Between 1939 and 1983, the Leeuwenhoek letters were translated into English by large committees of Dutch scientists, yield and 11 volumes with English and Dutch texts on opposing pages. These volumes include extensive annotation.

Leeuwenhoek was elected to the French Academy of Science in 1680. Throughout his life, he sent 27 letters to the French Academy for publication (Antonie van Leeuwenhoek, 1684) Leeuwenhoek became very famous in his time and was visited by the Queen of England, who went to Holland only to see his microscopes. Peter the Great, of Russia, also called on him. In 1716, the University of Louvain in Belgium honored him with a silver medal engraved with a portrait of him for his cel-ebrated discovery in Natural Philosophy. Before his death, van Leeuwenhoek made 26 lenses mounted in sliver plates. He bequeathed these to his daughter to give to the Society after his death. She did send them but unfortunately, all of van Leeuwenhoek’s microscopes have since been lost.

Commentary

Leeuwenhoek never once associated his “wee beasties” with disease. Otherwise, modern medicines would develop much ear-lier. We may fairly call Leeuwenhoek “The first microbiologist”

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because he was the first individual to actually culture, see, and describe a large array of microbial life. He actually measured the multiplication of the bugs. What is more amazing is that he pub-lished his discoveries. Evidence shows that Leeuwenhoek was aware of the Micrographia published by Robert Hooke. Although Hooke first observed the existence of microorganisms and deserved to be considered a distinguished microscopist, but he did not identify the microorganism.

Leeuwenhoek should be ranked one of the great naturalists and explorers. Leeuwenhoek represents the naturalists who changes human attitudes about nature through written descrip-tions and illustration — or the explorers who expands the hori-zons. Leeuwenhoek invented the simple microscope or strictly speaking a magnifying lens and, more importantly, used it to explore uncharted territory. He was a naturalist who took to the field to study nature, as did Charles Darwin (1809–1882) and James Hutton (1726–1797).

There were much progress on the technology of micros-copy after Leeuwenhoek. For example, Edward Culpeper (1660–1738) in 1730 put a mirror in the optic axis and helped focus the microscope by sliding one tube in another one. Ernst Abbe (1840–1905) developed condenser and applied an oil immersion technique in 1878. In 1935, Frits Zernike (1888–1966) first developed phase-contrast microscopy. There are many microscope makers all over the world. Magnificent microbial worlds were revealed in great detail; more are still to be discovered. With the recent development of electron microscopy by Ernst A. Ruska (1906–1988) and Max Knoll (1897–1969) in 1932, microcellular constituents and even molecular structures can be visible. What microscopy could do in the past may give a hint of what it might do in the future. Modern developments in microscopy, which is still actively going on, will undoubtedly lead us into a new stage of micro-biological research. The refreshing memory of the history of microscopy and the pioneers who discovered microorganisms and studied these organisms are worthwhile indeed.

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Suggested Reading

1. van Leeuwenhoek, A. (1677). Concerning little animals observed in rain, well, and snow water, as also in water wherein pepper had laid infused. Philosophical Transactions of the Royal Society of London 1(33): 821–831.

2. van Leeuwenhoek, A. (1684). Microscopical observations about animals in the scurf of the teeth. Philosophical Transactions of the Royal Society of London 14(159): 568–584.

3. Clay, R. S. and T. H. Court (1985, First edition 1932). The History of the Microscope. The Holland Press, London.

4. de Kruif, P. (1926). Microbe Hunters. Chapter 1, pp.1–24. Harcourt Brace & World, Inc., New York.

5. Gest, H. (2004). The discovery of microorganisms revisited. American Society News 70(6): 269–274.

6. van Leeuwenhoek, A. (1977). The Select Works of Antony van Leeuwenhoek: Containing His Microscopical Discoveries in Many of the Works of Nature. Vol. 1& II. Translated by Samuel Hoole. Arno Press, Inc., New York.

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Chapter 2

Robert Hooke (1635–1703): The First to Observe the Existence

of Microorganisms

Source: https://en.wikipedia.org/wiki/Robert_Hooke(US Public Domain image)

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Introduction

Robert Hooke (1635–1703) and Antonie van Leeuwenhoek (1632–1723) played similar roles with the microscope and the opening up of the microbial world. This is significant when we realize the importance of the cellular structure of life and the impact of microbiology on disease and health.

Robert Hooke published his book, Micrographia in 1665. Today, we realize that what Galileo’s Sidereus Nuncius did for the telescope, Hooke’s book did for the microscope, although it took longer for his observations to be accepted because of prejudice.

Life of Robert Hooke

Robert Hooke was born in 1635. We know very little about his childhood. He was enrolled as an undergraduate at the Christ Church College at the University of Oxford, but he did not obtain a degree there. Nevertheless, he became associated with a brilliant group of scholars including Christopher Wren (1630–1723) and Robert Boyle (1627–1691), who met regularly to dis-cuss a broad range of scientific problems. In 1662, he was a founding member of the Royal Society and served as “Curator of Experiments”. His duties included conducting “considerable experiments” and doing other research projects officially rec-ommended to him. As a curator, he provided the main sub-stance of many meetings and gradually became a commending intellectual force in the Royal Society. In 1663, he lectured on the structure of plant tissues such as cork and moss, he first coined the term cell. In 1665, he published Micrographia and described the structure of cork in detailed in Obs. XVIII. “the texture of cork, and of the cells and pores of some other such frothy bodies”.

However, Hooke not only described his observations in words, but also published 57 truly great micrographs, including for the first time, the eye of the insect, a bee’s stinging organ,

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the anatomy of the louse and flea, and the structure of peacock feathers. He also illustrated the microscopic view of diverse bio-logical objects including sponges, wood, seaweed, leaf surfaces, hair, fly wings, eggs of silkworms, mites, a flea, and a louse as well as microfungus Mucor, a common bread mold, the first view of the microbial world. Hooke obtained the microfungus specimen from a small white spot of hairy mold, many of which he observed on the red sheepskin covers of a small book. We, therefore, should recognize him as the first to observe microorganisms.

Hooke’s contribution is beyond description. The publication, Philosophical Transactions, the official journal for the Royal Society of London, is the forerunner of the modern day scientific journal, which marked a new era of natural science. His scien-tific talents were broad and his genius had many sides. Physicist John Desmond Bernal (1901–1971) wrote of Hooke in 1965, “his interests ranged over the whole of mechanics, physics, chemis-try, and biology. He studied elasticity and discovered what is known as Hooke’s law. . . . He invented the balance wheel, the use of which made possible accurate watches and chronometers; he wrote Micrographia, the first systematic account of the microscopic world, including the discovery of cells; he intro-duced the telescope into astronomic measurement and invented the micrometer”.

Gest believed that Hooke’s major contributions to microbi-ology have been minimized or ignored because of the slanted perspectives fostered by two widely read books. The first more influential is the book by protozoologist Clifford Dobell (1886–1949) of 1932, Antony van Leeuwenhoek and His “Little Animals”. Dobell stressed the Leeuwenhoek’s observations of protozoa and bacteria and took limited view of the scope of microbial life. Dobell’s book omitted any mention of Hooke’s discovery of microfungi and his contribution to microscopy nor cited Micrographia, which was simply listed among “Other Reference and Sources”. The other is Paul de Kruif’s (1890–1971) popular 1926 book “Microbes Hunters”. This book mentioned

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Hooke only once, simply as a person who confirmed Leeuwenhoek’s discovery of bacteria. As a matter of fact, it is Leeuwenhoek who confirmed the discovery of Hooke, not the other around in view of the timing. The other factor is that the cell theory that has remained basic to biology and medicine, and was so overwhelming to science that it is credited to Hooke, and microbial world’s observation was comparatively minor in comparison to cell theory. Nevertheless, Hooke rather than Leeuwenhoek was the first to observe and document the existence of a microorganism.

Suggested Reading

1. Gest, H. (2004). The discovery of microorganisms revisited. American Society News 70(6): 269–274.

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Chapter 3

Lazzaro Spallanzani (1729–1799): Fighting Against the Odds

Source: https://it.wikipedia.org/wiki/Lazzaro_Spallanzani(US Public Domain image)

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Introduction

Leeuwenhoek, whom Paul de Kruif has called “the first of the microbe hunters” (see Microbes Hunters by Paul de Kruif, Harcourt, Brace & Co., 1926) died in 1723. The publication of his letters with micrographs of bacteria, molds, and protozoa, by the Philosophical Transactions, the official publication of the Royal Society of London, which was the forerunner of our mod-ern scientific journals, amazed the world. The Royal Society’s acknowledgement of his findings and their generous support brought him fame in scientific circles as well and to the public at large. He was visited by the Czar of Russia and the Queen of England. The world was aware of the “animalcules (microbes)”, but there was no immediate successor to Leeuwenhoek. There was no attempt to connect microbes in any way with diseases. As a matter of fact, not more than 200 years ago, people includ-ing scientists, philosophers, intellectuals, or the general public believed that toads, snakes, and mice could be born of moist soil; that flies could emerge from manure; and maggots, the larvae of flies, could arise from decaying corpses. Many scien-tists of that era believed that “to question that beetles and wasps are generated in cow dung is to question reason, sense, and experience”. Leeuwenhoek’s “animalcules” were simple enough to be generated from nonliving materials. Every living creature was thought to be spontaneously generated. This is so called “spontaneously generation theory”. However, Lazzaro Spallanzani was an exception. He would take off where Leeuwenhoek stopped in the study of microscopic life.

Life of Spallanzani

Lazzaro Spallanzani was born on 12 January 1729, in Scandiano in the northern part of Italy, probably into an affluent family. He had a brother called Nicolo and a sister whose name is not known. His father was a lawyer and he wanted him to be a lawyer too. As a boy, Spallanzani loved to roam through the

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woods. When he saw natural fountains spring from the earth, he was told by his priest that this was from the “tears of sad, deserted beautiful girls who were lost in the woods”. “The sci-entific concept of nature” was not general in that time. Spallanzani was a very clever man. He had unboyish thoughts challenging such concepts even as a boy. Although his father wanted him to study law, he secretly worked on mathematics, Greek, French, and Logic, hoping to get into science. He also went to a noted scientist, Antonio Vallisneri (1661–1730), and told him about his interest. Vallisneri persuaded his father to send him to a Jesuit college. He changed to Reggio de Modena in 1744 and finally studied at University of Bologna from 1747 to begin his career in science. He studied mathematics with his cousin, Laura Bassi (1711–1778), the famous woman professor of Reggio. He wrote a scientific paper on the mechanism of skipping a stone on water. It was now more respectable to go into science than in Leeuwenhoek’s day. The Grand Inquisition was now torturing heretics rather than persecuting scientists such as Gallileo Galilei (1564–1642). It was beginning to be fashionable to study science; Voltaire (1694–1778), the great French philosopher, even studied the discoveries of Issac Newton and popularized them in his country.

At the age of 25, Spallanzani was ordained as Catholic priest, but he was not a blind follower of the faith. He savagely questioned everything, taking nothing for granted except the existence of God. He secretly despised all authority, but he got himself snugly into the good graces of authority. Before the age of 30, he was made professor at the University of Reggio. He was a good and popular teacher.

Spallanzani had strong skepticism about spontaneous gen-eration theory. Although the prominent scientists of his day believed and taught the theory, but Spallanzani did not. First of all, he read report of a scientist named Franceso Redi (1626–1697) who put meat in two jars, one of them covered by cheesecloth. Redi observed that flies laid eggs on the uncov-ered meat, which hatched out onto maggots and became adult

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flies; the flies could only deposit eggs on the cheesecloth in the covered meat, which did not get infested with maggot. Spallanzani decided that instead of arguing, he would try an experiment with Leeuwenhoek’s “animalcules”. With difficulty, he obtained a microscope and learned how to use it.

Priests at that time made up a large proportion of the pro-fessors in the universities. Spallanzani became a participant in a great debate with an English priest and educator, John Needham (1713–1781). Needham was also an experimenter with the wee beasties. The big debate of that era was their origin. Needham brought to the Royal Society reports on a series of experiments with mutton gravy. He also used seeds, crushed almond, and other media. After closing and heating the flask, he opened it, and by using his microscope, found it swarming with “animal-cules”. The Royal Society and scientists generally, accepted the reports as proof of spontaneous generation of all microscopic life. It was believed that heating destroyed any life that might have been in the soup, but that, with air and warmth, micro-scopic life arose again, “spontaneously”.

Spallanzani did not believe this theory, and he was almost alone in his rejection. He knew that he must design experi-ments that would show that either he or Needham was right. He decided that Needham had not cooked the soup long enough. Spallanzani boiled the flask for an hour. He also devel-oped methods of sealing the necks of the flasks with heat, rather than using corks. After leaving the flasks for various lengths of time, Father Spallanzani opened them and examined the contents under the microscope. No microbes! We have to know that similar experiments were performed by Louis Joblot (1645–1723) on 13 October 1711, long before Spallanzani was born. Louis Joblot lived before the time of Leeuwenhoek; microbes were not known in his days. Joblot’s work was unfor-tunately not well discussed at Spallanzani’s time.

The great debate between the two priests began. Needham had a wealthy French supporter, the great naturalist Georges Leclerc count de Buffon (1707–1788) who enjoyed science and

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scientists. He asked Father Needham what caused the animal-cules to arise out of the mutton gravy and other soups. “My Lord, it is Vegetative Force”, Father Needham replied and the name stuck. All the scientists of Europe were now talking about “Vegetative Life Force”. The Royal Society accepted the experi-ments and the lectures and made Needham a Fellow of the Society. Needham and the wealthy count de Buffon published and lectured widely. The French Academy of Science made Needham as an associate. Buffon was elected as a member of the Academy in 1739.

Spallanzani was a priest who believed strongly in God. He also believed that God supported the truth. Father Needham had even stated that it was “Vegetative Force” that made Eve grow from Adam’s rib. All of this was nonsense to Spallanzani, but he had to find a way to meet his fellow priest’s pretty words with facts. He designed an experiment. He took large glass flasks, round bellied with tapering necks. He put various kinds of seeds into one, and peas and almonds into other, fol-lowed by pouring pure water in all of them. He boiled all of these flasks for an hour. He then melted the necks of his bottles shut in a flame, so nothing sneak in. He proved that in no soups of any kinds would spontaneously generate life; but when con-taminated air was admitted, life appeared. He did similar experiment many times and proved conclusively that no “Vegetative Force” was needed for life.

Spallanzani’s idea was finally accepted by the scientists of Europe; but the question still arose: “where do the microbes come from?” Observation by microscope often showed two bacteria or other organisms, side by side. Some thought there might be a form of mating, as in higher forms of life. Others thought that in their violent movements in the liquid broth a bacterium might strike another one and break it into two. One of the triumphs of Spallanzani’s experimental methods was the development of what we call today a “pure culture”. He set himself the task of trying to isolate a single bacterium in a clear medium and watch it divide under the microscope.

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Finally, he was able to segregate just one tiny bacterium under his lens. No other bacterium could jostle it or break it and there was no other to mate with it. His excitement knew no bounds when he saw the single cell begin to elongate, pinch off into two cells, identical but each slightly smaller than the original. Soon these two became normal in size and divided as did the first one. He repeated this experiment a dozen times before publishing the results to the astonishment of scientists of his time.

He also worked on how bats find their way in a cave. It has nothing to do with microbiology, but it was a great finding in animal behavior. Spallanzani died on 12 February 1799.

Commentary

Spallanzani was an innovative scientist. His life and struggles against the false concepts of leading scientists of his day have been eclipsed by the stories of the famed Louis Pasteur (1822–1895) and Robert Koch (1843–1910). But he was a worthy suc-cessor to the great Leeuwenhoek. His courage in the face of ridicule by scientific leaders and his creative skill as an experi-mental scientist in demonstrating clearly that microbial life arose from preexisting microbial life are examples worthy of emulation, even today.

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Chapter 4

Edward Jenner (1749–1823): The First and Greatest Success

of Immunization

“…to merit and to the immense services rendered by one of the greatest of Englishmen Jenner”.

— Louis Pasteur (1822–1895)

Source: https://en.wikipedia.org/wiki/Edward_Jenner(US Public Domain image)

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Introduction

Before the era of Louis Pasteur (1822–1895) and Robert Koch (1843–1910), founders of modern microbiology, lived a scientist who worked with microbial disease and perfected a vaccination technique against human smallpox. He probably saved more lives than any other single physician. He also greatly influenced the work of Pasteur and Koch.

The smallpox (variola) virus has killed more people than any other microorganism. The virus probably evolved from an orthopox virus in Africa. It is likely that Egyptian armies were responsible for transmitting the disease to the Middle East. In 166 AD, Roman soldiers brought smallpox to Europe where it killed up to 200 people a day. In 900 AD, the Persian physician Rhazes (865–925) recognized smallpox and measles as two dis-tinctly different diseases. Over the centuries, smallpox became the leading infectious disease, and epidemics ravaged the world.

European colonization brought smallpox to America and Sub-Saharan Africa. The Spanish conquest of Amerindians in the 16th century coincided with the epidemics of smallpox that reduced the Amerindian populations so they were unable to resist the army of Hernando Cortes (1485–1547) and Francisco Pizarro (1471 or 1476–1541). In 1721, 5,759 cases of smallpox were reported in Boston (population of 10,700) and one in every seven patients died. By the 18th century, smallpox was killing 200,000–600,000 people every year in Europe.

In the East, this disease was equally dreadful, although we had no record of the death toll. But there was great misery. In China, smallpox was first reported in the 1st century; and it was believed to be imported from somewhere else. Over the many centuries, Chinese physicians developed different techniques to prevent smallpox. Perhaps, the best technique was done by removing scars from drying pustules of a person suffering from a mild case of smallpox, grinding the scars to a fine powder, and inserting the powder into the nose of the person to be

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protected. The inoculated person would usually develop a mild case of smallpox, but would eventually develop immunity to it. This method of prevention had been practiced widely in the 17th century in all of China. The basic concept for the Chinese physicians was “poison against poison”. Although the concept of immunity was not developed at that time, the method worked effectively and saved millions. In 1688, Russian doctors were sent to Beijing to learn this technique of “human-pox inoculation” from the Chinese, so Russia also practiced this method of smallpox prevention. A similar method was also practiced in Turkey.

One recorded vaccination was practiced in 1774 by Benjamin Jesty (1736–1816), a farmer and cattle breeder in the Dorset vil-lage of Yetminster, England. Jesty believed that an attack of the relatively trivial cowpox, or vaccinia (vacca, a cow) in humans afforded a protection against smallpox. He himself had con-tracted cowpox by contagion from a cow. He inoculated his wife and two sons using a stocking needle with the virus from lesions on the udders of a sick cow. His family were later fre-quently exposed to infection from smallpox but never got the disease. In 1789, Surgeon Trowbridge of Cerne inoculated his two boys with smallpox; these two boys did not contract the disease. (Parish, A history of immunization, Livingstone, 1965.)

This procedure was known and done by some others too. But they did not understand the knowledge of immunity. It was Jenner who proved the validity of this practice and made this vaccination practicable, which saved many lives. This is why we recognize his contribution and want to learn how he did it.

Smallpox was a frightening disease that lasted two weeks or longer. Transmitted through the respiratory route, the virus infected internal organs before being transported through the blood to the skin. Viral growth in the epidermis caused small bumps on the face, upper body, and arms. Over several days the bumps filled with fluid, became inflamed, broke, and formed a soft yellow crust. Those who recovered from the disease carried disfiguring scars.

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In 1967, smallpox was endemic in 30 countries, infecting 10–15 million people with two million deaths annually. The World Health Organization (WHO) initiated an eradication campaign. The campaign was so successful that by 1971 the value of smallpox vaccination was questioned. Between 1951 and 1970, 103 cases of smallpox with 37 deaths were reported in Great Britain. During that same period, 100 people had died due to smallpox vaccination. The disease has not occurred in the United States in over 30 years so the United States discon-tinued vaccination of children. Only travelers to endemic coun-tries had to show proof of immunizations, yet physician used 2.5 million doses of the vaccine in 1978 (endemic countries in 1974 were Bangladesh, India, Pakistan, and Ethiopia). WHO predicted the last cases of smallpox would be seen in 1975, but the last 192 cases of smallpox cases of smallpox in the world occurred in a 1977 outbreak in Somalia.

The last case of smallpox resulted from an accident with a laboratory culture at the university of Birmingham medical school in 1978. Nearly all laboratory cultures were destroyed after that. By 1995, the viral genome had been mapped so the remaining cultures in Moscow and Atlanta were scheduled to be destroyed in 1996.

Smallpox is the first disease that has been eradicated. This was possible, in part, because humans are the only host for the virus. Smallpox virus was in a tenuous position for parasite; if all its hosts are killed or if they developed immunity, the virus had no place to propagate. Since the virus didn’t kill all of its host and new hosts are always born, evolution worked in favor of the virus — until human ingenuity provided another obstacle.

The Life of Edward Jenner

Edward Jenner was born on 17 May 1749, in Vicarage in Berkeley, a market town in Gloucestershire, England. He was the sixth child of an Anglican clergyman, Stephan Jenner. His mother was the daughter of a clergyman, Reverend Henry

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Head, who was at one time a church official in the Cathedral of Bristol. Both his parents died before he was five years old. He was raised by his brother, also called Reverend Stephen Jenner, who inherited his father’s position. His family was relatively affluent and held some property in Gloucestershire.

In his early years, Edward went to a school named Wotton-under-Edge. Edward’s first schooling was received from the Reverent Mr. Clissold. Later he went to Cirencester, an old Roman town of Gloucestershire. He was placed under tutorage of the Reverend Washburn. He became respectably proficient in the classics. He was interested in natural history. When other boys would be out playing, he would spend hours devoted to a search for fossils or other natural objects. He was often seen wandering alone in the meadows around his home. Later in life, he referred to these meadows as his natural dwelling place. They served as a school for natural self-training in accu-rate observation.

At the age of 13, Edward was apprenticed to Dr. Daniel Ludlow, an eminent surgeon apothecary at Bristol. Edward stayed with him for many years (1762–1770), then went to London in 1770 where he had the privilege of residing as a favorite student in the family of famed Dr. John Hunter (1770–1773). Hunter had been a surgeon at St. George’s Hospital; he was also studying the habitats and anatomical structure of ani-mals in a menagerie/laboratory. Edward respected and loved the man. The independence and boldness of Dr. Hunter’s char-acter had a deep and permanent influence on Edward’s mind. It was a fortunate that these two men were brought together since they both had an unquenchable desire for knowledge of truth.

In 1771, Edward helped Hunter arrange the zoological specimens gathered by Mr. Joseph Banks (1743–1820) who sailed with Captain James Cook (1728–1779) on his voyage of discovery to the Pacific. Jenner aided Hunter’s zoological stud-ies in many ways. Hunter’s insistence on exact observations exerted a great influence on Jenner’s pursuit of knowledge.

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Edward Jenner returned to Berkeley to practice medicine in 1773, but he kept in touch with Hunter through letters, which he carefully preserved in a special file. He became an experi-mental scientist himself and applied his science to the good of humanity. Through correspondence, Edward Jenner and Hunter studied many subjects together such as hibernations of animals. Hunter encouraged Jenner to study the habitat of cuckoos, the migrations of birds, and the distemper of hogs. He also studied tuberculosis, ophthalmia, and manures.

In his leisure time, he played piano and flute, made draw-ings, and also fancied himself as a poet. His best poems were “Address to Robin” and “The Signs of Rain”, which had a sim-ple beauty.

In 1778, Edward married Miss Catherine Kingscote. This was a happy marriage, resulting in two sons and a daughter who lived to adulthood. Edward believed strongly in God and also in the beauty of nature. He believed children should be taught the wonders of nature and his wife supported his belief. They established schools for the poor around them for the teaching of the scriptures. Many stories were written about Jenner and his wife as they helped the needy and less fortunate people.

Jenner was described as a handsome man of stocky built who liked to dress well. He was generally loved and admired for his kind personality and his determination to go to the sick through storms, snow, or mud. He did not mind to look at a farmer’s cow.

Jenner was a competent physician. He was always con-cerned with some medical problems. For example, at a small medical society meeting he pointed out that people who had rheumatism often later had a heart attack. He was perhaps the first to note the relationship between this disease and heart trouble later.

Smallpox was one of the great diseases of the world at Jenner’s time. Fatalities from the disease were enormous, and those who survived often were pitted with ugly and disabling scars. In 1717, when Lady Mary Wortley Montagu (1689–1762)

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returning from Turkey, she brought the method to England called inoculation. Most of the time inoculation resulted in a mild case and more rarely in a full-blown case of the disease. Jenner’s great contribution of a vaccination for the disease and immunity did not come by chance but was based upon his careful observation.

It was while aiding Dr. Ludlow that Edward Jenner was first associated with the question that would haunt him until it was answered. One day, Dr. Ludlow was summoned to West England to look at a young dairymaid who was ill with what seemed to be smallpox. However, upon mentioning of smallpox by the doctor, the young woman insisted that she already had the cowpox from cow in the dairy and “nobody caught smallpox after having cowpox”. This idea intrigued the curiosity of young Jenner. He discussed it with Dr. Ludlow who told him that there was no truth to what the dairymaid said. It was only an old wives tale, and to be a good doctor he must learn to ignore such sayings. However, this experience impressed Jenner.

When cows suffered cowpox disease, their teats and udders would be pitted with sores similar to those in human cowpox. People milking cows pick up the “vacciniae” as the cowpox pustules were known and came down with a mild disease, from which they recovered. As the dairymaid told Jenner, she did not know anyone working with cattle, who had cowpox to come down with human smallpox.

The only one way of avoiding a severe case of smallpox was the method brought back by Lady Montagu from Turkey. Jenner was familiar with inoculation. Edward still wished there was a better way of protecting people. In 1778, there was a seri-ous smallpox epidemic in England. People who had not been inoculated were afraid of getting sick. So Edward Jenner was busy giving inoculations. In most people, a mild case was devel-oped when Edward Jenner inoculated them with smallpox. However sometimes, especially among farmhands, the smallpox matter he placed into their skin did not affect them. When he asked them if they ever had smallpox, they always told him no, but that they once had cowpox. After hearing these remarks,

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Edward Jenner immediately thought of the dairymaid from years ago, who said cowpox protected people from smallpox. He might also have recalled the cowpox vaccination practiced by Benjamin Jesty in 1774. He began his work eagerly to find out all he could do about cowpox. He went to farms to look at cows or humans believed to have cowpox. Gradually, he learned that farmers did not really know whether a cow was suffering from cowpox or some other disease. Jenner had to learn himself that how to recognize the disease. He spent a considerable amount of time learning about cowpox. He observed all the types of milkers’ nodules produced upon exposure to various kinds of cowpox. He found that not all types conferred immu-nity and was able to identify those that did. The small hollow cysts on the cow’s teats and udder contained a clear fluid, which infected the hands of milkers, producing sores. Edward Jenner found that those pox cysts on the cow about the eighth day of the disease were most likely to result in immunity for the milker. Taking the fluid at other times might produce sores, but would not produce immunity. He came to the conclusion that both cowpox and swinepox were forms of human smallpox. Now, he was ready to prove his theory that cowpox inoculation would produce immunity.

By the time, Jenner had the time and preparation to test his theory, cowpox become rare. In 1796, a report came in that cows at nearby farm had the disease. Edward rushed over to the farm. He first examined the cows and found the marks of real cowpox. Eagerly, he asked if any of the farmhands had come down with the disease. He was glad to discover that Sarah Nelmes, a dairymaid, had a large sore on one hand. He immediately asked to see her. He examined the sore and removed some of the cowpox matter (lymph). On 14 May 1796, Jenner inoculated an eight-year-old boy named James Phipps with this cowpox matter. After seven days, James complained about a pain in his armpit. Two days later, he had lost his appe-tite, ran a slight fever, and had a headache. He spent that night restlessly but the next morning was perfectly well.

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Edward waited until July (six weeks later) to inoculate James with pustular material from a full-blown case of human small-pox. Every morning after that day, Jenner rode up to the cottage where James lived, and every morning James ran out to meet him. There was no need to ask how he was for Jenner could tell for himself. The boy remained well. He was immune from small-pox. Jenner had demonstrated the success of “vaccination” for smallpox. In 1798, Jenner inoculated several children with cow-pox again and tried to give them smallpox as well. Every single child was safe. He was able to substantiate his claims.

He also published his famous booklet titled An Inquiry into the Cause and Effects of the Variolae Vaccine. This study com-prised of records of 23 cases. However, Jenner’s great report at first evoked resistance, not acceptance. He failed to arouse much interest in physicians or patients. Then, Mr. Henry Cline (1750–1827), a surgeon with whom Jenner had left some dried cowpox serum, used a quill of it as a counterirritant in treating another disease and found later that his patient had become immune to smallpox inoculation. From interest created by Cline’s report, this type of vaccination began to spread. The children of the king were then vaccinated, in spite of the oppo-sition of many physicians.

Edward Jenner published two more pamphlets on vaccina-tion: Further Observation on the Variolae Vaccine or Cow, in 1799; and A Continuation in 1780. In 1800, Jenner was officially invited to London to vaccinate the 85th regiment of the British Army.

Neither Jenner nor anyone else knew, at that time, that immunity would wear off, requiring what we today call “booster shots”, or supplementary inoculations. This knowl-edge was not acquired until Jenner had been in his grave for some time. Nevertheless, smallpox vaccination saved thousands and thousands of lives.

Although Jenner had begun his practice of medicine at age of 23, it was not until 20 years later that he was granted an MD degree from St. Andrews University (1792). In 1788, he was

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elected as a Fellow of the Royal Society. Parliament voted him a sum of 10,000 pounds in 1802, and in 1806, another grant of 20,000 pounds. He spent most of the money on his work in cor-respondence, preparation of vaccine, and similar efforts. In 1805, Napoleon (1769–1821) ordered all French soldiers to be vacci-nated if they had not contracted the smallpox virus. Napoleon also ordered a medal to commemorate the discovery of vaccina-tion. And, at Jenner’s request, English prisoners that were cap-tured during the war between France and England were released.

Jenner continued to live humbly and practiced medicine in Berkeley. He purchased a home in Cheltenham and was listed as a resident physician. He was the founder of Cheltenham Literary and Philosophical Association. The honorary degree of Doctor of Medicine was given to him by Oxford at the age of 64 (1813), Jenner almost retired from public life even though he continued his work on vaccination and nature studies. In the midst of fame and fortunate, his health declined. His wife died in the year of 1815, which gave him a blow. Jenner suffered an apoplectic attack at age of 71 but still worked scientifically. In 1822, Jenner wrote On the Influence of Artificial Eruptions in Certain Diseases and in 1823, he presented a paper titled On the Migration of Birds to the Royal Society. He is also a pioneer in the field of animal behavior. At age 74, a cerebral hemor-rhage was fatal to Edward Jenner on 26 January 1823. The English government offered the family a burial plot in Westminster Abbey, but the expense of a public funeral pre-vented the acceptance of the offer. The burial in Berkeley was selected and held in the churchyard.

Commentary

Jenner’s cowpox (vaccinia) virus and this live vaccinia prepara-tion was used with little modification for the next 150 years. Jenner did not know that he did, in fact, provide the means to “take smallpox away from the world”. In 1881, at the International Medical Congress in London, Louis Pasteur coined

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the term vaccination “to merit and to the immense services rendered by one of the greatest of English men Jenner”. In 1980, WHO proclaimed global eradication of smallpox.

Jenner was also a great pioneer of animal scientist. Jenner’s most profound interest was birdwatching. His assay on the Migration of Birds is still a masterpiece thesis of animal lore. He discovered that it was the robin and not the lark that heralded the daybreak and determined the order of appearance of the songs of the raven. His pioneering study on the migration of birds is a significant contribution to animal behavior study. There is a statue of Jenner in Gloucester Cathedral. Jenner received many well-deserved honors in his day. He was received by the King and Queen of England, and his contribution was promulgated by Prince of Wales, a strong supporter. He was well liked by everybody. Jenner’s brilliant scientific investiga-tion and his immunology, before the term was even known, are still an inspiration. He deserved to be remembered by every microbiologist.

Suggested Reading

1. Baxby, D. (1981). Jenner’s Smallpox Viruses. General Britain Heinemann Educational Book, Ltd., Redford Square, London.

2. Dolan, E. F. J. (1960). Jenner and the Miracle of Vaccine. Dodd, Mead and Co., New York.

3. The McGraw-Hill Encyclopedia of World Biography; An International Reference Work (1973). McGraw-Hill, New York.

4. Parish, H. J. (1965). A History of Immunization. pp. 24–25. E. & S. Livingstone Ltd., Edinburgh and London.

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Chapter 5

Agostino Maria Bassi (1773–1856): Pioneer of Studying Contagious Disease

Source: https://en.wikipedia.org/wiki/Agostino_Bassi(US Public Domain image)

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Introduction

Agostino Maria Bassi (1773–1856) discovered that a microscopic fungal parasite was the cause of silkworm disease. He was the first established the doctrine of microbial parasitism and who postulated theories on, or worked in many other important areas of agriculture, science, and medicine. His work contrib-uted greatly to the understanding of contagious diseases. He was also the first to notice that a disease was transmittable between humans and animals. He was a pioneer to the field of infectious disease and zoonosis.

Life of Agostino Bassi

Agostino was one of twins born on 23 September 1773 in the village of Mairago in Lombardy, Italy. His father was Onorato Bassi and his mother Rosa Sommariva. We do not know any-thing about his twin brother’s life except his name is Giovanni Francesco. His parents were very concerned about Agostino’s education. He attended school at a very early age in Lodi and later entered the University of Pavia. He studied law but was interested in science. He took many science courses including physics, chemistry, mathematics, natural sciences, and some medicine. Among his teachers were Antonia Scarpa (1752–1832), anatomist; Alessandro Volta (1745–1827), a well-known physicist, and Giovanni Rasori (1766–1837), a professor of pathology. He also had the chance to attend classes of Lazzaro Spallanzani (1729–1799) who opposed the hypothesis of “spontaneous generation” and carried out experiments to disprove it. We can see that he got a very good education, which laid down the path for his great scientific discovery later in life.

He received his doctorate of jurisprudence on May 1798, and was named provincial administrator and police assessor in Lodi, Italy, which was under French control at that time. Later he held various positions in civil service. Because of his bad

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eyesight, he returned to Mairago where his father had a farm and remained there for most of the rest of his life.

In addition to the work of silkworm disease, Bassi was also interested in other agricultural related subjects such as breed-ing of merino sheep, potato cultivation, and wine making. He published among other things a book Il Pastore Bene Istruito (The Well Educated Shepherd).

The silkworm disease was known in Italy as mal del segno, calcino, or calcinaccio because of the white efflorescence and calcined appearance that developed after the worms had died of its ravages. The same disease was known to French as la mus-cardine. In the beginning, Bassi suspected that the silkworm disease might be generated spontaneously, and he studied vari-ous environmental factors such as food, atmospheric condi-tions, methods of breeding, and phosphoric acid, which might cause the disease. After intensive studies, he concluded that the disease was not due to any of these factors but rather due to external agents. He demonstrated that the disease was trans-mitted by foods, contact with the dead worms, and contact with the hand and the clothing of the silkworm breeders. The germs were also carried by animals/flies. Bassi reproduced the disease by inoculating healthy worms with the white dust or with matter from the diseased worms. He used a microscope to examine the specimen of the silkworm and showed that the disease was caused by a cryptogam, a fungal parasite of the silkworm. He also noticed that the fine efflorescence (mass of spores), which appeared following the death of the silkworm, was composed of a multitude of minute plants (mycelium or hyphae) bearing “seeds” (spores). Only after the seeds devel-oped, the disease became infectious. The seeds penetrated into the bodies of the healthy worms where they nourished them-selves. The seeds were transmissible in various ways. The worms would eventually die and the small plants would produce new seed. The seeds could be latent for a long time.

Bassi presented his finding at the University of Parvis in 1833. He also reported his experimental results in Del mal del

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segno, calcinaccio, moscardino and malattiacheaffligeibachi da seta e sulmodo di liberarne le bigattajeanche le piuinfestate (1835–1836). This was the first experimental proof that a living fungal parasite was the cause of a disease in animal. We now know that the white efflorescence actually is a mass of spores. The fungal mycelium grows at the expense of the animals dur-ing the life of the worm until their death. The hyphae, which penetrate through the skin, bear the fine dust-like spores, would then appear. In his papers, he also proposed that certain diseases of humans were caused by organisms. He also devoted a considerable part of his paper to illustrate practical methods of preventing the silkworm disease; for example, avoidance of contamination, disinfection of the rooms where the disease occurred, and boiling of implements. He also proposed to use fresh air and sunlight as an effective disease prevention measure.

The fungal parasite discovered by Bassi was later identified by Giuseppe Balsamo-Crivelli (1800–1874) as Botrytis paradoxa, and later named Botrytis bassiana in memory of Bassi. Now, it is renamed as Beauveria bassiana.

Bassi’s discovery was later confirmed by Jean-Victor Audouin (1797–1841) and others. It had a great impact on agriculture and affected subsequent investigations of eminent physician Johan Lukas Schoneleion (some spelled Schonlein) (1793–1864), Miles J. Berkeley (1803–1889), and others who consid-ered fungal parasites as a cause of diseases in both animals and plants. Famous German pathologist Friedrich Gustav Jakob Henle's (1809–1885) classical paper of 1840 Von den Miasmen und Conagien und von den miasmatisch-contagiosea Krankheiten clearly indicated that Bassi’s conviction that para-sites were actually involved in many contagious diseases was correct. Also, over the ensuring years, the clear understanding of the nature of parasitism affected the concept of infectious disease and contributed to the disproving of the misconcept of “spontaneous generations”. Bassi’s method of using experi-mental inoculation also influenced many investigators at a

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later date including Heinrich Anton de Bary (1831–1888), a pioneer mycologist.

Bassi’s eyesight was never good and eventually prevented him from further microscopic observation. But he continued to pay attention to the contagious diseases. He suggested that parasites, animals, or vegetables were the causes of diseases including plague, smallpox, syphilis, and cholera. He died on 6 February 1856 in Lodi.

Commentary

Bassi was far ahead of Louis Pasteur (1822–1895) and Robert Koch (1843–1910) in studying the etiology of diseases and their contagious nature. He advocated the importance of quarantine and uses of various methods of prevention and disinfection, employing both asepsis and antisepsis. Although he is mainly remembered for his study of silkworm disease, his vision greatly influences the development of microbiology, pathology, pre-ventive medicine, and zoonosis. He was also a humanitarian; he was the first to care about silkworm disease, which greatly affected the income of farmers who raised them since that was an important form of agriculture for Italy at that time.

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Chapter 6

Ignaz Philipp Semmelweis (1818–1865): Savior of Mothers

“The air of that labor room, loaded with putrid matter, found its way into the gaping genitals just at the completion of labor, and onward into the cavity of the uterus, where the putrid matter was absorbed, and puerperal fever was the consequence”.

— Ignaz Philipp Semmelweis

Source: https://en.wikipedia.org/wiki/Ignaz_Semmelweis(US Public Domain image)

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Introduction

During the 19th century, women and their babies were dying in birth at alarming rates in Europe and the United States. In some years, as many as one in every four women who delivered their babies in hospitals died from puerperal fever. In 1795, Alexander Gordon (1752–1799), a Scottish physician, wrote that puerperal fever was transmitted “. . . by practicians who had previously attended patients affected with the disease”. Gordon recom-mended that doctors and nurses wash their hands after attend-ing infected patients. The American physician Oliver Wendell Holmes (1809–1894) in 1843 advocated handwashing to pre-vent puerperal fever. Holmes was horrified by the prevalence of the disease in American hospitals, which he believed to be an infectious disease passed to pregnant women by the hands of doctor. He recommended that a physician finding two cases of the disease in his practice within a short time should remove himself from obstetrical duty for a month. Holmes’s ideas were greeted with disdain by obstetricians in the United States. The medical community was not ready to accept the recommenda-tions of Gordon and Holmes at that time.

Why were physicians reluctant to accept the importance of handwashing? Perhaps because they didn’t understand the nature of infectious diseases. Robert Koch’s (1843–1910) work on germ theory of disease would not come for another 30 years. Or perhaps the answer was more pragmatic: The lack of indoor plumbing made it difficult to get water. In order to get water comfortably warm, it would have to be heated over a fire. Additionally, contact with water sources was associated with diseases such as malaria and typhoid fever. A different motive was stated by physicians in New York who protested Sara Josephine Baker's (1873–1945) program to teach hygiene to child care providers. Thirty physicians sent a petition to the mayor complaining that the program “was ruining medical practice by keeping babies well”. Concern for the health of mothers and babies has been of concern to many — but

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perhaps no one was as passionate, or as consumed as Ignaz Philipp Semmelweis.

Semmelweis lived before the age of microbiology, when, in fact, even the elementary knowledge of Leeuwenhoek’s “ani-malcules” was associated in no way with disease. But with the intuition of a true genius, he was able to achieve results later obtained by microbiology. Even a rudimentary knowledge of history makes us aware that a genius ahead of his time may not be accepted: Leonardo da Vinci (1452–1519), Nicolaus Copernicus (1473–1543), Galileo Galilei (1564–1642), and Gregor Johann Mendel (1822–1884) are such examples.

Life and Accomplishment of Semmelweis

Ignaz Philipp Semmelweis was born on 1 July 1818, in either Buda or Pest, Hungary. He was the fifth son of a bourgeois fam-ily. His father was Jozsef Semmelweis (1778–1846) and his mother Terezia Muller (1790–1844).

After completing schooling at the Gymnasium of Buda and at the University of Pest, he was sent to study law at the University of Vienna in the fall of 1837. He was bored by his law studies, but was fascinated by what he saw when he visited the anatomy laboratory. Then he transferred to the Medical School in the following fall. The next year he was transferred to the University of Pest Medical School so that he could be close to his family. His parents had eight children to support and the savings incurred living at home might have been important. The facilities at Pest were out-dated and inadequate; therefore, in fall of 1840, Semmelweis returned to the University of Vienna Medical School. During the last two years of the medical pro-gram, Semmelweis was in close contact with the three promis-ing figures of the medical school. They were also the most influential figures in his life — Baron Carl von Rokitansky (1804–1878), Josef Skoda (1805–1881), and Ferdinand Ritter von Hebra (1816–1880). In addition to medicine, Semmelweis

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attended a school of midwifery, which was attended largely by women. He received both Doctor of Medicine and Master of Midwifery degrees in 1844. He then completed a fifteen-month course in surgical training with Skoda, who was famous for both diagnostic and statistical methods in surgery.

Following his graduation, Semmelweis obtained a position at the Lying-in (obstetrics) Clinic at Bienna’s Allgemeines Krankenhaus (Vienna General Hospital). It was a teaching hos-pital that accepted patients who were too poor to have private physicians. In 1840, Johann Klein (1788–1856), the new director of the Lying-in Hospital had made procedural changing. The hospital consisted of two clinics: formerly, medical students and midwives were instructed in both clinics. Subsequently, medical students were instructed in the First Clinic and midwives, in the Second. Medical students no longer used a model to learn their practice. Now they used adult female and newborn cadavers. The First Clinic became a source of gossip throughout Vienna because the death rate due to puerperal fever in the First Clinic ranged between 13 and 18%, four times that of the Second Clinic. Poor women had learned that they had a better chance of surviving childbirth if they gave birth before going to the hospital. (They were called street birth.) Upon arriving at the hospital, the women begged to be assigned to the Second Clinic although the Second Clinic had more patients and was over crowded.

When Semmelweis joined the staff of the hospital, he was only twenty some years of age at this time. He was prematurely balding and beginning to get fat. He appeared older than his years and had an air of authority. He studied with exceptional zeal. He spent each morning doing dissections in the pathologi-cal laboratory and each afternoon attending patients in the clinic. Here, he observed the diseased, which would become his obsession. He was keenly aware that his patients were terrified and suffering. The physicians of that day had no knowledge of microbial disease and none of the curiosity of Edward Jenner (1749–1823), Joseph Lister (1827–1912), or Florence Nightingale

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(1820–1910) to see the common sense value of ordinary cleanli-ness. Physicians coming from an operation wore their bloody aprons as “badge of honor”. They went with bloody hands from one patient to another. Scalpels and medical instrument were not even washed much of the time. Green pus coming from wounds was considered beneficial to healing; that it con-tained Pseudomonads and effectively a good sign since it con-tained pyocyannes was not understood.

Semmelweis had light colored but piercing eyes. He saw wards full of blood, filth and misery; today any person would retch with horror at the terrible odor and archaic practices of those doctors and attendants. Young Semmelweis wrote vividly of his daily experience, “The air of that labor room, loaded with putrid matter, found its way into the gaping genitals just at the completion of labor, and onward into the cavity of the uterus, where the putrid matter was absorbed, and puerperal fever was the consequence”.

There were several hypotheses on the cause of puerperal fever including suppression of lochial discharge and internal fibrin deposits. Unfortunately, no one realized that these were the result, not the cause of the disease. On autopsy, the pus in the cadavers led to the notion that milk accumulated in the body to cause the disease. Some physicians cited the nebulous “influences” of the obstetric hospitals as the cause of the dis-ease. It is unlikely that Semmelweis had read Holmes’s paper on puerperal sepsis published the previous year. Semmelweis was a sensitive young man who was deeply moved by what he observed. The poor mothers were terrified and suffering. Klein saw that Semmelweis was an industrious student and, in 1846, appointed him as Assistant in the First Clinic. The young Assistant’s duties included teaching the 44 medical students and performing all clinical examinations.

Semmelweis collected information from both clinics. According to Lajos Markusovszky (1815–1893), Semmelweis was haunted day and night by a determination to solve the mystery of puerperal fever. He observed every possible reason and

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tested different hypotheses. He observed one major difference between delivery of mothers in the midwifery clinic and the medical student clinic. He noticed that wealthy women and street births were not examined by the medical students who had spent their mornings dissecting cadavers; and the doctor went directly from performing autopsies to examining women in labor without washing their hands or changing their aprons, which were stained with several days of blood. He particularly focused upon the unwashed dirty hands of medical students and doctors.

The death of his good friend and a much-admired professor of forensic pathology, Jakob Kolletschka (1803–1847), in 1847, was a horrible shock to Semmelweis but also provided him a needed insight. Kolletschka had sustained an accidental lacera-tion during autopsy. He developed lymphangitis in his arm, pleuritis, pericarditis, and peritonitis, five days later he died. Kolletschka died with identical symptoms and pathology as seen in the puerperal fever patients. An earlier event had stayed on Semmelweis’s mind. In 1844, Semmelweis watched the obstetrician Johann Chiari remove a tumor from a patient’s cervix. The operation was considered simple as Semmelweis had seen it done many times. A few days later the woman died of puerperal fever. Neither patient had given birth and Kolletschka had not been in the First Clinic. Semmelweis had found his explanation, “… the transmission of cadaveric particles clinging to the hand, and also by ichorous discharges originating in liv-ing organisms”.

On 15 May 1847, he ordered that all medical men must wash their hands with a brush in a solution of chlorinated lime before entering the delivery room. Although his order received the objection of staff and students because the chlorine irri-tated their skin and washing took too much time, but generally students and staff complied with the rule. The mortality rate in his wards dropped from 18% to less than 3%. He also kept the wards scrupulously clean. In October, 12 women in a row of beds became ill and 11 died. Semmelweis looked at the first

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patient examined that day, a woman admitted not for child-birth but because she had a uterine infection. He modified the washing procedure to require everyone to wash their hands with chlorinated lime after examining each patient. He had clearly seen the link causing the disease and had developed an antiseptic procedure to correct the situation. Semmelweis’ suc-cess annoyed Klein who had seen the death rate rise at his administration and his ire caused him to demote Semmelweis.

Semmelweis’s discovery of the etiology of puerperal fever was not by chance. He was also benefited by technical (later called epidemiology) and scientific processes. Rokitansky allowed Semmelweis to pursue any study he liked, and he availed himself of this opportunity, day and night. All the maternity statistics from 1789 onward were open to him. A great maternity ward with all its patients was available to Semmelweis’ disposal. The two lying-in clinics working under similar conditions were also available for Semmelweis’ com-parative study. And Professor Skoda’s teaching greatly helped Semmelweis’ logical mind to develop. The Semmelweis’ discov-ery was the achievement of a man with a deep clinical power of observation, scientific honesty, and expert knowledge in combination with his deep sympathy with suffering women of the world.

Today, this would appear to be a very clear statistically proven “fact”, to be acted upon throughout the entire hospi-tal, but that was in a different time. Without microbiology and the knowledge of the “germ theory”, doctors did not see a clear reason for washing hands.

This is also influences of “personality” and “character”. The personality and character of Semmelweis were such that they stood in the way of acceptance of his sound theory and prac-tice. He was a man without tact. He very boldly asserted that the dirty hands of the doctors were carrying the disease to the women patients. In a rough way he even called “… the doctor a murder and a Nero (37–68 AD)”. His order to wash hands with chlorinated lime before entering the deliver rooms had already

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annoyed Johann Klein, his supervisor. His methods of communi-cation were a problem all his life and would be a problem to anyone even today. Today, microbiologists ought to consider that a good communication is important. Even great discoveries need to be communicated and understood by all.

To his credit, Semmelweis made a statement in 1847 to the Vienna Medical Society that the cause of puerperal fever could be found in the blood poisoning by infectious matter carried on the hands of the doctors and medical students. For a new physi-cian to assert that the dirty hands of his superior and their stu-dents were transmitting the disease was a terrible affront to the professional pride of his colleagues. His efforts to establish more sanitary practices were ridiculed. He was harassed by his medical colleagues. He was distrusted by his supervisor Professor Johann Klein and was attacked seriously by medical profession-als such as Friedrich Wilhelm Scanzoni von Lichtenfels (1821–1891). Joseph Skoda supported his view and urged him to prove his case by animal experiments. Ferdinand Ritter von Hebra (1816–1880) published an article supporting Semmelweis and discussed cadaveric fever, a condition peculiar to a general teaching hospital, which would not occur elsewhere. However, Semmelweis refused to communicate his method officially to the learned circles. He was not given a renewal of his appoint-ment of his assistantship by his superior Johan Klein. In 1848, a haughty and offended Semmelweis left Vienna and was consid-ered for work in Dublin, Ireland. This did not work out, and he returned in a huff of contempt for his fellow physician. He then took an unpaid position as a senior physician in the Obstetric Clinic of a Pest Hospital in Hungary. In 1849, his strong sup-porter Von Hebra published a second article, expanding on Semmelweis’s conclusion and referring to his finding as equal in importance to Edward Jenner’s discovery. He also let himself be convinced by friends that animal tests would convince the skep-tics. He conducted nine experiments that clearly showed that puerperal fever in rabbits were contaminated with cadaveric material. Then he abruptly stopped; it was obvious to him that

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using clinic statistics was a better way to prove the conclusion. But the opposition against him was hardened.

Semmelweis was averse to writing and stubbornly refused to publish his findings. His medical students and associates were promoting his doctrine. However, at one point in 1850, the Board of Studies of the Vienna Medical School voted to study the correlation between puerperal fever mortality in institutions that did not perform cadaveric dissection and those that did. Somehow, the powerful Klein scuttled this move, and Semmelweis was not appointed as Assistant in the First Clinic. He was cut off from clinic work completely.

He resigned and left Vienna for Budapest where he became head of the obstetrical division at the St. Rochus Hospital in 1851. At Rochus, Semmelweis introduced chlorine disinfection and puerperal fever mortality dropped between below 1%. The same year, Semmelweis’s successor in the First Clinic of Vienna, Karl Braun, reported to the Vienna Society of physicians that the handwashing did not prevent puerperal fever. Semmelweis responded in his typical brusque manner that Braun was incom-petent or, at best, negligent.

The disease reappeared under Semmelweis’s watch in 1856, but this time it was different. When new mothers were infected at parturition, the child was almost always infected as well; this time the children were fine. Semmelweis concluded that the infection was occurring after the birth. The women languished for nine days prior to discharge, lying on sheets still soiled from patients who had lain on them before. The cause was not nurses failing to change sheets. The laundry had been taking away bags of sheets and returning bags of unwashed sheets. Semmelweis gathered the smelly bags and barged into the office of the hospital administer. Dumping the sheets on Statthaltereirat von Tandler’s desk, Semmelweis announced that this was the cause of the new outbreak of puerperal fever.

Another puerperal fever outbreak occurred in the 1857–1858 academic year. This time the cause was a nurse who had not changed sheets after infected patients. Semmelweis

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reported this event in the new Hungarian medical journal OrvosiHetilap. The publication achieved the desired results — the nurse no longer worked in the hospital and Semmelweis became a regular contributor to the journal. Semmelweis’s doc-trine was being promoted in England by James Simpson (1811–1870) and in the United States, Holmes had published his paper on the cause of puerperal fever.

In 1861, Semmelweis published his only report on his find-ing concerning puerperal fever. The rambling 543-page mono-graph entitled Die Ætiology included all of his data and tables but did not reflect the brilliant logic he had shown in discover-ing the method of transmission of the disease. His book was largely ignored. In frustration, he began to publish open letters to opponents of his doctrine. One such opponent was Rudolf Virchow (1821–1901), a German pathologist, who told Berlin obstetricians that puerperal fever was the result of weather conditions. Semmelweis had charts showing that the mortality from puerperal fever varied widely during any single month and could not be due to weather. Instead of using his data, Semmelweis responded in an open letter to Virchow that “823 of my pupil midwives know better than Virchow [and are] more enlightened than the members of the Berlin Obstetrical Society …“.

On 1 June 1857, Semmelweis married 18-year-old Marie Weidenhoffer (1837–1910), daughter of Ignac Weidenhoffer but was cheated out of a happy life by fate. The unfortunate man, who had seen so much misery as an attendant of child-birth saw his first child, Ignac Semmelweis, born a hydrocepha-lus, dying within two days of birth (16 October 1958). A second child, Maria Gabriella Antonia Semmelweis was born on 22 November 1859. The child died of peritonitis in March of 1860. Semmelweis did enjoy a good marriage and a solid financial background of his wife’s family. They had three more children: Margret Antonia Adel Semmelweis (1861–1928), Bela Antal Semmelweis (1862–1885), and Antonia Padua Maria Semmelweis (1864–1942).

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When physicians disagreed with the practice of handwash-ing, Semmelweis took it personally. In the course of his family tragedies and controversies with unbelieving colleagues, Semmelweis’s behavior became increasingly erratic and eccen-tric. He was tormented by the continuing outbreaks of puer-peral fever. The rate of infection at Paris Maternité was 12% in 1861. Perhaps, he thought about his own role in spreading the disease through his many mornings spent in dissecting. His wife, embarrassed by his bitter and emotional displays, con-tacted his medical associates for assistance. He was persuaded to have himself committed to a sanitarium in Vienna. In the sanitarium, a physician noticed that Semmelweis had a cut on one finger, probably the result of surgery. The wound became gangrenous and septicemia spread the bacteria to other organs. He died on 13 August 1865, a victim of bacterial infec-tion of his hands. This was a final irony, and he actually died of a puerperal infection. His body was taken back to his old Vienna General Hospital and an autopsy performed, which showed extensive brain damage.

Twenty-four years later, a speaker at the Academy of Medicine in Paris expressed his skepticism that disease could be spread by the hands. An outraged member of the audience shouted at the speaker “The thing that kills women with puer-peral fever . . .is you doctors that carry deadly microbes from sick women to healthy ones”. The man who shouted was Louis Pasteur who had, that year, observed Streptococcus pyogenes in the blood of puerperal fever patients. It was proved by many microbiologists that puerperal fever is due to bacterial infections.

Commentary

Antibiotics and autoclaves have solved many of the early prob-lems of infection, but according to the Centers for Disease Control and Prevention (CDC), “handwashing is the single most important means of preventing the spread of infection”.

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Handwashing is taught at every level of school, advocated in the workplace, and emphasized during medical training. Yet recent studies indicate that lack of proper handwashing still contributes significantly to disease transmission. In 1997, a report published by researchers at the CDC revealed that hands were washed before an interaction 27% of the time in a long-term care facility. In a 1996 study by Ohio State University College of Medicine identified handwashing rates as low as 31% in an emergency department. Approximately two million nosocomial infections occur each year in the United States.

So a man died who knew nothing about microbial cause of disease but who preached asepsis from an intuitive and prag-matic point of view, and unquestionably saved the lives of many women. He was the first to demonstrate the importance of person-to-person spread of infectious disease and the effec-tiveness of washing hands in an antiseptic solution.

It should be noted that he never overcame a strong Hungarian accent when speaking German. Every microbiologist must learn the need for communication skills. Semmelweis had only a transitory effect for rather minute periods of time because of the defects in personality mentioned — but also because of the unscientific attitudes of the medical profession of his time and their severe persecution of this unlikable, per-haps, but nevertheless, sound, and genius physician.

Semmelweis’s prophylactic measurements were widely accepted and so was his credit recognized. Many medical or academic organizations commemorated the work of Semmelweis. For example, two Semmelweis Memorial Committees were formed in 1891. In 1901, the Hungarian Academy of Sciences offered a prize for the best monograph On the History of Asepsis and Antisepsis, With Special Reference to the Teaching of Semmelweis. At the same year, Foundation of Semmelweis Cup by the Medical Casino of Budapest was established. The United Nations declared 1965 as the Semmelweis’s year; celebrations were held by Vienna and Budapest to commemorate the anniversary of the death of

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Semmelweis on 13–14 August 1965. At the same time, Semmelweis’ monument in the house where he was born was inaugurated, and the Semmelweis Historical Museum was opened. In 1966, a memorial plague was placed in the house where Semmelweis spent his last years (10 Vaci Street, Budapest).

Throughout Semmelweis’s life, he perceived and viewed others as graceless outsiders looking in. Although by hard work and with a touch of genius, he made a monumental discovery and was praised by some of the highest stars of medicine.

In 1995, P. Stolley and T. Lasky stated that “iatrogenic dis-ease usually reveals not error, but ignorance”. This was undoubtedly true of Semmelweis’s colleagues. Semmelweis saw every critique as a rejections. Eventually, his theory and sense of self became one and the same. Semmelweis’ life was indeed a tragedy as was his death. But he left us with a great discovery and paved the way for many great men such as Pasteur, Koch, and Lister in the fields of medicine and microbiology.

Today, Semmelweis might believe that he was indicated by the acceptance of his idea. Would he still experience the same frustration with the medical community’s failure to adopt this behavioral change?

Suggested Reading

1. Gortvay, G. and I. Zoltan (1968). Semmelweis: His Life and Work. Akademiai Kiado, Budapest.

2. de Kruif, P. (1926). Microbe Hunters. Harcourt Brace and Co., New York.

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Chapter 7

Louis Pasteur (1822–1895): The Master of Microbiology

“Science is not the product of lofty meditations and genteel behavior, it is fertilized by the heartbreaking toil and long vigils-even if, only too often, those who harvest the fruit are but the laborers of the eleventh hour”.

“Nature is plebeian; she demands that one works; she prefers callused hands and will reveal herself only to those with careworn brows”.

“Peace, love, and science!”

—Louis Pasteur

Source: https://en.wikipedia.org/wiki/Louis_Pasteur(US Public Domain image)

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Introduction

Louis Pasteur died on 28 September 1895. It is fair to consider Pasteur the first modern microbiologist. To review what he did more than 100 years ago not only refreshes our memory of how microbiology began but is also an inspiration to the future microbiologists. Pasteur was a chemist with a difference!

Early Years

Pasteur was born on 27 December 1822, in Dole, a French town in the Jura mountains near Switzerland. His father was Jean Joseph Pasteur (1791–1865) and his mother was Jeanne Etiennette Roqui (1793–1848). His grandfather was Jean Henri Pasteur (1769–1796); his great grandfather was Claude Etienne Pasteur (1733–1798). The name Pasteur means “Shepherd” and came from a humble beginning. His father was a tanner, which was a family tradition for several generations. He had one brother Jean Denis Pasteur (1816–1817) and three sisters: Virginie (1818–1880), Josephine (1825–1850) and Emilie (1826–1853). They grew up in Arbois, where the family settled in 1827. As a child, Louis loved the outdoors and long walks in the woods. He liked fishing and was very fond of animals. He also enjoyed drawing and, while a boy, earned a reputation as an artist, but gave up his art at the age of 19 to devote more time to science. Pasteur’s family was poor, but his father had hopes that Louis would become a teacher at a local lycée. His knit family made real sacrifices to provide for his schooling. But Louis was not quick in learning. His sisters also received schooling (unusual for those days) and Louis helped and encouraged them.

In October 1838, 15-year-old Louis left his home for school in Paris. His ambition was to become a teacher. He wanted to attend the École Normale Supérieure founded in 1808 by Napoleon (1769–1821) to train faculty for France’s colleges and universities. However, young Louis was terribly homesick and

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returned home after about a month. He attended the College d’Arbois and then the Royal College in Besancon (only 25 miles from Arbois), where he earned his Bachelor of Letters in 1840 at the age of 18. Louis studied hard and did tutoring as well at Besancon.

In 1842, not quite 20 years old, Louis went to Paris again and took a highly competitive entrance examination for École Normale Supérieure, where he wanted to attend. He only ranked 15 out of 22 and was admitted, he did not want to enter with such a poor ranking. So instead, he went to the Institute Barbet, where Louis was strongly inspired by Jean-Baptiste Dumas (1800–1884), Professor at Sorbonne at that time. Louis became a disciple of Professor Dumas, who was one of the founders of organic chemistry and guided his initial interest in chemical research.

Louis Pasteur had three loves: his studies, family, and God. He was very close to his family and helped his sisters every chance he got. His love was so great for his sisters that he offered them his salary from Royal College (Besancon) to help them with their education. This was very forward thinking because at that time few women attended college. Louis was very thrifty himself. He hated to spend more than 50 cents on any meal and kept strict track of all his expenditures. He once needed a stove for his room because it was too cold to study. He rented one rather than buying one because it was cheaper.

Louis again attempted the test to enter the École Normale Supérieure and ranked fourth in his class. He now felt that he was ready to learn. He was free to take any classes that he wanted to. He took mathematics, chemistry, and physics there. Louis also took classes at the Sorbonne. He took classes such as glassblowing, carpentry, and metal working, which were neces-sary for most chemists to make their equipment. He also took mineralogy and crystallography, which became his favorite sub-jects. Of course, the microscope was an important tool for studying crystals.

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Chemist with a Microscope

One of Pasteur’s professors, Jean-Baptiste Biot (1774–1862), a physicist noted for his work on optical activity in fluids, men-tioned a report by the German crystallographer Eilhard Mitscherich (1794–1863) that tartaric acid contained two differ-ent crystals, both with the same composition but one optically active and the other inactive. Pasteur’s curious, rigorous mind began to grapple with what seemed an inconsistency: How could these crystals be the same yet act differently? With the encouragement of Dumas and Biot, he set out to work with his microscope to unravel this mystery. He continued these studies when he became an assistant to Antoine Balard (1802–1876), a chemistry professor at the school and later that year he discov-ered the optical isomers of tartaric acid. With his microscope, he found that although some of the crystals of tartaric acid looked alike, they were actually mirror images of each other. Pasteur was only 24 when he discovered the molecular asym-metry in tartaric acid. Louis completed his studies in 1847.

This discovery alone would have ensured Pasteur a place in the history of science, because it is discussed in every historical treatment of isomerism. He would have been awarded with the Nobel Laureate, however, Nobel prize had not been set up at that time. His discovery aroused the interest of leading chemists in France, and Pasteur was appointed Professor of Chemistry at the University of Strasbourg in 1848, where most professors were about three times his age. Pasteur spent five years at Strasbourg. He met Marie Laurent, daughter of the Rector (President) of the University, in 1848 and fell in love immedi-ately. He had known Marie for only two weeks when he wrote to her father asking for her hand in marriage. On his wedding day, 29 May 29 1849, his friends had to extract him from the laboratory and bring him to the church. Later that day he was back in the laboratory, his wife by his side. Marie was his wife, friend, collaborator, secretary, and supporter for the rest of his life. He enjoyed his family immensely. They had five children,

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four girls and one boy. Three of their children died young: Jeanne at 9, Cecile at 12, and Camille at 2. Only two lived to adulthood: Marie-Louise (1858–1934) who married Rene Vallery-Radot (1853–1933) and Jean-Baptiste (1851–1908). In 1853, he was awarded the Cross of the Legion of Honor.

Pasteur’s move to Lille in 1854 to become Professor of Chemistry and Dean of the Faculty of Sciences, had momentous consequences. At Strasbourg, he had concentrated on crystals, but at Lille, a major agriculture center, a businessman con-fronted him and told him bluntly that basic science might be all right, but it would be better if science could help business. He asked Pasteur if he would be able to raise the yield of sugar from sugar beets and increase the production of alcohol.

A short time earlier, Theodore Schwann (1810–1882) in Germany had declared that alcohol was produced by sugar fungi and Charles Cagniard de la Tour (1777–1859) reported that the fermentation of beer resulted from the action of yeasts. However, these discoveries received little attention at the time. Then an alcohol distiller came to Pasteur in great dis-tress. His vats were filled with sugar beets seeded with yeast, but some of the vats had soured and were producing no alco-hol and he was losing thousands of francs a day. The bad vats smelled like sour milk. Pasteur’s natural instinct was to take samples from the vats and examine them under the micro-scope. This would never have been done by any ordinary chem-ist, but Pasteur’s habitual use of the microscope was to have great consequences.

A careful scientist, he had sampled both good vats and sour ones. The sample from the good vats consisted golden globules smaller than any of his crystals. He remembered the papers of Cagniard de la Tour and recognized that the globules were yeasts. He saw immediately that Cagniard de la Tour was right — the yeast was alive and produced the alcohol from beet sugar. In the samples from the sour vats, Pasteur found no yeast cells but instead masses of the dark rods. He thought that if the rods were alive, like the yeasty cells in the healthy vats, perhaps

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they multiplied and caused the “sickness” of the beet-sugar fermentation. The yeast produced alcohol, but the rods pro-duced lactic acid and overran the yeast. This reasoning began the field of microbiology. Pasteur did not understand yet where the rods came from or how they got into the vats, but he was able to save the industrialist by telling him to examine his fer-mentations for rods and completely destroy any fermentation that showed even one rod.

Pasteur wanted to know more about these rods, but this was too difficult in the beet pulp mash. He realized that he needed what today is called a selective medium in which the rods could grow and be studied. After many unsuccessful attempts, he finally developed a medium that would sustain growth. All this was accomplished in the midst of teaching and other work as well as an (unsuccessful) attempt to be elected to the France Academy of Sciences. Pasteur’s work to grow cul-tures of bacteria and yeasts attracted little attention.

Fermentation

In 1857, Pasteur moved back to Paris to become Administrator and Director of Scientific Studies at the École Normale Supérieure. His space was very limited, but he set up a tiny labo-ratory with his incubators and microscope. He became embroiled in a debate with the great chemist of the brewing industry in France and Germany: Chemist such as Justus von Liebig believed Charles Cagniard de la Tour said that fermentation was a chem-ical reaction but Pasteur said no, that was not, and that yeast caused the fermentation that creates wine and beer. Liebig insisted that albumin in the vats produced the fermentation. Pasteur, after developing the necessary methods of microbiol-ogy for pure cultures, proper media, sterile equipment, and transfer methods, demonstrated in a succession of experiments that wines and beers were the results of fermentation by yeast. He coined the term “fermentation” for life without oxygen.

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Disproval of the Fallacy of Spontaneous Generation

Pasteur presented papers and gave speeches on his proof that it was primarily the work of millions of yeast cells that pro-duced wine and beer. The great Liebig was proved wrong, and Pasteur was finally given the credit he deserved. Pasteur contin-ued to work with bacteria and yeast and developed increas-ingly better methods for handling them.

One problem, contamination from the air, was quickly entangled in an important scientific problem not yet resolved. In spite of the work of Lazzaro Spallanzani, most scientists, like the general population, still believed in “spontaneous genera-tion”, that is, that all life, from mice to the microbial forms, spontaneously arose when there was moisture and warmth. A sealed culture that was boiled would show no life, but air was thought to be needed for spontaneous generation of microbes. This was a serious problem for Pasteur in his numerous debates. Balard, his old chemistry professor, suggested that heating the flask that contained the culture and bent the tube into a long goose neck shape “S” curve so that incoming bacteria would be deposited on the curved walls of the tube. The media in these unsealed flasks did not become contaminated, but cultures grew when these media were inoculated with bacteria depos-ited on the tubing walls. This experiment convinced the opposi-tion and in 1860, Pasteur’s painstaking experiments finally demolished the argument for spontaneous generation.

Pasteur continued to study the wine industry. He learned that different types of microbes spoiled wine in different ways and other organisms produced perfect wines of different types. Experts in the industry were mystified by his ability to judge the type of spoilage in various wines and to recognize good wine with his microscope. Pasteur’s work greatly benefited the wine industry and even the vinegar producers.

In 1864, he discovered that heating would prevent wine spoilage; this process became known as pasteurization. In 1867, he was awarded the Grand Prix at the Exposition Universelle for

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his work on pasteurization and in the same year, he became Chemistry Professor at the Sorbonne.

Silkworms, Microbes, and Disease

In or around 1865, Pasteur received an urgent request from Dumas to work on a disease endangering the important silk industry in the south of France, Dumas’s native area. Pasteur had more difficulty here than in solving the problems of wine industry. Pébrine, the disease killing the silkworms, produced the black spot resembling pepper on the silkworm larvae. Pasteur and his assistant, Désiré, Jean-Baptiste Germez, finally found that a parasite was responsible and showed the grower that how to select silkworm moths that were free of the para-site. (He later found the second disease called flacherie caused by microbes, was also killing silkworms and discovered that it was passed on through silkworm droppings ingested as the worms fed on mulberry leaves.) He showed the growers that how to prevent the spread of these diseases and to produce worms that gave splendid yields of silk.

The more Pasteur studied silkworm diseases, the more cer-tain he became that there was a connection between microbes and disease in humans. Although most physicians dismissed this idea, Pasteur was convinced they were wrong. His urge to study the relationship of microbes to human disease was undoubt-edly strengthened by the deaths of two of his daughters from typhoid fever and by his unsuccessful attempt to find the cause of the 1866 cholera epidemic that struck France. At the age of 46, in 1868 a stroke left Pasteur partially paralyzed. He recov-ered but walked with a limp and did not have use of his right hand. But he was impatient to continue his work.

The Franco-Prussian War (1870–1871) caused another diver-sion in Pasteur’s plan. Pasteur were then living in the Jura hills (1870) and they worried about their son, a sergeant in the French Army. Pasteur developed a resentment for the Prussians that resulted in a “revengeful” but useful project. Even he had

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to admit that German beer was far better than French beer, so despite a personal dislike for beer, he set out to reverse the situ-ation. He coined the slogan “Better beer for France”, solicited funding from French brewers, and set up a scientific brewery in his already crowded laboratory. He did help the beer industry, but it is doubtful whether French beer surpassed German beer as a result.

Pasteur’s research and publicity about microbes got him elected to the Academy of Medicine in 1873. An 1874 letter from Joseph Lister (1827–1912), the English surgeon who devised antisepetic surgical techniques and thereby reduced surgical mortality — from usual 50% to 3%, thanked Pasteur for having: “by your brilliant researches, demonstrated to me the truth of the germ theory of putrefaction . . . furnishing me with the principles upon which alone the antiseptic system can be carried out”. Pasteur was pleased with this recognition from the medical profession. He had alerted all of Europe to micro-scopic life, and he hoped to pioneer studies of the relationship of bacteria to human illness. But he was beaten by Robert Koch (1843–1910), a Prussian country doctor, who turned the world upside down by his 1882 demonstration that tubercle bacillus was the cause of tuberculosis. Koch had earlier (1876) found the germ caused anthrax, a dreaded disease of humans and livestock and formulated Koch’s postulates (briefly that one organism transmits one disease).

In 1850, the Hungarian physician Ignaz Semmelweis (1818–1865) had stated the childbed fever (puerperal sepsis) was con-tagious and that the obstetric wards in Vienna were “pestholes”. Physicians sneered at this idea for many years, often in the face of Lister’s work on asepsis in hospitals. In the late 1870s, Pasteur disrupted a meeting of the Academy of Medicine in Paris and scandalized the Academy by shouting at a prominent physician lecturing (conventional) on the causes of childbed fever. Interrupting loudly, Pasteur told him bluntly that he was wrong about the cause and that the physicians themselves were carry-ing the infection. When the famous doctor told him that he

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would never find the microbes, Pasteur limped to the rostrum, drew a chain of small circles on the board and announced, “I have found it already and here is how it looks!”

Pasteur had not gotten into the medical field earlier because aside from his occupation with other projects, he knew nothing of anatomy and physiology, did not like the smell of hospitals and was squeamish about using animals for experi-ments. Fortunately, in the mid-1870s he took on three newly graduated physicians: Jules Joubert (1834–1910), Pierre Paul Emile Roux (1853–1933), and Charles Edouard Chamberland (1851–1908). The team began to study anthrax, collecting and growing various strains. With this, Pasteur examined the effi-cacy of “cures” pronounced by veterinarians and disproved them all. But some cows survived the disease, and Pasteur stud-ied these animals. When injected with his most virulent strain of anthrax, these animals survived.

Concepts of Immunity and Attenuation

Pasteur now confirmed and expanded the principle of immu-nity described by Edward Jenner (1749–1823), who in 1796 introduced vaccination with cowpox to prevent smallpox. Between 1877 and 1880 Pasteur was working on chicken chol-era and a dozen other animal maladies, including anthrax. In 1879, on discovering that some very old cultures of chicken cholera germs still contained live organisms, he used them to inoculate chickens which, to his surprise, recovered. When rein-oculated with a fresh virulent strain, the chickens survived new challenges as well. Jenner had shown that immunity to one organism could be produced by a different organism, but Pasteur showed that a body that survived exposure to a patho-gen would resist reinfection by the same pathogen.

The same research showed that the virulence of a pathogen can be decreased by attenuating (weakening) the disease pro-ducing ability of the organism, for example, by repeated sub-culturing of the pathogen on laboratory medium. The use of

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cowpox as a vaccine for smallpox was known, but Pasteur real-ized that using an attenuated microbe of the same species might do better. He told his assistants, “Now I have found out how to make a beast a little sick, just a little sick, so he will get better from a disease. All we have to do is to let our virulent microbes grow old in their bottles…. When the microbes age, they get tame. Will give a chicken the disease, but only a little of it….and when she gets better she can withstand….the viru-lent microbes. This is a vaccine I’ve discovered much more, much more scientific…. than the one for smallpox, where no one has seen the germ. We’ll apply this to anthrax too”.

Public demonstration of anthrax vaccine

Pasteur soon announced the protection of sheep with a strain of anthrax that he had attenuated. This caused an uproar because he had many detractors and there was widespread doubt about the accuracy and validity of his work in veterinary medicine, particularly since he was not a veterinarian or physi-cian. Furthermore, he was now in an international confronta-tion with Koch, the discoverer of the anthrax bacillus. Probably no microbiologist or immunologist besides Pasteur would agree to a major experiment in public when failure could cost his reputation, a reputation which in Pasteur’s case had been made with much difficulty and aroused much prejudice. Pasteur’s enemies, spearheaded by a horse veterinarian who also edited a veterinary journal, persuaded a distinguished Nobleman from Melun to lure Pasteur into making a large-scale public experi-mental demonstration of his anthrax vaccine. The experiment would be expensive for Pasteur’s enemies, but they felt the chemist had overstepped his bounds and wanted to bring him down. They believed his “magic” cures were the result of luck, not science.

Against the advice of his colleagues, Pasteur assented at once. He assured his associates that “what worked on 14 sheep in our laboratory will work on 50 at the public experiment in

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Melun!” They went by train to Pouilly-le-Fort, where 48 sheeps, 2 goats, and several cattle were on hand for the test. With a jaunty limp, Pasteur marched past the crowd of scientists, sena-tors, spectators, and reporters, including one from the London Times. Alcohol lamps were lit and syringes flamed. Animals were inoculated with attenuated anthrax organisms that would kill mice but not guinea pigs, then 12 days later inoculated with a strain that would kill guinea pigs but not rabbits. The animals survived these vaccinations and when the time came for the final challenge with the virulent anthrax germ, unvaccinated animals were also injected. The huge crowd that appeared at the end of the final experiment. A few days after the challenge inoculation with enormous dose of deadly anthrax, all of the vaccinated animals were well, frisky, and eating, whereas all but two were dead, and the two survivors were staggering and oozing blood from their nostrils and mouth.

Pasteur was now considered the miracle worker of the day. No one before or since has performed such a public experi-ment. Physicians and veterinarians, previously his worst detrac-tors, now came forward to congratulate him. The newspapers rushed to report that public experiment was a “perfect and unprecedented success”. Pasteur’s little laboratory was turned into a vaccine factory, in an effort that today would be consid-ered impossibly crude. Huge kettles were filled with broth holding the same anthrax. Pasteur’s associates worked overtime to produce vaccines that would save the sheep industry, ladling a few ounces of bacillus broth at a time into glass bottles that were simply cleaned and not sterilized. Although they had to do this complex work without adequate equipment; in less than a year, hundreds of thousands of sheep had been pro-tected by the efforts of this small laboratory. There were prob-lems with the vaccine. It was sometimes not pure enough, sometimes too weak, and sometimes deadly. However, it did prevent anthrax in enormous numbers of animals and saved many farmers from ruin.

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Other Diseases Studied

Other diseases studied by Pasteur included gangrene, septice-mias, childbirth fever, chicken cholera, and human cholera. His studies of chicken cholera showed that attenuated cultures could produce immunity. He also studied swine erysipelas and devel-oped a vaccine for it. He sent one of his students, Louis Ferdinand Thuillier (1856–1883) to Egypt as a member of team to study a violent outbreak of cholera. Unfortunately, Thuillier came down with cholera himself and died in Alexandra, Egypt in 1883.

Pasteur and Rabies, a “Filterable Virus”

The genius of Pasteur and the growing skill of the new micro-biology profession is no better illustrated than in the story of rabies vaccination. Pasteur once said, “I have always been haunted by the cries of those victims of the mad wolf that came down the street of Arbois when I was a little boy”. In France, the fear of rabies was so great that laws had to be passed to stop the shooting of citizens suspected of dying of the disease. In the midst of an international debate over the quality of his anthrax vaccine and critical papers from Koch (for there had been impure batches from those kettles and some disastrous deaths of sheep), Pasteur plunged into a search for the cause of rabies (hydrophobia). He personally took samples of saliva from the mouths of sick and vicious dogs whose sudden lunge and bite could have meant a horrible death.

He also took samples from human victims and, as often hap-pened in his research, started out with a mistake. From one child, he cultured an organism, which he named “the microbe like an eight”, and before the Academy of Sciences he postu-lated that his “figure eight” germ might be the cause of hydro-phobia. Roux and Chamberlain, under Pasteur’s guidance, soon found that the “figure eight” bacterium was also present in the mouths of normal children.

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Capturing and caging rabid dogs and allowing them to bite normal dogs were a dangerous job for the researchers. The saliva of the mad dogs was also used to inoculate laboratory animals. The results were strange: Some animals came down with rabies immediately, but others did not succumb for weeks or months. In addition, unlike what had observed in other dis-eases. Pasteur was not able to isolate a bacterium that clearly caused rabies, nor could he find anything that would grow in or on any of his media. However, the inevitable convulsions of rabies victims indicated that the organism was probably located in central nervous system. He thought of growing the organism in animal brain but worried that the injected virus “would get lost in the body before it could travel to the brain”.

When Roux suggested that trephining would provide an easy and painless way to deposit rabies fluid directly on the brain of a dog, Pasteur forbid it because he said the operation would hurt the dog! Nevertheless, Roux soon did the procedure in Pasteur’s absence and told him the next day. Pasteur’s fears were allayed when Roux then showed him the frisky dog. Two weeks later the poor dog developed rabies symptoms and soon was dead, but the scientists now have a 100% reliable way of culturing and transferring the elusive organism.

We know now that rabies is caused by a virus, a filterable agent. Other diseases caused by filterable agents were studied much later. In 1892, Dmitri I. Ivanowski (1864–1920) discovered a filterable organism (virus) that caused tobacco mosaic disease. Martinus W. Beijerinck (1851–1931) described the intracellular reproduction of this virus in 1898, and Friedrich August Johannes Loeffler (1852–1915) and Paul Frosch (1860–1928) dis-covered the filterability of the virus that caused foot-and-mouth disease. The secret was to develop a filter with a known pore size to find the cutoff point at which no “filterable” virus would pass the filter. In 1884, Pasteur’s assistant Chamberland invented a kind of filter that served the purpose. For years after this era, these microbes were called “filterable viruses”. (We have now dropped the word “filterable”).

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After hundreds of dangerous experiments with dogs and other animals, the concept of how the rabies organism attacked the nervous system and finally reached the brain became clear in Pasteur’s mind. He and his assistants found a way to attenu-ate the virus for use as a vaccine by drying the spinal cord of an infected rabbit in a sterile bottle. He found that a series of 14 injections of rabies vaccine would protect every rabid dog inoculated. At one time, he had the “modern” thought of vac-cinating all dogs in France but wisely gave up because he did not have the facilities to carry this program.

From all over the world, even from the leader of Brazil, poured in frantic letters asking for the vaccine for human beings who faced death from rabies, but Pasteur held back from his ultimate test. The vaccine must work the same way in humans, but maybe it wouldn’t. He even considered starting on himself. He wrote to a friend that he would infect himself, then take the vaccine. Then one day a woman from Alsace brought her a 9-year-old son to Pasteur’s laboratory — Joseph Meister, who had been bitten in 14 places by a mad dog just the day before, and his mother begged Pasteur to save her little boy. Two physicians were consulted and when they saw the fester-ing wounds, they agreed that Pasteur should try his canine vac-cine because otherwise the boy faced certain death. Thus, the first injection of the attenuated rabbit spinal cord vaccine into a human being was made in July 1885. After the 14 daily sub-cutaneous injections, the boy went back to Alsace with no sign of the disease. Pasteur’s doubts vanished, and he proclaimed the triumph to the world.

Immediately afterward, people from outside France flooded into the little Paris laboratory. Nineteen days earlier, 19 peas-ants from Russia were bitten by a mad wolf and some of them so mangled they could not walk, caught the attention of all of France when they arrived at the laboratory despite knowing only one word of French “Pasteur”. Because it had been so long since the peasants had been bitten, Pasteur was fearful that few if any could be saved, but he gave them two injections per

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day to make up the lost time. Amazingly, all but three of the Russians survived. Paris went wild, and the Russians returned to a nation that greeted them with the awe one might have for a person raised from the dead. The Tsar of Russia awarded Pasteur the Diamond Cross of St. Anne plus a hundred thou-sand Francs to build a research laboratory, the initial fund for the Pasteur Institute.

The Later Year

A second stroke in 1887 made Pasteur unable to perform exper-iments himself, but his interactions with colleagues and his students continued. The Pasteur Institute was completed in 1888 and Pasteur spent much of his time there. The Sorbonne honored him with a Pasteur Jubilee on his 70th birthday (27 December 1892). Pasteur’s family was fond of the small house they had built long before at Villeneuve I’Etant, outside of Paris. The grounds contained the kennels where the rabid dogs were kept during his work on rabies. Pasteur often took a vaca-tion in his house where he could be in a small laboratory near the kennels. There he died at 5 o’clock in the morning of 28 September 1895. Before his death, he said, “Peace, love, and science!” He was surrounded by family and colleagues. France wanted to bury him in the Pantheon, but his family insisted he be buried at the Pasteur Institute, where his body rests in a beautifully designed tomb.

Commentary

His life serves as a good example of a truly good man. His love of his family, friends, country, sincere honesty, and devoted hardworking spirit exemplify the highest human virtues. Pasteur was very close to his family and helped his sisters at every chance he got. His love was so great for his sisters that he offered his salary from Royal College (Besancon) to help them

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with their education while he was a student. This was very for-ward thinking because at the period of time few women attended college. Pasteur was very thrifty himself. He hated to spend more than 50 cents on any meal and kept strict track of all his expenditures. He once needed a stove for his room because it was too cold to study. He rented one rather than buying one because it was cheaper.

During the revolution of 1848, Pasteur became enthralled in the magic of revolution and enlisted in the National Guard against his parent’s wishes. He never had to fight, and soon the revolution was over. It was shortly after Pasteur made his dis-covery in isomeric crystals of tartaric acid. Very soon after his discovery, Pasture’s mother died of apoplexy. He was greatly saddened because he did not make it home in time. He always wanted his parents to share in his accomplishments.

Pasteur was a devout Catholic and lay with a crucifix in his hand. Pasteur was a believer. All his life, he followed what he considered was his God-given mission to save lives from disease. We all are benefiting by Pasteur’s scientific contribution. Without Pasteur, many of us would not be living today. The author had the chance to visit the Pasteur Institute to look at Pasteur’s favorite microscope, the crystal of tartaric acid, his handwritings, and the beautiful tomb. A strong compassion emerges into the author’s mind and could not stop shedding tears. Pasteur is truly a “perfect man of all ages”, and he has nurtured the spirit of universal truth. All microbiologists should seek to emulate him.

The following Table 7.1 gives a chronology of important events in Pasteur’s life.

Acknowledgement

The authors would like to thank Dr. Hubert A. Lechevalier for his kind help with this manuscript. This paper is revised from Society of Industrial Microbiology News, 45(6):267–273, 1995.

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Table 7.1 Chronology of Important Events in Pasteur’s Life

Year Event

1822 Born on 27 December

1827 Move to Arbois

1838 Trip to Paris and returned to Arbois

1839–1842 College Royal de Besancon

1842–1843 Studied at Lycee Saint-Louis, Sorbonne and at the Institute Barbet in Paris

1843–1846 Studied at the École Nornale Supérieure

1846 Worked as Assistant to Balard, Chemistry Professor at the École Normale Supérieure. Discovery of molecular asymmetry (stereoisomerism) in tartaric acid

1847 Graduated from École Normale Supérieure

1848 Professor of Chemistry at the University of Strasbourg

1849 Married to Laurent

1853 Synthesized racemic acid. Awarded Legion of Honor

1854 Appointed as Professor of Chemistry and Dean of the Faculty of Science at Lille

1857 Awarded Rumford Medal by the Royal Society of London (for his crystallography studies). Became Director of Scientific Studies at the École Noramle Supérieure in Paris, a position he held until 1867

1862 Elected as member of the Academy of Sciences, Mineralogy Section

1863 Named as Professor of Geology, Physics, and Chemistry at the Ecole des Beaux Arts

1864 Demonstrated presence of germs in air. Invented pasteurization

1865–1869 Studied silkworm disease at Alais (southern France)

1867 Became Professor of Chemistry at the Sorbonne

1868 Suffered stroke (left hemiplegia) in October

1869 Studied silkworm diseases on the estate of the Prince Imperial at Villa Vicenitina, Austria

1871–1877 Studied beer and fermentation at the École Normale Supérieure

(Continued)

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Year Event

1873 Elected as associate member of the Academy of Medicine

1876 Defeated in election for the Senate

1877 Studied anthrax

1878 Studied gangrene, septicemia, childbirth fever

1879 Studied chicken cholera; discovered the phenomenon of attenuation

1880 Studied rabies

1881 Public field trial of anthrax vaccination

1882 Elected to Académie Francaise. Studied cattle pleuropneumonia and rabies

1883 Established laboratory at family home; official celebration at his birthplace. Originated vaccination against swine erysipelas. Studied cholera (his colleague Thuillier died of cholera in Egypt)

1885 First treatment of rabies

1886 Established kennels for study of rabies at Villeneuve L’Etang

1887 Had second stroke

1888 Pasteur Institute inaugurated on 14 November

1892 Pasteur Institute held a celebration at Sorbonne on 27 December for his 70th birthday

1894 Last day at Arbois (July to October)

1895 Died on 28 September at Villeneuve L’Etang

Source: Modified from Dubos, T. (1950). Louis Pasteur, Free Lance of Science. New York: Charles Scribner’s Sons.

Table 7.1 (Continued)

Suggested Reading

1. Birch, B. (1989). Louis Pasteur. Gareth Stevens Inc. Milwaukee, WI.

2. Brock, T. D., Ed. (1971). Milestones of Microbiology. American Society for Microbiology, Washington, DC.

3. Bullock, W. (1935). The History of Bacteriology. Oxford University Press, London.

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4. Collard, P. (1976). The Development of Microbiology. Cambridge University Press, New York.


6. Dubos, R. J. (1976). Louis Pasteur, Free Lance of Science. Scribner, New York.

7. Grainger, T. H. (1958). A Guide to the History of Bacteriology. The Ronald Press Co., New York.

8. Grant, M. P. (1959). Louis Pasteur, Fighting Hero of Science. McGraw-Hill, New York.

9. Lechevalier, H. A. and M. Solotorovsky (1965). Three Centuries of Microbiology. McGraw-Hill, New York.

10. Vallery-Rador, P. (1933–1939). Oeuvres de Pasteur. Reunies par Pasteur Vallery-Radot. 7 vols. Masson et Cie., Paris. (Collection of all of Pasteur’s publications as well as a large number of unpublished manuscripts and notes.)

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Chapter 8

Ferdinand Julius Cohn (1828–1898): Pioneer of Bacteriology

Source: https://en.wikipedia.org/wiki/Ferdinand_Cohn(US Public Domain image)

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Introduction

Ferdinand Julius Cohn was not as famous as Louis Pasteur (1822–1895) and Robert Koch (1843–1910) because he worked on the classification of bacteria, thus he did not attract as much the attention of the general public as those who worked on the relationships of microorganisms with human diseases. His con-tributions include systematic classification of bacteria, discovery of bacterial spore, help in disproving the fallacy of spontaneous generation, and establishing a journal Beiträge zur Biologie der Pflanzen, which served as an important vehicle for the publica-tion of many pioneer bacteriological papers. Cohn’s work also helped establishing the recognition of bacteria as a separate group of living organisms different from plants or animals. He is a pioneer in bacteriology.

Life of Ferdinand Cohn

Ferdinand was born on 24 January 1828, in Breslau, Lower Silesia (now in Wroclaw, Poland). His father, Issak Cohn, was poor and lived in Breslau’s Jewish ghetto when Ferdinand was born. But Issak became a successful merchant, and he cared very much about the education of his children. To his great joy, Ferdinand was a genius; he could read at the age of two and was interested in natural history at a very young age. He first attended school at the age of four. In 1835, he entered the Breslau Gymnasium (equivalent to high school) and did very well in all courses. Unfortunately, at the age of 10 or 11, he developed for an unknown reason a hearing defect, which slowed his incredible pace of learning. It was probably due to this defect that he became a shy, studious, and sensitive boy who also suffered an acute physical and emotional retardation, which he did not begin to overcome until his last year of gym-nasium. In 1842, he entered the philosophical faculty of the University of Breslau. During this period, he was influenced by Professors Heinrich Göppert (1800–1884) and Christian Nees

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von Esenbeck (1776–1858) and developed an interest in botany. Young Cohn finished all the requirements for graduation at the University of Breslau, but because he was a Jew, he was barred from taking the final examination. Therefore, Cohn went to the more liberal University of Berlin in October of 1846, and received his doctorate degree in botany on 13 November 1847, when he was only 19 years old. In Berlin, he was very much inspired by the teaching of Eilhard Mitscherlich (1794–1863), Karl Kunth (1788–1850), Johannes Muller (1801–1858), and Christian Ehrenberg (1795–1876), who introduced him to the study of microscopic organisms. But because he was sympa-thetic to the revolutionaries of 1848, his academic career in Berlin was not prosperous.

In 1849, he returned to Breslau and stayed there for the rest of his life. In 1850, he became a Privatdozent at the University of Breslau. In 1859, he was appointed as an extraordinary pro-fessor and in 1871, he became an ordinary professor. Cohn was a great inspiring teacher. His academic career and research find-ings were all accomplished at this university.

Because he was influenced by Matthias Schleiden’s (1804–1881) cell theory and Hugo von Mohl’s (1805–1872) description of protoplasm in plant cells, he began to focus on lower plants — microscopic organisms. His tedious observations on the unicellular algae Protococcus pluvialis led to his early fame. He found that the protoplasm in plant cells and “sarcode” in ani-mal cells were very similar. He suggested that the distinction between animals and plants should not merely be based on the fact that animals possessed differentiated organ systems or a contractile substance peculiar to themselves. He drew an explicit attention to the identity between the contractile contents of plant and animal cells. Cohn’s work on P. pluvialis confirmed and expanded the suggestions of Karl Wilhelm von Nageli (1817–1891), Hugo von Mohl, Alexander Braun (1805–1877), Max Schultze (1825–1874), and others, that the essential constituents of the cell was its protoplasmic contents. All these findings

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eventually led to the “protoplasm theory of life”, which was first published by Max Schultze in 1861. The protoplasm theory of life was a big advance step to the understanding of life.

In 1848, Cohn’s former teacher Goeppert asked him to devote himself to algae and hoped that he would contribute to the flora of the cryptogamous plants of Silesia. Cohn diligently accepted this assignment and published the first two volumes of the cryptogamous plants in 1876. This work alone is a signifi-cant contribution to plant science.

Cohn conducted a detailed study of microscopic algae, fungi, and bacteria. Of particular interest was the way he treated bacteria, generally called Vibrionia. At that time, bacte-ria were considered as animals primarily because of their active, apparently voluntary, movement. Cohn pointed out that the ciliated swarm cells of algae and fungi performed similar move-ments. He suggested that bacteria followed the same develop-mental course as algae, and that some large bacteria belonged to the plant kingdom and displayed an especially close relation-ship to the Oscillaria.

In 1855, he demonstrated the sexuality of the unicellular alga Sphaeropleaannulina. He observed in Sphaeroplea the formation of spermatozoa and followed the progress all the way to the egg. In 1856, he demonstrated the same phenome-non in Volvoxglobator, a motile alga. The same year, he was appointed chairman of the botanical sections.

Between 1856 and 1866, Cohn did some work on the con-tractile tissues of plant, and also pioneered the phototrophic studies of microscopic organisms. At his urging, the Institute of Plant Physiology of the University of Breslau was ultimately cre-ated in 1866. That was the first Institute of Plant Physiology in the world. In 1872, he became the director of the institute. In the institute, he installed a marine aquarium that yielded mate-rials for much of his later work.

He cultured marine plant and studied the classification of lower plants. In 1870, he founded a journal titled Bretragezur Biologie der Pflanzen, designed primarily to publish the work

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that came out of his institute. The journal became well known because many pioneer papers of modern bacteriology were published in this journal.

After 1870, Cohn turned his attention primarily to bacteria. He defined bacteria as “chlorophyll-free cells of spherical, oblong, or cylindrical form, sometimes twisted or bent, which multiply exclusively by transverse division and occur either iso-lated or in cell families”.

He divided bacteria into four groups based on their mor-phology: (i) Sphaerobacteria (spherical), (ii) Microbacteria (short rods or cylinders). (iii) Desmobacteria (longer rods or threads), and (iv) Spirobacteria (screws or spirals). He insisted on classifying bacteria as part of the plant kingdom because of their similarity with well-known algae.

He also studied the nutritional requirement of Bacterium termo Dujardin. He found that bacteria obtained nitrogen from ammonia compounds just the same as green plants, but bacte-ria could not assimilate carbon from carbonic acid, which is dif-ferent from green plants. He also made a clear distinction between putrefaction and pathogenicity of bacteria. Putrefying bacteria were not necessarily pathogenic. In a long series of experiments, he pointed out that a temperature of 80°C would effectively destroy the life of all bacteria and prevent their development in an organic infusion. However, he admitted that Bacillus subtilis behaved differently. B. subtilis was more heat resistant than were B. termo. He also discovered the formation of spores by B. subtilis. In 1876, Cohn discussed extensively the implication of the discovery of thermo-resistant spores by B. subtilis in the controversy over spontaneous generation. He explained the reason that in boiled infusions or hay and cheese could resume microbial growth was because they contained heat resistant spores.

He married a former student Pauline Reichenback in 1867. Apparently, he had a wonderful marriage, and she wrote his biography in 1901 (Ferdinand Cohn: Blatter der Erinnerung). Ferdinand Cohn passed away on 25 June 1898 in Breslau.

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Commentary

One very interesting event in Cohn’s life was his association with Robert Koch. After Koch became famous for his contribu-tion to bacteriology, a myth developed in Breslau to the effect that Koch had been a student of Cohn, and that his ideas owed much to Cohn’s influences. Cohn clarified his relationship with Koch in the newspaper Breslauer Zeitung, 17 December 1890. He stated that Koch was invited by him to Cohn’s institute at Breslau only to demonstrate results, which he had already reached on his own, and to ask Cohn and his colleagues for their judgments of his work. Cohn’s role was essentially that of stimulating and encouraging Koch’s work and of providing a place for its publication.

Cohn was a well-known botanist, but his work was closely related to microbiology. He published nearly 200 papers and books. Cohn received many honors in his life. He held an honor-ary doctorate from the faculty of medicine at the University of Tubingen and was named a corresponding member of the Academia dei Lincei in Rome, the Institut de France in Paris, and the Royal Society of London. In 1885, he was awarded the Leeuwenhoek Gold Medal and in 1895 the Gold Medal of the Linnean Society.

We generally consider Louis Pasteur, Robert Koch, Martinus W. Beijerinck (1851–1931), and Sergei N. Winogradsky (1856–1953) as major pioneers for the establishment of microbiology as an independent scientific discipline. But it is prudent to remember that Ferdinand N. Cohn was the true originator of bacteriology as an independent scientific discipline.

Suggested Reading

1. A Biographical Dictionary of Scientists (1974). T. I. Williams (Ed.), John Wiley & Sons, New York.

2. Dictionary of Scientific Biography (1971). C. C. Gillispie (Ed.), Scribner, New York.

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Chapter 9

Joseph Lister (1827–1912): Pioneer of Antisepsis

Source: https://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister(US Public Domain image)

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Introduction

In the mid-1800s, the likelihood of serious infections severely limited successful recovery from surgery. The Hungarian physi-cian-surgeon, Ignaz Semmelweis (1818–1865) urged surgeons to wash their hands with chlorinated water to reduce the risk of infection. Few surgeons followed his protocols, however, and Semmelweis’s career was marked by disappointment and frustra-tion. Semmelweis died on 13 August 1865, just one day before an 11-year-old boy was admitted to the Glasgow Royal Infirmary with a compound fracture of the left leg. Joseph Lister, a profes-sor of surgery, placed a dressing dipped in liquid carbolic acid (phenol) on the wound and set the broken bones. During the next two weeks. Lister inspected the wound and changed the dressing several times. In 1865, compound fractures, compared to simple fractures, often had disastrous consequences. In this case, however, Lister’s patient healed completely.

Joseph Lister was born on 5 April 1827 in Upton House, a mansion outside London. He was the second son of Joseph and Isabella Lister. The elder Joseph Lister was a wealthy wine mer-chant and amateur microscopist. He was elected to the Royal Society of London because of his invention of the achromatic lens. The family belonged to the Society of Friends (Quakers) and young Joseph attended Quaker schools. There, his talent for and interest in medicine were nurtured and before his six-teenth birthday, Lister wrote four essays on “The Human Structure — Osteology”. These essays were prophetic of his genius and obsession. In 1844, Lister was ready to go to college, because he was not a member of the Church of England, he could not attend Oxford or Cambridge universities. Thus, he enrolled at the University of London where there were only two disciplines, medicine and arts; unlike most aspiring pre-med students. Lister enrolled in the arts. In 1847, he received his Bachelor of Arts degree and began his study of medicine by auditing an anatomy class.

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In 1846, Lister’s elder brother died of smallpox and Lister himself contracted the disease a few months later. Although it was a mild case, the physical weakness and lethal potential scared him. He sought solace in religion and, to his family’s dis-approval, began preaching his religion and proselytizing. The combination of disease, self-doubt, and family disapproval led to a nervous breakdown in March 1848. During his recovery, he traveled through Europe for several months before returning to his studies. In 1852, Lister received his Bachelor of Medicine (MB) degree from the University of London and became a Fellow of the Royal College of Surgeons (FRCS). Lister had no desire to enter private practice and, with his family’s money, he didn’t have to; he did want to continue his studies and do research. In 1853, Lister went to the University of Edinburgh to study with James Symes (1799–1870), who was regarded as the most prominent British surgeon of the time.

In 1854, Lister became house surgeon at the Royal Infirmary of Edinburgh under Symes and fell in love with Symes’ eldest daughter, Agnes. He resigned from the Society of Friends and joined the Scottish Episcopal Church where Symes belonged. Joseph Lister and Agnes Symes were married on 23 April 1856. The Listers had no children and seemed to devote nearly all of their time to the advancement of science. One room of their home was always used as a laboratory. This was not unusual at the time because scientific equipment was relatively simple. It was common for them to work in the lab until four in the morning.

As the daughter of a surgeon, Agnes Lister had informal training in dissection and was familiar with laboratory work. Lab notes show that she was directly involved in all of Lister’s experiments and hundreds of pages of laboratory notebooks are in her handwriting with only an occasional note by Lister. Agnes Lister was not the first woman to work with her scientist-husband in their home laboratory and either she did not defy Victorian prejudices to get recognition, or he didn’t give her

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recognition. Years later, while defending his position by refus-ing women to either attend or allow his medical school lec-tures. Lister said his wife was unlike women who were excitable and unpredictable. Agnes Lister was “like a staid adult man”. Ironically, his letter opposing the admission of women into clinical training was written by Agnes Lister.

Apart from extensive traveling with his wife, Lister was completely focused on improving surgery and had few if any, contacts outside of university hospitals, scientific meetings and his immediate family. A colleague, Hector Cameron (1843–1928), described him as courteous, gentle, and modest.

Surgery

Surgeons separated from the barbers’ guild and came under the heading of medicine in 1815. In London, hospitals did not begin paying surgeons until 1848. Historically, physicians and surgeons were separated on the basis of education, physicians having had training in medicine. A physician was a diagnosti-cian and left bleeding and cutting to the surgeons. No doubt, this prevented physicians from being blamed for a patient’s death. Surgery was usually limited to procedures that could be completed in seconds such as amputations of diseased bones or crushed limbs because the patient experienced excruciating pain.

The American dentist, William Morton (1819–1868), intro-duced the use of ether in 1846 and the Scottish physician, James Simpson (1811–1870), used chloroform in 1847. Surgeons could now work at a more leisurely pace and do more invasive proce-dures such as tumor removal and ovariotomy, and spend more time with a patient’s viscera exposed. The patient, unfortu-nately, had a good chance of dying from hemorrhage or con-tracting an infection that would cause more pain and perhaps death. The surgical death rate at hospitals in Europe and the United States ranged between 10% and 60%. Patients must have been terrified by the appearance of a surgeon in his

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blood-stained black coat with silk ligatures, removed from a previous patient, dangling from the buttonholes.

Surgeons tied off blood vessels with silk threads and left the ends showing through the wound where they provided drain-age and could be removed. Lister suspected there was no rea-son to remove ligatures and was concerned that the free ends were a source of contamination. He disinfected silk threads and sealed them in an experimental ligature on the carotid artery of a horse. When the horse died of old age; six weeks later, he examined the ligatures and was pleased to find the thread cov-ered with new tissue. Nevertheless, an autopsy of a human whose death resulted from an aortic aneurysm revealed a prob-lem with the silk thread. The dead woman, who had survived for 10 months with silk ligatures on the external iliac artery, showed a fibrous knot at the silk noose that inhibited the flow of blood which led to the aneurysm. Even sterile silk thread had two distinct disadvantages: the tight noose damaged blood vessels so they could not heal and fragments of disintegrating silk inflamed the surrounding tissue. Lister began testing plant and animal tissues as ligatures and found that fibers from sheep intestines (catgut) did not disintegrate into sharp frag-ments that further damaged the blood vessel and if disinfected with carbolic acid, did not transmit infection.

The problem of hemorrhage was always a concern of sur-geons and the Lister’s conducted extensive experiments on coagulation. As a result of presenting this work on coagulation, Lister was elected a Fellow of the Royal Society of London (FRS) in 1860. During his career, Lister made several significant contri-butions to modern surgery. He invented a needle for silver-wire sutures, a hook for extracting objects lodged in the ear, slender-bladed sinus forceps, as well as catgut ligatures.

Aseptic Surgery

Lister enjoyed surgery but worried that he was contributing to many deaths. The pattern was always the same: the operation

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was a success but the patient died. To paraphrase Lister’s description of the symptoms with which every surgeon was familiar: At first, colored serum, tainted with the odor of decomposition, oozed from the wound. During the next two or three days, the smell became increasingly offensive until pus was discharged and after that, there was usually a death certifi-cate. Lister tried to make a connection between the pus and death.

At the time, it was believed that pus was a result of sponta-neous generation and there was no connection with contami-nation: but Lister’s thought were different. He noticed that when blood coagulates and forms a scab on a wound, “bad consequences are usually averted”. For a while, he believed that air caused pus formation and that the scab kept air out. Then, he read the work of Louis Pasteur.

Lister was impressed with Pasteur’s (1822–1895) research that demonstrated microorganisms were responsible for the chemical changes observed during decomposition of organic matter. Moreover, Pasteur had proven that these microbes were in the air. Lister reasoned that the microbes described by Pasteur in decomposing plant matter could also be responsible for the chemical changes seen in infected human tissue. The Listers repeated Pasteur’s S-neck flask experiment using urine in place of sugar-yeast water to verify the presence and growth of microbes.

Lister averred that all that was necessary to prevent infec-tion was to dress surgical wounds with something capable of killing the microbes. In 1864, Lister heard “of the remarkable effects produced by carbolic acid upon the sewage of the town of Carlisle, the admixture of a very small proportion not only preventing all odour from the lands irrigated with the refuse material, but, as it was stated, destroying the entozoa which usually infest cattle fed upon such pastures”. Lister’s first two attempts to use carbolic acid to prevent infection of broken skin were failed — both patients developed infections. He had disinfected the skin but didn’t have a way of preventing

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subsequent entry of microbes. On his third case, an 11-year-old boy who had been run over by a cart, Lister covered the wound with “lint” clipped in carbolic acid. Lister reported his success in treating this and other compound fractures with carbolic acid in a series of papers published in The Lancet in 1867. Lister was not unaware of the danger of carbolic acid. He experimented with crystalline as well as aqueous and oil dilutions of carbolic acid to find a preparation that would disinfect but not irritate or inflame tissue. At first Lister called his innovations as “anti-septic procedures”, but later he introduced the word aseptic.

Baron Joseph Lister, MB, FRCS, FRS

Lister liked to start his fall semester with new lecture material. Characteristic of his intellect, dedication, and compulsion, this new material was original research. For the Fall 1876 semester, Lister developed the technique of serial dilution to get a single bacterial cell from which to grow a pure culture.

Lister’s most famous patient was Queen Victoria (1819–1901). In 1871, he was called by the Queen’s private physician to lance a somewhat ignoble six-inch abscess in the Queen’s arm-pit. After opening the sore, Lister sprayed carbolic acid onto the wound before it was bandaged. Lister took a personal interest in the comfort and recovery of all of his patients and visited them daily. On one of these visits to The Queen, Lister saw that the wound was not draining properly and it occurred to him to use drainage tubes. He manufactured the necessary tubes from the rubber tubing used in the spray atomizer and slid them into the wound. (Lister did not know that the French surgeon, Pierre Chassaignac (1804–1879), used drainage tubes in 1859.)

In 1876, Lister spoke at the International Medical Congress in Philadelphia. Lister was the most distinguished medical per-son at the Congress and was seated next to Ulysses S. Grant (1822–1885), President of the United States, at the Medical Congress banquet. After listening to Lister’s two and one-half hour presentation, Joseph Lawrence (1836–1909), a Missouri

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physician, returned to his lab and developed an antibacterial mouthwash. The mouthwash, named Listerine®, was manufac-tured by Lambert Pharmacal Company. Robert Johnson (1845–1910), a New York pharmacist, was also in the audience in Philadelphia. Johnson listened to Lister’s description of a nine-layer gauze dressing covered with a layer of rubber. Lister made these dressings himself, soaking the gauze in carbolic acid and dying the rubber pink so it could easily be identified and placed away from a wound. Johnson and his two brothers, James and Edward, founded Johnson and Johnson Company to manufac-ture and sell sterile cotton and gauze dressings.

Lister spent his professional career in teaching hospitals. He lectured easily and his students appreciated his knowledge and dedication. Over the years the number of Listerian students, as disciples of aseptic techniques were called, began to outnum-ber the non-Listerians. Although British physicians had diffi-culty accepting Lister’s ideas (and Pasteur’s for that matter), physicians from Germany, France, Denmark, Italy, and the United States visited Lister at Edinburgh to learn his methods. Lister’s antiseptic procedures received nearly universal accept-ance following Robert Koch’s publication of the Germ Theory of Disease. In 1877, Lister was made an honorary member of the Budapest Medical Society. The Hungarians were probably bewildered later when Lister wrote that when he first applied the antiseptic principle to wounds and he had not heard of the Hungarian-born Semmelweis.

By 1892, Lister had been Professor of Clinical Surgery at King’s College of Medicine for 15 years and had reached the mandatory retirement age. Lister received many honors during his long career including the reprinting of his articles in a spe-cial issue of The British Medical Journal (BMJ) (5). In 1893, he was the first physician elevated to the peerage and henceforth, was known as Joseph Baron Lister. Lister was extraordinarily lonely after Lady Lister’s death in 1893. He tried to remain pro-fessionally active and served as President of the Royal Society from 1895 to 1900 and President of the British Association for

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the Advancement of Science (1896). As the years went by, he became reclusive. In 1903, he suffered a stroke from which he never recovered. He eventually went blind and almost totally deaf but continued to discuss aseptic procedures with col-leagues who visited. His last publications were in 1909 in The Lancet and BMJ.

Lister died in his sleep on 10 February 1912. Lister’s funeral at Westminster Abbey was attended by the British Prime Minister, many government officials, and representatives of the Monarchy. Burial at Westminster Abbey was offered but refused because Lister had wanted to be buried at West Hampstead Cemetery with his beloved Agnes.

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Chapter 10

Heinrich Anton de Bary (1831–1888): Pioneer of Mycology

Source: https://en.wikipedia.org/wiki/Heinrich_Anton_de_Bary(US Public Domain image)

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Introduction

Heinrich Anton de Bary is a pioneer in the study of fungi and algae. His extensive and careful studies of the life history of fungi and contribution to the understanding of algae and higher plants were landmarks of biology. He is regarded as the founder of modern mycology and plant pathology (phytopathology).

Life of de Bary

Heinrich de Bary was born on 26 January 1831 in Frankfurt, Germany. He was one of 10 children of August Theodor de Bary (1802–1873) and Emilie Meyer de Bary. August de Bary was a physician. He encouraged Heinrich to join the excursions of the active group of naturalists who collected specimens in the nearby countryside. De Bary’s youthful interest in plants and in examination of fungi and algae were inspired by George Fresenius (1808–1866), a physician, who also taught botany at Senckenberg Institute. Fresenius was an expert on thallophytes. In 1848, de Bary graduated from the Gymnasium at Frankfurt and began to study medicine at Heidelberg, continued at Marburg. In 1850, he went to Berlin to continue pursuing his study of medicine. During this period of time, he had the opportunity to study under Alexander Braun (1805–1877), a well-known botanist; Christian Gottfried Ehrenberg (1795–1876), known for his work on Infusoria; and Johannes Muller (1801–1858), an expert on plant physiology and anatomy. Despite the fact that his major discipline was in medicine, he continued to explore and develop interest in plant science. In 1853, he received his degree in medicine at Berlin, but his dis-sertation title was “De plantarum generatione sexuali”, a botanical subject. The same year, he published a book on the fungi that caused rusts and smuts in plants.

After graduation, de Bary practiced medicine in Frankfurt, but only for a very short period of time. He was drawn back to botany and became Privatdozent in botany at the University of

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Tubingen, where he worked as an assistant to Dr. Hugo von Mohl (1805–1872) for a while. In 1855, he succeeded the well-known botanist Carl von Nägeli (1817–1891) at Freiburg in Breisgau, where he established the most advanced botanical laboratory at the time and directed many students.

De Bary married Antoni Einery in 1861; they raised four children. In 1867, de Bary moved to the University of Halle to succeed the Professor Diederich. F. L. von Schlechtendal (1794–1866), who, with Hugo von Mohl, cofounded the pioneer botanical journal Botanische Zeitung. De Bary became the coeditor and later the sole editor of this journal. As an editor and a contributor of this journal, he exercised a great influence on the development of botany.

After the Franco-Prussian war (1870–1871), de Bary was appointed professor of botany at the University de Strassburg, and also elected as the first rector (president) of the reorgan-ized university. He conducted much research in the university botanical institute and attracted many students from Europe and America, and made a great contribution to the develop-ment of botany.

De Bary was devoted to the study of the life history of fungi. At that time, various fungi were still considered to arise through spontaneous generation. He proved that pathogenic fungi were not the products of cell contents of the affected plants, and they did not arise from the secretion of the sick cells.

In de Bary’s time, potato blight had caused sweeping crop devastation and economic loss. He studied the pathogen Peronospora infestans and made a great contribution by eluci-dating its life cycle. The origin of plant diseases was not known at that time. Much as Miles J. Berkeley (1803–1889) had insisted in 1841 that the fungus found in potato blight was the cause of diseases, de Bary declared that the rust and smut fungi were the causes of the pathological changes in diseased plants. He concluded that Uredinales and Ustilaginales were parasites.

De Bary spent a considerable amount of time studying the morphology of fungi and noticed that certain forms, which had

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been classified as separate species, were actually successive stages of development of the same organism. De Bary studied the developmental history of Myxomycetes (slime molds) and thought it was necessary to reclassify the lower animals. He first coined the term Mycetozoa to include lower animals and slime molds. In his work on Myxomycetes (1858), he pointed out that at one stage of their life cycle (the plasmodial stage), they were little more than formless, motile masses of the substance, and that Felix Dujardin (1801–1860) had called sarcode (proto-plasm). This is the fundamental basis of the protoplasmic theory of life.

De Bary was the first to demonstrate sexuality in fungi. In 1858, he had observed conjugation in the alga Spirogyra, and in 1861, he described sexual reproduction in the fungus Peronospora sp. He saw the necessity of observing the whole life cycle of pathogens, and he attempted to follow it in the living plants.

De Bary published his first work on fungi in 1861, then, spent more than 15 years studying Peronosporeae, particularly the P. infestans and Cystopus (Albugo), parasites of potatoes. In his published work in 1863 titled “Recherches sur le developpe-ment de quelques champignons parasites”, he inoculated spores of P. infestans on healthy potato leaves and observed the penetration of the leaf and the subsequent growth of the mycelium that affected the tissue, the formation of conidia, and the appearance of the characteristic black spots of the potato blight. He also did similar experiments on potato stalks and tubers. He watched conidia in the soil and their infection of the tubers. He observed that mycelium could survive the cold winter in the tubers. From all these studies, he concluded that organisms could not be generated spontaneously.

He did a thorough investigation on Puccinia graminis, the pathogen of the rust of wheat, rye, and other grains. He noticed that P. graminis produced reddish summer spores called “urediospore”, and dark winter spores called “teleutospores”. He inoculated sporidia from the winter spores of the wheat rust

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on the leaves of the common barberry (Berberis vulgaris). The sporidia germinated and led to the formation of aecia with yel-low spores — that was the familiar symptoms of infection on the barberry. De Bary then inoculated aeciospores on moisture retaining slide, then inoculated these to the leaves of seedling of rye plants. In time, he observed the reddish summer spores that appeared in the leaves. The sporidia from the winter spores germinated, but only on barberry. De Bary clearly dem-onstrated that P. graminis required different hosts during the different stages of its development (a phenomenon he called “heteroecism” in contrast to “autoecism”, when development take place only in one host). De Bary’s discovery explained why the eradication of the barberry plants had long been practiced as a control for rust.

De Bary also studied the formation of lichens, which are the result of an association between a fungus and an alga. He traced the stages through which they grew and reproduced, and the adaptations that enabled them to survive drought and winter. He coined the word “symbiosis” in 1879 in his mono-graph “Die Erscheinung der Symbios” (Strasbourg, 1879) as “the living together of unlike organisms”. He carefully studied the morphology of molds, yeasts, and fungi, and he basically established mycology as an independent science.

He died of a tumor of the jaw having boldly submitted to extensive surgery on 19 January 1888 in Strassburg, Germany (now Strasbourg, France).

Commentary

De Bary’s concept and methods had a great impact on the growing field of bacteriology and botany. He published more than 100 research papers. His personal influence was immense. Every one of his numerous students was enthusiastic in admira-tion of his kind nature and genial criticism, his humorous sar-casm, and his profound insight, knowledge, and originality. He directly impacted students who later became distinguished

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botanists and microbiologists such as Sergei N. Winogradsky (1856–1953), William Gilson Farlow (1844–1919), and Pierre-Marie-Alexis Millardet (1838–1902), and so on. He was one of the most influential of the 19th century bioscientists. To call him a pioneer of mycology is very appropriate.

Suggested Reading

1. Heinrich Anton de Bary. http://www.nndb.com/people/ 142/000096851/. Accessed 16 November 2016.

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http://www.nndb.com/people/142/000096851/

http://www.nndb.com/people/142/000096851/

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Chapter 11

Thomas Jonathan Burrill (1839–1916): Pioneer of Microbe and Plant Diseases

“Were I a heathen, not knowing the true God, I would not worship the sun — but would bow in adoration to the trees and herbs of the fields”.

—Thomas J. Burrill

Source: https://en.wikipedia.org/wiki/Thomas_Jonathan_Burrill(US Public Domain image)

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Introduction

We have learned so far that animal and human diseases are caused by many microbes, but microbes can also cause plant diseases. One of the pioneers of studying the relationship between microbes and plant is Thomas J. Burrill (1839–1916), who was a pioneer in the study of the plant disease “fire-blight”. He represented the American pioneer spirit of the time.

Life of Thomas Burrill

Burrill was born on 25 April 1839 near Pittsfield, Massachusetts. His father was John Burrill and his mother was Mary Francis. John Burrill was a native of Penrith, England, and came to the United States in 1818, settling first in Pawtucket, Rhode Island, where he worked in Slater’s Cotton Mill. The family was influ-enced by the tide of westward emigration at the time and moved to Stephenson County in northern Illinois when Thomas was only one year of age. They settled on a farm. Thomas helped his father on the farm and attended a country school a few months of each winter. At the age of 19, he entered high school in Freeport, Illinois. Because he was a painful self- conscious boy, he felt miserable in school and quit. The next year, he tried again in the Rockford school, where he graduated and entered the Illinois State Normal University at the age of 23 and graduated in 1865.

Thomas was studious. At Illinois State Normal University, he came in contact with good teachers. His teacher of botany was Dr. Joseph A. Sewall (1830–1917), curator of the Museum of the State Natural History Society located in Normal, Illinois. Thomas was greatly inspired by him. Because he attended the meetings of the Society of Natural History held in nearby Bloomington, Indiana, Thomas was further stimulated in his love of natural history.

Thomas’ career was a rather lucky one. From 1865 to 1868, Thomas held the position of Superintendent of the Urbana

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public schools. But he spent a lot of his spare time studying the flora of the Urbana area. In the summer of 1867, he was selected as a botanist to accompany Major John W. Powell (1834–1902) to explore the Grand Canyon and surrounding regions. The next year in the spring of 1868, he was called to the Illinois Industrial University (now the University of Illinois in Champaign/Urbana) to teach algebra. He was made Assistant Professor of Natural History and Botany later. Two years later, in 1870, he was promoted to the professorship of Botany and Horticulture. Academic advancement in those days was much easier and quicker than now. He then became established for a life of active and influential service on the faculty of the University of Illinois. He was a Professor of Botany and Horticulture in 1870–1903, and Professor of Botany from 1903 to 1912. He took charge of the university library as its first librarian and was secretary of the faculty from 1870 to 1883. He was Dean of College of Science from 1877 to 1884; dean of general faculty, 1894–1901; Vice President of the university from 1879 to 1912; and botanist of the agricultural experiment station, 1888–1912. Upon his retirement in 1912, he was made professor emeritus until his death in 1916.

Thomas Burrill began his academic career by teaching very fundamental courses. He taught most of the day in many sub-jects. He was a horticulturist in an experimental station. Most of the trees on the campus of the University of Illinois were planted with his own hands. He had an extensive love for trees. He was frequently heard to say, “Were I a heathen, not know-ing the true God, I would not worship the sun — but would bow in adoration to the trees and herbs of the fields”. In an Oriental old saying, “It takes tenths of years to plant trees; it takes hundredth years to nourish human talents”. He indeed planted the trees and also nourished human talents.

He was a hardworking man. He worked from sunup to mid-night or until all work was completed for that day. He was very much interested with the natural history of the state of Illinois. In 1869, he organized a group, including himself, that collected

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a large number of natural history specimen from Chicago to Cairo in the state of Illinois. He brought back specimens includ-ing 582 plants for preservation and study. In 1875, he made a collection of the woods of the state for the Centennial Exposition. In addition to teaching, he wrote reports, collected specimens from everywhere in the state of Illinois, served on numerous committees, nourished young scientists, and con-ducted experiments. His administrative skills also greatly con-tributed to the growth and the development of the University of Illinois. With little remnant of his time, he was charged by the Board of Reagent with the sale of mules. He lived a strenu-ous life indeed.

The most significant contribution of Burrill to science is in the field of Phytopathology, in which he is indeed a pioneer. Burrill became interested in the disease of pear blight. He noticed the pear blight might be a bacterial disease. Although the bacterial disease in animals was common, bacterial disease in plants was hardly heard of. Burrill presented his preliminary study on this malady to the State Horticultural Society in 1878. He described the organism seen in the tissues. He transferred the disease by exudation from the diseased plant to the bark of healthy trees. Finally, he conclusively proved that bacteria caused disease in plants and identified the organism as Micrococcus amylovorus. These results were published in the Report of Illinois Industrial University in 1882 and also in the American Naturalists in 1883. His findings were generally accepted by American botanists but were doubted by his con-temporary colleagues in Europe. However, his theory was even-tually proved to be totally correct.

He continued to study the bacterial diseases of sorghum and brown corn in 1886 and 1888. From 1885 to 1891, he became active on taxonomic work and planned to publish a volume on the cryptogenic flora of Illinois. Unfortunately, he did not realize his publications, because he was appointed as acting president for the university; the administrative duty diverted much of his time. But instead, he published two

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monographs: one was on rust, the other on mildew of Illinois. Both of them were valuable mycological publications. Beginning in 1902, he carried on extensive investigation on the bitter rot of apple, which caused a great damage to southern Illinois. He also engaged in the study of ear rots of corn, potato scab, rust of blackberry and raspberry, and peach “yellow”. All of these works established him as a pioneer in the field of phytopathol-ogy. Burrill was devoted to agriculture for the benefit of better living in the state of Illinois. His contribution to the Illinois College of Agriculture is immense.

Burrill was granted the degree of Master of Art (AM) by the Northwestern University in 1876. In 1881, the University of Chicago conferred the honorary degree of PhD to him. The Northwestern University also granted him the honorary degree of Law (LLD) in 1893. In 1912, just before his formal retirement, the University conferred him with the honorary degree of LLD. There were many honors that he received from the various sci-entific societies of which he was a member. He was elected President of the National Bacteriological Association in 1916. He was a member of the American Association for the Advancement of Science and elected Vice President in 1885. He was President of the American Microscopical Society, 1885–1903, and secretary, 1886–1889. He was also a member of Botanical Society of America, International Botanical Society, American Society of Naturalists, American Academy of Arts and Sciences, Illinois Academy of Science, and the University Club, Urbana, Illinois.

Burrill’s public service is also very impressive. In 1889, he was invited as one of three Commissions by the US Department of Agriculture to settle a controversy concerning a prevalent bac-terial disease of pigs. In 1895, he was appointed by Governor John P. Altgeld (1847–1902) of Illinois to investigate and report on the subject of tuberculosis in the state prisons.

After his retirement, he became engaged in active research on symbiosis between nodule bacteria and leguminous plants. He carried this research until the end of his life in 1916.

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One more thing worth mentioning: Burrill was among the first American botanists to introduce and use the compound microscope to biological laboratory. He was also the first to organize a bacteriological laboratory in an American State University; and one of the two or three American writers on plant diseases.

Burrill married Sarah Helen Alexander (1842–1920), daugh-ter of Ephraim Alexander of Schenectady, New York, on 22 July 1868 at Urbana, Illinois. Two children were born to them. He died in Urbana, Illinois, 14 April 1916.

Commentary

Burrill was an ideal companion and a resourceful person, and always was capable of seeing the amusing side of situation. His life was full of vigorous activities. He also enjoyed amateur photography and camping.

Burrill was a self-taught scientist. He laid the foundation of phytopathology, which extends the horizon of the contribution of microbiology. He published more than 100 research papers on scientific and educational subjects. He was initially employed to teach algebra by Illinois Industrial University. Most of the people would be happy to have that kind of teaching job, and probably would stay such a position for life. Yet, he was inter-ested in natural history and botany and developed into an expert of plant pathologist, and finally became a pioneer in phytopathology. All of these accomplishments were done dur-ing his unselfish service as a busy administrator. His scholastic efforts were impressive. He was the first to find that fire blight was caused by bacteria. He was indeed an American pioneer and researcher and teacher of plant pathology before the sci-ence even had a name. If not for the excessive time on admin-istrative and public service, his scientific contribution would be even greater.

His administrative and public service was superb. Look at the present status of the University of Illinois as an evidence. Is

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the university as one of the outstanding universities in the world? Burrill helped lay the profound foundation for this uni-versity. A Greeks proverb says, “Let not a man boast that he has had a happy life until the day of his death”. “Burrill would not have boasted of anything since he was quiet and unassuming rather than loud and aggressive. He lived unobtrusively, serenely, and usefully, and because he died full of years and of honor, he was loved by all his intimates, and respected by all who knew him”, said by Erwin F. Smith (1854–1927). Thomas Burrill is by all means a precious human jewel.

Suggested Reading

1. Glawe, D. A. (1992). Thomas J. Burrill, pioneer in plant pathology. Annual Review of Phytopathology 30: 17–25.

2. Smith, E. F. (1916). In memoriam: Thomas J. Burrill. Journal of Bacteriology 1: 269–271.

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Chapter 12

Gerhard Henrik Armauer Hansen (1841–1912): Pioneer of Leprosy Studies

Source: https://en.wikipedia.org/wiki/Gerhard_Armauer_Hansen(US Public Domain image)

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Introduction

Gerhard Henrick Armauer Hensen was well known for his pio-neer study of leprosy — a prominent disease in the history of human sufferings.

The term of leprosy came from the Hebrew. In both the Old and New Testament, the leprosy is given to a number of physi-cal conditions. These conditions were considered as a punish-ment from God for sin. The victim was said to be in a state of tsara’ath, or defilement. The Hebrew term was later translated as lepros, from which came the word leprosy. Leprosy is a chronic infectious disease that primarily affects the skin, mucous membranes, and nerves. This disease is caused by a rod-shape bacillus, Mycobacterium leprae, which was identified by Gerhard H. Armauer Hansen in 1874.

Armauer was born on 29 July 1841 in Bergen, Norway. He was the eighth of the 15 children (10 sons and five daughters). His father, Claus Hansen, was a wholesale merchant, who, fol-lowing bankruptcy after 1851, became a cashier in a bank. His mother, Elisabeth Concordia Schram, was a member of a family of master joiners long established in Bergen, Norway.

We know very little about the childhood of Armauer, but not at all, and he had a pleasant childhood. He was a good student; he did not have to study hard, and had no problem entering the university. He was also an excellent athlete. He entered the University of Christiania (now the University of Oslo) to study medicine in 1859. He had to earn a living while he was a student. He first taught at a school for girls; later, he worked as a substitute for the prosector of anatomy for a year. Then he began his tuition courses in anatomy. He worked so hard that as he later said he did not know that there were such physical or mental fatigues. He found the most efficient time to work was between six and eight o’clock in the morning. Hansen obtained his degree in medicine in 1866, and completed his internship at the National Hospital in Christiania. Then he went into medical practice at Lofoten, located on a group of islands

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of northern Norway. Lofoten was a community with population of 6000. They were mostly fishermen.

In 1868, Hansen entered the service of the leprosy hospitals in Bergen, and began his career in leprosy research. He worked under Dr. Daniel Cornelius Danielssen (1815–1894) at Saint Gorgen Hospital, and later became his son-in-law. Dr. Danielssen was an authority on leprosy. He published a book entitled Om Spedalskecd (On Leprosy) with Dr. Carl Wielhelm Boeck (1808–1875) in 1847. He helped to establish Bergen as the European center for leprosy research. Danielssen had discarded the idea that the disease was contagious. He purposely injected himself with material from leprosy patient and did not develop leprosy. He regarded leprosy as a hereditary disease, a viewpoint com-mon to other investigators at that time.

Hansen, on the other hand, after learning the epidemiology of leprosy, and in a meticulous series of 58 studies over a num-ber of years, he quickly realized that leprosy was a specific dis-ease which must have a specific cause. He showed that a unique bacillus was associated with the disease in every leprosy patients. His findings were in contradictory to Dr. Daielsson’s theory. Fortunately, Danielssen and Hansen had great mutual respect to each other and both were broad minded.

We must realize that bacteriology was still in its infancy at that time. The Koch's postulates had not yet been proposed. In 1870, Hansen managed to obtain a grant that allowed him to travel to improve his knowledge in histopathology in the European mainland. He went to Bonn and later to Vienna. He also visited Switzerland and Venice. Upon returning to Norway, he used primitive staining methods to stain the biopsy speci-mens from patients with leprosy. In 1873, he discovered rod-shaped bodies later identified as M. leprae in those specimens. He published his observations in 1874. By improving his staining technique in 1879, he was able to show great numbers of rod-shaped bacteria typically aggregated in parallel. He believed that the bacillus was the etiological agent of leprosy, which proved to be true. He was the first scientist to suggest that a

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chronic disease could be caused by microorganisms. This was a few years ahead of Robert Koch (1843–1910), who discovered the tuberculosis bacillus to be the cause of tuberculosis in 1882.

Hansen put a lot of effort into trying to prove that his bacil-lus (people at that time often called it Hansen’s bacillus) was indeed the cause of the disease. He attempted to isolate M. leprae on artificial media, but without success. He tried to transmit the disease to rabbits, but failed. He had not suc-ceeded in any experimental reproduction of the disease. How could he study the disease further? In an ill-advised effort, Hansen inoculated the eye of a woman suffering from the neu-ral form of the disease with material drawn from a leprous nodule of a patient suffering from the cutaneous form. There was no clinical consequence following the inoculation. But, Hansen because of not asking for permission to perform such an experiment, met with a legal problem. As a result of this, he lost his position as resident physician of the Bergen leprosy hos-pitals in 31 May 1880.

Nevertheless, Hansen in 1875 became the leprosy medical officer for the entire country of Norway. He was on this post until his death. Through his effort, he implemented changes in the control methods of leprosy. Many of the changes were based on his hypothesis that the etiological agent of the dis-ease is M. leprae. As a result of his tireless effort, the Norwegian Leprosy Act was passed in 1877, and amended in 1885. Under the new regulations, patients of leprosy could live in precautionary isolation away from their families. This disease quickly and steadily declined in Norway. There were 1752 known cases in Norway in 1875, and decreased to 577 cases by the end of the 19th century. The word “hansenarium” was suggested to replace the standard “leprosarium”. The contributions of Hansen were obvious, and he won the respect of his people.

A very interesting episode occurred to the discovery of Hansen’s leprosy organism. In 1879, Albert Neisser (1855–1916) from German visited Hansen’s laboratory. Neisser tried to apply

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new staining techniques developed by Robert Koch, that is, staining the tissues of leprosy patients, which was provided by Hansen. Neisser did not succeed, but took some tissues back to Germany where he succeeded in staining them. Therefore, Neisser published a paper of his discovery. In the following year, Neisser published a second article in which he claimed the honor of discovering a microbe that caused the disease. Neisser totally discredited Hansen who had willingly shown him every-thing that he could and had surely been instrumental in provid-ing Neisser’s material. This episode was later reviewed by G. L. Fite and H. W. Wade, and they commented that “Far from genuinely giving Hansen any credit, Neisser spent much effort to assert the importance of his organisms as against Hansen’s”. Neisser’s behavior had not been appreciated by scientific com-munities or the general public.

Because of his leprotic studies, Hansen received many hon-ors in his lifetime. He was elected as honorary chairman of the first Conference Internationale de la lepre, held in Berlin in 1897, and president of the second such conference in 1909, also held in Bergen. He was the honorary chairman of the International Leprosy Committee and a corresponding or hon-orary member of numerous scientific societies. His contribu-tions were so well-recognized that contributions toward a portrait bust of Hansen were solicited internationally in 1900, and the bust was unveiled with great ceremony in 1901.

Hansen was also interested in the doctrine of Darwin. He learned the doctrine of Darwin during his trip to Vienna some time in 1870. He decided to study Darwin’s book, and sought to emulate Darwin’s methods. He considered Darwin’s methods as a model of dispassionate observations. Although he was not interested in philosophical speculations, he did have apostolic zeal for Darwin’s work. He gave numerous lectures and pub-lished articles in the popular press about Darwin’s doctrine. These actions evoked a serious attack from clergy and religious groups. Hansen, however, ignored the attacks made against him, and continued to carry on his regular work.

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Hansen married Stephanie Marie, daughter of Dr. Danielssen on 7 January 1873. Unfortunately, Stephanie died of pulmonary tuberculosis on 25 October of the same year. In honor of his love, Hansen named a new marine organism that he had discovered, Stephanostoma hansenii. Hansen married again on 27 August 1875 to Johanne Margrethe Tidemand. She was a widow and was related to the many commercial noble families in Bergen. They had one son, Daniel Cornelius Armauer Hansen (1876–1950), who also became a medical doctor who specialized in tuberculosis. Armauer Jr. became the chief of tuberculosis hospi-tal in Bergen in 1929 and also was a distinguished leprologist.

Hansen was a man of many interests, ranging from music, religion, and polar exploration, to marine biology. He was also fluent in several languages. He was very knowledgeable with scientific advances of his days, and he was well known and was friendly with Norwegian contemporaries including Edvard Grieg (1843–1907), the famous composer, Henrik Ibsen (1828–1906), a world renowned playwright, and Bjornstijerne Bjornson (1832–1910), a Nobel prizewinning writer and a poet, and a leader in the movement for an independent Norway. In 1900, Hansen suffered the first of a series of heart attacks, which con-fined him to periods of bed rest. But, he still continued to carry on his job and traveled in the country on his official inspection duty. In February of 1912, he traveled to Floro, a fishing village north of Bergen, and stayed at one of his friend’s house. Unfortunately, a heart attack occurred, and he died there on 12 February. He was given a State Funeral and he is remembered as a hero who fought against leprosy. The government of Norway established a museum in his memory. Today, the museum is an attractive site for tourists.

Now, we do not fear leprosy as much as it was feared in the Middle Ages. But there are still millions of people, mostly in Africa and Asia, who suffer from leprosy. In the United States, approximately 5500 known cases of leprosy exist, and nearly 200 new cases of leprosy are reported each year although there is a gradual increase. Leprosy is not as contagious as other

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diseases. Contamination usually occurs only between people in fairly intimate and prolonged contact. The time from infection to the appearance of symptoms is usually measured in years. Patients with leprosy are no longer kept in isolation, because now they can be made noncontagious within a few days by the administration of sulfone drugs. Chaulmoogra oil was used for the treatment of leprosy for many years. Present day, the sul-fone drug, “dapsone”, has been the mainstay of treatment. But recently rifampin and a fat-soluble dye, clofazimine, have been used in combination with “dapsone” to fight drug-resistant strains. Although M. leprae was identified as the etiological agent for leprosy, it has never been grown in artificial media. Experimental reproduction of the disease can be done in the footpad of armadillos. Vaccines for leprosy are still in develop-ment. Interestingly, the Bacillus Calmette-Guérin (BCG) vaccine for tuberculosis seems to be effective against leprosy.

Suggested Reading

1. Fite, G. L. and H. W. Wade (1955). The contribution of Neisser to the establishment of the Hansen bacillus as the etiologic agent and the so-called Hansen-Neisser contro-versy. International Journal of Leprosy 23: 418–428.

2. Jay, V. (2000). The legacy of Armauer Hansen. Archives of Pathology & Laboratory Medicine 124: 496–497.

3. Vogelsang, T. M. (1976). Hansen, Gerhard Henrik Armauer. In Dictionary of Scientific Biographies, p. 101.

4. Hansen, G. A. (Translator) (1976). The Memoirs and Reflection of Dr. G. Armaur Hansen. German Leprosy Relief Association (DHAW).

5. Patrix, J.-M. (1997). Gerhard Armauer Hansen, Leprabasillens oppdager. Edie Forlag A/S.

6. Gonzalezprendes, M. A. (1964) Leprosy Review 35:127–147. 7. Hansen, G. A. (1910). Livserindringer og Beragtninger,

English translation from G. Fite. Forlagt af H. Aschehoug and Co., Kristiania.

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8. Truman, R. W. (2011). Probable zoonotic leprosy in the Southern United States. The New England Journal of Medicine 364: 1626–1633.

9. Ryan, K. J. and C. G. Ray (Eds.) (2004). Sherris Medical Microbiology (4th Edition). pp. 451–453, McGraw Hill, USA.

10. Yawalkar, S. J., A. C. McDougall, J. Languillon, S. Ghosh, S. K. Hajra, D. V. Opromolla and C. J. Tonello (1982). Once monthly rifampicin plus daily dapsone in initial treatment of lepromatous leprosy. Lancet 1(8283): 1199–1202.

11. World Health Organization (1995). Leprosy disabilities: magnitude of the problem. Weekly Epidemiological Record 70(38): 269–275.

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Chapter 13

Robert Koch (1843–1910): The Great Medical Microbiologist

“One should first investigate the problems with attainable solutions. With this knowledge thus gained, we can proceed to the next attainable objectives”.

“I have undertaken my investigations in the interest of public health and I hope the greatest benefits will accrue from there”.

“If I think today of all of praise which you have heaped upon me, I must, of course, immediately ask myself if I deserve it. Am I really entitled to such homage? I guess that I can, with a clear conscience, accept much of the praise you have bestowed upon me. But I have really done nothing else than what you yourself are doing every day. All I have done is work hard and fulfill my duty and obligations. If my efforts have led to greater success than usual, this is due, I believe, to the fact that during my wanderings in the field of medicine, I have strayed on the paths where the gold was still lying by the wayside. It takes a little luck to be able to distinguish gold from dross, but that is all”.

— Robert Koch

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Source: https://en.wikipedia.org/wiki/Robert_Koch(US Public Domain image)

Introduction

Robert Koch is an important figure in the history of combating human diseases. His “Koch’s postulates” serve as the gold standard for identifying disease agents. Many infectious dis-eases such as acquired immunodeficiency syndrome (AIDS), Hanta virus pulmonary syndrome, and the avian flu are threat-ening us today, as well as reemerging threats from the past such as tuberculosis and hemorrhagic diarrhea. Therefore, it is worthwhile to recall how we battled diseases in the past. The past may give leads for the future.

Background and Education

Robert Koch was born to Hermann Koch and Mathilde Julie Henriette Biewend on 11 December 1843 in Klausthal, Harz, an

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old mountain town of some 10,000 inhabitants, 50 miles south of Hannover, a part of Prussia. Robert was the third son of 10 brothers and sisters. Koch’s parents and family encouraged his interests in nature, science, and photography.

Robert attended the Clausthal Gymnasium (equivalent to high school in the United States) in 1851. In 1853, Koch entered the Georgia Augusta Universität in Gottingen. Initially, he was interested in philology (linguistics) but his adviser suggested that he study medicine or mathematics and natural sciences. After studying botany, physics, and mathematics for two semes-ters, he transferred to medicine.

Between 1860 and 1870 when Louis Pasteur (1822–1895) was discovering the microbiology of the wine, beer, and vine-gar industries, and controlling the parasites of diseased silk-worms, Robert Koch was a small nearsighted medical student. One of Koch’s favorite teachers in the university was Professor Friedrich Gustav Jacob Henle (1809–1885), who aroused Robert’s interest in bacteriology. He was also influenced by Georg Meissner (1829–1905), an anatomist, and Karl Ewald Hasse (1810–1902), a clinician who taught him pathology and thera-peutics. In 1865, Koch was an assistant to Professor Wilhelm Krause (1833–1910) in the pathological institute of the univer-sity. During this tenure, Koch conducted research that he reported in a prizewinning paper “Umber das Vorkommen von Ganglienzellen an den Nerven des Uterus (On the presence of ganglion cells on nerves of the uterus)”, which he dedicated to his father. He received his MD (Eximia cum Laude) degree in 1866 when he was 23 years old. After his graduation, he worked as an assistant physician in the hospital of Hamburg and later in Langenhagen.

Early Career

Robert really wanted to become a ship’s doctor and sail around the world; however, in 1867 he married Emmy Adolfine Josephine Fraatz (1847–1913), the youngest daughter of the Superintendent General of Clausthal, whom he met in the

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hospital. In 1868, following the wedding, the Koch’s left for Langenhagen to practice medicine in Niemegk, near Potsdam. Their only child Gertrude was born in September of that year. In 1869, the family moved to Rakwitz (now in Poland).

Life was dull for Robert. He was a poor existence of riding horseback through the mud and waiting up nights to deliver the babies of farm wives. His fate seemed to be merely that of attempting to alleviate the pain of sick people and to save as many lives as possible.

During the Franco-Prussian War, Robert served as an Army surgeon (1870–1871). In 1871, Koch was discharged from the army and, unfortunately, lost his mother in the same year. He went back to practice medicine in Rakwitz. However, boredom and disappointment persisted because he knew neither the cause nor the cure for so many ailments. He carried a little hand lens with him and often looked at boils and rashes, trying to see if he could observe something that might give him a clue to the disorder. Emmy had observed how he turned his little hand lens on everything and she bought him a microscope for his 28th birthday in December of 1871. This gift would influence Koch’s future work.

In 1872, the Koch family moved to Wolsztyn, East Prussia. His job title was “kreisphysikus”. Dr. Koch was a good doctor, and he became highly respected locally. However, in his contin-ued pursuit of trying to understand the causes of disease, Koch kept perfecting his skills with the microscope. He also became particularly interested in the etiology of infections. In order to pursue his interests, Robert made a small laboratory and primi-tive dark room in his surgery suite and began to look at drops of blood from sheep and cattle, which had died of anthrax.

Using his beloved microscope, Koch looked at the black blood from animals dying of anthrax and always saw some tiny “sticks” and “threads”, finer than silk. By intuition, he also looked at the blood of normal healthy goats, sheep, and cows. He never found any of these sticks and threads in their blood. Did the little sticks cause anthrax? Had he been Louis Pasteur,

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he would have immediately said “yes” to this question and would have proceeded with his experiments. However, not being aware of Pasteur’s work, Koch developed a different, more methodical approach to study as compared to Pasteur.

While observing the “sticks” and “threads” on a slide of blood from an anthrax victim, Koch wondered: “They don’t move. Were the “sticks” and “threads” microbes, or does the sick blood form the ‘threads and sticks’?” Others in France, such as Casimir Davaine (1812–1882) and Louis Pasteur had made similar observations and had announced they were bacilli, liv-ing microbes that were probably the cause of the disease anthrax. However, Koch tended to be a “lone wolf” researcher. He did not care what others believed; he wanted to find the truth for himself. Faced with the reality of not being able to afford sheep or goats for experiments; he found that he could get children to catch white mice. He soon had what we would call a colony for his experiments. Being unaware of Pasteur’s work with anthrax, Koch developed the method of inoculating the mice with sterile wood slivers, soaked in blood from an ani-mal, which died of anthrax. He cut a tiny slit with his scalpel and stuck the sliver into the slit in the tail. Koch studied the experimental disease in mice and found that it was entirely similar to the disease in larger animals; at necropsy, the most noticeable feature was an enormously enlarged spleen. He found that the blood and spleen were good sources of the little “sticks and threads”. But he wanted to see what was reproduc-ing in the blood, free of debris in the blood.

The Hanging Drop Slide

Every microbiologist today is taught routinely how to make a hanging drop slide to study bacteria in isolation. Koch was the first to do this, and at that time the only one to use the method. He used fluid from the eye of an ox as a medium, which would approximate blood and would be clear for observation. By inoculating the drop of ox eye fluid with the smallest amount

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of infected blood or spleen possible he was able to culture anthrax bacilli and observe their multiplication free of other debris. He was now able to transfer the bacillus from one hang-ing drop to another and so achieve a pure culture. After eight such transfers, Koch placed a drop of the fluid, swarming with bacilli, on a sterilized wood sliver which he inoculated it into a slit in a mouse’s tail. The next day Koch had a dead mouse, and to his immense joy, he found the same bacilli from the spleen. He had proved that the same little rods that killed cattle, sheep, and mice were the same bacillus, which he had so carefully cul-tivated in his ox-eye fluid. Even ahead of Pasteur, Koch demon-strated that one certain kind of microbe caused a specific disease.

It is almost unbelievable, today, to think that such magnifi-cent and original work would go unreported in scientific jour-nals and meetings. But, Koch never gave this a thought. He kept on inoculating various species of animals with the bacilli from his hanging drop cultures until he found that they would kill sheep. But one big question worried him. Why did the dis-ease disappear and reappear? Why were certain fields sure to bring down cattle or sheep at any time? Koch was aware of the superstitions surrounding some areas in France where no flock of sheep could go without dying of anthrax because the areas were “cursed”. However, he also observed that his little bacilli could die and that animals exposed to cultures swarming with thousands of anthrax germs were not always killed.

Then one day, he found the answer to the “cursed” fields and the reappearance of anthrax in flocks and herds. Inadvertently, he had left a hanging drop culture at body tem-perature for more than 24 hours. Thinking it would be full of long threads of bacilli, he was surprised to see that the threads were not as clear and each one contained small specks resem-bled glass beads. They looked almost like strings of tiny pearls. At first he feared contamination of his culture, but by careful observation he discovered that the beads were inside of the threads. An experiment came quickly in mind, and he took

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some of the fluid from the eye of a steer and made a new hang-ing drop in which he put some of the threads with “pearls”. He also put bovine eye fluid on dried smears of anthrax and watched these in the hanging drop cultures.

Spores!

To his surprise, Koch observed the transformation of the little microscopic beads into typical anthrax bacilli! His experiments with spores kept him in his laboratory for several months, dur-ing that time he found that the spores never developed in liv-ing animals, nor did they ordinarily develop at low temperatures. They usually developed in dead animals.

Finally, Koch felt that he had all of the pieces of the puzzle together and decided to reveal his findings to the scientific community. He wrote to Professor Ferdinand Julius Cohn (1828–1898) at the University of Breslau (now Wroctan). In 1876, Professor Cohn set up a “seminar” for Koch with the medical faculty. To their surprise, the stuttering Koch did not lecture. Instead of telling, he showed them in three days and nights what he had taken months and years of learning. He did not argue or harangue, but with great skill he slipped his inocu-lated wood sliver into the mouse tails. The distinguished pro-fessors were amazed to see how he handled his hanging drop cultures, his bacilli and spores, and microscopes.

Professor Julius Friedrich Cohnheim (1839–1884), considered him the most skilled scientist in Germany, rushed through the building and told all of the students and professors to go to see Dr. Koch. By this time, all professors and students, including the famous Paul Ehrlich (1854–1915) came to see Koch. Cohn and Cohnheim did their best to get Koch to come to Breslau, but their efforts failed. Cohn also arranged for the publication of Koch’s work on anthrax. After a short stay in the city, Koch returned to Wollstein and continued his microbiological research.

In 1878, Koch also published his work on the isolation and cultivation of staphylococci from surgical infections, and he

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described staphylococci seen in microscopic examinations of substances exude from infected wounds. Koch’s friends at Breslau had not forgotten him. In 1880, the government awarded him the position of Extraordinary Associate of the Imperial Health Office in Berlin. Finally, Koch had a real labora-tory and two assistants: Friedrich August Johannes Loeffler (1852–1915) and Georg Theodor August Gaffky (1850–1918).

Photomicrography!

Today, it is almost unbelievable that Koch took photomicro-graphs with the equipment of the 1880s. We know that Koch had discussions with microscopists, Carl Zeiss (1816–1888) and Ernst Abbe (1840–1905) in l878. Koch had observed that two people could look into a microscope and see different things! Few people believed the hand drawn micrographs were real. “No one will believe there are germs until they see photo-graphs. Then ten men can see the same image at the same time and come to an agreement on it”. Koch was making a true sci-ence of microbiology.

Flourishing of Microbial Diseases Research

The discoveries of Koch now became known in both Europe and America and a wave of enthusiasm swept over the medical world. Everyone wanted a microscope; however, at the same time, a fear of germs caused some researchers to work in a mist of germicides which resulted in much questionable research.

Koch was not influenced by these concerns. He decided that the most important task was to isolate and culture single germs in the absence of air-borne contaminants. With this goal in mind, he looked at the surface of half of a boiled potato — and was surprised to see small colored round spots on the surface: a violet one, a grimy one, and a red one. When he used a plati-num wire loop to pick up a bit of one of the spots and examine it under the microscope, he was surprised to find that every

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bacterium from a drop were the same. The potato served as an excellent solid medium for some bacteria. But, Koch pressed on and developed a medium with gelatin and beef broth. Later, Professor Walther Hesse (1846–1911) used agar-agar, a seaweed product for solidifying media. Today, this product is commonly used in microbiology laboratories all over the world and is sim-ply called “agar”. Koch also developed the methods of using dilutions and then placing the dilutions on solid media to achieve a pure bacterial culture.

Koch published his “Methods for the Study of Pathogenic Organisms” in 1881. During this time, he also had the opportu-nity to demonstrate his culture methods to Louis Pasteur and Joseph Lister (1827–1912). With his successes, Koch forged ahead to new challenges.

Consumption, or what is now called tuberculosis, was ram-pant at that time around the world. It was known that the disease could be transmitted from sick people to cows and other animals. Professor Cohnheim, Koch’s friend, had found that he could infect a rabbit in the eye and watch the tubercles grow. Koch also began to use rabbits and guinea pigs, and infecting them with tubercles from men who had died of tuber-culosis. He studied the tissue of animals and men (obtained from sanitaria where people had died of consumption), but failed to see any microscopic organisms.

Pathologists also studied the tissues of patients. They stained the tissues in hopes of observing the bacteria, but the results were mixed. Stained tissues, however, gave Koch the idea that he might need to stain the organism in order to be able to see it. He tried a variety of stains without success until he discovered that methylene blue, a stain Paul Ehrlich was using, would dye the tubercle bacillus — but only when the slides were left in the stain for a long time. Now he was able to see tangled masses of the little curved bacterium in all diseased tissues.

Growing the tubercle bacillus on his usual media proved dif-ficult. Koch decided that because the organism was almost a

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complete parasite and was so closely associated with the tissue of the body, he needed a special nutritive medium for it. Going to the butcher shops he obtained the straw colored serum that was left when blood clotted. He sterilized this serum and poured it into test tubes, to form what we now call “slant tubes”. The cultures on the “slant tubes” did not seem to work; no growth was seen on them in a day or even a week. But Koch was not the one to give up and after 15 days he found very tiny specks. These proved to be colonies of the elusive tubercle bacillus! Koch tried the bacillus on a wide range of animals, including monkeys and even goldfish, and found that mammals were highly susceptible to the bacillus.

Koch experienced many successes in 1882. He presented his work on the discovery of the tuberculosis bacillus to the Berlin Physiological Society (This work won him a Nobel Prize in 1905). In this article, he outlined the four steps necessary to prove that a given organism was the cause of a given disease. These were the famous “Koch postulates”.

During this time, Koch also discovered that Asiatic cholera was caused by the “comma bacillus” and that the disease was transmitted by contaminated water. In 1883, Koch announced the development of a vaccine against cholera and headed the “German Cholera Commission” in Egypt and India. In 1884, he reported the discovery of Cholera vibrio. In 1885, he won an appointment as a professor at the Berlin University and shortly thereafter was named to two directorships simultaneously — the Prussian Board of Health and the Hygienic Institute of Berlin.

Continuing his work with tuberculosis, he successfully dem-onstrated that tuberculosis could be transmitted by aerosols. Lacking a modern laboratory, Koch made a crude box and put in his garden. He ran a lead pipe from a window of his house and with hand billows; pumped a deadly aerosol of tubercle bacilli into the box housing a variety of animal species. The ani-mals all succumbed to the bacilli at varying rates. Based on this work, Koch contributed to the field of public health through

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his communications and by offering courses in public health at the university.

Tuberculin Incidence

In 1890, when Koch reported his work on the discovery of tuberculin, a product of the tuberculosis bacillus, many of the most eminent scientists in Germany were present. He announced that an injection of tuberculin into guinea pigs arrested the generalized diseases.

Rudolf Virchow (1821–1902), who had been previously criti-cal of Koch’s work was there and when Koch finished, many expected the “normal” criticisms. However, Koch, by his thor-ough investigations had preempted any questions.

A clinical trial was conducted later that year using the tuberculin isolated from the culture of Mycobacterium tubercu-losis. Unfortunately, the tuberculin vaccinations produced a cell mediated, delayed type of hypersensitivity, which resulted in some deaths. In spite of the disappointment, the tuberculin test is still a powerful test for the diagnosis of tuberculosis.

Further Infectious Disease Research

In 1891, Koch was appointed director of the Institute for Infectious Disease, where he continued his research, and offered the first postgraduate course in bacteriology. Many of his disci-ples, either as students, assistants, or trainees, later became remarkable scientists who made significant contributions to medicine and/or microbiology. Some notable examples include Carl Fraenkel (1861–1915), Wilhelm Dönitz (1838–1912), Richard Friedrich Johannes Pfeiffer (1858–1945), and Emil Adolf von Behring (1854–1917) from German; Kitasato Shibasaburo (1853–1931) from Japan; and William Henry Welch (1850–1934) and Theophil Mitchell Prudden (1849–1924) from the United States.

In 1892, Koch became involved with the European cholera epidemic; in 1896, he visited South Africa to investigate disease

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called rinderpest, against which he developed a vaccine. He also investigated black-water fever, malaria, red-water fever, and sleeping sickness. In 1897, he visited India and German East Africa to study bubonic plague, Texas fever, and malaria. He designed measures to fight leprosy in the district of Memel. In 1898, he returned from Africa to Berlin. In the same year, he went to Italy to study children’s malaria where he applied the quinine prophylaxis treatment. Next year (1899), he went to Java and New Guinea to study malaria. In 1901, during the Tuberculosis Congress in London, Koch announced his findings that bovine and human tuberculosis were different. In 1903, he began his studies on cattle infectious diseases in Africa, where he celebrated his 60th birthday in Bulawayo. Koch was the first to observe spirochetes in the blood of persons with relapsing fever. He discovered ticks were carriers of the relapsing fever agent. He also studied the development of Piroplasam. One of the bacteria, Borrelia kochii was named after him. In 1904, he relinquished his directorship of the Institute for Infectious Disease, and concentrated on studies of sleeping sickness dis-ease in Dar es Salaam. In 1905, he returned from Africa and received Germany’s Order of the Crown, and was awarded a gold medal at the Paris International Tuberculosis Conference. In December 1905, he was awarded the Nobel Prize. In 1906–1907, he revisited Africa to study sleeping sickness, a parasitic disease, and discovered that the tsetse fly transmitted the caus-tic parasite. In 1907, he returned from Africa and was granted a title of “Excellence” by the Kaiser.

Visits to the United States and Final Years

Robert Koch visited the United States twice in 1908. The first time was in April when he and his wife were welcomed by the German Medical Society and other distinguished scientists and famous individuals including Andrew Carnegie (1835–1919), Simon Flexner (1863–1946), William Henry Welch (1850–1934), and Theobald Smith (1859–1934). He was welcomed in Japan by

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his former student and a colleague, Kitasato Shibasaburo. Koch had intended to return to Germany by way of China and India, but his plan did not materialize because he was ordered by the German government to participate in the International Tuberculosis Congress, which was held in Washington, DC on September 1908. In this Congress, Koch was involved in the controversy of the transmission of the bovine tubercular bacilli to humans. Koch had previously (1901) stated that the bovine tuberculosis was not transmissible to man, and he held firmly to his view. However, there was a transmission of bovine tubercu-losis from cattle via milk. The controversy boiled down to the fact that Koch was concerned with the classical, pulmonary tuberculosis. However, the opponents were concerned with serious infections of people with tubercular bacillus, which occurred most commonly in children and tended to be intesti-nal versus pulmonary. Unfortunately, after the Washington meeting of the same year, Koch’s influence in America was sig-nificantly diminished since his view on bovine tuberculosis was in error.

Koch returned to Berlin on 21 October 1908. Although he intended to initiate research on tuberculosis, his health deterio-rated, and he was not able to conduct further research. Due to a heart attack, he spent most of his final three years at Baden-Baden, a health resort. On 27 May 1910, Robert Koch died from heart disease. His ashes were deposited in a mausoleum at the Institute for Infectious Diseases, which the Kaiser ordered to be named after Robert Koch. A shrine was also dedicated to him in Japan; on which Élie Metchnikoff (1845–1916) placed a com-memorative plaque from the Pasteur Institute.

Honors and Awards

Many honors and awards were bestowed upon Robert Koch. Notably, he received the Harben Medal in 1901 and was given a foreign membership into the Paris Academy of Science in 1902. In 1905, he was awarded with the Nobel Prize in

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Physiology or Medicine. In 1906, he received the Prussian Order Pour le Mérite and the title Wirklicher Geheimer Rat with the predicate Excellenz. Early in 1908, he received the first Robert Koch Medal which was a series of honors intended to com-memorate the greatest living physicians. He proposed to estab-lish a Robert Koch Foundation to combat tuberculosis, and it was approved, and a million marks were quickly accumulated for it. The Kaiser (Wilhelm II 1859–1941) contributed 100,000 marks and Andrew Carnegie donated 500,000 marks. There were many other honors bestowed upon him in his lifetime.

Personal Caliber

Koch was a remarkable person and worked hard during his entire life. His laboratory attracted distinguished scientists from all over the world. In addition to his numerous contributions, Koch’s other interests ranged from the arts to astronomy and mathematics. He was also interested in anthropology, ethnol-ogy, and geography. He admired Johann Wolfgang von Goethe (1749–1832), and loved to play chess as an entertainment. He disliked didactic lecturing and was not a good speaker; he pre-ferred to convince people by demonstrating facts rather than brilliant rhetoric. Nevertheless, he did have a good sense of humor within his intimate circle of friends.

Commentary

Koch’s research has been of the greatest importance to the sci-entific world; many of the laboratory practices he started are still followed today. He not only discovered the specific organ-ism responsible for the specific disease, he also developed sani-tary measures, such as water filtration, for controlling the spread of various diseases. Robert Koch left a legacy of knowl-edge and understanding for all mankind.

Koch and Pasteur had long scientific arguments over anthrax. Koch, with meticulous skills, let Pasteur knew that the

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anthrax cultures, which were supposed to kill only mice, also killed rabbits. He found other discrepancies related to the primitive state of Pasteur’s microbiology. There were also batches of Pasteur’s anthrax vaccine, which killed sheep. Koch and Pasteur also competed in discovering the cause of Asiatic cholera. Although there was strong nationalistic feeling in each of these scientists, when a French microbiologist, Louis Ferdinand Thuillier (1856–1883), died of cholera, Koch laid a wreath of laurel on the grave, such as “should be given to the brave”. Although he had the fate of Thuillier (died of cholera infection) in mind, Koch continued to work on cholera and finally isolated the causative agent and developed methods of handling the causative bacteria of the disease. For this discovery, he was received like a conquering hero when he returned from India to his native Germany.

The lives of Pasteur and Koch reveal how different approaches succeed! Pasteur amazed the world by developing a vaccine for rabies and the successfully treating Joseph Meister (1876–1940) and 16 Russian peasants who were bitten by a mad wolf. Koch brought world attention to Germany, as Pasteur’s work did for France.

A present day microbiologist can learn a great deal from a study of the human side of these two truly pioneer microbiolo-gists. It is clear that there is no single way to pioneer a scientific discovery. Pasteur was to follow his intuition; he had flashes of insight, and often his ideas were completely unsound (we sel-dom hear of these). However, Pasteur was able to pursue the sound insights and drop the unsound.

Koch was the epitome of the logical and the systematized. He carefully thought through his problems and approached each one in a sequence of logical steps. He also used the “sci-entific method” to an extent often ignored by Pasteur. He actu-ally isolated his organisms, grew them in pure cultures outside the body of the animals, recreated the disease in normal ani-mals and then, again recovered the organisms in pure culture (Koch’s Postulates!). At each step, he demonstrated conclusively

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that he was in control of the causative organism and that there was freedom from contamination. The methodology of microbiology today is largely derived from the work of these two men.

We also might consider the motivation of both men in their tireless work to rid the world of tragic diseases that caused untold sufferings and death, with no hope of alleviation until their marvelous experiments. In view of his continuous quest for studying the causes of diseases and expounding the means of prevention, Robert Koch was uncomparable in the history of fighting against infectious diseases. He was indeed the father of medical microbiology. We are now living in an era of bioter-rorism, in which emerging and/or reemerging infectious dis-eases continue to threaten our lives, the life and the work of Robert Koch is even more worthy of memory.

Suggested Reading

1. Barlow, C and P. Barlow (1971). Robert Koch. Heron Books, Geneva.

2. Brock, T. D. (1999). Robert Koch: A Life in Medicine and Bacteriology. American Society for Microbiology Press, Washington, D.C.

3. Dolman, C. E. (1973). Koch, Heinrich Hermann Robert. In Dictionary of Scientific Biographies, C. Gillispie (Ed.). Vol. 7, pp. 420–435, Scribner, New York.


5. Knight, D. C. (1961). Robert Koch: Father of Bacteriology. Franklin Watts, Inc., New York.

6. Reid, R. (1975). Microbes and Men. Saturday Review Press, Dutton & Company, Inc., New York.

7. Heinrich Hermann Robert Koch. http://www.whonamedit.com/doctor.cfm/2987.html. Accessed 16 November 2016.

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Chapter 14

Élie Metchnikoff (1845–1916): Phagocytosis and Immunology

Source: https://en.wikipedia.org/wiki/%C3%89lie_Metchnikoff(US Public Domain image)

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Introduction

“It was, so to speak, a salon of a new kind — a European scientific caravansary. The visitors flooded at all hours, bring-ing in news from the whole world, more true than that of the newspapers. For them, the laboratory became an observatory. Who was not seated near the desk full of books, magazines, and manuscripts or in front of the white enamel table on which the microscope reigned? Scholars, statesmen, artists, journalists (so many!), actresses, singers, women-of-the world, enlightened ones, and how many Russians: Ministers of the Czar, noblemen, balalaika players, ardent and skinny stu-dents, and so many hangers-on of all nationalities. These few square meters of Metchnikoff’s laboratory, truly one of the summits of Europe, was a free space, a cell gifted with immu-nity against prejudices, vanity, egotism, and fantasy”. This description by Etienne Burnet (1873–1960), one of Metchnikoff’s numerous students at the Pasteur Institute, ably indicates the intense vitality of the scientist and the com-plex nature of his character.

Metchnikoff was a far different figure than the aristocratic Louis Pasteur (1822–1895) or the severe Robert Koch (1843–1910). He seemed to be a creation of Fyodor M. Dostoyevsky (1821–1881): highly emotional, passionate, impulsive, and, as a youth, irritable to noise and prone to violent temper and sui-cidal depression. Yet he was known for his generosity, liveliness, and fondness for joking. He spoke frankly and truthfully in simple, unpretentious language. He was always the 19th cen-tury liberal, deploring violence yet supporting his students in university protests. (One of his brothers, Lev, was sympathetic to revolutionaries and fought with Garibaldi in Italy.) Although Metchnikoff was outspoken in his advocacy for admission of women to academic as well as medical colleges, he did not believe that women could accomplish creative work in science and that genius was a male trait.

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Life of Metchnikoff

Metchnikoff’s life began in the steppes of Kharkov, the Ukraine, where he was born. Élie Ilyich, born on 16 May 1845, the fifth child of a military officer, Ivanovitch Metchnikoff, was an aris-tocratic landowner. His mother Emilia Nevahovna, was the daughter of a Polish Jewish agricultural officer and writer Leo Nevahova. Raised in the Russian Orthodox Church, Metchnikoff recognized his Jewish roots, but as for religion, he became an atheist.

Attracted to nature and the pursuit of its knowledge at age eight, by the age of 15 he was a scientific prodigy, auditing lectures at the University of Kharkov, receiving private instruc-tion in histology, and founding a journal club at his school. Borrowing a microscope from a medical student, he began to examine protozoa, and beginning in 1862, at age 17, he sent his observations to journals. One, concerning the contraction of the vorticella stem, was translated by England’s prominent zoologist, Edwin Ray Lankester (1847–1929), for a microscopy journal. On an ill-fated trip to Leipzig to study at the German university, Metchnikoff obtained a copy of Darwin’s Origin of the Species. The book changed his life and countless other young Russian biologists. Darwinism would continually guide Metchnikoff’s research.

He continued his scientific education at the University of Kharkov, with semesters in German laboratories under Friedrich Gustav Jacob Henle (1809–1885), Karl Rudolf Leuckart (1822–1898), and Karl von Siebold (1804–1885), among others. Earning his bachelor’s degree at age 19, he went to the University of St. Petersburg for his master’s. Metchnikoff next traveled to the new University of Odessa, where he lectured, becoming popu-lar with his slightly younger students. He took leave to return to St. Petersburg to complete his doctorate in invertebrate zoology, conducting field work in Italy, Spain, and France.

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By now he had married Ludmilla Federovitch, more out of convenience than love. She was ill with tuberculosis, misdiag-nosed as chronic influenza. His financial situation became grave, Ludmilla’s condition worsened, he was continually frustrated with the absence of adequate laboratory facilities, and when he wanted to resign in protest of the persecution of the Poles, he found that he could not afford it. Metchnikoff sank into pessi-mism and despair. More wounds followed. He failed by one vote to obtain a professorship at the Medical-Surgical Academy. He had hoped to receive the Baer Prize for zoology but was refused contention on the pretext that his article was presented in man-uscript form instead of being printed. When Ludmilla died in 1873, his spirit totally collapsed; he could not even attend the funeral. His wife had been given morphine in her final days. To escape his vexation, Metchnikoff began to use the drug himself, ultimately breaking the addiction after an overdose.

As a therapeutic change of pace after detoxification, he went to the Kirgiz and Astrakhan Steppes for anthropological study of the Mongol Kalmuks. Metchnikoff explored the rela-tionship of racial characteristics and progressive physical evolu-tion and was tempted to travel with one of their Buddhist lamas to Tibet. He returned for follow-up studies in 1874 and 1876.

Returning to Odessa, he met and married Olga Nicoleavana Belokopytova in 1874. She was 17; he, 29. Olga was his compan-ion, technician — she was interested in zoology, and friend; at times substitute mother. On her own, Olga would make a sig-nificant contribution to the study of germfree animals. She was also fond of art both as an observer and practitioner. Later in Paris, she became part of Auguste Rodin’s (1840–1917) circle of friends.

Metchnikoff, along with his friend Alexander Kovalevsky (1840–1901), was creating a new scientific discipline: compara-tive embryology. Fritz Muller’s (1821–1897) For Darwin sug-gested that embryology could provide proof of evolution; thus, Metchnikoff searched for order among the invertebrates, espe-cially the starfish and medusae. He examined the origin of the

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middle embryonic layer, the mesoderm, and the amoeboid cells that acted in metamorphosis, nutrition, and tissue repair.

This was a period of intense philosophical debate on the ramifications and details of evolution. Metchnikoff chastised Darwinians for applying natural selection only to derivations from their preconceived program of developmental parallelism epitomized by Ernst Haeckel’s (1834–1919) maxim, “Ontogeny recapitulates phylogeny”. Metchnikoff pondered how the same structure among the phyla may have different functions and why closely related animals can vary so widely in their development. He wondered if evolution could take place within the embryo. One of the great perplexities was why organic matter progressed toward both perfection and com-plexity in organization — why, indeed, it progressed at all. Moreover, Darwin defined natural selection as inducing only gradual change. Metchnikoff recognized the power of macro evolution, declaring, “Abrupt variation has more chance for durable existence than small individual peculiarities”. Metchnikoff also supported collective variation, regarding it necessary to have a certain sum of individuals to change before the beginning of selective activity. These ruminations were set-ting the stage for Metchnikoff’s carriage of Darwinian princi-ples into medicine.

Meanwhile, Metchnikoff began his first work in microbiol-ogy. In 1878, to arrest the massive destruction of cereal crops by weevils and beetles, he considered biological controls. He determined that an epidemic among these insects by the fun-gus Metarhizium anisopliae was possible. He was able to cul-ture it on sterilized beer mash and prepare spore preparation. Metchnikoff recommended developing nurseries for cultivating strains of increased virulence and activity, and later helped establish entomological commissions in Kharkov and Odessa. In 1884, his protege Isaak Krasilschikov carried on the labors by dispersing some 50 kg of spores on some fields around Kiev. Within two weeks, up to 80% of weevil larvae were killed. Then the studies ended. Some historians hold that the decline of the

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threat on sugar beets made the work unnecessary; others point toward the inconsistency of results.

Politics was now interfering with Metchnikoff’s academic work. The government required the ratification of professors elected to the University Council. This soon produced a shift toward conservatism among the faculty, placing them in oppo-sition to the far more liberal and socialistic student body. The conservatives soon rose against a liberal professor who accepted a law student’s thesis critical of the status quo. The dean pro-posed that all such theses be rejected and the majority of the faculty concurred. After the students naturally protested, the curator implored Metchnikoff and other liberal professor to intercede and stem the student disorder. Although a compro-mise was arranged that would return the students to their classes and remove the dean, the administration broke the agreement. Student leaders were severely punished.

Metchnikoff took these matters personally. His health suf-fered under the stress, and he fell into depression with insom-nia, inappropriate giddiness, and cardiac trouble. When Olga was then infected with typhoid, Metchnikoff decided to com-mit suicide by infecting himself with a species of Borellia, the spirochetes of relapsing fever. After a prolonged illness, he recovered with a bright, joyful spirit and the resolve to resign from academia.

It was when he and his family (Olga and her siblings) were on a working vacation at Messina that Metchnikoff’s ideas on the origin of the mesoderm and its function of digestion, on evolution, and on pathology crystallized. A pilot test of intro-ducing a rose thorn under the skin of some starfish larvae brought about a practical approach. He had been well versed with the phenomenon of white cells capable of engulfing debris, foreign particles, living microorganisms, and atrophied tissue, and the arguments whether the action was active or pas-sive. Now his epiphany was not merely the recognition that white cells had the power to defend the animal against foreign invaders but also that white cells were indeed active and fun-damentally independent of organismal agency.

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This observation was important because at this time causa-tion was regarded as downward from an almost Platonic ideal of selfhood and organism. The organism was assumed to be naturally harmonious and that any break from this order was disease. Although the prevailing philosophy described that nature acting on the whole on behalf of its parts, Metchnikoff saw an independent part acting on behalf of the whole. Instead of Rudolf Virchow’s (1821–1902) cellular subsystem restoring the integrity of the organism, Metchnikoff saw the integrity of an organism arising from the activity of certain constituents.

Armed with the term phagocyte, meaning “devouring cell”, from Carl Claus (1835–1899), a zoology professor at Vienna, Metchnikoff went back to Odessa for the 1883 Congress of Physicians and Naturalists to present the first paper on the sub-ject. Later visiting his friend Kovalevsky, Metchnikoff noticed that the water fleas (Daphnia) in the aquarium were sluggish. Inspection showed them to be infected with a fungus, and so began a pro-found investigation on the natural history of an infectious disease from the standpoint of both host and parasite. This 1884 paper, A disease of Daphnia caused by a yeast. A contribution to the theory of phagocytes as agents for attack on disease causing organism, described dose kinetics, giant cell formation, intracellular destruc-tion of spores and hyphae, and on the other side, the rupture of leukocytes. It is a milestone in medical microbial ecology.

In another report that year, Metchnikoff proposed that since bacteria in infections were especially abundant in splenic white cells, the organ’s function — then unknown — is prophy-laxis, the removal of septic bodies from the organism. He also suggested that lymphatic glands and bone marrow are of simi-lar importance in immunity.

With Louis Pasteur’s successful vaccination against rabies echoing around the world, and especially after his treating a group of Russian peasants bitten by a rabid wolf, the officials of Odessa decided to create its own institute for the manufac-ture of vaccines. Nikolai Gamaleia (1859–1949), a student of Metchnikoff, was sent to the Pasteur Institute to learn the pro-cedure, and Metchnikoff himself was appointed director of the

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biological station. Despite the importance and prestige, Metchnikoff decided to resign in 1886. A PhD in an MD world, the opposition of the local medical society, and the lack of cooperation of the district administrator and the technical staff were too much to bear.

The search for a scientific refuge led to the newly opened Pasteur Institute, where Pasteur was supportive of Metchnikoff’s comparative approach. Pasteur also felt that a zoologist and evolutionist would be beneficial to the young institute. Thus, rejecting a last minute offer by the St. Petersburg Institute of Experimental Medicine, Metchnikoff emigrated to Paris on 15 October 1888. In 1890, Metchnikoff had only nine students, including Olga and a former student in Odessa, Waldemar Haffkine (1860–1930). That situation would change, and Metchnikoff would be received as the most popular instructor of numerous budding scientists, including the two future Nobel Laureates, Jules Bordet (1870–1961) and Charles Nicolle (1866–1936). With Emile Roux (1853–1933), Metchnikoff instituted the course in bacteriology modeled after Robert Koch’s. After the death of Pasteur, Metchnikoff became subdirector of Medical Research and Chief of Service.

Like any novel scientific concept, Metchnikoff’s phagocyte theory of immunity had to be developed fully and, of course, be marketed through publications, collaborations, and presen-tations. Toward this end, he gave a series of lectures in April and May of 1891, which were compiled as Lectures on the Comparative Pathology of Inflammation (the French edition appearing in 1892, the English version in 1893). Metchnikoff recast inflammation, which most saw in a negative light as being associated with disease, as a routine positive physiologi-cal activity, typically without overt signs, that maintains organ-ismal integrity through the agency of phagocytes. The engine of his process was disharmony, which became a general meta-physical element in Metchnikoff’s final philosophical treatises.

The discussion closed with an astonishing and prescient mention of the role of mind with respect to chemotaxis and

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sensory awareness among phagocytes. He included the state-ment that “the psychical acts of the higher animals are [not] fundamentally different in their nature from the more simple phenomena peculiar to the lower organisms”. Metchnikoff’s student Serge Metalnikov (1870–1946) would found the field of psychoneuroimmunology with his work on the conditioned response.

The chief opposition came after 1890 with the discovery of the antitoxic capacity of the blood, later the presence of particulate antitoxins, by Emil von Behring (1854–1917) and Kitasato Shibasaburo (1853–1931) and others. Active produc-tion or passive acquisition of antiserum, devoid of cells, could protect against diphtheria and tetanus. Soon, immunity to those bacteria that were not associated with toxins was claimed also to be a humoral activity. Although Metchnikoff wrote that he never declared that phagocytes are unaided in their elimination of microorganisms and accepted that serum factors could weaken the bacteria, he knew that he was now on the defensive.

The polemic was formally manifested at the Bacteriology Section of the 1891 International Congress of Hygiene at London, where the leaders of bacteriology and immunology summarized their positions and attacked the opposition. Metchnikoff emphasized the need for phagocytes to eliminate living, reproducing pathogens. By 1894, at the Congress of Hygiene at Budapest, compromises were achieved. Metchnikoff proposed, correctly, that macrophages could produce comple-ment, and that elements of the phagocytic organs produced the protective substances that pass into the plasma. Roux, recalling this latter debate, described Metchnikoff as the “Demon of Science”, with red face, burning eyes, and disheveled hair who nonetheless brought applause from the audience. In 1897, Metchnikoff separated macrophages from microphages (neutrophils) and distinguished their different protective functions. The body of his research was finally brought together in 1901 in the major treatise Immunity in Infective Diseases.

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Whereas self, health, and immunity were customarily regarded as discrete qualities or conditions, Metchnikoff found them to be ever variable processes. Despite the common meta-phors of combat and defense, in accord with his embryological principles, phagocytes do not protect the self because there is no given organismic integrity that is to be protected. As he wrote in 1892 in his essay, The struggle for existence between the parts of animal organisms, the developing organism is a composite of competing lines of cells and tissues for which the motile white cell serves as mediator and integrator.

Metchnikoff next began a major study on aging and death and offered an immunological basis, the first such theory. Microbial antagonism, normal flora, and the ecology of the intestine were of special interest to him. It stemmed from his studies of cholera wherein he had developed a suckling rabbit model of the disease. Success depended on the inoculation of intestinal flora favorable to the proliferation of the cholera vibrio. Now, he believed, like Charles Bouchard (1837–1915), that the intestinal flora of people release toxins that gradually produce a generalized atrophy; Metchnikoff gave phagocytes a role in aging by removing the damaged cells.

Noting that breast-fed infants had a fermentative rather than putrefactive intestinal flora, mainly lactobacilli, and that the long-lived people of the Balkans and the Caucasus con-sumed lactobacillus-containing sour-milk products, such as yogurt and kefir, he advocated a rigorous regimen of these favorable bacteria (a probiotic concept). He believed that it would promote a full life for the individual, perhaps up to 100 years, without the premature infirmities associated with senil-ity. His two philosophical books brought the subject to the public and the resulting demand created the commercial yogurt industry. Despite accusations, Metchnikoff had no financial stake in any company.

Metchnikoff and Roux studied syphilis from 1903 to 1906 as a model of arteriosclerosis. Their work led to another triumph:

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an animal model for syphilis using macaques or chimpanzees. Confirming Fritz Schaudinn (1871–1906) and Erich Hoffman’s (1868–1959) discovery of the spirochetal agent, they discovered that Calomel, mercurous chloride, proved efficacious as a prophylaxis when applied to the penis up to 30 minutes after inoculation. Thinking that here was a good topic for a thesis, a medical student, Paul Maissoneuve, volunteered to test the procedure on himself. After 94 symptom-free days, he and the treated chimpanzees were declared free of syphilis.

Metchnikoff felt that the decade of 1895–1905 was the hap-piest period of his life. He and Olga lived in a suburb of Paris, Sevris, and came to the Pasteur Institute every morning by train. His work was productive and wide ranging. He enjoyed teaching and mentoring, and through many public lectures he offered people frank knowledge and advice in hygiene, tuber-culosis, and syphilis.

In 1908, after political wrangling, the Nobel Committee decided to award both Metchnikoff and Paul Ehrlich (1854–1915) the Nobel Prize for their contributions in immunology, although by this time the concept of immunity through anti-bodies dominated the field. On his celebratory grand tour the following year, Metchnikoff met Leo Tolstoy (1828–1910). (Metchnikoff’s brother Ivan Ilyich was the model for the author’s story The Death of Ivan Ilyich.) The visit at Tolstoy’s estate Iasnaia Poliana was a summit meeting of optimistic medical science versus social ethics and spiritual concerns inter-fused with Russian politics. World War I was a profound shock. The Pasteur Institute was closed, all scientific work ceased, and the laboratory animals were slaughtered. Its younger men now moved to the front lines or served in medical units. To no sur-prise, Metchnikoff’s chronic cardiac condition worsened. The carnage of the battlefield crushed the optimistic age of the nineteenth century and manifested the dark side of science and technology. The life flowed out of Europe and Metchnikoff, too, was dying. Taken to Pasteur’s old flat for his final days, he

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died on 15 July 1916. In accordance with his wishes, his body was cremated and the ashes kept in an urn in the library of the Institute, where it remains on the cabinet today.

Commentary

Metchnikoff was one of the giants of science. Although a zool-ogist, embryologist, pathologist, immunologist, bacteriologist, gerontologist, and philosopher, he was foremost a biologist, a student of life. Although the quality of his experiments did not lead to a vaccine or cure, his creative genius led to theories that seemed ridiculous or were misconstrued by his contemporaries but were ahead of his time. Metchnikoff is the true father of immunology, the science.

Suggested Reading

1. Bernstein, H. (1913). With Master Minds. Universal Series Publishing Co., New York.

2. Bibel, D. J. (1988). Elie Mechnikoff’s bacillus of long life. ASM News, 54: 661–665.

3. Metchnikoff, O. (1921). Life of Metchnikoff 1845–1916. Houghton Mifflin, Boston.

4. Stillwell, C. R. (1991). The Wisdom of Cells: The Integrity of Elie Metchnikoff’s Ideas in Biology and Pathology. A disser-tation. Notre Dame, Indiana.

5. Tauber, A. I. and L. Chernyak (1991). Metchnikoff and the Origins of Immunology. From Metaphor to Theory. Oxford University Press, New York.

6. Zalkind, S. (1959). Ilya Mechnikov, His Life and Work. Foreign Languages Publishing House, Moscow.

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Chapter 15

Charles Louis Alphonse Laveran (1845–1922): Discoverer of the

Malaria Parasite

“The knowledge of these new pathogenic agents has thrown a strong light on a large number of formerly obscure questions. The progress attained shows once more how just is the celebrated axiom formulated by Bacon: “Bene estscire, per causasscire”.

—Charles Louis Alphonse Laveran

Source: https://en.wikipedia.org/wiki/Charles_Louis_Alphonse_Laveran(US Public Domain image)

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Introduction

Malaria is caused by protozoa (a kind of microorganism), and it was one of the most serious human diseases in the last century. It is still a terrible disease and is estimated that 2–4 million peo-ple have this diseases today. There were many scientists who studied this disease. Alphonse Laveran (1845–1922) stood out as one of the distinguished scientists and contributed greatly to the control of this disease. He imposed an important microbio-logical principle. This is why we have featured him here.

Alphonse Laveran was the son and grandson of distin-guished physicians. On his mother’s side, he came from a line of celebrated soldiers. His career showed the influences of both sides of the family. His father was Dr. Louis Laveran. While at the military hospital at Metz, Louis married Mademoiselle de la Tour of the famous Lalleman family (on her mother’s side). Alphonse was born in Paris on 18 June 1845. His parents went to Algeria, where his father served in the Army. From the age of five he spent his childhood in Algeria, running freely among his orange groves. He retained the delightful memories of that country all his life. He also benefitted by the teaching of his father, both regarding the place of the Army in society also the duties and responsibilities of a medical officer.

When the family returned to Paris in 1856, he attended Sainte-Barbe and Louis-le-Grand schools. He decided to choose his father’s profession and applied for entrance in the Ecole du Service de Santé Militaire at Strasbourg in 1863. He was accepted and completed the medical courses in 1867. While still a medical student, in 1866, he served as resident medical officer at the civil hospital in Strasbourg. Following graduation, he took an advanced course at the Val-de-Grâce in Paris and was then appointed Aide-Major at the military hospital of St. Martin (now called Hospital Villemin). He served during the Franco-Prussian War in 1870, seeing active battle in the besieged town of Metz. He functioned as a surgeon and witnessed the real horrors of war. This was before the knowledge of antiseptic

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surgery. Septicemia, erysipelas, and gangrene were common, and surgery was not performed on many of the wounded patients. There was a shortage of food and medicine.

Alphonse Laveran was present at the surrender of the gar-rison and the town of Metz to the triumphant Germans. After the surrender of Metz the German sent him back to the French Army, and he served the rest of the war in the military hospital of Lille.

He was next designated to the 10th Hussars at Pontivy. His advanced medical studies were continued and the following year, he took the examination for a fellowship at the Val-de-Grace. He passed this with great distinction and took the posi-tion of professor agrégé des Maladies et Epidemies des Armees. In 1873, he was appointed to the chair his father held at Val-de-Grace. His father had studied malaria, as had many other army physicians. No one was able to discover the causative organism. Laveran began the use of a microscope and taught microscopy to his students. Students attending his lectures and working in the laboratory were greatly impressed with his vigorous teach-ing and “practical demonstrations with the microscope”. He demanded of his pupils the same high degree of application that he required of himself. He was described as having “inflex-ible discipline” and “unrelenting training in the search for truth”. His professional life and scientific work were indivisible. Laveran began a search for the cause of malaria. He suspected that the material cysts with their black pigment contained the parasites; the general consensus of opinion, however, was that these were caused by a degeneration of the blood tissues of the body. When promoted to the rank of Surgeon-major, he was transferred to Biskra, in Algera, where no malarial fever was present. Here he worked on what was known as “Biskra Boil”, which he published later.

In 1879, he was transferred to the Military Hospital at Constantine, where there was a great deal of malaria. On the 6th of November 1880, the memorable day in the annals of tropical medicine occurred when he recognized the malaria in

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the blood of a soldier. He had long suspected the parasites, but on this day he saw the parasites that have the appearance of protozoa with threads (flagellae) thrashing the body cells. He knew that he was seeing parasites and he placed them at once in the Hematozoa. Subsequent work showed that these para-sites were found only in malarial victims. When in Italy in 1882, Laveran found the same parasite near Campagna. He named the organism Oscillaria malariae and reported it to the Académie de Médicine on the 23th and 28th of November 1882. In 1884, a final report was published under the title Traité des Fìevrers Palustres. This report summarized observations on 432 cases of malaria in which the organism was seen. The organism was later given the name Plasmodium malariae.

It should be remembered that all of his work was done when microbiology was in a elementary stage. Also, it was later known that his work was compounded by the fact that in Algeria at that time, several forms of malaria abounded — quartan, tertian, and malignant fever — each a separate “race” of organism. Laveran was not able to trace the complete life history of the organism [done later by Camillo Golgi (1843–1926) in Italy, where only one form — quartan — existed]. Also, there had been many false reports of finding the organism. Laveran and others continued the research and Laveran’s dis-covery was vindicated, although it was not until 28 years had passed that a memorial tablet was placed in the hospital in Constantine where Laveran made the initial discovery.

In 1885, Laveran married Mademoiselle Pidancet of Montoy-Flanville in the village church at Noisseville, very near Metz. His marriage was a very happy one. His wife appreciated his dedi-cation to both science and the Army. She brought him peace of mind and freedom from any cares that would distract a scien-tific investigator. From a year before his marriage and for nine years following marriage, he held the chair of Military Hygiene at the Val-de-Grace in Paris; the years 1884–1994 were devoted to “sound teaching in science”, emphasizing both the practical and the theoretical. In addition, he founded a Museum of

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Hygine and published a textbook. His book, Traité d’ Hygiene Militaire, was greatly appreciated by the Army.

Laveran’s initial reports of malaria were received with skep-ticism, although his work was sound. In 1889, the Académie des Sciences awarded him the Bréant Prize in belated recognition of his discovery of the Plasmodium organism. In 1891, he published his second work on malaria, Du Paludisme et de son Hématozaire. This work was received with great enthusiasm — actually more than when the initial announcement was made eleven years before. In 1893, he was elected a member of the Sociétéde Biologie and the Académie de Bedecine (correspond-ent). In 1895, he was made a commander of the Legion d’ Honneur and also a Fellow of the Académie des Sciences. When the fellowship in the military program at Val-de-Grâce, Paris, terminated in 1894, Laveran hoped to be appointed to another Army post where he could continue his research on malaria. In spite of his efforts, the Army did not yet have control of the disease. The military authorities completely ignored his great research talent and progress he had made. For the next two years he was Director, first of a military hospital at Lille and then another at Nantes. However, he was not satisfied because he had no opportunity to continue his research. When troops were sent to Madagascar, where malaria was endemic, he fully expected that he would be sent to protect the soldiers and fur-ther the control of the disease. This was not done, and many of the troops died as a result.

Finding that all hopes of research in the Army were not to be his, he reluctantly resigned from the service at the age of 50. He refused to reconsider this decision in spite of great pressure for him to continue the tradition of his family. He had given himself unstintingly to the military, but they shortsightedly had lost one of their most brilliant research men. But Alphonse Laveran was not one to allow himself to be defeated, some-thing every microbiologist needs to learn. The Army did not desire malarial research, but he was given a most enthuiastic welcome by the Pasteur Institute. Two of Pasteur’s distinguished

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collegues Pierre Paul Emile Roux (1853–1933) and Pierre Emile Duclaux (1840–1909) gave him a special welcome and unlimited assistance. For 25 years, he reported by eight o’clock every morning to his laboratory and allowed nothing to interfere with research on the love of his life, protozoa as causes of dis-eases. Without losing sight of the unfinished work on malaria, he worked on numerous new parasitic diseases. With assistant, Felix-Etienne-Pierre Mesnil (1868–1938), he investigated various microscopic parasites and published accounts of new diseases. One of these was sleeping sickness in men and animals. He was reponsible for the program of the French Commission on Sleeping Sickness in the Congo. David Bruce (1855–1931) had identified Trypanosoma brucei as the parasite that caused the disease, and Laveran did a great deal of research on the proto-zoan. He published a massive work on the disease with Mesnil in 1904 and a second and larger edition in 1912.

World-wide work on malaria was underway, and Laveran kept in touch with all aspects of it. Sir Ronald Ross (1857–1932), in India, discovered the external cycle of Plasdmodium malariae in the mosquito, a big advance. Even before this, the possibility that the mosquito might be the vector had been surmised. Sir Patrick Manson (1844–1922) published the account of insect transmission of Chyuria, another disease. Manson also fomu-lated a well-known hypothesis on transmission of malaria by mosquitoes. The great contribution of Ross, however, was his discovery the importance of the species. In 1898, Manson lec-tured in Edinburgh on the Anopheles species of mosquitoes that took the microbes out of the patients with malaria, further developed the organism in their own body, and then put back the infective organisms into the human patient.

The data met resistance, as it took some years for numerous scientists to bring in coherent and identical reports. But Laveran, from his immediate experience with malaria was among the very first to support Ross. This support was based upon facts, as mosquitoes were sent to the Pasteur Institute from all malarious countries. At that time, Laveran showed that

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malaria was only found in those countries where the Anopholes species of mosquitoes lived. Ross’ work established the impor-tance of malarial research. In 1901, Laveran read a paper before the Académie de Médecine in which he outlined the most appropriate measure for protection against malaria as well as the suggestion of establishing a malarial control society for Corsica Ligue Corse Contre le Paludisme. Laveran was made Honorary President and he went to Corsica in 1902 to study the local habits of the anopholene mosquitoes and to make sure that preventive measures were properly applied. When it was reported that malaria was present where no Anopholes mos-quitoes could be found, Laveran himself made field trips and quickly demonstrated the insect and its eggs. In 1903, he pub-lished the small but concise and accurate Prophylaxi du Paludisme, which gave details for control and preventive meas-ures against malaria.

From 1903 until 1917, Laveran worked on protozoan para-sitic diseases known as leishmaniases [Sir William Boog Leishman (1865–1926) had first identified these causative agents of such diseases as Indian and Mediterranean kala-azar and the Delhi and Biskra “Boils”]. These were thought to be separate and distinct diseases until Laveran clearly established their identity and relationships. In 1917, he published a treatise, Traité des leishnmaniases, which was of great value. Because of the expansion of his work, Laveran was given the entire second floor of a new building. Laveran used his Nobel Prize funds to provide the equipment for his laboratory. A vast number of sci-entists and students came from all over the world to do research and study in his laboratory. Many foreign scientific societies now recognized his work. In 1902, he was given the Jenner Medal of the Epideomiological Society of London. In 1906, the Moscow Prize was given at the International Congress at Lisbon. In 1907, the Noble Prize for Medicine and Physiology was awarded for his work in protozoology. In 1976, he was made a foreign member of the Royal Society, as well as a member of several other British scientific societies in the same period.

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During the years 1919–1921, with Dr. M. G. Franchini, he organized the knowledge of flagellate parasites of insects, giv-ing the methods of obtaining them in pure culture and outlin-ing the diseases they caused. With remarkable vigor, this work was concluded and published in 1921. Beginning in 1920, it was noticeable that Laveran was decling in strength. In that year, he addressed the Académie de Medécine at its 100th year anniver-sary. He passed away on 18 May 1922.

Commentary

Microbiologists, and indeed, all scientists, need to think about Laveran’s work in relation to microbiology. Sir Ronald Ross has emphasized something all need to understand: “Laveran’s great discovery of 1880 was not connected in any way with the science of medical bacteriology — which had been recently founded by Louis Pasteur (1822–1895), Joseph Lister (1827–1912), and Robert Koch (1843–1910) — but with much older science of animal parasiotology”. In this book, we have seen how many microbiologists followed the lead of the great bacteriologists. But we have also seen Barbara McClintock (1902–1992) using the microscope to study the subcellular particles, such as chromosomes and genes, quite apart from bacteriology. Here we find that Laveran was a parasitologist — with a microscope, and also apart from the new science of bacteriology!

Alphonse Laveran was indeed a microbiologist, but one who developed the specialty within bounds of his own science: parasitology. To the growing numer of microbiologists who were mainly baceriologists and virologists, Laveran contributed a large part of the vast block of protozan microbes.

Aphonse Laveran was a stiff, aloof, and quiet man. Those who were closely acquainted with Laveran knew that under-neath a somewhat distant and reserved exterior, there lay hid-den a very sensitive soul. His character was invariably upright. His speech was slow and thoughtful, enhanced by precision and

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free of solemnity. His physionomy, the clarity of his gaze reflected the serenity and honesty of his intelligence. He sur-rounded his research with silent discretion up until the time, he decided to publish his results. He was very astute both as a physician and scientist. He refused to be interviewed by jour-nalists. Public hardly knew of him, and he could not have cared less. For a long time, he suffered from the indifference, hostil-ity, or distain with which his discoveries were met. The igno-rance and ingratitude of military leaders, who obstinately barred the way of his reaching the higher ranks of the army and achievement of research were paticularly painful for him. However, he was welcome by the Pasteur Institue, where he was offered a laboratory, the Academy of Sciences, The Royal Society of London, all the scientific associates of the world, and honors. He was awarded the Nobel Prize by the Carolin Institue, and the Academy of Medicine wanted him to preside over their 100th anniversary.

We are lucky that his contributions were recognized by the world. Laveren, for at least 27 years, did not cease to work on pathogenic protozoa, and the field he opened up by his discov-ery of the malarial parasites has been increasingly enlarged. Protozoal diseases constitute one of the very interesting chap-ters in both medical and veterinary pathology today.

In spite of the advances of Laveran and those following him, the disease still takes its toll. It is estimated that malaria affects 300–500 million people world wide and causes 2–4 million deaths annually. Actually, there are probably more people dying of malaria today than 30 years ago. Although pesticides have been used, pesticides-resistant mosquitoes have fre-quently returned in force. The most basic and patient biological controls devise of Laveran still seem best.

Suggested Reading

1. Alphonse Laveran. Protozoa as Cause of Diseases. Nobel Lecture, December 11, 1907. https://www.nobelprize.org/

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nobel_prizes/medicine/laureates/1907/laveran-lecture.html. Accessed 16 November 2016.

2. Charles Louis Alphonse Laveran (1845–1922). https://www.cdc.gov/malaria/about/history/laveran.html. Accessed 16 November 2016.

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Chapter 16

Martinus Willem Beijerinck (1851–1931): Pioneer of General

Microbiology

“Fortunate are those who now start”.

— Martinus W. Beijerinck

Source: https://en.wikipedia.org/wiki/Martinus_Beijerinck(US Public Domain image)

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Introduction

On 10–14 December 1995, microbiologists, historians, and digni-taries participated in a conference, “Beijerinck Centennial — Microbial Physiology and Gene Regulation: Emerging Principles and Application”, in The Hague, The Netherlands, to commemo-rate the appointment of Martinus Willem Beijerinck, the great microbiologist as a professor at Delft School. Despite his discover-ing the filterability of the infectious agent responsible for tobacco mosaic disease, identifying the first microbial sulfate reducer, and conducting pioneering research on nitrogen fixation, very few current students of biology are aware of Beijerinck’s fundamen-tal contributions to the development of microbiology. Martinus Willem Beijerinck was called “one of the big four in bacteriology (along with Winogradsky, Pasteur, and Koch) by Thomas H. Grainger Jr. (1908–1994) in his Guide to The History of Bacteriology.

One reason that Beijerinck is not better known is that his studies of plant- and soil-associated microorganisms were less dramatic than those of his contemporaries Louis Pasteur (1822–1895) and Robert Koch (1843–1910). Moreover, their work related to familiar human and animal diseases.

Beijerinck, with his keen ability for observation, was a prime mover in establishing general microbiology as a major disci-pline. Well before most universities recognized microbiology as a distinct discipline, he established the Delft School of Microbiology, which set the cornerstone for developing many such departments and institutions worldwide. Soon this school attracted a steady stream of insightful researchers, including the botanist and microscopist Gijsbertus van Iterson (1878–1970) and N. L. Söhngen, the first to describe microorganisms that use methane as their sole source of carbon and energy. Afterward, Albert Jan Kluyver (1888–1956), who inherited the chair of Beijerinck in 1921, developed the theory of metabolic unity and diversity for microorganisms. And Cornelis B. van Niel (1897–1985), one of Klyuver’s students, migrated to the United States, where he influenced an entire generation of microbiologists

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with his enthusiasm for unusual biochemistry and physiology. Together with Pasteur, Koch, and the Russian Sergei N. Winogradsky (1856–1953), who specialized in soil microorgan-isms, Beijerinck is regarded as a founder of microbiology as a distinct discipline.

The field of microbiology is a major building block in the edifice of the biological sciences. Whether in natural sciences or social sciences, scientific progress is made by fellow human beings, but the human side of history of science is often neglected by modern textbooks. It is important for us to refresh us memory of how microbiology first started and who were involved with those pioneering work. This is why we would like to retrace what Beijerinck did more than a century ago and how the human side of him influenced his work.

Beijerinck’s Humble Family Origins

The family of Martinus Willem Beijerinck, who was born on 16 March 1851, in Amsterdam, The Netherlands, came from Twente in the province of Overijssel. His father, Derk Beijerinck (1805–1879), and mother, Jeannette Henriette van Slogteren (1811–1875) had one other son, Frederik Leonard Beijerinck (1844–1883), and two daughters: Henrietta Wilhelmina Beijerinck (1847–1937) and Johanna Hermana Alida Beijerinck (1849–1923).

When Derk Beijerinck’s tobacco business in Amsterdam failed, he and his family moved to Naarden in 1851, where he worked as a clerk in the Haarlem booking office of the Holland Railway Company (Hollandsche I. Jzeren Spoorweg Maatschappij). Although Martinus’s family was too poor to send him to school, his father taught him not only all the sub-jects of elementary school but also French, English, a little German, artwork, and some astronomy, and physics. His father was especially fond of him among all his children because he was very bright.

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At the age of 12, Martinus went to the elementary school of Master Knoop and subsequently, the Hoogere Burger school (secondary school) at Haarlem. Despite an inferiority complex that undermined his self-confidence, intelligence and hard work led him to the top rank in his class. Moreover, during this time, Frederik Willem van Eeden (1860–1932), his science teacher in botany, greatly influenced Beijerinck. The two of them spent considerable time wandering the sand dunes near Haarlem nurturing a love of plant life. In 1866, at the age of 15, he won a contest on the basis of his collection of 150 species of plants.

With the help of his brother and uncle, Beijerinck entered the Delft Polytechnical School, where chemistry became his major subject but plants remained his major interest. While a student, he met J. H. van 't Hoff Jr. (1852–1911), who later won the first Nobel Prize in chemistry. They undertook many experi-ments together, and van 't Hoff later became Beijerinck’s life-long adviser. Whenever a new opportunity arose for Beijerinck, he consulted with van 't Hoff before taking action.

In 1872, Beijerinck graduated and enrolled in the University of Leiden. On 7 June 1873, he passed the candidate examina-tion, magna cum laude, and was hired to teach at the Agricultural School in Warffum in the province of Groningen; however, his teaching was poor, and this job lasted for only a year. Meanwhile, he continued to study biology at the University of Leiden. In 1875, he became a part-time teacher at the State Secondary School at Warffum while continuing graduate school. When his mother died that same year, he was very sad.

Research and Teaching Become Beijerinck’s Focus

In 1876, he took another teaching position at the Agricultural High School in Wageningen. This time, he liked his job greatly because he enjoyed teaching botany, a subject that really inter-ested him. He continued research and in 1877 published his first

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paper on plant galls. In the same year, he received his doctorate from the University of Leiden. His dissertation was dedicated to his father. He remained on Wageningen, teaching and studying plant galls. In 1883, one of his papers was accepted by the Amsterdam Academy, a great honor for him.

According to L.E. Den Dooren de Jong (1890–1980) one of his students during this period, Beijerinck was a demanding and unpopular teacher who shouted his demands and often berated students for their mistakes. Their attitudes disap-pointed Beijerinck, who did not seem to understand the out-look of life of these “normal” students.

To Beijerinck, there was nothing more interesting than science. Botanical research was the love of his life. His work was increasingly reorganized by the scientific establishment in The Netherlands, which in May 1884, elected him to the Royal Academy of Sciences in Amsterdam. During this period, he also came into regular contact with many influential scholars, such as the botanist Hugo Marie de Vries (1848–1935), who redis-covered Mendel’s rules of inheritance; the astronomer A. C. Oudemans (1827–1906); the ophthalmologist and optician F. C. Donders (1818–1889); and the German physiologist and micro-biologist T. W. Engelman (1843–1909). In later years, he became quite close with famous physiologist C. A. Pekelharing (1848–1922). At Wageningen, with Adolf Mayer (1843–1942) who first discovered the transmissible nature of tobacco mosaic disease, he founded the Society of Encouragement of Natural Science (Natuurwetenschappelijk Gezelschap.)

Beijerinck Moves from Academic Life

In 1885, Beijerinck became a microbiologist at the Netherlands Yeast and Alcohol Manufactory in Delft at the invitation of its farsighted director, J. C. van Marken (1845–1906). Paid an extraordinarily high salary and provided with excellent equip-ment to establish an outstanding laboratory for microbiology, Beijerinck really came into his own.

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Just before the new laboratory was completed, Beijerinck visited several important researchers elsewhere in Europe, including the founder of mycology, Heinrich Anton de Bary (1831–1888) at Strasbourg, France, and the other expert on fungi, Emil Christian Hansen (1842–1909), whose specialty was the study of yeasts at Copenhagen, Denmark. Although admired for his scientific enthusiasm, Beijerinck’s fondness for dispute did not always make him welcome. For instance, his plans to visit Koch in Berlin were unfulfilled because of his reputation. From then Beijerinck developed a pronounced dis-like for medical bacteriology. In contrast to most microbiolo-gists at the time, who followed the lead of Pasteur and Koch by focusing on human and animal diseases, Beijerinck devoted his research to plant microbiology.

During this period in Delft, Beijerinck continued to work on plant galls and related microbiological problems. He made an important discovery, which later affected agriculture greatly, which he isolated nitrogen fixing root nodule bacteria in pure culture. But, this work was one of a multitude of projects. Increasingly absorbed in research, he spent long hours in the laboratory, sometimes spending entire nights without sleeping. Much of Beijerinck’s research was relevant to production needs at the factory. For instance, he studied the butyl alcohol fermentation of Schizosaccharomyces octosporus and the nitrogen fixing of Bacillus radicicola. He also studied luminous bacteria and green algae.

Despite such work-related successes, however, Beijerinck was deeply withdrawn and lived an extremely lonely life. Some of his writings reflect a concern that he might not live up to the expectation of the company. In addition, he did not get along well with colleagues. Fortunately, he enjoyed strong support from director Jacob Cornelis van Marken (1845–1906), who con-vinced him several times not to resign. He also formed a close friendship with a young technologist, F. G. Waller, a future chairman of the board of the Yeast and Spirit work.

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Beijerinck’s Scientific Reputation Grows

In 1894, Beijerinck earned his place in history with his paper on Spirillum desulfuricans, a sulfate reducer. He showed that this bacterium extracts energy by metabolizing sulfur compounds. This interest in esoteric sulfate reduction arose from practical problems in yeast factory, which used canal water for its steam boilers. Bacterial extracted minerals on the water caused a buildup of calcium sulfate that fouled the boilers. Like that of Pasteur, Beijerinck’s research led to solutions for industrial problems but also produced valuable insights into basic microbiology.

Recognizing Beijerinck’s many achievements and realizing that the scope of his work could expand in better surroundings, Dutch government officials created a special position for him at the Delft Polytechnic School in 1895. Because the school had no formal program in biology at that time, he was given a position in the chemistry department. Beijerinck was delighted with this new opportunity, which meant returning triumphantly to the school where his career started.

Although he was still unpopular, Beijerinck was a big inspi-ration to those who understood his vast knowledge and dedi-cation to science. Most of his few doctoral students became eminent scientists and made significant contributions to science and in industry.

Beijerinck appreciated the quality scientific research, giving credit to his assistants and allowing them to publish important papers on their own. A. H. van Delden, his assistant from 1895 to 1904, did important work on nitrogen fixation on Bacillus oligocarbophilus, and on the retting of flax. He also helped work on sulfate reduction, Gerrit van Iterson, Jr. (1878–1972), his assistant from 1902 to 1907, obtained a doctoral degree under Beijerinck’s guidance in 1907. By far his most brilliant student, van Iterson studied denitrification and decomposition of cellulose and later became an eminent professor of botany. Yet another student, Ir. N. L. Söhngen (1878–1934), was the first

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to describe organisms that use methane as an energy and car-bon source by demonstrating conversion of hydrogen to meth-ane and obtaining the first enrichment cultures of methane-producing bacteria.

Beijerinck’s persistence was amazing. According to den Door de Jong, he would sometimes begin to examine inocu-lated plates in the morning and continue late into the evening on the same plates. His keen observations enabled him to dis-cover many important microorganisms and their functions. His contribution to science includes aspects of bacterial diversity, botany, genetics, industrial microbiology, microbial taxonomy, microbial techniques, and virology. Beijerinck’s scientific high-lights are listed in the Table 16.1.

Beijerinck developed the principles of enrichment culture. Through these techniques, he and others could more readily uncover the role of microorganisms in natural processes and better harness these activities to benefit agriculture and indus-try. His unusual application of these principles made him per-haps the first microbiologists to emphasize an ecological approach to microbiology. “In an experimental sense, the eco-logical approach to microbiology consists of two complemen-tary phases which give rise to an endless number of experiments”, he noted, “On the one hand, it leads us to investigate the con-ditions for the development of organisms that have for some reason or other, perhaps fortuitously, come to our attention; on the other hand, to the discovery of living organisms that appear under predetermined conditions, either because they alone can develop, or because they are the more fit and win out over their competitors. Especially this latter method, in reality noth-ing but the broadest application of the elective culture method, is fruitful and truly scientific, and it is no exaggeration to claim that the rapid and surprising advances in general microbiology is due to this methodology. Beijerinck was awarded many honors, including the Leeuwenhoek Medal by the Royal Academy

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Table 16.1. Scientific highlights of Beijerinck’s career.

1. Demonstrated the filterability of the infectious agent of tobacco mosaic disease and coined the term “filterable virus”. Described the intracellular reproduction of tobacco mosaic virus in 1898, a pioneering contribution in virology.

2. Isolated Bacillus radicicola and proved that it forms nodules on the roots of Leguminoase species. Later isolated Rhozobium species, studied nitrogen fixation, and demonstrated nitrogen fixation by free-living microorganisms, particularly Azotobacter chroococcum.

3. Isolated and described in detail the denitrification process of Bacillus sphaerosporus and Bacillus nitrous.

4. Isolated sulfate producing Thiobacillus species and demonstrated their chemoautotrophic nature. Also studied hydrogen sulfide production by Azotobacter species.

5. Contributed to the understanding of lactic acid bacteria involved in producing kefir and yogurt. Demonstrated the significance of catalase-negative reaction and proposed the generic name Lactobacillus.

6. Introduced the generic name of Acetobacter, described pigment-producing Acetobacter melanogenum, and studied butyric acid and butyl alcohol fermentation.

7. Pioneered the study of luminescent bacteria and isolated Photobacterium luminosum (1889). Pioneered the study of yeast, isolated Schizosaccharomyces octoporus from raisings, and discovered the saccharolytic enzyme lactase of Sacharomyces tyrocola.

8. Was the first to obtain pure culture of algae, zoochlorellaen and gonidia of lichens.

9. Studied urea decomposition, microbial variations (mutations), and oxygen relationships among bacteria.

10. Observed Sarcinaventriculi in media of high acidity and under anaerobic conditions.

11. Studied plant galls and did extensive morphological work on adventitious structures and regeneration phenomena in plants.

12. Studied phyllotoaxis, the arrangement of leaves on plant stems.

13. Investigated the fungus Clasterosporium carpophilum (later named C. beijerinck), the potent cause of gummosis.

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of Sciences at Amsterdam, honorary membership in the Royal Botanical Society of Edinburgh, candidacy for a Nobel Prize in chemistry, commandership in the Order of Oranje-Nassau, cor-responding membership in the Czech-Slovakian Botanical Society, The Danish Emil Christian Hansen Medal, and election as a foreign member of the Royal Society of London.

Beijerinck never married. In fact, he never dated and had few, if any, female friends. He believed that marriage would interfere with his work. He was so reclusive, that he even refused to have his picture taken. After his father’s death, he lived in virtual seclusion with his sisters, Henriette and Hohanna except for socializing with the chemist van 't Hoff and his wife.

Although Beijerinck retired in 1921, he never gave up research. He died quietly on 1 January 1931 in Gorssel, The Netherlands.

Commentary

It is best to quote Joan Bennett’s (1942–) comments of Beijerinck. “For Beijerinck the allure of science never waned. When he was 75, he wrote to Allbert Jan Kluyer, his successor in Delft: “Fortunate are those who now start.” This motto was later inscribed in the entrance to the Kluyver laboratory in the Polytechnical Institute in Delft. This phrase does not translate aphoristically into English, but the philosophy it expresses in universal: young scientists are among the most fortunate peo-ple alive. They can look forward to a lifetime of discovery. There is no better way to honor the memory of Martinus Beijerinck than for each of us to convey this message to our students”.

Despite the, sometimes, unseemly character of Beijerinck, his persistence and dedication to research made him a great pioneer microbiologist. His work laid down the foundation of microbiology and biotechnology for today. His almost single minded crazed obsession for science is still a great inspiration to all microbiologists.

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Suggested Reading

1. Bennett, J. W. and H. Phaff (1993). Early biotechnology: The Delft connection. ASM News, 59(8): 401–404.

2. Bennett, J. W. (1996). Martinus Willem Beijerinck: Dutch father of industrial microbiology, SIM News 46(2): 69–72.

3. Grainger, T. H. (1958). A Guide to History of Bacteriology. The Ronald Press Company, New York.

4. Van Iterson, G., L. E. den Dooren de Jong and A. H. Kluyver (1940). Martinus Willem Beijerinck, His Life and Work. Martinus Nijhoff, The Hague.

5. Van Niel, C. B. (1949). The “Delft School” and the rise of general microbiology. Bacteriology Reviews 13: 161–174.

6. Waksman, S. A. (1931). Martinus Willem Beijerinck. Scientific Monthly 33: 285–288.

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Chapter 17

Walter Reed (1851–1902): Yellow Fever Fighter

Source: https://en.wikipedia.org/wiki/Walter_Reed(US Public Domain image)

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Introduction

Yellow fever is an acute viral hemorrhagic disease transmitted by infected mosquitoes. The “yellow” in the name refers to the jaundice that affects some patients.

Walter Reed is remembered as a hero in fighting against yellow fever, a disease, which created human miseries in many parts of the world including the United States. Walter was born in a small town, now vanished, called Belroi, near Gloucester, Virginia on 13 September 1851. His father was Lemuel Sutton Reed and his mother Pharaba White, both North Carolinians of English descent. Reed’s ancestry can be traced back to a sturdy country family of Northumberland, England. His father was a Methodist minister, who was called to a new parish every year or so. So Walter grew up in a number of communities including Gatesville and Farmville, Virginia, and Murfreesboro, North Carolina. He had one sister named Laura who married to a Methodist minister James Blincoe in 1859, and three brothers, Tom, James, and Christopher. He was the youngest. He was raised together with them with love and affection, but with stern admonitions about duty, and encouragement to read. His brother Tom and James fought for the South during the American Civil War (1861–1865). James lost a hand in a battle engagement, but he showed a gallant disregard for what must have been excruciating pain. When the shattered hand was amputated in a crude field hospital, he was reported to have said, “Thank you, Doctor, you have left me enough to hand the girls on”. He saw further active duty and was outraged at General Robert E. Lee’s (1807–1870) surrender.

Walter matured early because his mother died at the age of 49 when the Reed family settled in Charlottesville, Virginia in 1865. The 14-year-old Walter was devastated. On the day of her burial, he and his brother Christopher officially joined the Methodist Church, perhaps to keep a promise to their mother, but the motive was not truly known. He later referred to his mother as “a dear sainted spirit — a poor suffering mother”.

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In 1866, his father married to a wealthy and generous Harrisburg widow Mary Catherine Byrd Kyle (1826–1899) whom the children grew to love.

Walter did not have regular schooling until late in 1865 when a Confederate Lieutenant William R. Abbot, who later became headmaster of Bellevue School near Bedford, Virginia, tutored Walter for about two years. Walter studied the tradi-tional subjects of Latin and Greek, English composition, gram-mar and rhetoric, and history and the humanities. He also learned much about art, music, and other subjects from Abbott’s wife. He was determined to get a good education, so he studied very hard. He was qualified to enter the University of Virginia at the age of 15. (He had reported himself being 16 because the school allowed only 16-year olds to register.) After a year, he was permitted to take medical courses. At age 18 (in 1869), he received an MD, standing third in his class of 50, and he was the youngest in his class. He then went to New York to study at the medical school of Bellevue Hospital. He took the competitive examination for the position of assistant physician at the Infants Hospital at Randall’s Island and ranked first in December of 1869. He earned a second MD in a year, but the degree was not awarded to him officially until he was twenty one.

At Bellevue Hospital in New York, he saw the poverty, ill-ness, and misery that overflowed into one of the world’s big-gest, bloodiest, and busiest hospitals. Perhaps, it was his experience in this hospital that created the idealism in him that challenged the widespread filth, despair, and agony he found around him in medical work.

Reed then became assistant physician at the New York Infant’s Hospital. In 1871, he was resident physician at Kings County Hospital, Brooklyn for four months, and in 1871–1872 at Brooklyn City Hospital. At Brooklyn City Hospital, he became involved with Dr. A.N. Bell, who had been concerned with com-munity health and sanitation. Dr. Bell had influenced Walter’s mind regarding disease. Dr. Bell’s influence led Walter Reed to

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turn his attention from bedside medicine to the possibility of fighting disease by preventative actions at the community level. With reference to Yellow Fever, Dr. Bell believed that this dis-ease was transmitted by materials, not by the mosquito, a view-point, which was strongly contradictory to Walter Reed’s latter conclusion on the mode of transmission of Yellow Fever.

At the same time, Walter served as a district physician for the New York Department of Public Charities. His work had been noticed by a famous New York Surgeon, Dr. Joseph C. Hutchison (1822–1887), who was also a member of the Board of Health. Dr. Hutchison appointed Reed to the job of public sanitary inspector of the Brooklyn Board of Health in June of 1873. Dr. Reed worked long hours for very little money. He could have made more money by building a private practice, but he got more personal satisfaction from working with the poor and underprivileged. He felt that the people in New York in general did not care for these common underprivileged peo-ple, who needed mainly a clean place to live and food to eat. As a public sanitary inspector, Reed was not able to set up a private practice because of his poor salary. So the young doctor took an examination held by the Army Medical Corps to select doctors to serve in the Army.

In June of 1875, he received a commission as an assistant surgeon with the rank of first lieutenant in the Army Medical Corps and was ordered to a post in Fort Yuma, Arizona. In April of 1876, he married Emile Lawrence, whom he had met and became engaged to while on a series of visits to visit his father in Murfreesboro, North Carolina, where his father was a minis-ter. He was forced to leave his wife behind and left for his post in Arizona. The trip to the West proved to be very hot and unadventurous. Finally, Reed was moved to Fort Lowell in Tuscon, Arizona, after only a couple of months at Fort Yuma. He was able to send for his wife Emile.

From Fort Lowell, Reed moved with his wife to Camp Apache, a post near the White Mountains north of Tuscon, where there was an Apache Indian reservation. There he and

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his wife, Emile, had a son in 1877, Walter Lawrence Reed (1877–1956). When Lawrence was only four months old, the Apaches escaped from the reservation led by their outlaw chief, Geronimo. Soldiers were sent from Camp Apache to recapture the Indians. One night, a couple of soldiers showed up at Dr. Reed’s door with a little Indian girl. The little girl was the only one captured when the United States soldiers raided the Apache’s campsite of Geronimo. Dr. Reed saved the little girl’s life but could not return her to the Apaches because of the outbreak of the war with them, so he and Emile adopted the little girl and raised her as if she were their own daughter. The little girl was named Susie.

In 1880, the Reeds went back East to Washington, DC for a short period of time. While in Washington, DC with the Army, Dr. Reed was told of how there were advances in medicine while he was away out West. New discoveries in Europe had found their way to the United States, and now the medical field was learning the new science of Bacteriology, the study of tiny microorganisms and their relationships to the cause of disease. Dr. Reed could not go back to medical school because he was soon assigned to another lonely fort near Omaha, Nebraska. But he decided to learn for himself. He taught himself to read German and Italian, and he sent away for medical journals from all over Europe, where the new discoveries were being made about the bacteria and their relationships to disease. Dr. Reed yearned to look for himself at these tiny microorganisms through a microscope. While during his stay in Omaha, Nebraska, he had another daughter named Blossom born in 1883.

During this period of life, Dr. Reed became restless. He was aware of new discoveries in Bacteriology and Pathology, but the garrison life afforded him no opportunities for further study. He therefore applied for a leave of absence to do advanced work; instead, he was ordered to go to Baltimore, Maryland as an attending surgeon. He was authorized to study at Johns Hopkins Hospital where he completed several clinical courses. He undertook the study of pathology under Dr. William

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Henry Welch (1850–1934) who recognized his abilities. Dr. Reed did autopsies, learned pathological techniques and performed experiments.

The posting at Baltimore was for only one year, so Dr. Reed was sent to another frontier army outpost, Fort Snelling, Minnesota. While there, he felt that he would have to give up his hope of studying Bacteriology. He was soon to have a stroke of luck. A young high school Biology teacher in St. Paul, Minnesota, where the Reeds lived, had set up a crude labora-tory and offered to share the laboratory with Dr. Reed if he would teach him Bacteriology. Dr. Reed and the biology teacher worked diligently to discover the bacterial cause of diphtheria.

In 1890, Walter was allowed to report to Johns Hopkins Hospital for a postgraduate session in pathology and bacteriol-ogy. His stay in Johns Hopkins was interrupted by the outbreak of the American Indian War. He was called back to active duty to Fort Keogh, Montana, to take care of the victims in this Army/Indian confrontation. In 1892, Walter was assigned to St. Paul, Minnesota. He worked on diagnosis of diphtheria. Diphtheria was a terrible disease at that time, particularly for children.

In 1893, Reed was promoted to major, and was brought back to Washington as curator of the Army Medical Museum and professor of Bacteriology and Clinical Microscopy at the newly established Army Medical College when George Sternberg (1838–1915) was appointed Surgeon General of the United States. Reed worked closely with John Shaw Billings (1838–1913) in the old Surgeon General’s Library and Army Pathology Museum. He had an enormous laboratory. He helped students doing their research and did research on his own. When new bacteria were discovered, he would obtain specimens to study under his microscope. He would study bacterial habitats and their relationships to the cause of human disease. Walter stud-ied diphtheria, rabies, and other diseases. His work during this period of time can be seen in the Reports of the Surgeon General of the Public Health Service of the United States.

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Beginning in 1896, Dr. Reed’s talent and skill as a distin-guished medical investigator were gradually revealed. At that time, malaria was rampant in the Washington Barracks and at Fort Myer in Virginia. It was generally believed to be caused by bad drinking water. Walter investigated this case and showed that there were no outbreaks of the disease in other areas of Washington that, like the military bases, obtained drinking water from the swampy Potomac. Moreover, both officers and enlisted men shared much of the same food, water, and housing; malaria was rife among only the enlisted men, not the officers. So what was the difference? Reed found that enlisted men would, often, surreptitiously leave camp at night and go to the city along a trail through the swamps. Therefore, Reed concluded that not the drinking water, but rather the bad air — “malaria” in the ancient sense — was responsible for the disease. When these enlisted soldiers stopped going along the trail through the swamps, the outbreaks of malaria ceased. Until 1904, Dr. Reed’s report was not published.

In 1898, the United States declared war against Spain. The war was over in four months, but many soldiers died after the war in their own army camps due to fever that reached epi-demic proportions. Walter Reed was appointed as chairman of the board to investigate these outbreaks of the fever. Since the symptoms of the fever were similar to typhoid fever or malaria, he talked to most of the doctors and also tested samples from several soldiers in his laboratory that he set up in a railroad car in which he was traveling. After thorough examination, Dr. Reed concluded that it was a typhoid fever. This confused some of the medical doctors at the camp because the only cause known for typhoid fever at the time was unclean drink-ing water, and it was proven the soldiers drank clean water before their outbreak with the fever. Dr. Reed thought there must be some other cause for the fever, and set out to find it. He traveled extensively all over the South, touring Army camps and testing and retesting everything that came into contact with the typhoid patients. Finally, he discovered that there was

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another cause for typhoid fever besides contaminated drinking water. He found it was transferred through personal contact with a patient who had fever. This discovery led to typhoid patients being quarantined for the first time. Dr. Reed also found that flies from the latrines were landing on the plates of the soldiers at mealtimes at one of the camps in Tennessee. He took samples of the food and found that the typhoid bacteria were present in the food contaminated by the flies. He had the latrines cleaned and screens placed on the windows of the mess hall, and the typhoid fever did not happen again. The work of Dr. Reed made it possible to stop the typhoid epidemic that had killed between 50 and 100 times as many soldiers as had died in the actual combat in Cuba.

Dr. Reed’s reputation as a medical investigator grew. In 1900, he was appointed as head of an army board to investi-gate the causes of yellow fever, which had broken out among the American troops in Cuba. Yellow fever is a terrible disease caused by a virus, which can be transmitted by mosquitoes. But at that time, no one knew the agent that caused the disease. In the United States, yellow fever was an annual scourge; the dis-ease swept up the eastern seaboard and often spread to the Gulf states and up the Mississippi River valley. Its occurrence was predictable. The season of its occurrence, and the tempera-ture coordinated its spread, but no one knew its cause. In 1878, yellow fever was at epidemic levels in Memphis. Over 18,500 people fell victim of the disease and 5,000 of them died. In addition, over 30,000 people fled the city.

As early as 1848, the Alabama physician Josiah Clark Nott (1804–1873) suggested that an insect — perhaps the mosquito caused the disease, but he had only tenuous evidence. In 1881, Cuban physician, Dr. Carlos Finlay (1833–1915), working closely with the United States Fever Commission in Havana, suggested that yellow fever was transmitted by a mosquito, Culex fascia-tus (now called Aedes aegypti). This hypothesis was generally considered laughable by most modern medical practitioners of that day. In 1897, the Italian bacteriologist Giuseppe Sanarelli

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(1864–1940) stated that the organism Bacillus icteroides was the specific causative agent, but there was no convincing proof.

Dr. Reed was assigned by Dr. George Sternberg (1838–1915) who also believed yellow fever to be caused by a bacterium. Dr. Sternberg wanted Reed to prove whether Bacillus icteriodes was the cause of yellow fever. Dr. Reed was accompanied by James Carroll (1854–1907), Jesse W. Lazear (1866–1900), and Aristides Agramonte (1868–1931). Reed and Carroll soon dem-onstrated that Sanarelli’s organism bore no relationship to yel-low fever. Reed and his coworkers turned to the theory that the mosquito was the vector of the disease.

Finlay had been very helpful in this investigation. He pro-vided the team with mosquitoes, mosquito eggs, and instruc-tions for raising the insects in the laboratory. Finlay also aided in the clinical verification of the disease.

Surgeon General Sternberg felt that the only way to test the mosquito theory was on humans. Reed proposed that the Board members be the first to be tested since he was apprehen-sive of testing humans and felt that they should be willing to be tested before others. In fact, while Dr. Reed was on his way to New York, Dr. Carroll and Dr. Lazear, took mosquitoes from test tubes that had bitten yellow fever victims and let them bite themselves. But no one got the disease. One day, Dr. Reed got word from Cuba that Dr. Carroll and Dr. Lazear had contracted the fever after being bitten by mosquitoes that had also bitten yellow fever patients. By the time Reed arrived in Cuba, Dr. Lazear died on 25 September 1900, but Dr. Carroll recovered. Dr. Reed was terribly depressed. He studied Dr. Lazear’s note-book and found that the fever was spread by mosquitoes that had bitten disease victims in the first three days of the disease. When Reed’s experiments had required human volunteers from the enlisted men, he was very concerned about their safety. He very carefully described the risk to them. The mortality from yellow fever was high; in Havana that year it had reached 20%. Those that did not die from the fever were temporarily devastated. The statement of risk of the experiment was written

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and signed by the volunteers and witnessed. This experiment was the first of its kind, the first thoroughly controlled clinical research on humans. It established the highest standards of informed consent. This part of the contribution that was made by Dr. Reed seemed to have never been mentioned again.

Dr. Reed and his team produced 22 other experimental instances of yellow fever, of which none was fatal. Through experimentation, he proved that the female Aedes aegypti mosquito can become infected by biting a victim of yellow fever only during the first three days of the illness; the mos-quito does not become infectious until two weeks after but may then remain infectious for up to two months in a warm season. The mosquito does not become ill, nor her eggs influ-enced. The eggs do not develop until she has fed on blood, which harbors an infection that will affect the human. The period of incubation of the disease is three to five days after the victim has been bitten by an infected mosquito. Once hav-ing had the disease, an immunity was provided in the human against subsequent attacks. It was established that blood from an infected person early in the course of the disease will cause yellow fever upon injection into a susceptible person. They also found that blood from an infected person could be passed through a Pasteur filter and still remain infectious. This is exper-imental proof that a filterable virus can cause a human infec-tion. Dr. Reed had convinced the medical community of his theory. The US Army then controlled the mosquitoes causing the fever near the camps, and the epidemic was ended. Within a year, Havana was freed of its age-old plague of yellow fever.

Dr. Reed was exhausted by the experiments. He had always been short of money, plagued by the daily routine of examin-ing boards, and even charged erroneously by personnel officers of being absent from duty without leave. He had never been rewarded for what he deserved. Nevertheless, he was now world famous for finding the cause of yellow fever. Some peo-ple recognized the significance of his work. For example, Harvard University awarded him an honorary MA, citing him as

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“Walter Reed, Medical Graduate of Virginia, the Army Surgeon who planned and directed the experiments in Cuba that have given man control over the fearful scourge of yellow fever”. The University of Michigan awarded him the LLD degree. He only enjoyed this for a short time; however, Walter was depressed in mind, body, and estate. On 23 November 1902, he suffered a ruptured appendix and abscess of the cecum that perhaps followed from an amoebic infection that he might eas-ily have contracted in Cuba, and he died. He was buried in Arlington National Cemetery and honored with a gold medal and a postage stamp bearing his likeness. Walter Reed Army Hospital in Washington, DC is named in his memory.

Dr. Reed’s work enabled the men trying to build the Panama Canal to complete their work that had been halted by yellow fever. When complimented on finishing the Panama Canal and his success in dealing with yellow fever, the man who had destroyed the mosquitoes there, Major William Gorgas (1854–1920), replied that he himself was not a great man, but was merely following in the footsteps of a great man, Dr. Walter Reed.

As we respect the work of what Walter Reed had done on yellow fever, we should remember how he had constantly edu-cated himself. While other medical doctors might have enjoyed good livings, Walter Reed diligently did research, kept up with new scientific discoveries, and learned new medical informa-tion. Success favors the prepared mind. Walter Reed’s life serves as a good example of a self-educated person. Walter had dedi-cated his whole life preparing to fight against human maladies and his experiments saved many lives. We should also remem-ber the sacrifice of Dr. Lazear.

Yellow fever is still a serious problem; there is no specific treatment for it. There was a vaccine produced in 1927 by Max Theiter (1899–1972) and the first official global disease eradica-tion effort began, endorsed by the governments of the Americas. However, it was later discovered that several species of monkey and ape could carry the virus, both in Africa and in South America. In America, capuchin monkeys were unharmed

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by the virus, but they carried yellow fever and could be a source of the virus for feeding mosquitoes. In contrast, when yellow fever hit Central America, epidemics virtually exterminated the nonimmune Aeteles and Alouatta monkey populations. It was also discovered that Aedes aegypti was not the only mosquito that could carry yellew fever; A. africanus, A. simpsoni, and A. albopictus, could also carry the virus. The virus could be passed from one mosquito generation to the next in the insect’s eggs; this allowed for long periods of time — and several insect gen-erations, during which time the disease seemed to disappear. But the virus was actually silently residing in generations of monkeys and mosquitoes, ready to reappear in human epi-demic form under proper conditions. By the late 1950s, scien-tists realized that there were two types of yellow fever: the urban form associated with A. aegypti and the forest or sylvan form that could be found in a variety of monkeys and wild mos-quitoes. Eradication of the urban form might be possible through vaccination, covering all water sources, and dichlorodi-phenyltrichloroethane (DDT) spraying of insect breeding sites. But forest or sylvan Yellow Fever could not easily be eliminated. Although World Health Organization (WHO) and Pan American Health Organization (PAHO) tried to eliminate the A. aegypti from this hemisphere, the task was never thoroughly executed.

In 1960, an enormous yellow fever epidemic broke out in western Ethiopia. Over 100,000 people had suffered the disease by the end of 1962 when the epidemic died down. Yellow fever killed one out of three infected Ethiopians. The virus is endemic in tropical areas of Africa and Latin America, with a combined population of over 900 million people. The number of yellow fever cases has increased over the past two decades due to declining population immunity to infection, deforestation, urbanization, population movements and climate change.

There is no cure for yellow fever. Treatment is symptomatic, aimed at reducing the symptoms for the comfort of the patient. Vaccination is the most important preventive measure against

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yellow fever. The vaccine is safe, affordable and highly effec-tive, and appears to provide protection for 30–35 years or more. The vaccine provides effective immunity within one week for 95% of persons vaccinated.

In recent years there has been a steady increase in the num-ber of yellow fever epidemics, cases, and deaths in tropical Africa. These epidemics have become an annual occurrence in Africa, and this disease is endemic in 33 of the continent’s 46 countries. Up to 50% of severely affected persons without treatment will die from yellow fever.

There are an estimated 200,000 cases of yellow fever, caus-ing 30,000 deaths worldwide each year. On the other hand, in Latin America, the mosquitoes that carry the virus are returning in force. “Will Yellow Fever be another emerging disease on our planet”? Is a question for all of us ponder.

Suggested Reading

1. Bean, W. B. (1982). Walter Reed: A Biography. University Press of Virginia, Charlottesville.

2. Garrett, L. (1994). The Coming Plague. Pp. 66–70. Farrar, Straus and Giroux, New York.

3. Groh, L. (1971). Walter Reed: Pioneer in Medicine. Gerrard Publishing Company, Champaign, Illinois.

4. John, M. (1994). The Fall and Rise of Yellow Fever. Pp. 48. World Press Review.

5. Hill, R. N. (1957). The Doctors Who Conquered Yellow Fever. E. M. Hale and Company, Eau Claire, Wisconsin.

6. Truby, A. E. (1943). Memoir of Walter Reed: The Yellow Fever Episode. Paul B. Hoeber, Inc., New York.

7. Wood, L. N. (1943). Walter Reed: Doctor in Uniform. Julian Messner, Inc., New York.

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Chapter 18

Emile Roux (1853–1933): Diseases Fighter

Source: https://en.wikipedia.org/wiki/Pierre_Paul_%C3%89mile_Roux(US Public Domain image)

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Introduction

Emile Roux was a close associate of Louis Pasteur (1822–1895). He helped Pasteur conduct a lot of research and made many significant contributions to science, particularly disease fight-ing. Dr. Roux was the director of the Pasteur Institute from 1904 to 1933, and Pasteur Institute was the foremost biomedical/research microbiological center in the world during Roux’s tenure as director.

Life of Roux

Pierre-Paul-Emile Roux was born on 17 December 1853, at Confolens, France. Confolens is a small ancient town on the banks of the Vienne in region of Charente. His father was head of a college in Confolens, but his father died young and left nine children. Two brothers of Roux joined the army and fought in the Franco-Prussian War and died in the battle.

At a young age, Emile entered school at Confolens and took mostly classic courses. Later, he went to Aurillac (a commune in the Auvergne region in south-central France, capital of the Cantal department) and spent some time at the Lycée at Le Puy (Haute Loire) where he obtained his bachelor of science degree. In 1872, he entered the “Ecole preparatoire de médecine et de pharmacie” in Clermont-Ferrand where he met Dr. Emile Duclaux (1840–1904) who was his mentor and a lifelong friend. Duclaux had a great influence on Emile Roux’s career.

Duclaux had been an “agrégé préparateur” (assistant) to Pasteur and later became a faculty member in science in Clermont and as a “suppleant” (assistant) to the chair of chem-istry in 1866. Roux did his research in Dr. Duclaux’ a laboratory on “Des variations dans la quantited’ ureeexcretee avec une alimentation normale et sous l’ influence du the et du café”, which was published in 1873.

From Clermont, Roux went to Paris to finish his medical studies, and then entered the military hospital of Val-de-Grace,

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http://en.wikipedia.org/wiki/Auvergne_(r%C3%A9gion)

http://en.wikipedia.org/wiki/Regions_of_France

http://en.wikipedia.org/wiki/Cantal

http://en.wikipedia.org/wiki/Departments_of_France

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where Alphonse Laveran (1845–1922) was his teacher for a time. Roux did not like military life. From 1874 to 1877, he worked at the Hotel-Dieu where he acted as “aide-de-clinique” for part of the time in the service of the famous clinician profes-sor L. J. Behier (1813–1876). In 1877, Roux again worked for Dr. Duclaux, who by now was a professor of Meteorology at the Institute Agronomique in Paris. Dr. Duclaux did not have a labo-ratory in his own institute but had a research space specially arranged in Pasteur’s laboratory at the École Normale Supérieure. Roux worked as a “preparateur” for Dr. Duclaux. Pasteur noticed his laboratory skills and took him along with Charles Chamberland (1851–1908) and Jules Joubert (1834–1910) as coworkers. After this, Roux had a close association with Pasteur until Pasteur’s death in 1895.

In 1883, Roux obtained his MD degree; the title of his dis-sertation was “Des nouvelles acquisitions sur la rage”. He worked closely with Pasteur. The new Institute Pasteur (in Rue Dutot) was inaugurated in 1888. (The old quarter was in Rue d’Ulm.) From 1888 to 1897, Roux was the “chef de service” of the Institute. From 1897 to 1904, he was the “sous-director” (deputy director). (The director was Dr. Duclaux.) In 1904, Roux became the director of the institute when Duclaux died. Roux held this position until his death in 1933.

When Roux began to work with Louis Pasteur in 1878, Pasteur had just started to work on infectious diseases in humans and animals. Pasteur was a chemist, and he needed help for this new research adventure. At this time, Emile Roux and Charles Chamberland were Pasteur’s assistants and cowork-ers. Chamberland was a physicist; Roux was the only trained medical doctor in Pasteur’s team. Roux’s help was indispensable for Pasteur’s research. From 1879, they began to attack the problem of anthrax and together published about 10 important research papers on the etiology of the disease, viability and spread of anthrax spores, and attenuation of the anthrax bacil-lus (Bacillus anthracis). They found that inoculation of sheep and cattle with attenuated anthrax bacillus would immunize

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them against subsequent infection of the virulent form of bacil-lus. A similar immune response also occurred with attenuated chicken cholera. In 1881, Pasteur accepted the challenge of his professional enemy and carried out the remarkable public experiment of anthrax vaccination in Pouilly-le-Fort. Roux and Chamberland were the true actors who actually did the experi-ment for Pasteur. Later (1888), Roux demonstrated that produc-tion of immunity against anthrax could be achieved by the inoculation of the sterilized liquid culture of Bacillus chauvoei.

In 1882, Isidore Straus (1845–1898) and Louis Ferdinand Thuillier (1856–1883) joined Pasteur’s laboratory. Thuillier helped Pasteur to study swine fever and rabies. In 1883, Pasteur appointed Straus to be the leader of the French expedition sent to study Asiatic cholera which was epidemic in Africa. Roux, Edmond Nocard (1850–1903), and Thuillier were also there. Unfortunately, in the September of 1883, Thuillier caught the disease in its most acute form and died in Alexander, Egypt. Roux and Straus also had severe cholera in Toulon in 1884.

Roux and Chamberland helped Pasteur in many areas of research. They helped Pasteur study rabies. They discovered the virus fixe and developed the method of attenuation of the virus in the spinal cord. Roux demonstrated the passage of rabies virus along the nerves, and the virus could be conserved for weeks in glycerin at room temperature. Roux participated in the early inoculations of human beings bitten by rabid dogs. In all endeavors, Roux never disappointed Pasteur.

Roux also helped Alexandre Yersin (1863–1943) in their work on diphtheria bacillus. They confirmed and extended the discovery of Friedrich August Johannes Loeffler (1852–1915) in 1884. Roux and Yersin proved that the diphtheria toxins caused typical paralysis and laid the foundation for bacteriological diagnosis of diphtheria.

Roux was also involved in many aspects of microbiological research including the cultivation of anaerobes, microphotogra-phy, the action of heat and light on anthrax spores, and the use of potatoes in microbiological cultures. He was the chief contributor

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to the Annales de l’Institute Pasteur. His name occurred on the title page of the first number in 1887 and was on every single number down to December 1933, a total of 51 volumes.

Roux collaborated with many colleagues, assistants, and friends within the Pasteur Institute. He collaborated with Edmond Nocard on the cultivation of tubercle bacillus and found that glycerin was an important food component for the tubercle bacillus. They also demonstrated the protective inocu-lation of ruminant against rabies by intravenous injection of the rabies virus. Roux and Nocard proved experimentally the appearance of rabies virus in the saliva of inoculated dogs.

In collaboration with Louis Martin (1864–1946), Roux eluci-dated the production of diphtheria antitoxin by the immuniza-tion of horses. Antitoxin could be used to treat diphtheria. The work on the production of diphtheria antitoxin led to the crea-tion of a great establishment near Paris at Garches in the for-mer historical imperial chateau of Villeneuve-l’Etaing for the production of diphtheria antitoxin. This establishment has sup-plied France with enormous quantities of antitoxin of different kinds as well as other bacterial products for diagnostic or thera-peutic purposes since then.

In 1898, Roux and Amédée Borrel (1867–1936) reported the occurrence of cerebral tetanus. In the same year, Roux with Nocard, Borrel, Taurelli-Salimbeni and Edouard Dujardin-Beaumetz (1868–1947) discovered the infective agent of bovine pleuropneumonia.

Roux worked closely with Élie Metchnikoff (1845–1916) on the problem of syphilis. In 1905, Fritz R. Schaudinn (1871–1906) and Eric Hoffmann (1868–1959) identified that Treponema pallidum was the caustic agent of syphilis, but no animal model could be used for syphilis because syphilis did not occur to the common experimental animals. Roux and Metchnikoff showed that apes (chimpanzees, baboons, and macaques) could develop syphilis with artificial infections. This was a milestone break-through since syphilis inflicted about 10% of the population in Paris at that time. He was awarded with the Osiris Prize of the

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Institute de France in 1906 for this work, and then was appointed director of the Institut Pasteur.

Roux was also a great teacher. In 1888, he initiated a course in microbiology at the Institut Pasteur and taught it every year. He had personally taught more than 3,000 students from all over the world. He was an eloquent lecturer with great enthusiasm.

Roux was a great administrator and scientist. He kept in close contact with everything in the laboratory, and was in complete sympathy with the aims of all the workers whom he helped in every way not only by his knowledge but also by the constant display of his altruistic nature.

During World War I (1914–1918), he mobilized the whole of the bacteriological service under his control to help win the war. After the war, he was very emaciated and suffered much from cold weather. He was most commonly seen in a long pel-erine with a warm muffler tied carelessly around his neck. As age advanced, he became more feeble, but his spirit triumphed over his frail body. He had never been married, and lived an ascetic monastic life in two small rooms in the hospital of the Institut Pasteur. He was looked after by one of his sisters. Several months before his death, he had been ill and confined to bed through sheer weakness. He was visited every day by his former student, Albert Calmette (1863–1933). Although Calmette looked healthy, Calmette died after a short illness on 29th October 1933. When Roux heard the news, he was shocked and said “Alores rien ne me sera épargné (Then nothing will be spared me)”. He became a changed man and lived only four days longer and died on the 3rd of November, 1933. The last words he was heard to whisper were, “Travaille-t-on dans les laboratoires? Il faut travailler! (Do we work in the laboratories? You have to work!)”

Before his death, Roux expressed that he wished to be bur-ied in his native Charente at his death, but the clamor of the public and the press made the government impose on his remains a national funeral. The funeral ceremony took place at Notre Dame and was attended by President Lebrun and all the

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great officers of the State. At the end, his coffin was taken back and placed in a tomb in the garden of the Institut Pasteur where he lived and worked for almost half a century.

Roux received many honors in his life. He was twice Laureate of the Institut de France (1884–1896) and twice Laureate of the Academie de Medecine (1886–1896). He was the recipient of the well-known Osiris Prize of l’Institut. He was a member of the Académie des Sciences and of the Académie de Médecine. In 1889, he was invited to deliver the Cronian Lecture at the Royal Society in London with the title “Les inoculations preven-tives”. When Joseph Lister (1827–1912) passed away in 1912, Roux attended the funeral in Westminster Abbey in London. He was elected a foreign member of the Royal Society in 1913 and received Copley Medal in 1917. Dr. Roux was never awarded the Nobel Prize, but his contribution to science and medicine is far more worthy than a Nobel Prize.

Commentary

Roux devoted his 50 years of life to the study of various diseases and made a great contribution to the study of each of them. An article possibly written by Miss F. Mable, a sister of Mrs. Emile Duclaux, appeared in Time, 13 November 1933. The article said, “his large heart was ever open to individual distress, and among the thousands who clamored for a national funeral, there were many poor widows, lonely women, past worker, and deserted mothers whom he had succored. In youth, he must have been handsome; he retained his sparkling eyes, bright, clear, encouraging, fierce, kind, sparkling as a boy’s according to his mood, and the classic form of his head and his features though thin and worn were still noble”.

Perhaps, we can understand better what kind of person he was by looking at how he respected his mentor, Dr. Duclaux, and his master Pasteur, the way he treated his colleagues and how he nurtured his students. He is indeed a human treasure for all to remember.

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Chapter 19

Emil von Behring (1854–1917): Pioneer of Serology

Source: https://en.wikipedia.org/wiki/Emil_von_Behring(US Public Domain image)

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Introduction

In 1901, the first Nobel Prize for Physiology and Medicine was awarded to Emil von Behring for “his work on serum therapy against diphtheria”. Diphtheria is a rare disease now because diphtheria vaccination is available to almost every child born today. But over a century ago, thousands of children died of diphtheria. Behring and his associates were responsible for the cease of this disease. Furthermore, Dr. Lloyd G. Stevenson (1918–1988) has well stated, “for his work on serum therapy, especially its application against diphtheria, he has opened a new road in the domain of medical science and therapy placed in the hands of physician a victorious weapon against illness and death”. The life of von Behring is one for us to know.

Life of von Behring

Emil Adolf von Behring was born on 15th March 1854 in Hansdorf, West Prussia (Deutsch-Eylau, now Germany) as one of eldest son of August Georg Behring, a schoolmaster with thir-teen children, and his second wife, Augustine Zech Behring. His father wanted him to study theology, so he prepared himself for the priesthood. But this was not realized because a military doctor, a friend of his family intervened in this decision. Instead, Adolph entered the Army Medical Academy in Berlin (the Pepiniere). He received medical degree in 1878, and two years later he passed the state examination that allowed him to practice.

The army sent him to Posen (now Poznan, Poland), then to Bonn in 1887, and finally back to Berlin in 1888. Even at this early time, his effective, diligent and hardworking attitude toward life could be seen and he was very methodological. His lecture notes on the subject matter were not only systemati-cally arranged but also enriched by his own ideas. In 1888, he became a lecturer at the Army Medical College. He also took time to participate enthusiastically in the amusement of his

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fellow students and was known for his incisive speech and even caustic remarked. The military style of training had already been stamped on Behring’s life and remained right to the end.

He got up at four o’clock every morning, and ate a juicy steak for breakfast. When other colleagues arrived in the labo-ratory, he had already finished half of the day’s work. He main-tained a very high working efficiency for whole life. By this means, he was laid down the foundation for his success later in life.

To begin with, he worked as army doctor with various mili-tary units in Poznan, East Germany, next at the Pharmacological Institute in Bonn, and finally (at the age of 35) he became an assistant to Robert Koch (1843–1910), the well-known physi-cian, in 1889, at the University of Berlin. Two years later, he accompanied Koch to the Institute for Infectious Diseases where he was made professor at the age of thirty nine. From 1889 to 1994, Behring’s main effort was to fight against diph-theria and tetanus. Both of these diseases are caused by bacte-ria that do not spread widely through the body, but produce generalized symptoms by excreting toxins. Diphtheria, nick-named the “strangling angel” because of the way it obstructs breathing, was a terrible killer of children in the late 19th cen-tury. Its toxin had first been detected by others in 1888. Likewise, tetanus was often fatal to patients infected. The teta-nus bacillus was cultivated in 1889 in its pure state for the first time by Kitasato Shibasaburo (1856–1931), who came from Japan only the year before they had met.

In the beginning, He thought of how the body could be sterilized against the infection of bacteria. He used techniques from army experiences by employing iodoform, acetylene, and mercury compounds as antiseptic wound dressings to kill infec-tive bacteria. But all these attempts were failures. He treated cultures of diphtheria bacillus with iodine trichloride, with which he then inoculated a number of guinea pigs. He found that some survived and they became immune to diphtheria. What had happened? During his experiments, he noticed that

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rats were never attacked by anthrax. He discovered that rat serum was able to destroy the anthrax bacillus. This became the basis of his further experiments.

Was this due to the chemical agents or some reaction of their body? Behring made a series of experiments. He was able to show that minute amounts of diphtheria toxin were able to immunize the animals. By taking blood serum from an immunized guinea pig and injecting it into another animal, he found that he was able to transfer the protection to the second guinea pig. An animal already suffering from diphthe-ria toxin could sometimes be cured. He discovered that serum did not affect diphtheria bacilli, but it neutralized the toxin they produced. He used the word antitoxin (we now know that this is antiserum), which was directed against the poison produced by bacteria not against the bacteria themselves. Then he treated animals with serum of animals, which had survived the disease. The results were that the serum of an animal that had been cured could cure other animals. This was called serum therapy.

It should be mentioned that discovery of Behring was not by accident or made by von Behring alone. It was led by some pre-vious experiment which von Behring and Kitasato Shibasaburo worked together. Together, von Behring and Kitasato were able to demonstrate that rabbit could be immunized against tetanus in the same way as could be against diphtheria. On 4 December 1890, Behring and Kitasato published their first classic paper Ueber das Zustandekommen der Diphtherie-Immunität und der Tetanus-Immunitätbei Thieren (“The Mechanism of Immunity in Animals to Diphtheria and Tetanus”). A week later, Behring published a second paper but this one carried his name alone. The second paper was dealing with immunity against diphtheria and outlining five ways in which it could be achieved. These reports announced that injections of toxin from diphtheria or tetanus bacilli led animals to produce in their blood substances capable of neutralizing the disease poison. The name “antitoxin” was used. “Antitoxin” was used

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by him probably it meant “to use toxin against toxin”, a con-cept common to the Orientals. This term was ridiculed by the scientific communities at that time because to them it explained nothing. But von Behring had certainly made his mark in his antitoxin work. Kitasato, without whom the serum might never have come into being, deserved just as much credit as Behring for the work, but apparently was neglected by the Nobel Prize Committee. And we have learned racial equality at that time was not so well recognized as today.

Behring had then to get enough blood serum containing his diphtheria antitoxin to begin trials on human beings. He started with immunized guinea pigs. From there, he progressed to rabbits and from rabbits to sheep. By Christmas of 1891, he had enough antitoxin to take a needle and inject a small child lying in a Berlin hospital. The immunization was remarkably success. After careful resting in Berlin’s Charite Hospital and later in Leipzig, the serum was made available to doctors. Finally, von Behring was successful in establishing a method for the immunization of human beings against diphtheria and pro-duced the vaccine called “T.A.” for diphtheria.

Another person should be mentioned in the work of diph-theria antitoxin. This was Paul Ehrlich (1854–1915), another of the talented associate in Koch’s laboratory, who was chiefly responsible for standardizing the antitoxin, thus making it practical for wide spread therapeutic use. Ehrlich discovered a method of measuring the amount of antitoxin in blood, and calculated how much of it was needed to effect a safe cure in human beings, and also made the suggestion to use goats and horses to obtain antitoxin many times more powerful than von Behring had succeeded in preparing. Of course, the major con-tributions were made by von Behring and credit went to him. But Paul Ehrlich did not receive the credit of diphtheria work to the extent as he would have liked to. Nevertheless, Ehrlich was later recognized and awarded the Nobel Prize for his other work (in 1908) in immunology.

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Behring was also able to develop commercialization of the antitoxin in 1892. He was approached by the representatives of the Lucius and Bruning dye works at Hochst near Frankfurt. Behring agreed to have his product developed and marketed by the Hochst firm, and von Behring obtained benefits. The suc-cess of diphtheria gave him both fame and fortune. He won a professorship and also some ennoblement with the right prop-erly call; himself: the “von Behring”. He established his own chemical factory: the “von Behring Works”. In 1896, he married 18-year-old Else Spinola, the daughter of the director of the Berlin Charite’ Hospital. They had seven children.

In 1894, von Behring became professor of Hygiene at Halle and shortly after, at the University of Marburg (Germany). A year later, he was named a professor and director of the Institute of Hygiene at the University of Marburg. Thereafter, he focused much of his attention on the problem of immuniza-tion against tuberculosis, a terrible disease at that time. He donated all his properties including his Nobel Prize awards and many other awards to the institute. Many well-known scientists came to work for him. Although Behring failed to discover a tuberculosis vaccine, he proposed a theory that the disease was spread by infants drinking milk contaminated by tuberculosis bacilli and devised methods to disinfect the milk. He tried immunization calves with a weakened strain of the human tuberculosis bacillus, but the results were disappointing. His basic idea of using a bacillus from one species to benefit another influences the development of later vaccines. For years von Behring worked tirelessly day and night. Unfortunately at the age of 50, he contracted tuberculosis himself.

For years von Behring worked tirelessly day and night. Behring also did not abandon his work on diphtheria during this period. In 1913, Behring invented a new toxin-antitoxin preparation that gave longer-lasting immunity to the diphthe-ria, and in 1914, Behring established his own company to manufacture serum and vaccines. He used profits from this to

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keep a large estate at Marburg, on which he grazed cattle used in experiments, and also for a gathering place of society. He did not succeed in combating tuberculosis. Behring was subject to frequent bouts of serious depression, some of them required sanatorium treatment. In addition, a fractured thigh led to a condition that increasingly impaired his mobility. Finally, he contracted pneumonia in 1917 and died on 31st March of that year, two weeks after his 63rd birthday.

Commentary

The first drop in diphtheria mortality was due to the antitoxin therapy introduced by von Behring and this is the contribution he is primarily remembered. In Germany alone, an estimate 45,000 lives per year were saved. It is also why he was awarded with Nobel Prize. He also shared a sizable cash prize from the Paris Academy of Medicine with Emile Roux (1853–1933), who was one of the men who had the diphtheria toxin in 1888. There were other financial rewards as well. He was also granted honor memberships in societies in Italy, Turkey, France, Hungary, and Russia.

During the period, when von Behring and Paul Ehrlich both worked in the Koch’s laboratory, there was much competition, both racing to be the first. Paul Ehrlich did not speak to von Behring nor give him any good comments. Behring’s style might be considered personal, acquisitive, and aggressive in scientific work. But his military training made him go straight forward to the goal. Because of his military background, he might have cultivated a good relationship with the Ministry of Culture and other governmental officers of Germany, which might have helped von Behring to be successful more rapidly than Ehrlich. The hard, efficient working habits of von Behring, and generous in spending money was a big contradiction to Ehrlich’s habits. Despite the conflict and the bad comments from Paul Ehrlich during his life time, at the funeral of Paul Ehrlich in 1915, von Behring was one of the scientists who

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walked behind Ehrlich’s coffin to his grave where he said, “If we have hurt you, forgive us”. Behring was not vindictive. He lived during the World War I (1914–1918), when the waves of national enthusiasm were running high. Behring never took a chauvinistic attitude. He dedicated his last book, published in 1915 to Élie Metchnikoff (1845–1916) (a Russian scientist who lived in Paris, please see Chapter 14) with the words “Travaillona!” Behring was a great human being indeed.

Behring’s vaccines helped to save the lives of millions of injured soldiers in Word War 1, as well as countless others threatened by tetanus and diphtheria. The contribution made by von Behring to the signal triumph of conquering such a ter-rible disease was great. His hard working, efficient dedication to science gives much for us to learn. A Russian woman in Moscow wrote a letter to von Behring and said “I do not know if any mothers have expressed their gratitude to you. But my child is saved because you though wisely and fought for science — your name is blessed by happy mothers like me”. This short statement surely described well the great contribution made by von Behring. In this spirit, we should respect and emu-late such a pioneer of microbiology and medicine.

Suggested Reading

1. Hermon, A. and K. Jurgeo (1968). German Nobel Prize Winners. Pp. 43–44. Heinz Moos Verlagsgesellsachaft, Munich, Germany.

2. Reid, R. (1975). Microbes and Men. Pp.109–123. Saturday Review Press, Dutton & Company, Inc., New York.

3. Medical Discoveries, Berhring, Emil von. http://www.discov-eriesinmedicine.com/General-Information-and-Biographies/Behring-Emil-von.html. Accessed 16 November 2016.

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Chapter 20

Erwin F. Smith (1854–1927): Father of Plant Pathology

“Those who dwell in the clearer light of the next generation will build better than we have done and will scarcely realize how slowly and painfully many of us have groped about for what seems to them so plain”.

“Be then my scroll; lies one beneath this sod to whom all nature voiced the living God”.

— Erwin F. Smith

Source: https://en.wikipedia.org/wiki/Erwin_Frink_Smith(US Public Domain image)

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Introduction

There is always a building called Smith Hall in American universi-ties, especially those with agricultural colleges. That is in mem-ory of Erwin F. Smith. There is a bacterial genus called Erwinia that is also in memory of Erwin F. Smith. From these instances, microbiologists would probably understand why Erwin F. Smith is an important figure in the history of microbiology.

Life of Erwin F. Smith

Erwin Frink Smith was born in the little village of Gilbert Mills, New York, on 21 January 1854. His father was Ransellor King Smith, and his mother was Louisa (Frink) Smith. The Smith family were of Anglo-Saxon stock, some of the lines going back among the earliest of the New England settlers. They were pio-neers who settled in eastern Massachusetts and then moved on to Connecticut, and afterward into central New York. Later they moved to southern and central Michigan and then farther west. His immediate forebears on both sides lived in central New York in small farming communities, and his family had set-tled in Gilberts Mills shortly before he was born. The town of Gilbert Mills was one with many flour mills. A very keen interest in Smith’s boyhood was the functioning of these mills. He won-dered how God could run the mills. When he heard the grind-ing of the mill wheels he pictured in his mind a “prisoned God” at work! The mills also often had adjacent mill ponds. He liked to spend time to these mill ponds and their streams in the coun-try side. He always fell a consciousness of the presence of the Creator there, as well as in the grinding of the mill wheels. He also felt an obligation to serve his creator, who created the world and all that was in it. Smith’s social life centered around the church and school, and there was a strong element of piety in his home life.

There was much hard work to be done. In general his was a happy boyhood, with all the interests and activities connected

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with farming, to which he early on added an interest in books, nature, science, art, and music. He seemed to develop a fond-ness for all forms of arts and a desire for perfection.

Erwin was left without his father at the beginning of the American Civil War (1861–1865) as his father enlisted in Company, K. of the New York 184th Infantry. During those years, young Smith had the blessing of the assistance of a school teacher friend, Miss Ida Holmes. Noting his love of nature, she guided him with the loan of books and magazines of a scientific nature. In addition, he read works by Alfred Tennyson (1809–1892), Charles Dickens (1812–1870), and Henry Longfellow (1807–1882). He chose science as his major interest, but his reading was so broad and his interest in all subjects so great that he had a difficult time selecting a major among such sciences as botany, chemistry, medicine, physics, and geology.

Smith, with his family migrated in 1970 to a farm home in Hubbardston in southern Michigan. He was now 16, and entered with the public schools there. His formal schooling was inconsequential, partly because of financial need and his shy-ness. Later, the family moved to a farm near Ionia, Michigan. Smith was now 22 and appeared mature for his age wearing a full beard. He entered Ionia High School, and was immediately recognized by the principal, Anson P. Dewolf, as an exception-ally intelligent student. In addition to his regular high school courses, he was granted permission to “come and go as the spirit moved him”. By the time he graduated high school, he was already 26 years old. He soon mastered French on the side and also learned botany through tutoring by the local pharma-cist, Mr. Charles F. Wheeler (1842–1910). Mr. Wheeler was a gifted amateur botanist. His botanist job was no sinecure as just a year after graduating from high school, he published his first book: The Flora of Michigan in 1881.

After some studies at Michigan Agricultural College, he entered the University of Michigan and obtained his bachelor’s degree in 1886. He then immediately began graduate work and was granted a doctor’s degree in 1889. His PhD research work was

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on a plant disease, “peach yellow”. He continued working on this research for several years. At all times, Smith was fortunate in having advisors and colleagues who recognized his abilities. They gave him encouragement in his irregular method of study and research. During this period, he also married. His wife was Charlotte May Buffet Smith (1871–1906), who had an extended period of illness, dying in 1906. With her, he had a great love of nature and studied mycology and bacteriology. In 1915, he pri-vately published a book dedicated to his wife titled For her Friends and Mine: A Book of Inspiration, Dreams and Memories.

In 1913, he married again. His second wife was Ruth Warren Smith. She was his confidant and assistant until his death.

In his initial work of peach yellow disease he was not able to isolate and culture the mycotic agent. In 1892, he was greatly influenced by Dr. M. B. Waite, who had been an assistant in the laboratory of Dr. Jonathan Burrill (1839–1916) and had learned from him the methods of the pure culture of bacteria. He also studied the papers of Louis Pasteur (1822–1895) and in addi-tion, learned to read papers in German and Italian. He also learned from Theobald Smith (1859–1934) and Veronus A. Moore who worked near him in the US Department of Agriculture on diseases of animals.

This was early in the development of microbiology and not all scientists, especially botanists, were convinced that diseases of plants were caused by bacterial and mycotic agents. One eminent scientist from Germany, Alfred Fischer (1858–1913), wrote a book in 1897: Vorlesungen uber Bakterien (Lectures on Bacteriology). In this book, he stated that bacteria and molds had not been proven to cause plant diseases. Smith had both the needed experimental knowledge and the ability to write in German. He did not hesitate to engage Fischer in scientific debate. The debate, at this time, did not settle the question, but greatly enhanced the world opinion of Dr. Smith. The situ-ation was that European scientists distrusted the American papers, while Americans accepted them and did not go along with the hypothetical ideas of the Europeans.

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Smith’s work covered a wide range of plant diseases and was done with very painstaking exactness. His work, and that of his assistants, was later proven to be essentially correct and was accepted. He had a highly analytical mind and great ability to organize and synthesize knowledge. His work in plant pathology culminated in a three-volume treatise published in 1905, 1911, and 1914. The latter part of his life was devoted to research in cancer-like diseases in plants, particularly that of crown gall. He worked on all phases of its tumoricity and mor-phology. He was convinced that both plant and animal tumors had similar etiologies. In 1913, he received a certificate of honor from the American Medical Association for his work on “Cancer in Plants”.

Erwin Frink Smith was not only a scientist but also a leader in his field. He was president, at different times, of the follow-ing associations: Society for Plant Morphology and Physiology (1902); Society of American Bacteriologists (1906); American Association for the Advancement of Science, Section G (1906); Botanical Society of America (1910); American Phytopathological Society (1916); and the American Association for Cancer Research (1925).

He was also elected fellow of the American Academy of Arts and Sciences and was became a member of the National Academy of Sciences. Although others worked on microbial diseases of plants, under the auspices of the US Department of Agriculture (USDA), his work was the most outstanding. Great credit should be given to him for leadership and ingenuity in furthering the work in agriculture.

Typical of the honors given him was the dinner in his honor by the American Phytopathological Society. There were 172 signatures of scientists affixed to a plaque, which read: “To Erwin Frink Smith, scientist, linguist, poet, and friend, which for forty years has devoted his life’s service to the broad field of pathology, in grateful appreciation we the members of the American Phytopathological Society dedicate this testimonial”.

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Smith thought of himself as the “Pasteur” of plant microbi-ology. He was a devoted student of Pasteur’s work and pub-lished material on Pasteur. He wrote several papers on Pasteur and translated with the help of his assistant, Miss Florence Hedges, into English from Emile Duclaux's (1840–1904) “Pasteur: Hostorie d’une Espirit (Pasteur: History and Spirit)” It is believed that evidence of his love of Pasteur the reflected in the beard, which he wore.

Erwin Frink Smith died on 6 April 1927, in his own home in Washington, DC. He was 73 years old and survived by his second wife of 13 years. His interest in science is demonstrated by his desire to have ashes scattered over the waters of the Woods Hole, Masschusetts, from promontory where he loved to sit and think of science. He wrote his own epitaph: “Be then my scroll: lies one beneath this sod to whom all nature voiced the living God”.

His work continues as the pioneering foundation of plant pathology.

Suggested Reading

1. Campbell, C. L. (1983). Erwin Frink Smith — Pioneer Plant Pathologist. Annual Review of Phytopathology 21: 21–27.

2. Jones, L. R. Biographical Memoir of Erwin Frink Smith, 1854–1927. National Academy of Sciences of the United States of America, Biographical Memoirs, Vol. 21.

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Chapter 21

David Bruce (1855–1931): Pioneer of Veterinary Microbiology

“We are all children of one Father. The advance of knowledge in the causation and prevention of disease is not for the benefit of any one country, but for all …”

—David Bruce

Source: https://en.wikipedia.org/wiki/David_Bruce_(microbiologist)(US Public Domain image)

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Introduction

David Bruce was well known for his study of Malta fever (bru-cellosis) and sleeping sickness (trypanosomiasis). He identified Micrococcus (Brucella) melitensis as the etiological agent of brucellosis in cattle; and proved that a Trypanosoma brucei was the agent causing sleeping sickness and tsetse fly (Glossina mor-sitans) was the vector transitions the disease. He was a great physician, and a pioneer of veterinary microbiology.

Life of David Bruce

David Bruce was born on 29 May 1855 in Melbourne, Australia. His father was also called David, and his mother was Jane Hamilton. They originally lived in Edinburgh, Scotland, and migrated to Australia during the gold rush of the early 1850. When David was five years old, they decided to return to Scotland and settled in Stirling. He attended high school till the age of 14 when he decided to work for a warehouse firm in Manchester. He dreamed to be a professional athlete. But his dream was not realized because he had a terrible pneumonia at the age of 17. He decided to go back to school. In 1876, he gained admission to the University of Edinburgh.

David was interested in natural history, particularly animals. He loved birds and he took courses in zoology. But he was per-suaded by a physician to study medicine and he graduated in 1881 with MBCM degree. As he was working as an assistant to a doctor in Reigate, he met Mary Elizabeth Steele, daughter of a previous owner of the practice. Mary was six years older than David, and they were married in 1883. Although they never had any children, they had a very successful marriage. Mary was an indispensable helpmate at home and social occasions for David. And above all, Mary helped David’s scientific research, which made him a great scientific pioneer.

Dr. Bruce was not fond of general practice. In August of 1883, he was commissioned as a surgeon captain in the Royal

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Army Medical Corps. The following year, he was sent to Malta, stationed in the Valetta Hospital. Despite no facility for research at the hospital, Dr. Bruce was impressed by Dr. Robert Koch’s (1843–1910) recent discovery of the tubercle bacillus, he began to do some investigations on a disease called Malta fever, which was quite common among soldiers in the British garrison in Malta. He purchased a microscope and observed an enormous number of micrococci in the spleen of a fatally ill patient. He isolated the micrococci by following the method of Robert Koch and reported his observations in 1887. He continued to study the properties of this organism and proposed the name of M. melitensis. This organism was renamed as Brucella melitensis by Feusier and Meyer in 1920, because that the organisms was not a Micrococcus. The epidemiology of Malta fever was not clear until 1905 when Themistocles Zammit (1864–1935), one of the Maltese members of the Commission for the Investigation of Mediterranean Fever headed by Bruce, found that goat milk was the disseminating vehicle. When goat’s milk was eliminated from the diet of the Malta garrison, the disease disappeared. This disease is now called brucellosis. The names “Malta fever” or other names such as “Mediterranean fever” or “undulant fever” are no longer used.

Bruce left Malta in 1889. Following a stage in Dr. Robert Koch’s laboratory in Germany where David and Mary learned the latest techniques in microscopy, staining and media mak-ing, and so on. Dr. Bruce taught pathology at the Army Medical School at Netley where he introduced the most advanced knowledge and experimental procedures of Louis Pasteur (1822–1895), Joseph Lister (1827–1912), and Robert Koch to his students. In 1894, Bruce was posted to Natal. The Governor of Natal whom he knew while he was in Malta asked him to inves-tigate a disease called nagana — a sleeping illness affecting cattle in the Northern Zululand.

The Bruces went to Ubombo by an oxen wagon. It took them five weeks. It must be an extraordinary experience for them. They lived in a creede hut for two months, and used a veranda

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as a laboratory. Time after time, they examined the specimen and the animals affected with this nagana. Initially, the bacterio-logical examinations of affected oxen proven negative, but extensive microscopic study of blood specimens revealed a motile, vibrating hematozoon. Bruce inoculated the blood from the infected cattle into healthy horses and dogs; they became acutely ill, and the blood swarmed with hematozoa. The hema-tozoon was confirmed to be a trypanosome. The Bruces discov-ered that the mode of transmission of the disease was the tsetse fly. Bruce proved that nagana was identical with the tsetse fly disease described by David Livingstone (1813–1873) in 1858. Bruce stayed at the Zululand bush for almost two years, some-times temporarily recalled to Natal. He published his observa-tions entitled Preliminary Report in 1895 and followed by Further Report in 1897. These were the classic documents which described the hematozoa of nagana, established the tsetse fly, G. morsi-tans, as the vector, and also implicated regional wild animals such as antelope and buffalo as the trypanosomal reservoirs.

In 1899, Bruce was elected as a fellow of the Royal Society of London, the highest scientific honor in England. Bradford and Plimmer published a paper naming the trypanosome as T. brucei.

During the Boer War (1880–1902), Dr. Bruce was a director of a hospital at Ladysmith, and he successfully performed many surgeries. Mary Bruce worked as a nurse in her husband’s oper-ating theater; she received the Royal Red Cross. They returned home in October 1901 when the Boer War was almost over.

In 1903, Bruce was appointed with another mission. He was chosen to head the Royal Society’s Sleeping Sickness Commission to Uganda. As a matter of fact, a similar commission was formed in 1902 on behalf of the Foreign Office and at Patrick Manson’s (1844–1922) urging. But the mission of 1902 had not been success-fully directed, and its two members had returned home. The Bruces reached Entebbe (capital of Uganda) in March 1903. Along with him were Dr. David Nabarro (1874–1958) and a sergeant technician. A young very productive Italian bacteriologist, Dr. Aldo Castellani (1877–1971) who was on the previous mission, remained.

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Sometimes earlier, Patrick Manson had suggested that Filaria perstans was the etiological agent of sleeping sickness. But it was soon proved not to be the case. Castellani observed trypanosomes in the cerebrospinal fluid taken from five victims of sleeping sickness. He had also previously cultured strepto-cocci from cerebrospinal fluid and heart blood from more than 30 patients who died of sleeping sickness. He was well aware of the potential significance of the trypanosomes. He was further unhappy about the arriving of Dr. David Nobarro (1874–1958) who was a little older than him and who was due to replace him. Castellani imparted his observations to Bruce, asked per-mission to temporarily continue working on this disease, and also to publish his finding as the sole author. Dr. Bruce agreed to his request. Thus, Dr. Castellani continued to make observa-tions and demonstrated trypanosomes in 20 additional cases. He also taught Dr. Bruce’s techniques of lumbar puncture and examination of cerebrospinal fluid for trypanosomes.

At first, Dr. Bruce was skeptical that trypanosomes caused human sleeping sickness, because Dr. Joseph Dutton (1874–1905) in 1902 had reported that T. gambiense in the blood of a febrile Englishman in Gambia, and in 1903, Dr. Baker had diag-nosed similar cases of trypanosome fever in Uganda. Their reports showed no connection between these conditions and human sleeping sickness. However, soon after the departure of Castellani, Bruce and Nabarro accumulated an ample evidence that human sleeping sickness was caused by T. gambiense. Dr. Bruce returned to England in August 1903. The progress report by Bruce and Nabarro did acknowledge the discovery made by Castellani. But the following report written by Bruce, Nabarro, and E. D. W. Greig betrayed a changed attitude. The later report encouraged Bruce’s supporters, particularly Ray Lankester (1847–1929) to minimize Castellani’s contribution. This caused a controversy carefully reviewed by Jack N. P. Davies (1915–1998) in his articles published in 1962 and also in 1968.

Dr. Bruce rejoined the Royal Society’s Continuing Commission in Uganda between 1908 and 1910. He was then in charge of

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research concerning the conditions affecting the transmissibility of T. gambiense by Glossina palpalis. They also found that cattle and other animals were potential reservoirs of the parasites. In 1911, he was appointed as director of another Sleeping Sickness Commission in Nyasaland (now Malawi) to study trypanosomia-sis. Between 1912 and 1913, they identified T. rhodesiense as the main regional pathogen and G. morsitans as the vector. They also characterized other trypanosomal species pathogenic to domestic animals. The commission concluded that T. rho-desiense and T. brucei were identical species. But this view did not hold because later studies by German group proved that they were different.

From 1914 to 1919, Bruce was commander of the Royal Army Medical College. He was the chairman of a committee responsible for scientific research of tetanus and trench fever which were quite common during the war time. His administra-tive abilities were fully utilized. He retired in 1919. In his last years of life, he suffered recurrent lung infections, and had to stay in Madeira (in Portugal) in winter. He died on 27 November 1931. His beloved wife, Mary, died four days before him. Mary stayed with him all these years, accompanied him in all adven-turous trips, worked self-effacingly beside him as a technician, microscopist, and draft woman. Bruce requested that her out-standing assistance should always be emphasized in any bio-graphical account of him. Wherever there was a story about a successful man, there was always a great woman behind him. Mary Bruce was a typical example of this kind of woman.

Dr. Bruce received many awards and honors in his life. He was elected member of the Royal Society and the Royal College of Physicians of London. He received honorary doctorates from the universities of Glasgow, Liverpool, Dublin, and Toronto. He was also an honorary member in several foreign academies and societies, and received numerous medals of honor. He was appointed under the order of bath as the companion (CB) in 1905, knighted in 1908, and made Knight Commander (KCB) in 1918.

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Commentary

Some accounts mentioned that Dr. Bruce had an abrupt manner and egotistical personality, and that he often made blunt speeches. But he unselfishly dedicated his great energies and talents to mankind’s health and welfare. He lived in humble conditions and died not wealthy. He cared about future of humanity and had great compassion as expressed in his presi-dential speech “Prevention of Disease” delivered to the British Association meeting at Toronto in 1924. He said: “We are all children of one Father. The advance of knowledge in the causa-tion and prevention of disease is not for the benefit of any one country, but for all — for the lonely African native, deserted by his tribe, dying in the jungle of sleeping sickness, or the Indian or Chinese coolie dying miserably of beriberi, just as much as for citizens of our own towns”. He is a great human being.

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Chapter 22

Sergei N. Winogradsky (1856–1953): Founder of Soil and General Microbiology

Source: https://en.wikipedia.org/wiki/Sergei_Winogradsky(US Public Domain image)

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Introduction

If we look back at the history of microbiology, it is not dif-ficult to see that its establishment as the third greatest divi-sion of biology (the other two are zoology and botany) was surely due to the efforts of several early pioneer microbi-ologists. Among the host of early pioneer microbiologists, four of them are given the greatest credit for the establish-ment of microbiology as a science in its own right. They are Louis Pasteur (1822–1895), Robert Koch (1843–1910), Martinus W. Beijerinck (1851–1931), and Sergei N. Winogradsky (1856–1953). These four especially laid the foundation of microbiology as it is taught today and prac-ticed in the laboratory.

Now, let us look at Winogradsky and see what we can learn from his life story. You may have found it difficult to learn about Winogradsky. His name is not in many encyclope-dias. What is the reason for this surprising omission from the great American and British encyclopedias? The reason is sim-ple. It is because the species of microbial life with which Winogradsky worked were unfamiliar to writers and editors. Both Pasteur and Koch worked on species, which affected the health of human beings and their domestic animals. Pasteur and Koch also were involved in fearful epidemic diseases, such as rabies and cholera, well known to editors and jour-nalists. Beijerinck and Winogradsky worked on soil bacteria. As we noted about Beijerinck, who lived from 1851 to 1931, he was a contemporary of Winogradsky. Both lived at a time when Pasteur and Koch were famous; both were known to Pasteur and well recognized by him as outstanding bacteri-ologists. Winogradsky also took part in one of the great debates of biologists, related to the establishment of micro-biology as a formal sector of the biological sciences. This was the debate on “monomorphism and pleomorphism”. Winogradsky took the side of Koch and Pasteur, about which more will be said.

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Life of Winogradsky

Sergei Nikolaivich Winogradsky was born 1 September 1856 in the city of Kiev, Russia. His father was Nikolai Konstantinovich; he was a banker. His mother, Natalia Viktorovna Skoropadskaia, was from an old Ukrainian German family in the state of Tchernigov. Winogradsky spent his childhood in the city of Kiev. His family was a rich one with a dozen servants; the family owned cattle and horses. As a boy, he liked the out of door — orchard, gardens, and pastures where the cattle were kept. As a boy, he followed the dictates of his family and attended the Eastern Orthodox Church, of which there were many in Kiev (Orthodox belief did not carry over his adult life). He wrote: “I simply forgot everything; all mysticism completely disap-peared as I grew older”. Winogradsky and his brother Alexander entered the second class of the second gymnasium (equivalent middle school) when he was ten years old. His father chose the second gymnasium because it offered both Greek and Latin. At that time, educators believed, as did his father, that classical languages were a sound basis for education. But, young Winogradsky did not enjoy the classes and said they were “not only uninteresting and unpleasant, but depressing, both physi-cally and mentally”. Alexander became a lawyer, and Sergie studied law for two years at the University of Kiev, starting in 1873. He was not interested in the subject and transferred to the division of natural science. He found this instruction boring and ceased attending lectures or doing laboratory work.

Both Alexander and Sergei were interested in music and for a while this became his major interest. He entered the famous Conservatory of Music in the capital city of St. Petersburg, and became a piano student of the great teacher Leshetitski. Although music left an indelible imprint on his personality, he was not satisfied with music alone. His feeling was that music “had aesthetic emotions alone, without any activity of the brain”. When Leshetitski left Vienna, Winogradsky quit music and returned to the natural science faculty of the University of

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St. Petersburg in 1877. Here, he had the opportunity to study various scientific subjects under eminent scientists, such as Dmitri I. Mendeleev (1834–1907), Nikolai A. Menshutkin (1842–1907), Alexander K. Butlerov (1857–1861), Alexander A. Inostrantzev (1843–1920), Beketov A. S. Famintzin (1815–1881), and Karl F. Kesseler (1815–1881). All were excellent scientists; from them he began to appreciate science. He developed a keen interest in analytical chemistry and worked under the guidance of Professor Menshutkin. Although still an undergraduate stu-dent at the University of St. Petersburg, in 1879, he married Zinaida Tichotzkaia. She was his close companion for 60 years.

They had a happy marriage, with four daughters. During this period, he decided to specialize in plant physiology and entered the laboratory of the famous professor Famintzin. In 1881, he graduated with a diploma in science. Recommended by Professor Famintzin, he became a candidate in the University in prepara-tion for a professorship. The great discoveries of Pasteur and the new science of bacteriology were becoming known and also the work of Heinrich Anton de Bary (1831–1888) and Mikhail S. Voronin (1838–1903) in mycology. Winogradsky made a decision to study microorganisms and began his career as a “microbe hunter”. He was also to become known for his great contribu-tion to the development of “General Microbiology”.

There has been almost no available encyclopedic knowl-edge of Winogradsky, unlike Koch and Pasteur, with whom his work relates. The division of his life was outlined into seven periods by another greater microbiologist, Selman A. Waksman (1888–1973). However, we would add one more period to become a total of eight periods. Some were short and intense; we can also see how the changes in a country can affect scien-tific endeavors and how a scientist responded to the vicissitudes of life in turbulent periods. These eight periods follow.

1. The first four years (1881–1885) was called “The First St. Petersburg Period”. There was a turbulent period in the life of Russia, and there were likewise personal difficulties in the

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life of Winogradsky. There is an example of high-level accom-plishment in spite of such problems. Ferdinand Julius Cohn (1828–1898) and Robert Koch started the classification system of bacteriology and worked out several life cycles, as well as developing methods of isolating and culturing bacteria. Winogradsky was involved in the work of several botanists, including H. de Bary, M. S. Voronin, and Julius Oscar Brefeld (1839–1925). He was particularly interested in yeast of wine, Mycoderma vini. He followed closely the work of Koch, but Pasteur had an even greater effect on his life and work. Pasteur’s brilliant work on the biochemistry of bacteria gained Winogradsky’s attention. Pasteur’s work on fermentation, par-ticularly, stimulated Winogradsky to undertake pioneer research in microbiology in which the influence of the environment and nutrition were paramount. His detailed studies included both organic and inorganic nutrients, including the effects of such elemental components as magnesium, rubidium, and potas-sium. He repeated Carl von Nägeli’s (1817–1891) work and, starting with a single cell, growing the yeast in Geissler cham-bers, was able to confirm that rubidium could take the place of potassium, but that claims made for calcium could not be sub-stantiated. Young Winogradsky checked all his results not only by direct microscopic examination, but he also recorded them by a unique system of photomicrography, which he developed. A lesson can be learned from this work. Photomicrography was highly difficult during this period of time, but Winogradsky was convinced that anyone studying microbiology needed to see as well as read about the organisms. He presented a paper on Christmas day, 1883, before the Botanical Section of the University, which was attended by the great botanist Ivan Borodin (1847–1930). Borodin published a summary of the paper and added that he “admired the purity of the cultures exhibited by the speaker in physiologist Famintzin’s labora-tory”. But, typical of much undergraduate work, the paper was never published. (A preliminary abstract only was in “Contributions of the St. Petersburg Society of Natural Sciences

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for 1884”.) Readers need to keep in mind the necessity of pub-lishing any pioneer work — even undergraduate work of high quality. Although undergraduate work, this was truly pioneer microbiology and worthy of publication. Another reason for failure to publish was the turmoil in Russia at the time. Russia was in a revolution headed by Tsar Alexander II (1818–1881), who liberated the serfs. This was succeeded by a reactionary movement led by Tsar Alexander III (1845–1894). Under him, a highly reactionary set of laws was made for the universities; everyone who did not fit was persecuted, including Jews and especially dissenting students and professors. Another serious personal problem had to do with the health of his wife. The cold weather in the north had a detrimental effect upon the health of his wife and, in spite of his outstanding botanical and microbiological success, he was forced to leave. He did, how-ever, achieve the degree of Master of Science in Botany from the University. Upon leaving the University, he settled for a time in the Crimea in the south, which was much more beneficial to his wife’s health. In spite of personal and public problems, he continued his research in a small laboratory in his home. Winogradsky was well aware of the political difficulties in Russia of the time and the impossibility of carrying on satisfac-tory research in the Universities. He, therefore, decided to go to Western Europe where he could work under more favorable circumstances.

2. The second period of his life has been called “The Strassburg Period” and took place from 1885 to 1888. He entered the labo-ratory of the great botanist, Anton de Bary. Microbiology was in its beginning stages, but was the theater of a great debate as to whether there were many and varied species of bacteria (monomorphism) or just a few species, which changed their characteristics frequently (pleomorphism). Koch and Pasteur favored monomorphism, and this was also the side which Winogradsky took. He was especially impressed with the writ-ings of de Bary on the differentiation of bacterial species. He

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later wrote these words on the subject: “Bacteria, in spite of their elementary composition, are, like other organisms, char-acterized by morphological types that can and should be sys-tematically grouped into genera and species”.

The sulfur bacteria were a group, which both sides of the conflict used as illustrations. De Bary was very pleased that Winogradsky became interested and that Winogradsky was making a detailed study of the sulfur bacteria in relation to the problem of classification. He was able to demonstrate that Zopf’s results, at variance with what he found about the species of bacteria, was caused by a lack of pure cultures. Zopf had unknowingly worked with mixtures of cultures.

The investigations of separate bacteria in pure cultures, such as the “thread-forming bacteria”, sulfur and iron bacteria, were very exhausting at times. When Winogradsky was most discouraged he became impatient. He finally got away and took a long walk. While he was out walking the entire solution of the nutrition problems of Beggiatoa and similar bacteria came to him. When this solution was clarified in his mind; in 1887, he soon completed the investigations and presented the results in print. Sometimes, a microbiologist needs to get away from an “insoluble problem”, for a while, and allow his intui-tive forces to work!

During the early period, even, Winogradsky was not afraid to encounter prevailing opinion when he thought those ideas were wrong. For instance, little was known about the nutrition of the sulfur bacteria, and the majority of microbiologists believed that hydrogen sulfide was produced through the action of certain sulfide bacteria upon sulfates. Winogradsky differed and did not resort only to words, but through experi-ments demonstrated that bacteria brought about the disap-pearance, rather than the formation of hydrogen sulfide, which he found was oxidized to sulfuric acid. He, more than anyone else (for instance, C. Engler and E. Hoppe-Seyler [1825–1895]) clarified the role of sulfur as the source of energy for these

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bacteria, enabling them to use the carbon in carbon dioxide, as well as bicarbonates, to synthesize cellular materials.

In the autotrophic bacteria, Winogradsky will always be remembered for this pioneer work with sulfur-oxidizing organisms, iron bacteria, and the nitrifying bacteria. (We will find that nearly a half century later, he returned to his earliest studies to fill in gaps not completed earlier.) He completed his work in 1888 and published the results in a monograph in Leipzig, the same year. This publication aroused the scientific world and even focused attention on Winogradsky as an origi-nal investigator. Unfortunately, in 1887, de Bary became ill and died. For Winogradsky, his loss was decisive and he decided to leave that city. Although he investigated going to a university in Kiev or St. Petersburg in Russia, he had left his family in Germany and finally decided to rejoin them and remain in Western Europe.

3. From 1888 to 1891, Winogradsky carried out work in Zurich, Switzerland, and this has been called “The Zurich Period”. Much work was done at the University of Zurich and a mi crobiologist can learn much from it. Two things are impor-tant to note: (1) Important work can be done in a short length of time, if it is adequately organized and (2) It may be necessary to work, alone, in the face of much opposition. During the two years of this period, he did work that “yielded perfect results”. He solved one of the greatest problems in microbiology, put-ting him in the front ranks of biologists the world over. His prestige from this relatively short period of work on nitrifica-tion has remained unchallenged since that time. He confirmed R. Warrington’s observations that nitrification proceeded in two steps but went much further and established that two dis-tinctly different groups of organisms were responsible. He iso-lated the bacteria in pure culture and worked out their physiology in detail. He demonstrated that process was compa-rable to photosynthesis in the higher plants which utilized oxy-gen. The nitrifying bacteria, he found, were obligate autotrophs;

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they were totally unable to use any sources of energy except that, which were liberated in the particular oxidation processes. Organic matter was found to be completely unnecessary for their metabolism and in fact, injurious to some species. These experiments were published in the Annales Flustitut Pasteur (1890–1891). These were followed by additional morphological descriptions of the organisms involved in other publications.

During the latter part of this Zurich period, Winogradsky was faced with extremely difficult decisions as to the direction of his research in the immediate future and where he should live. It is probable that he could have remained at the University of Zurich. However, his fame among microbiologists was world-wide as a result of these 2 years of intensive work. Through his fellow Russian, Élie Metchnikoff (1845–1916), who was now associated with Pasteur, the great Pasteur offered him a posi-tion in his new institute. At the same time, the Russian prince, Alexander P. Oldenburg (1844–1932), offered him the position of chief of the division of general microbiology of the newly formed Institute of Experimental Medicine in St. Petersburg. The microbiologists need to be aware of the “human side” of science. Winogradsky was greatly impressed with the freedoms of the West and the scientific spirit of the Pasteur Institute. But he realized that if he made this choice, it was probable that he would remain in the West and perhaps never return to his beloved Russia. He was well received, personally, by the great Pasteur, who repeated the offer made previously through Metchnikov. Family affairs also entered into his decision. The estates of his father were being distributed, and he was given the estate at Podol, which he had always loved. He finally decided to return to Russia.

4. From 1891 to 1905, he entered “The Second St. Petersburg Period” of his life. Microbiology, in spite of the worldwide rec-ognition of Winogradsky, was not the major field at the new institute. The chemist W. M. Nencki (1847–1901) was well known and came from a professorship at the University of Bern,

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in Switzerland. Even better known was the physiologist Ivan Petrovich Pavlov (1849–1936), who would later receive the Nobel Prize (1904) for work on the conditioned reflexes of ani-mal. Microbiology was given old and inadequate buildings and never adequate funds. The personality of Winogradsky was an important factor. Whereas, Pavlov was outgoing and attracted students and supporters, Winogradsky was retiring and attracted no collaborators. He had only two assistants during this entire period and attracted few graduate students, without whom it is difficult to get results.

Perhaps even more important, Prince Oldenburg saddled him with administrative burdens, which robbed him of research time and did not bring him new knowledge or ideas. One example was the threat of Asiatic plague in Russia, which Oldenburg greatly feared. He placed Winogradsky in charge of defenses against the plague. He was forced to carry work away from his laboratory in the fort of Alexander I. Successful, but laborious work on the plague was carried out by Winogradsky and his group, but the Minister of Finance, Court Witte, and enemy of Prince Oldenburg, attacked the work of the scientists. Through the personal efforts of Winogradsky these unfair criti-cisms were deflected, but he said that he remembered this “as a typical example of my relations with the ruling clique of the Russian Empire”.

In the laboratory, he was able to demonstrate the experience in the soil of anaerobic bacteria, which were capable of fixing atmosphere nitrogen. It is interesting that he failed to recognize the importance of aerobic bacteria in this process. Eight years later, Beijerinck described Azotobacter chroococcum, which functioned in this capacity. Winogradsky worked on the nature of the nitrogen-fixing bacteria in the cycle of life. He and others made little progress in this work and he dropped it. However, after 40 years we will find that he returned to the problem with success. This is an important lesson for any microbiologist.

Retting was a method used in Russia and many countries in the processing of flax, the plants of which were covered with

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mud or clay and placed in streams to soak. Omitting the fungi, which also are used in flax retting, anaerobic and aerobic form of bacteria were used in the primitive process. Winogradsky and his associates were able to demonstrate retting with a pure culture of the anaerobe. In 1902, he was made director of the institute, which absorbed so much of his time that little was left for research. During this period at the institute, another major drain of his time was the publication of a journal, the Archives of Science. Prince Oldenburg insisted that the journal be put out in both Russian and French. Winogradsky said that the work on this journal was not without “unpleasantness”. The necessity of editing in two languages was an enormous drain in time. Through the hard work of Winogradsky, this soon became the foremost scientific journal in Russia and well known in Europe. As director, he continued these editorial activities. During this period he did a great deal, both as a laboratory scientist and as an administrator to advance the cause of micro-biology in Russia and Europe.

He was a member of the Medical Council of the Empire and also of the Scientific Committee of the Ministry of Agriculture. In 1901, Winogradsky was elected a member of the Moscow Society of Natural Science; in 1902, he was named as a corre-sponding member of the French Academy of Science. He initi-ated contacts, which resulted in the formation of the Rus sian Microbiological Society. His activities and reputation as a micro-biologist were big factors in the establishment of this society, which was formally organized in 1903. He served as president during the first two years. Among the severe personal problems of this period was his own health; in 1898, he first became ill with influenza, and this was followed by nephritis. Finding the northern area unhealthful for himself as well as his wife, he began spending time at his estate in the Ukraine.

In spite of his great successes at the Institute, he began to find that his own interests and those of the organization were not compatible. The Institute moved almost entirely in the direction of human medicine. As in the difficulties over the

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plague, he found himself irritated with the frictions he had with officialdom. A combination of such factors led to his retirement in 1905. However, he did not, completely sever rela-tions for seven years. This took place in 1912.

5. The next period, from 1905 until 1922 was termed “The Period of Transition and Rest”. This was indeed a period of transition, but much less a period of rest! Winogradsky retired from the directorship of the institute and settled on his estates in the Ukraine. He developed large-scale farming to a high degree of excellence — the rearing of horses for the Army and the management of a large forest and nursery. In his “spare time”, he continued his beloved music. Not wanting an audi-ence, his daughters were forced to listen to his piano sessions behind stair wells or screens!

Some of the severest fighting of World War I (WWI; 1914–1918) took place not far from his home. Winogradsky did his part for Russia by supplying horses and farm produce for the Army. Many of his immediate family served in the Armed Forces, his daughters as nurses. In 1917, the March Revolution in Russia resulted in the establishment of a provisional govern-ment. The government under Alexander Kerensky (1881–1970), attempted to go on fighting with the hope that winning bat-tles would strengthen the government against the growing Bolsheviks. But they were not successful, and the Bolsheviks captured the government on 7 November 1917. There was a great strife in Russia at this time. The city of Kiev, for instance, had had twenty different governments. Many of the old wealthy families of Russia were forced to leave the country, Winogradsky and his family among them. Because of health, his wife had to remain in Kiev. He was greatly concerned with getting her out of Russia but was not able to do so. He left on a French warship and went to Switzerland. Here, he found that friends had taken care of his villa and he had only a small amount of money in a local bank. Kiev was now the scene of major battles between the Bolsheviks and the Polish Army. But

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Winogradsky’s concern for his wife caused him to go to Warsaw where his daughter Katherine lived with her husband Blevdzvitch. He found that it was not possible to contact his wife in Kiev and so was forced to return to Switzerland.

To try to help his wife, he joined with other Russian profes-sors in the new country of Jugoslavia, accepting a professorship at the Agricultural Institute of the University of Belgrade. Here, he was able to hire smugglers to spirit his wife in a wagon out of Russia. Although he feared that she would not be able to stand the trip, in fact, she was able to join him, after a very dif-ficult journey.

Unfortunately, he found that the Institute had no labora-tory for him and not even a library where he could prepare lectures. There was one bright spot! In the director’s office he found a complete set of Centralblatt fur Bakteriologe, which was the standard journal for all nonmedical bacteriologists. No one has ever discovered just how this journal got in the office, but it was a “Godsend”. It had been 17 years since he had worked on soil bacteria. Now he was able to discover what had been done during that time.

To his surprise, he found that very little had been done with the autotrophic bacteria. He immediately prepared a paper, “Iron Bacteria as Inorganic Oxidants”, which was accepted and published in the Centralblatt in 1922. He quickly followed this with papers on the nitrifying bacteria, which were published in the French journal Comples Rendus. Beijerinck has earlier opposed his ideas and during Winogradsky’s period of inactivity made a misdirected attack on Winogradsky. He immediately took issue with Beijerinck and eventually demonstrated that he, Winogradsky, was right. He did not fail to meet Beijerinick’s opposition head-on stating “One hesitates to attribute to the eminent Dutch bacteriologist — a faulty technique. I can be rather explained by his original manner of treating the subject in question as virgin ground, paying so little attention to what others have already accomplished. Mr. Beijerinck does not attach any

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importance to the method of control of the purity of the culture established by us”.

Winogradsky and his wife lived modestly in two small rooms and gradually adjusted to their new life in the West. It was not long before Winogradsky was again the center of attention in the microbiological world. The paper he had just written attracted immediate attention and in February of 1922, he heard from the head of the Pasteur Institute in Paris, now Émile Roux (1853–1933). Roux repeated the offer from Pasteur, made many years before, for him to organize a division of agricul-tural bacteriology at the Pasteur Institute. The microbiological world had lost track of Winogradsky, but with his Belgrade writing and acceptance of the position at the Pasteur Institute, honors were “heaped upon him”. The French Academy of Science made him a foreign associate in the Royal Academy, a foreign member. In an unusual move recognizing his impor-tance, the All Union Academy of Sciences of Soviet Russia elected him to honorary membership.

6. Winogradsky now entered the sixth period of his life, termed by Waksman, “the Brie-Comte Period” in which he worked at the Pasteur Institute (1922–1931). However, since he did not like the city, his laboratory was in the nearby location of Brie and he was able to live in the country. After he went to the Institute, other papers followed those on the iron and nitrify-ing bacteria he had written previously. He started his laboratory work at the Pasteur Institute by the direct study of soil. He had a thorough review of all work done in this field and found that, although “hothouse” varieties of soil bacteria had been studied to some extent, there was almost no progress in the study of actual bacterial complexes in the soils themselves. He demon-strated that soil is a very complex medium, which could not be explained solely by the isolation of individual organisms and their growth in pure culture. Soil bacteria were also not very accessible to study by microscope. Microorganisms taken from the soil and grown in isolation were materially modified.

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Winogradsky knew that the complexity of the study of soil bacteria needed to have methods of auxiliary culture, which would supplement study directly of the soil. His concept was that in the soil there was competition for nutritive elements by various cells. He also realized that perhaps a majority of organ-isms in the soil are there in a dormant state. In the soil he affirmed that it was necessary to study both static and dynamic microbiology. He utilized the methods of an American bacteri-ologist, Harold J. Conn (1886–1975) to make his studies. These methods, including making dilute suspensions of soil on glass slides by means of agar or gelatin, and were modified by Winogradsky in the study of nitrogen-fixing bacteria.

Another subject of importance which he studied was the decomposition of cellulose. Previously, he had initiated this work and turned it over to assistants, one of them V. S. Omeliansky. The work had not been done properly and wrong conclusions had been drawn. In 1928, he reopened the question and began a detailed study of the problem. He particularly was interested in the role of aerobic bacteria at this time. In some instances, he took issue with Selman Waksman (1888–1973).

7. Winogradsky did not stop with cellulose studies; these were followed in the 1931–1933 period with detailed studies of the agents of the nitrification process. In his earlier studies, he had written that only two groups of organisms were involved in the formation of nitrite and nitrate in nature by the oxidation pro-cess. In these later studies, he found that the soil carried a much more abundant flora of nitrifying bacterial organisms capable of varied adaptations. Under purely laboratory conditions, he found that the bacteria form types, which could not be consid-ered normal. He emphasized the importance of studying newly isolated organisms from the soil. He further emphasized that general microbiology must be distinguished from ecological microbiology. The former deals with pure cultures and labora-tory refinements, the latter with spontaneous cultures and organisms in natural habitats.

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The Pasteur Institute period was one of intensive work over a long period. A microbiologist can learn a great deal from his life during this period. The work was done between the ages of 66 and 84 — ages when most men have retired. It is highly interesting that his mind was as active during this period as it was in his younger days. He lived long enough to examine his earlier work critically, and he was able to reexamine the nature of the soil processes. As late as 1924, Beijerinck was adhering to his early ideas that bacteria in the soil were not directly active and that they only produced hormone like substances, which made the plants capable of the fixation processes. Winogradsky, conversely, argued that the bacteria were active agents. He demonstrated that the fixation process centered in the nodule, even when the latter was severed from the rootlets. This work greatly aided the development of commercial cultures of nitri-fying bacteria by Ira L. Baldwin (1895–1999) at the University of Wisconsin in the United States.

His last memoir at the Pasteur Institute took up the critical analysis of the synthesis of ammonia in the soil and in water basins. In this, he emphasized the major role of microorganisms in this process. These last years were also difficult ones for him due to the severe illness of his wife, who died in September of 1939. This was a severe blow to him, but, nevertheless, at the age of 85 he was still a vigorous man, with a mind as alert as one of a youth. Even in his advanced years he was as eager as when he was younger to tackle the most difficult problems. During his long life time, he made great advances on three fields: general microbiology, soil microbiology, and general physiology.

8. The last period of Winogradsky’s life was from 1941 until 1953. For the second time in his life, at Brie, near Paris, and as a part of the Pasteur Institute, he once again became isolated from the rest of the world. France was overrun by Germany in 1941 at the onset of WWII (1939–1945). The hardships of this war were heavy on him. The invasion of Poland cut off contact

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with his daughter Katherine, who was then a widow living with a young son, Wladishaw. During the German occupation, the home of Winogradsky was commandeered by the Nazis several times, with both ordinary and very bad experiences. At one time, the explosion of an ammunition dump not far away broke out every open window in his house. Throughout the German occupation, Waksman, who attempted to keep in con-tact with Winogradsky, said that “a black curtain descended upon Winogradsky”. His daughter Helen was able to make some contact with him and to communicate with Waksman. She sometimes went by bicycle to Brie from the Pasteur Institute in Paris to see her father and to note the status of his work and health. She wrote that it was necessary for him to remain in his home during the occupation to make sure that everything was not stolen.

After the occupation came the evacuation, which was still worse. During this period, Winogradsky was forced to stop all laboratory work because of the shortage of all supplies. Although they welcomed the “American boys”, laboratory work was no longer possible. Winogradsky, characteristically, when he had to desert his beloved laboratory, started working on a book he had planned earlier: Half a Century of Microbiological Research. He assembled all his papers and wrote revisions, abstractions, and commentaries. He also wrote a preface to the book. The director of the Institute, M. Jacques Trefouel (1897–1977) promised to do his best to publish the book. On his 90th birthday, Winogradsky turned the manu-script over to his daughter Helen. Helen worked at the Pasteur Institute but went home to see her father as often as she could. Selman Waksman’s son Byron was stationed with American troops in France and visited Winogradsky and his daughter Helen. Waksman himself visited with Winogradsky on the lat-ter’s 91st birthday and three months before the 94th birthday. He found that Winogradsky had deteriorated physically but not mentally. He was working hard to translate all his papers into French and to write his book. The Pasteur Institute was

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interested but without funds to help or to publish it. This was no easy task at the end of WWII. The publishing house of Masson in Paris was ready, but did not have adequate paper. Paper could be obtained only from America. Paper could not be purchased on the open market anywhere, but only obtained from the stock allotted to a printer. Waksman tried to find paper without success; also, it was found that some cash was necessary. He took the problem to Dr. A. N. Richards, president of the National Academy of Sciences in the United States. Dr. Richards felt that much was owed scientifically to both Winogradsky and to the Pasteur Institute. The National Institutes of Health set aside some of its own paper, and Dr. Richards was able to induce the Rockefeller Foundation to supply some funds and Rutgers for other funds (which were given to Waksman, but which he made available for the book). A cash gift from the American Academy of Science also enabled the publisher to consider the cash as a donation and thus reduce the price of the book. Winogradsky had received offers from the Russian Academy of Sciences in Moscow to publish the book, but he preferred to have it published in France. The book appeared in 1950. Waksman was able to visit him, with three of his daughters at Brie right after its publication. Waksman’s visit in 1950 was to be his last. Sergei Winogradsky died on 24 February 1953, at the age of 97.

Commentary

Born and educated in Russia, almost all of his scientific work was done outside of his native land by necessity. He carried out the most highly productive initial scientific activities done in both Germany and Switzerland. He had two highly active peri-ods of scientific work interspersed with a long period of largely administrative and editorial duties. But he never lost the spirit of science and an understanding of the internationalism of sci-ence. It is worth noting also, the interest of microbiologists in the work and interest of each other and the generosity of

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microbiologists in establishing another scientist, such as Winogradsky, in a new position when problems in a country presented obstacles.

Winogradsky approached microbiology in a very different way. His research metholodogy was chiefly by enrichment cul-ture techniques. However, Koch and Pasteur preferred in pure culture systems, which became the mainstream of microbiology we practice today. If Winogradsky’s approach of enrichment culture became the mainstream, microbiology would then have taken totally different direction. We should appreciate what enrichment approach can do in our understanding of microbi-ology, particularly the role of microbes in the natural habitats.

Suggested Reading

1. Waksman, S. A. (1953). Sergei N. Winogradsky, His Life and Work; the Story of a Great Bacteriologist. Rutgers University Press, New Brunswick, New Jersey.

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Chapter 23

Kitasato Shibasaburo (1853–1931): First to Isolate Clostridium tetani

and a Pioneer of Serology

“Improvement of human’s public health is an important mission in my life”.

—Kitasato Shibasaburo

Source: https://en.wikipedia.org/wiki/Kitasato_Shibasabur%C5%8D(US Public Domain image)

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Introduction

Kitasato Shibasaburo was well known for his isolation of Clostridium tetani and its antitoxin (antiserum). Antiserums were capable of curing many diseases, including diphtheria and tetanus. Antiserum therapy was a development of a new disci-pline in immunology, called serology at that time. Antiserum led to the development of vaccine for diseases. Nowadays, injection of the tetanus vaccine is always a mandatory practice of baby deliveries or any other complicated surgeries. The teta-nus vaccine injection is still required for people who are work-ing in a veterinary environment such as barns. He also assisted Emil von Behring (1854–1917) in exercising serological therapy to fight against diphtheria, which was a serious contagious ill-ness that claimed many thousands of lives in Europe and America (possibly other continents too). He also participated in important research including the fighting against bubonic plagues and helped Robert Koch (1843–1910) in battling of tuberculosis. He was one of the trailblazing pioneers of preven-tive medicine. In addition, his numerous administrative posi-tions allowed him to build many medical, microbiological, and/or hygienic establishments. His work significantly contributed to the public health of humankind, particularly in Japan.

Biography

Kitasato was born on 29 January 1853, in a humble family in a mountain village called Ogunigo Village in Kiushu, Higo Province (present day called Oguni Town, Kumamoto Prefecture, Kyushu) in southern Japan. As a bright student in high school, he was admitted to the Kumamoto Medical School and entered Governmental Medical School in Tokyo, which is the forerunner of the School of Medicine of Tokyo Imperial University (now called the National Tokyo University). He completed his medical studies in 1883.

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Kitasato was chosen by the Japanese government to do research under Robert Koch. He worked at Dr. Koch’s labora-tory in the University of Berlin from 1885 to 1891. An everlast-ing association was established between Kitasato and Koch. In 1889, he was the first person to isolate the pure culture of C. tetani, the causative agent of tetanus. He also noticed that the attenuated C. tetani would induce immunity in experimental animals. For instance, he found the rabbit that had been injected with the antiserum survived the infection of the live cultures of C. tetani. This is immunity. But in Kitasato’s lan-guage, he called it antitoxin.

About the same time as Kitasato worked on tetanus, Emil von Behring worked on diphtheria under Robert Koch also. With the help of Kitasato, von Behring also demonstrated that a similar phenomenon occurred with the diphtheria bacillus. But, we were very sure that the antitoxin term was first used by Kitasato because antitoxin or the toxin against toxin concept was a common term in oriental traditional medicine. Kitasato published his work on tetanus with coauthor von Behring on December 4, 1890, with the title Ueber das Zustandekommen der Diphtherie-Immunität und der Tetanus-Immunitätbei Thieren (“The Production of Immunity in Animals to Diphtheria and Tetanus”). A week later, von Behring published a second paper without including Kitasato. The second paper was deal-ing with immunity against diphtheria and outlining various ways in which it could be achieved. These reports announced that injections of toxin from diphtheria or tetanus bacilli led animals to produce substances capable of neutralizing the dis-ease poison in their blood. The name “antitoxin” was first coined by Kitasato. Without Kitasato’s initial finding, the anti-serum might never have come into being. It was conceivable that Kitasato at least suggested to, if not personally helped, von Behring to apply the antitoxin (or antiserum) harvested from animals and injected into the test animals that were infected with diphtheria to see the effect of antiserum. However, von Behring did not include Kitasato as a coauthor in

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his paper. Therefore, Kitasato and von Behring demonstrated the value of antitoxin in preventing disease by producing a pas-sive immunity in an animal that received graded injections of blood serum from another animal infected with the disease. Kitasato also used antitoxin for prevention of anthrax caused by Bacillus anthracis. This was the foundation of serotherapy or called a serological therapy. This was the major finding in explaining the workings of the immune system and in the developing of vaccines for diseases. Kitasato deserved just as much credit as von Behring did. But apparently was neglected by the Nobel Prize Committee. In 1901, von Behring was awarded the first Nobel Prize for Physiology and Medicine alone; Kitasato was left out. Many historians still consider that to be one of the most regretful events in the history of the Nobel Prize.

Kitasato spent seven years helping Robert Koch in the study of another terrible disease, “tuberculosis”. In 1891, Kitasato returned to Japan. Before returning to Japan, the German gov-ernment conferred upon him the title of honorary Professor of Berlin in 1891. This was the first honor given to a foreign scien-tist by the German government.

In Japan, Kitasato devoted to establishing a laboratory to study bacteriology. He was greatly assisted by the famous Japanese educator Yukichi Fukuzawa (1835–1901) who helped bring Japan into modern society by the so called “Meiji Restoration”. This was the first modern scientific institute (labo-ratory) ever established in Japan. This laboratory was under the supervision of the Hygiene Society of Japan initially. With enlargement and new services, this laboratory had been strengthened by new buildings. And the laboratory to study bacteriology was under the supervision of the Minister of Interior of Japan in 1899. With continuous growth and expan-sion of the laboratory’s functions, this laboratory became an institution and was eventually incorporated into the Imperial Institute of Infectious Disease in 1905 when a new modern facility was built at Shirokane-Daimachi. Both the laboratory

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(institution) and the Imperial Institute of Infectious Disease were under the directorship of Kitasato. Kitasato and the new institute staff members contributed greatly to the eradication of many infectious diseases in Japan and helped establish the modern public health management for the Japanese society. In the meantime, Kitasato continued his studies of microbiology and infectious disease. Numerous research papers were pub-lished from his institute under his direction. For example, one of his early students, August von Wassermann (1866–1925), who, along with Kitasato, demonstrated how dead cultures could be used in vaccination. In 1898, Kitasato and his another student, Kiyoshi Shiga (1871–1957), discovered Shigella dysen-teriae, the causative agent for bacillary dysentery (shigellosis). Dysentery is an infection of the lower intestinal tract producing pain, fever, and severe diarrhea. Shigellosis was the most com-municable enteric disease at that time. He also studied the mode of action in tuberculosis. This period was a productive period during Kitasato’s scientific career.

In 1894, an outbreak of the bubonic plague (a contagious disease spread by the flea of contaminated rats) occurred in the British occupied Hong Kong. Kitasato was invited by the British authority to investigate the case. With the help of a large team of Japanese scientists, Kitasato did isolate a bacterium that he concluded was the cause of the disease. However, it was found that Kitasato reports were vague and somewhat contradictory. A thorough analysis of the morphology of the organism discov-ered by Kitasato was investigated by other microbiologists, who claimed that Kitasato's samples were likely contaminated. These reports, which led Kitasato’s identification of the cause of bubonic plague, became dubious.

Alexandre Yersin (1863–1943), a Swiss born French bacteri-ologist, was a director of Pasteur Institute located in Saigon (now called Ho Chi Minh city), also arrived in Hong-Kong, three days after Kitasato to investigate the bubonic plague. Yersin conducted a vigorous investigation and reported to the British authorities his discovery of a bacterium that appeared to have

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the characteristics of a disease causing bacterium only one week after his arrival in Hong Kong. He proved that it caused a plague like disease when injected into rats. He successfully cul-tured the bacteria in pure culture and proved that the disease was transmitted from one rat to another. The bacteria he iso-lated, now called Yersinia pestis, was used to a make an anti-plague vaccine, and in 1896 the antiserum was prepared against the disease causing pathogen provided the world’s first cure of a patient with plague.

It seemed that credit of isolation of the bubonic pathogen was given to Alexandre Yersin. Most scientific communities credit Yersin as sole discoverer. The pathogen was named Y. pestis in honor of Yersin. However, others had different view-points. Bibel and Chen (1976) in their investigation published in the Bacteriological Review, 40(3), 633–651, stated that, “there is little doubt that Kitasato did isolate, study, and reasonably characterize the plague bacillus and should not be denied this credit”. Kitasato had been called a plague fighter. The fact that Kitasato was not rewarded the Nobel Prize in 1901, could be part of the reason for Kitasato’ popularity. Many scientists wished that Kitasato’s work on the bubonic pathogen would be credited with some kind of honor. So, the scientific community was happy to see that Kitasato did truly isolate the pathogen so he could be awarded some kind of honor to compensate his disappointment. However, history did not work that way.

In 1910, an outbreak of bubonic plague occurred in Manchuria, China. This turned out to be the beginning of the large pneumonic plague pandemic of Manchuria and Mongolia, which ultimately claimed about 60,000 victims. China sent a St. Mary’s Hospital School trained physician and scientist Dr. Lien-Teh Wu (1879–1960) to eradicate the disease. Dr. Wu quickly eradicated the disease by burning all the corpses. Dr. Wu also organized an international conference by inviting experts and scientists from international countries, including United States of America, Great Britain, France, Germany, Japan, Italy, Austria-Hungary, Netherlands, Russia, and Mexico to discuss

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measures to prevent future such pandemic. Japan, at the time, was an Asian giant in military power and occupied the majority of territory of Manchuria. Japanese military authority did not like to see China be able to organize an international confer-ence in order to cease the episode of bubonic plague pandemic. Kitasato met a strong opposition of Japanese military authori-ties’ to get into Manchuria, China, to help stopping the spread of the disease. However, he ignored the Japanese military authorities’ opposition, did come to China to attend the International Plague Conference that was held in Mukden (Shenyang) on 11 April 1911, by the invitation of the conference organizer and chairman of the conference, Dr. Lien-Teh Wu. Kitasato was elected as the honorable president of the confer-ence and actively chaired three sessions of discussion on how to establish preventive measures for fighting against plagues.

In 1914, the Japanese government incorporated the Institute of Infectious Disease into the Tokyo Imperial University and transferred the administration to the Minister of Education, Japan. This change displeased Kitasato because he was not con-sulted before this action. In protest, he resigned his position as a director.

In order to continue his research work, Kitasato established a private laboratory known as Kitasato Institute, which was located at the corner of Shirokane Sankocho. This institute grew gradually into a large establishment. Later, the Kitasato Institute developed into Kitasato University, where he became the President for the rest of his life. In 1910, Kitasato was invited by Yukichi Fukuzawa to establish a medical college in Keio Gijuku University and was the first dean of Medicine College. With his leadership, this medical college became one of the most prominent centers of medical education in Japan today. In September 1921, Kitasato together with several Japanese medical scientists, found the Sekisen Ken-onki Corporation with the intention of manufacturing the most reli-able clinical thermometer possible.

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Kitasato served many important organizations. He was the first president of the Japan Medical Association. In 1917, Kitasato was appointed a member of the House of Peers by the Japanese Emperor. He also obtained approval of the Diet (Japanese Congress) for a bill to organize the Medical Practitioners Association of Japan and served as the first President. He did everything possible to improve Japanese pub-lic health. For this work, he was elevated to the peerage (noble ranks) and was made a baron (danshaku) in 1924.

Kitasato established many institutions in Japan, which were dedicated to the study of microbiology and public health. He strongly believed that his mission in life was to devote himself to the scientific investigations and advancement of public health. He traveled broadly and obtained many honors. He was a member of the Imperial Academy of Japan, foreign member of the Royal Society of London, Ehrenmitglied der Preussischen, Akademie der Wissenchaften of Berlin, and Associe’ Etranger de l’Academie de Medecine de France. He was awarded the Harben Gold Medal of the Royal Sanitary Institute of London.

On 13 June 1931, he died of intracranial hemorrhage at the age of 75 at his home in Azabu, Tokyo. His grave is at the Aoyama Cemetery in Tokyo.

Commentary

It should be noted that in 1890, von Behring and Kitasato were the first to discover antibody molecules in the blood serum of immunized animals, and to demonstrate that these antibodies could neutralize diphtheria toxin and tetanus toxin. They also demonstrated the specificity of antibodies; tetanus toxin can-not neutralize diphtheria toxin and vice versa.

Sachi Sri Kantha (1953–) stated that the possible reasons why Kitasato lost the first Nobel Prize for medicine to von Behring as presented are as follows: (1) The Nobel selection committee lit-erally interpreted Alfred Nobel’s will to award the prize to “the person who has made the most important discovery”. (2) In the

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late 19th century, diphtheria was a serious contagious disease, which claimed many thousands of lives in Europe and America; and von Behring’s solely authored paper on diphtheria antitoxin clinched the award for him. (3) The merit of tetanus antitoxin to humans, which was the focal point of the 1890 paper on tetanus jointly authored by von Behring and Kitasato, was not recog-nized at the time of the award in 1890; it became apparent only during the World War I (1914–1918).

Kitasato lived until 1931, and hence he was eligible for Nobel recognition for almost another decade after the resump-tion of the Nobel awards in 1919, but his 1890 contribution again failed to receive the Nobel merit. Between 1919 and 1930, two more unshared prizes were awarded to research on serology and immunology. Jules Bordet (1870–1961), who fol-lowed Kitasato in the field of serology, was a recipient of the 1919 Nobel Prize in Medicine “for his discoveries in regard to immunity”, Karl Landsteiner (1868–1943) “for his discovery of the human blood group” in 1900. The reason that Kitasato’s 1890 discovery was becoming “too old for recognition” in the 1920s is difficult to accept, since Landsteiner was recognized by the Nobel committee three decades after the appearance of his original publication. Historian James R. Bartholomew, com-mented that the exclusion of Kitasato from 1901 Nobel Prize in Medicine has not yet been satisfactory explained.

Whatever the reasons for why Kitasato’s contribution failed to receive Nobel Prize committee’s favor consideration, Kitasato’s original serological discovery was as significant as any other Nobel Prize receivers’ contribution, and Kitasato certainly deserved to be a Nobel Prize Laureate.

Kitasato was not only a hardworking scientist, but also was a humanist. He was extremely devoted in his affections toward his parents and his mentor. He respected profoundly to his mentor, Robert Koch. When Robert Koch died, Kitasato built a private shrine in the inner court of the Institute in remem-brance of his master. Each year, Kitasato commemorated the day of Koch’s death with a ceremony in his memory. Kitasato is

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a humanity fighter also. Living in the Japanese military emperi-orist dominant social atmosphere, he remained calmly of not agreeing to the Japanese military expansion policy. Surely, he considered the improvement of human public health as his most important mission in life. Indeed, he lived to that high standard. He was unselfishly devoted to the improvement of the public health not only to his native land, but also to the welfare of general human beings. He left a big legacy to humanity. Kitasato was certainly a human treasure whom we all are proud to venerate.

Suggested Readings

1. Bibel, D. J. and T. H. Chen (1976). Diagnosis of plague: an analysis of the Yersin–Kitasato controversy. Bacteriology Reviews 40(3): 633–651.

2. Cunningham, A. (1992). The Laboratory Revolution in Medicine. Pp. 209–244. Cambridge University Press, Cambridge.

3. Howard-Jones, N. (1973). Was Shibasburo Kitasato the co-discoverer of the plague bacillus? Perspectives in Biology in Biology and Medicine 16: 292–307.

4. Solomon, T. (1997). Hong Kong, 1894: the role of James A. Lowson in the controversial discovery of the plague bacillus. Lancet 350 (9070): 59–62.

5. Jerne, N. K. (1985). The generative grammar of the immune system (Nobel Lecture). Angewandte Chemie International Edition 24: 810–826.

6. Kantha, S. S. (1991). A centennial review; the 1890 tetanus antitoxin paper of von Behring and Kitasato and the related developments. The Keio Journal of Medicine 40(1): 35–39.

7. Bartholomew, J.R. (1989). The Formation of Science in Japan: Building a Research Tradition, Yale University Press, New Haven and London.

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Chapter 24

Theobald Smith (1859–1934): The Captain of American Microbe Hunters

“The joy of research must be found in doing since every other harvest is uncertain”.

“Research is fundamentally a state of mind involving continued reexamination of the doctrines and axioms upon which current thought and action are based. It is, therefore, critical of existing practices”.

“Great discoveries, which give a new direction to currents of thought and research, are not, as a rule, gained by accumulation of vast quantities of figures and statistics. — The great discoveries are due to the eruption of genius into a closely related field, and the transfer the precious knowledge there found to his own domain”.

—Theobald Smith

Source: https://en.wikipedia.org/wiki/Theobald_Smith(US Public Domain image)

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Introduction

Theobald Smith was one of the most distinguished early American microbiologists and comparative pathologists in the world. Devoted to the study of the causes of many infectious animal diseases and a search for their controls, he made many contributions to bacteriology, immunology, and parasitology. Smith elucidated the causative agent and mode of transmission of Texas cattle fever and had other significant impacts on vet-erinary medicine and public health. Unlike many scientists of that day, Smith received no training in Europe; he was a typical, American born and educated scientist.


Theobald Smith was the son of German immigrants. His father, Philipp Schmitt, and mother, Theresa Kexel, came to America in 1854 and settled in Albany, New York. Philipp Schmitt was a tailor and earned very little money; the family worked extremely hard and lived a very simple life. Smith was born on 31 July 1859. He spoke fluent German at home and began his education at a German-speaking private school. This strong German background equipped him later to be able to read the original papers of Robert Koch (1843–1910) and Paul Ehrlich (1854–1915), whose work greatly influenced Smith’s later research.

In 1877, Smith entered Cornell University. During his college years, he was very industrious, versatile, and interested in scientific subjects. He established durable friendships with the physiologist and comparative anatomist Dr. Burt G. Wilder (1841–1925) and the microscopist, Dr. Simon H. Gage (1851–1944). In 1881, he gradu-ated with a PhD with honors and enrolled at the Albany Medical College, where he received his MD in 1883. His dissertation was entitled Relations Between Cell-activity in Health and Disease.

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Achievement

Early work

Although Smith wanted to go to Europe to further his studies in medicine, funding was unavailable. With the help of profes-sor Gage, he obtained a position as an assistant to Dr. Daniel Elmer Salmon (1850–1914), chief of the veterinary division of the US Department of Agriculture in December 1883. In 1884, only six months later, he became an inspector in the newly established Bureau of Animal Industry to work with Salmon against diseases that threatened the development of the agri-cultural industry in the United States. These diseases were bovine pleuropneumonia, glanders, infectious diseases of swine, and Texas cattle fever.

This was a challenging job since Salmon was not a specialist in microbiology and could not offer much help. Smith taught himself how to use Robert Koch’s culture-plate methods. He found that hog cholera and multiplex swine plague (Schweinesouche) were two different diseases caused by differ-ent bacteria. These findings were reported in two monographs, Hog Cholera: Its History, Nature and Treatment (1889) and Special Report on the Cause and Prevention of Swine Plague (1891), and were included in the annual reports of the Bureau of Agricultural Industry from 1885 to 1895. He also isolated a bacillus that he thought was responsible for hog cholera and demonstrated that immunity was developed by pigeons inocu-lated with heat-killed cultures of this bacterium. This work opened a new approach to bacterial vaccine production. The genus Salmonella (named after Salmon) was created in 1900 by Lignières. This organism originally named Bacillus choleraesuis by Smith in 1894. Some 20 years later after Smith’s discovery of Salmonella choleraesuis, the etiological agent of hog cholera proved to be viral, and S. choleraesuis was shown to be only a secondary invader.

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Texas cattle fever

At the same time, Smith was engaged in another important study against Texas cattle fever. This disease was at the heart of the “Missouri War”. The war also ranged between the North and the South and across the entire United States. At that time, the less expensive land was in the South, where cattle were raised, but a good part of the population needing the meat was in the North. The South bought “feeder calves” from the northern states to fatten up and ship back as steers. However, the calves from the North usually got sick and died within a few days or weeks after entering the southern states. This issue was compounded by the more serious problem of northern cattle becoming sick and dying after southern stock were driven through the North. As a result of this strife, mutual distrust developed and the “Missouri War” began. Farmers everywhere were encountering the disastrous losses of cattle. New York City went into a panic when cattle shipped there from the west died by the hundreds in their cattle cars. The distinguished doctors of the New York City Metropolitan Board of Health went to work to try to find the microbe but had no luck whatsoever. No one could stop the cattle from dying.

Salmon had charged Smith and his colleague Cooper Curtice (1856–1939) to find the cause of the Texas cattle fever. The first year they were given the spleens and livers of four cattle that died of Texas cattle fever. Smith quickly realized that, even though he found a veritable menagerie of microbes in the tis-sues, the samples smelled and looked bad because they were spoiled. He also brushed aside other claims that the responsible germs were associated with manure. Smith then decided that he needed to go into the field in order to conduct the appropri-ate work. In June of 1887, seven thin but healthy cattle were brought in from farms in North Carolina. These cattle were loaded with several thousand ticks of many sizes, some so small a magnifying glass was needed to see them.

Frederick L. Kilborne (1858–1934), superintendent of the experimental farm out in the field, had heard farmers say, “No

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ticks, no disease. You always find it when there are ticks”. Smith discussed this theory with other doctors and veterinarians. It was laughed at; whoever heard of a tick causing a disease? Smith proceeded to start four field experiments and put four southern cattle, loaded with ticks of all sizes, with six healthy northern cattle, free of ticks and disease into Field No. 1. This was the first experiment, one that most scientists at the time would have thought stupid. Smith and Kilborne then set out the second experiment, which involved removing every tick from the three remaining southern cattle. When they were sure that all the ticks had been removed, they put these three south-ern cattle with healthy northern cattle. Smith said, “You can be sure they won’t get any ticks from these southern animals!” But, they would use the same drinking water, and there would be the effect of saliva and manure to observe. They also observed the cattle every day and picked off any fresh ticks that hatched. In another field (third experiment), they put northern cattle inoculated with ticks from the southern cattle. In yet another field (fourth experiment), they put the healthy north-ern cattle out on the grass, inoculated with ticks imported from the southern states where the disease was present.

The results were significant. No northern cattle put in with the tick-free southern cattle died. However, all northern cattle placed with tick-infested southern cattle and/or in fields infested with the ticks soon contracted the fever and died.

During the hot months of July, August, and September, Smith studied the biology of ticks. Through these efforts, Smith characterized the life cycle of the tick and discovered that it took about 20 days, for the “seed ticks” to mature and start another generation. In the meantime, Smith looked for the responsible organism. He decided that the place to look was not only the spleens and livers, but also the blood. He knew that the cattle with the disease became anemic and decided that the organism must be attacking the blood. Sure enough, he found some strange pear-shaped spaces in the red corpus-cles of the sick cows. At first, he thought they were only “holes” but he discovered they were living organisms.

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The pear-shaped organism was a protozoan of the order Sporozoa, the suborder Haemosporidia, of the subclass Pyroplasma. The protozoan was initially named Pyrosoma bigeminum and later identified as Babesia bigemina with the disease termed “babesiosis”. It was also called “red water dis-ease” from the color of the urine of the animals. The effective way to eradicate the disease was by killing the ticks. Cattle were dipped into a soluble chemical, which killed the ticks. Theobald Smith’s work effectively eradicated the continent-wide scourge (Texas fever) and the cause of the “Missouri War”.

The elucidation of the etiology of Texas cattle fever was a milestone of medicine. For the first time it was proved that an anthropod was the vector of a microbial disease. All together Smith and his crew spent only four hot summers finding the answer, perhaps it was a record of achievement considering the magnitude of the problem. Smith’s report, titled Investigation into the Nature, Causation, and Prevention of Texas or Southern Cattle Fever was published in 1893 (written with F. L. Kilborne. Government Printing Office, 1893. U. S. Bureau of Animal Industry Bulletin, No.1. Reprinted in Medical Classics, 1:372–597, 1936–1937). Initially, farmers resisted the mandatory dipping and without the solid knowledge of the cause, the government would not have had the strength to force cattlemen to dip every head of cattle driven or shipped North.

The work on Texas cattle fever significantly enhanced Smith’s reputation. He was promoted to chief of the division of animal pathology of the Bureau of Animal Industry in 1891. Even though his successes were not appreciated by Salmon, Smith was not deterred from pursuing other interests. He was intrigued with the novel protozoan disease of turkeys (black-head disease) and tuberculosis in cattle.

Sanitary bacteriology

Smith also developed an interest in the bacteriology of water supplies. Beginning in 1885, he observed the total bacterial

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counts of samples from the Potomac River and later he system-atically examined the microflora of the Hudson River for fecal bacteria. Smith cited his findings on the fecal contamination in rivers in a report he submitted in 1893 in which he advocated a quantitative assay of Bacillus coli communis (coliforms) as an index of fecal pollution. He also introduced the use of fermen-tation tubes (Durham tube) as a method to detect the presence of gas-producing coliforms. His techniques and detailed studies were later incorporated into recommendations from the com-mittee of American Bacteriologists (now called the American Society for Microbiology) appointed in 1895. This recommenda-tion was also later incorporated into the first edition of the Standard Methods of Water Analysis of the American Public Health Association published in 1905.

Smith also recognized the importance of improving sanita-tion of sewage, milk, and water supplies. Although he never liked publicity, he constantly addressed these issues with farm-ers and sanitarians and regularly attended the meetings of the Biological Society of Washington, DC. Although still working at the Bureau of Animal Industry, Smith became professor of bac-teriology in the Medical Department of Columbian University (now known as George Washington University) in 1886, a posi-tion he retained until 1895.

Antitoxin development

In 1895, Smith resigned from the Bureau of Animal Industry and accepted the appointment of director of an antitoxin labo-ratory for the Massachusetts State Board of Health and profes-sor of zoology at Harvard University. In six months after he took the job, he produced potent diphtheria antitoxin that was in great need by the country. In 1896, through a cooperative arrangement with Dr. H. P. Walcott, chairman of the State Board of Health, and Dr. Charles Eliot (1834–1926), President of Harvard University, Smith became the newly founded George F. Fabyan, chair of comparative pathology (endowed by a wealthy

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Bostonian). Smith retained his directorship of the antitoxin laboratory. In the same year, Smith made a trip to Europe and met the world’s leading microbiologists Robert Koch and Paul Ehrlich. He discussed with them his preliminary observations on the two varieties of mammalian tuberculosis bacilli, one for human, and the other for bovine. To that time, it had been assumed that the human strains and the bovine strains of tuberculosis bacilli were identical. This work was expanded and published in another classic report titled A Comparative Study of Bovine Tubercule Bacilli and of Human Bacilli From Sputum (Journal of Experimental Medicine, 3: 451–511, 1898). Koch confirmed his observations three years, later, but did not acknowledge Smith’s priority until 1908. Koch differed with Smith and thought the bovine bacilli did not play any signifi-cant role in human tuberculosis.

In 1903, Smith made another trip to Europe to inspect vac-cine producing facilities before enlarging his anti-diphtheria antitoxin producing laboratory. He adopted Paul Ehrlich’s standardized antitoxic unit and introduced many improve-ments in titration methods. He also was the first to observe the hypersensitivity response of the body to foreign body or drug (anaphylaxis) later referred to as “Theobald Smith’s phenome-non” by R. Otto and Paul Ehrlich. This phenomenon was also called Richet’s phenomenon, named after Charles Robert Richet (1850–1935).

Other accomplishments

Smith also studied the agglutination relationship between cer-tain members of typhoid and para-typhoid groups of Salmonella and observed the nonidentity of the flagellar and somatic agglutinations. These findings formed the basis for the subse-quent development of the Kaufmann-White scheme for the serological identification of the Salmonella species.

Malaria was also endemic in parts of Massachusetts. Smith postulated that malaria was mosquito borne, but the State

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Board of Health was reluctant to support his hypothesis. He did not pursue the subject due to lack of support and because Massachusetts was at an unfavorable latitude to study malaria. Instead, he studied other parasitic diseases, such as murine sar-cosporidiosis, coccidiosis of mouse kidney and rabbit intestine, and amebiasis in the pig. He continued his studies with turkey blackhead disease, which he started to study while he was in Washington, DC. Overall, Smith made significant contributions to the understanding of numerous animal infectious diseases.

Smith’s scientific contributions became well known and the function of his laboratory was expanded. He was consulted by bacteriologists, veterinarians, and sanitarians. However, he had to defend his animal experimentation during an antivivisec-tionist campaign. Around 1909, he was persuaded by Eliot to give eight Lowell lectures to popularize the importance of com-parative pathology. In 1912, he was an exchange professor at the University of Berlin, staying for six months. His inaugural address was titled Parasitismus und Krankheit (Parasitism and Disease) (Deutsche medizinische Wochenschrift, Berlin, 38:276–279, 1912). His convictions about the importance of compara-tive pathology were permeated by the concept of host-parasite interrelationships, which dominated his whole life’s research.

In 1914, he was invited by Dr. Simon Flexner (1863–1946), director of the Rockefeller Institute for Medical Research, to head the newly endowed department of animal pathology to be established at Princeton, New Jersey. This was an acknowl-edgment of his leadership in comparative pathology and his reputation as a productive investigator. At this time, he was 55 years old and in poor health. In June 1915, Smith was honored by his distinguished colleagues worldwide at a testimonial din-ner. He was now chiefly responsible, in consultation with Rockefeller Institute representatives, for the plans of the new division and design of their laboratories and animal quarters.

Since 1917, due to Smith’s efforts, anti-pneumococcus and anti-meningococcus sera were manufactured in horses. In 1920, Smith and Harry W. Graybill (1875–1938) showed that the

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protozoan responsible for turkey blackhead disease was trans-mitted by embryonated eggs of a small nematode Heterakis papillosa, which was a parasite in the caeca of infected birds. Smith, when still in Washington DC, named the infective agent Amoeba meleagridis. It was recognized by E. E. Tyzzer to be a unique flagellate Histomonas meleagridis. Also, in collaboration with R. B. Little, he studied the bacterium Brucella abortus, which caused bovine contagious abortion. Together they published 12 papers and a monograph in this field. In 1926, they described the protection induced by vaccinating heifers with a living low viru-lent strain of B. abortus.

Smith also did other significant research. For example, he studied Vibrio (Campylobacter) fetus that caused cattle abor-tion, and Bacillus (Actinobacillus) actinoides that caused calve bronchopneumonia. He also studied the protective effect of colostrum from newborn calves (Journal of Experimental Medicine 36:181–198, 1922).

In 1929, Smith resigned the directorship of the division of animal physiology at the Rockefeller Institute. He was suc-ceeded by Dr. Carl Ten Broeck (1885–1966), his longtime associ-ate. However, he remained active as a member emeritus of the Rockefeller Institute. In 1933, he succeeded William Henry Welch (1850–1934) as the president of the board of scientific directors (he had been vice-president since 1924). However, his health deteriorated rapidly and Smith was admitted to the hos-pital in November 1934. He was diagnosed with intestinal can-cer and passed away on 1 December 1934.

Honors and Awards

Smith received many honors, mostly rather late in his life. He was given a dozen honorary doctorates from renowned American and European universities. He delivered the Herter, Mellon, Pasteur, Gross, De Lamar, Milbank Memorial, Welch, and Thayer Lectures between 1917 and 1933. He also gave an excellent series of five Vanuxem lectures at Princeton in 1933.

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The lectures were later published in a book as Parasitism and Disease (Princeton, New Jersey, 1934), which described his scien-tific philosophy and endeavor. Smith was a member of many scientific societies and was elected president of the Society of American Bacteriologists (1903), The National Tuberculosis Association (1926), and the Triennial Congress of Physicians and Surgeons (1928). He was a foreign member of the Royal Society of London and 11 other societies. He received the Mary Kingsley, Flattery, Kober, Trudeau, Holland, Sedwick, Manson, and Copley medals.

Personal Calibers

Smith married Lilian Hillyer Egleston on 17 May 1888, and, by all accounts, had a wonderful marriage. Lilian was very intelli-gent with high principles and social graces; she helped to fur-ther Smith’s work and life’s aim. They had two daughters and a son.

Smith’s contributions derived from a farsighted, dispassion-ately critical intelligence, extremely concerned for detail, tech-nical inventiveness, and most of all his unsparing industrious hard work and persistence. His relaxations were very modest; when tired, he sought solace in reading, piano playing, or cal-culus. Besides preparing manuscripts, he enjoyed boating and working around the house. He was extremely concerned about his staff. He insisted on his staff taking their vacations. He was also reticent. As Hans Zinsser (1878–1940) said, “There were about him an unobtrusive pride, a reserve tinged with austerity, which did not invite easy intimacy”. As a leader, he was very fair in judgment and very conscientious. He would strongly defend great ideas even at a high cost in a hostile environment. Behind his back, some of his friends and staffs called him “Three Balls Smith!”

He was never happy under Salmon, perhaps because of Salmon’s professional jealousy, or other unknown reasons. Smith was also involved with the unfair treatment of Dr. Alice

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C. Evans (1881–1975). When Evans discovered that B. abortus contamination in milk was responsible for human undulant fever and called for mandatory pasteurization of milk before its sale in the market, Smith strongly opposed the idea that brucel-lae in cow’s milk might cause human diseases. Smith published reports in 1915 and 1919 that disagreed with the premise that brucellae in milk might be hazardous to health. He was not cor-rect in this case, because drinking unpasteurized milk was indeed the major way of contracting brucellosis. In 1925, when Smith learned that Evans was to become a member of the National Research Councils’ Committee of Infection Abortion, he declined to become chairman of the committee.

Commentary

In Smith’s life, he was serious about his teaching, research, and other duties. His industrious working morale intensified his determined pursuit of new knowledge on the etiology, pathol-ogy, and prevention of communicable diseases. Smith was a true leader of the microbiological research in this country; therefore, it is fitting to name him the “pioneer of veterinary medicine”. Many of his discriminating contemporaries consid-ered him comparable to Pasteur and Koch. Although Smith was an MD, his work was largely on veterinary science. No doubt, if Smith’s work had been on human diseases, he would have been awarded a Nobel Prize. The contribution of veterinary medicine is often ahead of human medicine; however, the reward for the contribution for veterinarian scientists is hardly comparable to those of medical scientists.

Smith’s portrait in oil was placed at the entrance to the Theobald Smith Building of the Rockefeller Institute, now known as the Rockefeller University, where he served so faith-fully in various capacities for 33 years. He should be cherished today as a great builder of the prosperity of this country both in wealth and wisdom.

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Suggested Readings

1. de Kruif, P. (1926). Theobald Smith: Tick and Texas fever. Pp. 234–251. Microbe Hunters, Harcourt Brace and Co., New York.

2. Conklin, E. G. (1935). Theobald Smith. Proceedings of the American Philosophical Society 75: 333–335.

3. Zinsser, H. (1936). Biographical Memoir of Theobald Smith, 1859-1934. Biographical Memoirs, National Academy of Sciences 17(12): 261–303.

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Chapter 25

Alexandre Yersin (1863–1943): Pioneer Plague Fighter

Source: https://en.wikipedia.org/wiki/Alexandre_Yersin(US Public Domain image)

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Alexandre Emile John Yersin was well known for his isolation of the causative agent of bubonic plague which played an extremely important role in the episode of human misery in our history. This disease killed millions in Europe in medieval time and had also struck the French as recently as 1720. He was also a very interesting person though he is a relatively unknown contributor to the conquest of infectious diseases. His life career was full of excitements and challenges which most scien-tists do not experience.

Alexandre was born on 23 September 1863 in a village called Lavaux in Aubonne, near Lausanne, Switzerland, where his fam-ily lived in a gun powder factory. His father was the director of the factory and a self-taught insect expert. His father died three weeks before Alexandre’s birth. So Alexandre was raised by his mother. As a young boy, he developed an interest in science. He accidentally discovered, in an attic trunk of their house, a micro-scope and some dissecting instruments of his father. That gave him a great excitement and enhanced his interest in medicine. A family friend, a physician, also influenced Alexandre, so he decided to pursue a career of medicine.

Yersin received his secondary education in Lausanne before entering the Academy there; he then attended the University of Marburg, and then moved to the Faculty of Medicine in Paris. During his school year in Paris, he accidently cut himself while performing an autopsy on a patient who had died of rabies. Yersin immediately got in contact with Dr. Emile Roux (1853–1933) a coworker of Louis Pasteur (1822–1895). Dr. Roux gave him an injection of a new therapeutic serum, and his life was saved. This incidence brought him in close contact with Dr. Roux. They established a close life-long relationship. Roux recognized his talent and hired him as his assistant in 1888, while he was in his fourth year of medical training. Together, they conducted research on rabies. Yersin also spent some time in Dr. Robert Koch’s (1843–1910) laboratory to learn more about medicine, particularly the tubercle bacillus. Upon return-ing to Paris, Yersin began his own research with Roux on the

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toxic properties of the diphtheria bacillus. As Yersin became increasingly recognized for his work at the Pasteur Institute, he became bored with research. He wanted a change, but did not know what to do. He did not want to practice medicine because he thought it was wrong for physicians to make a living from the sickness of others.

In 1889, Yersin abruptly left the Pasteur Institute and was employed as a physician on a ship sailing from Marseilles to Saigon (now called Ho Chi Minh City) and Manila. At that time, most of Indochina was under French control and largely unex-plored. After arriving in Saigon, Yersin fell in love with the land of Saigon and its people. He returned to Paris and left again for Indochina. He had made three dangerous expeditions into inte-rior of the Indochina. He explored the crocodile-infested rivers in a dugout canoe, and made expeditions on elephants into jungles where tigers roared. He adapted Vietnam as his home country, and made many contributions to this region of the world.

Yersin was concerned with the health of the people and studied the diseases that affected them. In 1894, Yersin was appointed a medical officer in the French colonial service and conducted research on the bubonic plague epidemic that was sweeping through China. In the summer of 1894, he went to Hong Kong where an outbreak of plague had occurred. Yersin wanted to study the cause of the plague and find a way for preventing the spread of the disease to Vietnam. However, things were not easy for him in Hong Kong. Kitasato Shibasaburo (1853–1931), the famous colleague of Robert Koch, had arrived in Hong Kong three days before Yersin did. Kitasato brought a large team of scientists from Japan. The British authorities in Hong Kong had given the Japanese team access to patients and full use of the laboratory facilities there. Yersin did not speak English, so the communication with the British authorities was a problem. Although Yersin and Kitasato could speak German to each other, the Japanese team treated him coolly. This was probably due to the reflection of the intense rivalry between Pasteur and Koch.

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Yersin was reduced to setting up a laboratory in a bamboo shack, and he had no access to get the samples from the plague victims. In order to get samples for investigation, he had to bribe the British sailors responsible for disposing of the bodies of plague victims. Nevertheless, Yersin did vigorous investiga-tions, and only a week after his arrival in Hong Kong, he reported to the British authorities his discovery of a bacterium which appeared to have the characteristics of a disease causing bacterium, which was invariably present in the bodies of the victims of the plague. He cultured the bacteria and proved that it caused a plague like disease when injected into rats. He also later proved that the disease was transmitted from one rat to another. He clearly proved that the bacterium was the cause of bubonic plague according to the principle of Koch’s Postulates. The bacterium he isolated (now known as Yersinia pestis) was used to make an antiplague vaccine, and in 1896, antiserum prepared against the organism provided the world’s first cure of a patient with plague.

Kitasato worked with blood from the heart of victims of the plague. He also announced that he isolated the bacterium that caused the plague. However, it was subsequently proved that the organism he isolated was a laboratory contaminant. Nevertheless, Kitasato was named codiscover of the plague bacillus because of his great prestige, or because of his major contributions on diphtheria antitoxin and tetanus studies. He had not been properly rewarded by the scientific community before.

In 1904, Yersin was called back to Paris where he continued his research at the Pasteur Institute, of which Roux had become director. With the help of the colleagues, Drs. Albert Calmette (1863–1933) and Amédée Borrel (1867–1936), he discovered that certain animals could be immunized against the plague through the injection of dead Y. pestis. He quickly returned to Nha Trang, Vietnam, where he established a branch of Pasteur Institute and directed the research work himself. Although it was only a modest laboratory, he perfected the production of

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antiplague serum, which made it possible to reduce the death rate of plague from 90 to about 7 percent.

By obtaining the assistance of Paul Doumer (1857–1932), governor general of Indochina of France, Yersin founded a medical school in Hanoi. He directed the study and research there for many years. Through his lead, Indochina was able to control several epidemics that beset the country, especially malaria. In recognition of Yersin’s medical achievement, the French government appointed him honorary director of the famous Pasteur Institute.

In addition to his contributions to the medical field, Yersin was interested in the welfare of the people. He introduced improved breeds of cattle which were a main part of rural farming. He conducted research in agronomy such as improving the cultivation of rubber and quinine-producing trees. His interest in the cultivation of grains and soil conditions led him to initiate a series of ecological studies. He also reflected on the natural history of Indochina, and became fascinated with the flora and fauna of the land. Yersin had become deeply con-cerned over the needs of the sick and the poor and fought hard against the exploitation of the lower classes.

During the early days in Indochina, Yersin discovered the high plateau of Langbiang, and founded a small colonial village there. That village was later developed into the city of Dalat. The municipal authorities of Dalat established a Lycee Yersin (a public high school) in 1935 in memory of Yersin’s con-tribution to the city. Yersin devoted his whole life to his adopted country, and died on 1 March 1943 in Nha Trang, Annam, Vietnam.

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Chapter 26

Albert Leon Charles Calmette (1863–1933): Antituberculousis

and BCG Vaccination

“I hope that it will be given to me to work until my eyes are closing to the light and that I will fall asleep my soul in peace, conscious of having done that I have been able”.

— Albert Calmette

Source: https://en.wikipedia.org/wiki/Albert_Calmette(US Public Domain image)

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Early Life of Albert Calmette

Albert Leon Charles Calmette was the first to develop an antivenom serum. His work revolutionized the treatment of snakebite of men and domestic animals. He was also well known for his work on the prevention of tuberculosis (TB) by inventing the Bacillus Calmette Guerin (BCG) vaccine against this terrible disease.

Albert Calmette was born on 12 July 1863 in Nice, France. Young Albert wished to be a sailor, but at the age of 13, he contracted a serious bout of typhoid, which prevented him from entering the Naval School. Perhaps, because he had expe-rienced such a terrible malady, he decided to be a medical doctor to help fighting against diseases. He managed to enter the Naval Medical Corps in 1881 at Brest, France. In 1883, he was assigned to serve as an assistant doctor in a squadron in the China Sea as Vietnam was under the control of France at that time. During this time, he met Dr. Patrick Manson (1844–1922) at the new School of Medicine in Hong Kong. Dr. Manson pio-neered the study of filaria in the mosquito Culex pipiens by demonstrating the various stages in the life history of the para-sitic worm responsible for elephantoid disease. Calmette stud-ied under the direction of Dr. Manson on the insect transmission of the disease. On his return to France in 1885, he completed his MD degree at the University of Paris by presenting a doctoral thesis on filariasis. Through these experiences, Dr. Calmette was deeply moved with the human sufferings of malaria, dysentery, cholera, and other diseases; he developed a strong desire to study further the pathology of these tropical diseases.

In 1886, he was posted to Gabon in the French Congo as a colonial surgeon. He continued his interest in tropical pathol-ogy and publishing papers which included a description of sleeping sickness, which involved the histological examination of the central nervous system. On returning to France in 1887, he married Emilie de la Salle. Shortly afterward, he and his wife left the islands of St. Pierre and Miquelon in the Atlantic Ocean,

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south of Newfoundland. In these islands, Calmette was involved with the medical and surgical care of 6000 fishermen working in the waters of the Grand Banks. At this time, he was inspired by the articles of the new journal Annales des l’Institut Pasteur, and he taught himself the technique of culturing bacteria. Calmette isolated in pure culture the causative rouge de morue bacillus of cod. The rouge de morue occurred in the fresh salted cod which were packed in barrels. The cod developed red spots which rendered the fish unsaleable. He also demonstrated that the problem was due to the contamination of microbial spores from the crude salt imported from Cadiz, and the problem could be solved by adding small amounts of sodium sulfite to the preserving solution which prevents the growth of the spores.

In 1888, the newly established Institut Pasteur in Paris was inaugurated. When the Colonial Health Service was formed in 1890, Calmette decided to transfer to the new corps. On his return to Paris from the Atlantic Ocean, Calmette obtained a leave to attend a course of 25 lectures in General Microbiology which was initiated by Emile Roux (1853–1933) and Élie Metchnikoff (1845–1916) at the Pasteur Institute in Paris. He also had the chance to meet the great pioneer microbiologist, Louis Pasteur (1822–1895), who was deeply impressed by Calmette’s work in the Islands of the Atlantic Ocean. Calmette had become deeply interested in microbiology. In 1891, Louis Pasteur recommended him to the Colonial Health Service to become the director of the research laboratory in Saigon, Vietnam (Birkhaug, 1934). The research laboratory was later called Saigon Pasteur Institute, which was officially opened on 1 April 1891.

The Saigon Years (1891–1893)

In Saigon (now called Ho Chi Minh City), Calmette demonstrated his exceptional ability to organize. He quickly adapted

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laboratory procedures for the tropical environment and worked on problems related to the local population. First, he improved the production of the antirabies serum. Treatment of rabies required inoculation with an attenuated virus present in the spinal cord of rabid rabbits. Rabbits were scarce and harbored the rabies virus. Calmette introduced the practice of storing the excised spinal cords in glycerol. This allowed the virus to retain its virulence for a long period of time. This practice became widely adapted. Second, he observed that the incidence of small-pox in the local population was high. Calmette experimented with local cattle and discovered that an excellent yield of the small pox virus could be obtained by inoculating the skin of young water buffalo with vaccinia virus. Over the next two years, half a million people in Indochina were vaccinated with smallpox vaccine (Gelinas, 1973). He was also interested in local customs and became curious as to the properties of “Chinese yeast” used to ferment rice without the necessity of malting. As a result of this, he isolated the Chinese yeast later named Amylomyces rouxi. This discovery later lead to an industrial exploitation of the yeast. He also investigated diseases such as dysentery and cholera which were common in those areas at that time.

He did research on snake venoms, because venomous snakes such as Naja tripudians and Cobra capel were common in the tropical area where he was located. Calmette studied the physi-ology of envenoming and the effectiveness of antiseptics and diverse substances as a means of treatment. Potassium perman-ganate was considered the best neutralizing substance for snake venom following the work of de Lacerda in Rio de Janeiro, but it was not effective to neutralize cobra venom once it had passed into the tissues from the site of inoculation. Calmette found that 1% gold chloride was effective in prevent-ing intoxication. But the most effective treatment lay in the antiserum. In 1888–1889, Emile Roux and Alexandre Yersin (1863–1943) found that the symptoms of diphtheria were due to soluble exotoxins produced by diphtheria bacillus. In 1890, Emil von Behring (1854–1917) and Kitasato Shibasaburo

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(1856–1931) reported that rabbits and mice could be immu-nized against diphtheria and tetanus. Calmette in Saigon attempted to produce an immunity to cobra toxin in animals. Various methods such as successive inoculation of cobra venom either heat treated or mixed with gold chloride followed by progressively increasing doses of virulent venom had been tried. After two years of extensive research, he failed to pro-duce the effective antiserum. Exhausted with the extensive work, he contracted dysentery and finally was called back to France. However, the Saigon Institute served as a model later for the Institute of Bacteriology in that part of the world. A bronze bust of Dr. Calmette was erected on the Grounds of the Pasteur Institute in Saigon in his memory. That bronze bust is still there today.

Studies on Antivenomous Serum and Antiplague Serum

In Paris, Calmette joined Dr. Roux’s laboratory and continued looking for ways of raising anticobra serum for therapeutic use. In 1894, he finally achieved his goal by developing an antivenom serum, which provided animals with an immunity to snake bite. Calmette also studied the preparation of the ven-oms from the European viper, the Australian tiger snake Hoplocephalus curtus (Notechis scutatus) and black snake Pseudechis porphyriacus. Calmette’s protocol was later shown to induce hyperimmunization with the formation of specific immunoglobulin G (IgG) capable of in vivo protection against snakebites.

In June of 1894, the bubonic plague occurred in Hong Kong. Alexandre Yersin was sent to Saigon to study the disease. Yersin isolated the causative agent of the plague (later called Yersinia pestis) and sent the culture to Dr. Roux’s laboratory. Calmette and his associate Amédéé Borrel (1867–1936) immediately explored ways of producing an antiplague serum. Calmette and Borrel joined by Yersin on his return from Hong Kong, found that if a culture of Y. pestis was preheated to 58°C, it would

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slowly produce antisera after being injected into guinea pigs and rabbits. They also found that a horse after it was injected with a culture of live bacilli produced a serum, which after 6 months, still protected experimental animals from a virulent dose of toxin. This method provided antisera of therapeutic value when tested by Yersin during an outbreak in China in 1896. At the same time, Yersin was commissioned to establish another Pasteur Institute in Nha Trang, North of Saigon.

In 1895, another Pasteur Institute in Lille, France, had been established at the request of the people of northern France. Calmette was recommended by Pasteur to become head of that institute. Reluctantly, Albert and his wife Emilie moved to Lille in 1895. He held the position from 1895 to 1919.

In Lille, he continued his study of snake venoms. At the end of 1895, horse antivenom serum was used to successfully treat human snakebite. In 1896, he was invited by the Royal Colleges of Physicians and of Surgeons in London, where he demon-strated lucidly the potential of antivenomous serotherapy to treat snakebite. His demonstration of anticobra serotherapy revolutionized the treatment of snake bites in men and domes-tic animals.

Research Concerns Public Health

In 1897, Calmette was joined by Dr. Camille Guerin (1872–1961) a veterinary bacteriologist. Together, they tackled the problem of the deterioration of the potency of the antivariolic vaccine when the vaccine was continuously transferred from calf to calf. The two workers showed this to be a dilution effect due to an accumulation of saprophytic microbes derived from the skin of a calf. They discovered that the rabbit was sensitive to the vaccinia virus and developed an alternative inoculation for calf and rabbit. The method has proved to be of great service and was later helpful in the development of a lymph vaccine.

Calmette was also interested in the problems of public health. He worked vigorously to improve social hygiene. He

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initiated the first course in bacteriology and experimental therapy in Lille in 1896. He studied ankylostomiasis, an anemia resulting from an infestation of hookworm, which raged in the coal miners of northern France. With the help of Maurice Breton, Calmette made a thorough study of the biology of the parasite, and devised prophylactic measures to prevent infec-tion. The prevention involved education in social hygiene and required assistance from the infected miners. Calmette was also concerned with the problem of purification of water polluted with city and industrial wastes. He visited the water purification plant in England, and with the help of Rolants, Boullanger, Constant and Massol, he created at La Madeleine near Lille the first French station of biological purification of sewage based on the technique of using beds of bacteria.

Development of BCG

However, the most important contribution while he was in Lille was that he and his associate made an intensive study of tuber-culosis (TB). He spent more than 30 years of sustained, dedicated efforts in finding a means of eradicating TB which was rampant at that time. For example, in a city of 220,000 inhabitants, 6000 of the poor miners had TB. Of the 6000 miners, from 1000 to 1200 died each year, and the infant mortality rate reached 43%. Calmette set up an antitubercular dispensary, the first in conti-nental Europe, for the early diagnosis and treatment of TB for those who were not able to go to a sanitorium and to provide advice on how to minimize the familial spread of the disease. The dispensary also provided practical help for the sick and their families. It provided them with food, a laundry, and chaise lounges to be used during the respiratory exercises.

Robert Koch was a pioneer in the studies of TB. He identi-fied Mycobacterium tuberculosis to be the causative agent of the disease. In 1882, he attempted by using the tuberculins isolated from the culture of M. tuberculosis as a vaccine. Unfortunately, the tuberculin vaccination caused a disaster,

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because it produced a cell mediated delayed type of hypersen-sitivity. In 1903, von Behring proposed that pulmonary TB could be acquired from the intestinal tract when young. Calmette and Guerin followed the proposal of von Behring and demon-strated that young calves which ingested at two different times a dose of less virulent human strain of tubercle bacilli, devel-oped an immunity against a later dose of the virulent bovine strain. Calmette found that heat-modified bacilli might be effective as a vaccine. Along this line, he discovered that bile was useful in attenuating the virulence of tubercle bacilli. He persevered in his research for a living, nontubercle forming, antigenic strain of bacilli for use as a vaccine. At this critical time in 1915, his work was interrupted by the occupancy of Lille by the German army (World War I). During this period, his life was very difficult, for example, his wife and 24 other women were held by the German soldier outside Hanover for 4 months as hostages. Calmette could not do experiments. He spent some time writing the manuscript of his work which was published later in 1920. The title of his monograph was L’Infection Bacillaire et la Tuberculose, which was a milestone contribution to TB research.

In 1917, he was appointed as assistant director of the famous Pasteur Institute in Paris. However, he was not able to leave the northern France because Lille was still occupied by German. Finally, he managed to go to Paris. In Paris, Calmette and Guerin were joined by L. Negre and A. Boguet to continue his work on a vaccine for TB. Experiment after experiment, finally a vaccine called BCG (Bacillus Calmette Guerin) was developed. The vac-cine contains a strain of Koch’s bovine bacillus that had been attenuated by repeated culture on a nutritive medium mixed with bile. It is effective for both animals and man, because the tubercle bacilli of the bovine strain and human types are geneti-cally related, and they can provide cross immunity. The BCG vac-cine is a safe vaccine because the live bacilli it contains remain in low virulence. The first clinical trials were made successfully in 1921. Dr. Roux was so impressed with the potential of BCG

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vaccine as a prophylactic measure against TB that he convinced the administrative council of the Institute to build a five story building designed by Calmette for the research of TB. In 1931, when the building was finished, over 100,000 new-born babies were being treated with BCG vaccine annually in France alone. Calmette’s work was convinced and BCG was used worldwide.

However, there were incidences of failures of the vaccine. In 1929, 72 infants at Lubeck, Germany, died of TB after receiving locally-prepared BCG that had been locally and accidentally con-taminated with a human tubercle bacillus strain (the kiel strain). The incidence depressed Calmette profoundly, and caused a public stir. BCG was originally administered orally, shortly after birth, but later following the improvement of Negre and Bretey, it was administered by injection. The authors can still remember vividly the injection of BCG during their childhood. It caused local lesions, but they were always minor. BCG is still adminis-trated to children in many countries of the world.

Calmette also found that immune calves inoculated with BCG became sensitive to tuberculin. He discovered that the per-sistence of sensitivity to tuberculin was correlated with the presence of living bacilli within the lymphatic system, and a positive tuberculin reaction (later called Calmette reaction) still is a skin test indicating the cellular immune response of infec-tive or vaccinated persons and cattle.

Final Years

In the last year of his life, Calmette went back to study the use of cobra venom in the treatment of cancer in mice. Calmette was also a good teacher. He trained many good students such as L. Negre and A. Boquet, whose works obtained the attention of the entire world. Calmette received many honors in his life. He was awarded with the LLD from Cambridge University. He was an honorary fellow of the Royal Society of London in 1921. He was a member of the Royal Society of Medicine, the Academy of Sciences (section of medicine and surgery), the Academy of

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Medicine, and the Academy of Colonial Sciences. In his career, he published several books and many research papers.

Calmette died after a few days of illness on 29 October 1933 in Paris in the midst of his scientific work. Two years before his death, Calmette reflected on his life in a letter to his children, he said: “I hope that it will be given to me to work until my eyes are closing to the light and that I will fall asleep my soul in peace, conscious of having done that I have been able”. His wishes had been realized. He was a cheerful man. He was always unselfish and full of kindness and faith. His enthusiasm often brought him into controversies, but he was never unkind even irritated greatly by his unintelligent opponents. He was a true Pastorian. He lived simply, and never asked for any mate-rial advantages. He was an industrious and methodical worker, and also a rapid thinker.

Recent Findings of BCG

BCG was later found to have anticancer effects and has been shown to be active in treating leukemia, malignant melanoma, and a variety of others cancers including superficial bladder tumor. Numerous literatures are available which were not to be reviewed here.

However, TB is still a big human malady. The incidence of TB 7.5 million case per year in 1995, and is expected to increase 11.9 million in 2005. One-fifth to one-third of humanity is infected with a mycobacterial entity, the annual death rate due to TB being about 3 million. The care-fatality rate is estimated at 55% in untreated people and 15% in the chemotherapy treated patients. Even BCG vaccination had been administrated more than 50 years ago why TB is still prevalent?

Ferru criticized the Calmette’s experimental design to set up to prove the efficacy of BCG and the misuse of the statistics exploiting the result. Smith thought that the protective efficacy of the vaccine was wildly exaggerated. BCG vaccine was found to provoke in the guinea pig a general lymphatic diseases that

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was claimed to heal spontaneously. BCG itself was shown to multiply in various organs of the guinea pig during months of inoculation, which Calmette did not examine. 1988, Tardieu reported that BCG induced TB meningitis in health children vac-cinated with BCG. The contamination of BCG with M. tuberculosis did occur with Lubeck incidence. But this was later served as an explanation for all cases. Lottealso reported that complications occurred by intradermal BCG vaccination. The adverse effects were reported to be 10-folds superior to those commonly reported. Fine and Rodrigues (1990) reported that the excess of TB cases observed during the first 5 years following BCG vacci-nation of children in the Southern India trial. BCG was not effective in fighting against TB in monkey. Maes (1996) states that BCG might in fact favor subsequent mycobacterial infections.

BCG is an attenuated mycobacterial strain and capable of inducing cellular and humoral immune responses against non-peptidic antigen, lipoarabinomannan which are present in the cell wall of M. tuberculosis. These immune responses protect the patients effectively only if the production of interleukine-2 and g-interferon is not impaired. However, Constant and colleagues (1995) indicated that BCG strain hither analyzed were poor pro-ducers of phosphorylated nonpeptic antigens. BCG does not mobilize neutrophils and is not responsive to granulocyte colony-stimulatory factors (G-CSF); neither depletion of neutro-phils nor administration of G-CSF affects the delayed type hyper-sensitivity elicited with BCG in amice. BCG had also been shown to increase lipopolysaccharide (LPS)-induced lung injury inde-pendently of neutrophils. BCG did stimulate dendritic cells, which are potent antigen-presenting cells capable of initiating primary immune response, resulting in the secretion of a-tissue necrotizing factor (a-TNF) 50-fold above basal level. BCG acti-vates antigen-specific CD4+ cytotoxic cells that kill antigen-pulsed presenting cells, resulting in suppression of interleukin-2 production. In some cases, IgM output against proteinic antigens is detected but IgG antibodies against proteinic antigens are

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rarely produced. During the BCG infection, it is found that inter-leukin-6 is specifically produced in a significant amount which suppresses T-cell responses. Therefore, BCG seems to elicit a response essentially to it surface antigens lipoarabinomannan and glycolipid. In some vaccinated patients, a substantial immu-nosuppresion is likely to be the result of the infection, which explains the increase in frequency of parasitic mycobacterial infections among vaccinated infants.

For a good immunoprotection, the formation of IgG-type antibodies and of cellular immunity against mycobacterial pep-tidic and proteinic antigens is essential. This is achieved by only a small proportion of the people vaccinated with BCG. These facts explain that BCG may indeed be beneficial to some groups living in some environment, but not to most of others. There are serious shortcomings of the vaccine, which need to be prudently addressed.

It is interesting to note that the process of BCG vaccine to be imposed worldwide in 1950 by medical and other organiza-tions was rather a political manipulation. The immunological advances of the last few decades seem to be neglected in an effort to develop an effective immunoprotection against TB. TB is certainly a complicated disease. Many factors including envi-ronmental and socioeconomical factors are involved. It will be wise to invest more studies to the total immunoprotection of mycobacterial diseases, which are health treat today. There are many reasons for the reemerge of TB in modern world which are not the scope of this review.

BCG is not used in the United States because the controversy mentioned above. Even now generally considered safe and effective, the incidence of TB is not sufficient to warrant wide-spread use. The vaccine’s use is limited to high-risk individuals such as health-care workers regularly exposed to TB patients, or to personnel specializing in pulmonary and chest diseases. World Health Organization (WHO), however, does recommend immunization at earlier ages in underdeveloped countries to combat serious contagious diseases.

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Commentary

In light of these shortcomings with BCG, it would be most depressed to Calmette if he would still be alive. However, these findings are not necessary to discredit the contributions of Calmette to medical/biological sciences. Imposing BCG as a worldwide vaccine against TB was done long after the death of Calmette. Calmette would probably be disappointed to the way it was handled. Calmette’s work such as the development of prevention of rouge de morue, isolation of Amylomyces rouxi, social hygiene, establishment of the biological purification sta-tion of sewage, and development of antivenom serum and antiplague serum are all incredible pioneering contributions to science and it applications.

As Cornelis B. van Niel (1897–1985) always pointed out to his students to appreciate the frequently slow and difficult pro-cess of moving from clearly erroneous to more correct, but never immutable conclusion. BCG is an example for this case.

As one of the authors always thought “we are indeed children of these tireless devoted pioneer microbiologists and/or physicians”. Because of their work, we are able to sur-vive or to be born healthy. Since the average life span was only 40 before the turning of the 20th century, it was not an easy job to just remain alive. The work of Calmette gives us a lot of meditation as to what we shall do in our life time which was so preciously made possible through pioneers such as Calmette?

Suggested Reading

1. Aarestrup, F., S. Goncalves and E. Sarno (1995). The effect of thalidomide on BCG-induced granulomas in mice. Brazilian Journal of Medical and Biological Research 28: 1069–1076.

2. Akaza, H., S. Hinotsu, Y. Aso, et al. (1995). Bacillus Calmette-Guerin treatment of existing papillary bladder cancer and carcinoma in situ of the bladder. Cancer 75: 552–559.

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3. Behring, E. A. and S. Kitasato (1890). Uber das Zustandekommen der Diphtherie-Immunitat und der Tetanus, Immunitat bei Thieren. Deutsch Medizinisch Wochenschrift 49: 1130–1145.

4. Bernard, N. (1962a). Extracts from La Vie et L’oeuvre Albert Calmette 1863–1933. La Presse Medicale 70: 1323–1325.

5. Bernard, N. (1962b). La Vie et L’oeuvre Albert Calmette 1863–1933. Pp. 120–141. Editions Albin Michel, Paris.

6. Beyazova U., S. Rota, C. Cevheroğlu, T. Karsligil (1995). Humoral immune response in infants after BCG vaccina-tion. Tubercle and Lung Disease 76: 248–253.

7. Birkhaug, K. E. (1934). Albert Leon Charles Calmette (1863–1933). Annals of Medical History, New Series 6: 291–300.

8. Black, J. (1999). Microbiology: Principles and Exploration. 4th Edition. Pp. 483. Prentice Hall, New Jersey.

9. Bon, C. (1996). Serum therapy was discovered 100 years ago. In: C. Bon and M. Goyffon (Eds.). Envenomings and Their Treatments. Pp. 3–9. Fondation Marcel Merieux, Lyon.

10. Brisou, B. (1995). Les pionniers de la peste medecins colo-niaux et pasteuriens: Yersin, Simond, Girard et Robie. Histoire des Sciences Medicals 24: 327–336.

11. Calmette, A. (1892). Contribution a Ietude des ferments de I’amidon. La levure chinoise. Annales de I’Institut Pasteur 6: 604–620.

12. Calmette, A. (1896). The treatment of animals poisoned with snake venom by the injection of antivenomous serum. Lancet ii: 449–450.

13. Calmette, A. and C. Guerin (1906). Sur la vaccination contre la tuberculose par les voies digestives. Comptes Rendus de I’Academie des Sciences 142: 1319–1322.

14. Calmette, A. (1922). L’infection Bacillaire et la Tuberculose chez l’Homme et les Animaux, 2nd Edition, Masson, Paris.

15. Calmette, A. (1927). La Vaccination Preventive Contre la Tuberculose par le “BCG”. Pp.73–79. Masson, Paris.

16. Calmette, A. (1931). Epilogue de la catastrophe de Lukeck. Presse Med 2: 17.

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17. Calmette, A., A. Saenz and L. Costil (1933). Effets du venin de cobra sur les greffes cancereuses et sur le cancer spon-tane (adeno-carcinoma) de la souris. Comptes Rendus de I’Academie des Sciences 197: 205–209.

18. Constant, P., Y. Poquet, M. Peyrat, F. Davodeau, M. Bonneville and J. J. Fournie (1995). The antituberculous Mycobacterium bovis BCG vaccine is an attenuated mycobacterial producer of phosphorylated nonpeptidic antigens of human gamma delta T cells. Infection and Immunity 63: 4628–4633.

19. Coplen, D. E., M. D. Marcus, J. A. Myers, et al. (1990). Long term follow up of patients treated with 1 or 6 week courses of intra-vesical bacillus Calmette-Guerin: analysis of possible predictors of response free of tumor. Journal of Urology 144: 652–657.

20. Ferru, M. (1995). La faillite du BCG. Temoignages d’hier et d’aujourd’hui, 2nd Edition., France, Saint Gratien.

21. Fine, P. E. M. and L. C. Rodrigues (1990). Modern vaccines: mycobacterial diseases. Lancet 335: 1016–1020.

22. Gelinas, J. A. (1973). Albert Calmette, The Saigon years 1891–1893: a historical review. Military Medicine 138: 730–733.

23. Gheorghiu, M. (1996). Antituberculosis BCG vaccine: lessons from the past. In: Plotkin, S. and Fantini, B. (Eds.). Vaccinia, Vaccination and Vaccinology: Jenner, Pasteur and Their Successors. Pp. 87–94. Elsevier, Paris.

24. Harland, S. J., C. R. Charig, W. Highman, et al. (1992). Outcome in carcinoma in situ of bladder treated with intra-vesical bacilli Calmette-Guerin. British Journal of Urology 70: 271–275.

25. Hawgood, B. J. (1996). Sir Joseph Fayrer MD FRS (1824–1907), Indian Medical Service: snakebite and mortality in British India. Toxicon 34: 171–182.

26. Hawgood, B. J. (1999). Doctor Albert Calmette 1863–1933: founder of antivenomous serotherapy and of antitubercu-losis BCG vaccination. Toxicon 37: 1241–1258.

27. Herr, H. W., C. M. Pinskym, W. F. Whitmore, et al. (1983). Effect of intravesical bacillus Calmette-Guerin (BCG) on car-cinoma in situ of the bladder. Cancer 51: 1323–1326.

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28. Herr, H. W., V. P. Laudone, R. A. Badalament, et al. (1988). Bacillus Calmette-Guerin therapy alters the progression of superficial bladder cancer. Journal of Clinical Oncology 9: 1450–1455.

29. Herr, H. W., D. D. Wartinger, W. R. Fair and H. F. Oettgen (1992). Bacillus Calmette-Guerin therapy for superficial bladder cancer: a 10-year follow up. Journal of Urology 147: 1020–1023.

30. Herr, H. W. (1997). Summary of effect of intravesical bacillus Calmette-Guerin (BCG) on carcinoma in situ of the bladder. Seminars in Urologic Oncology 15: 80–85.

31. Kervran, R. (1962). Albert Calmette et le B.C.G. P. 98. Libraire Hachette, Paris.

32. Lamm, D. L., B. A. Blumenstein, E. D. Crawford, et al. (1995). Randomized intergroup comparison of bacillus Calmette-Guerin immunotherapy and mitomycin C chemotherapy prophylaxis in superficial transitional cell carcinoma of the bladder, a southwest oncology group study. Urologic Oncology 1(3): 119–126.

33. Larsson, L. O., M. Magnusson, B. E. Skoogh and A. Lind (1992). Sensitivity to sensitins and tuberculin in Swedish children. IV. The influence of BCG-vaccination. The European Respiratory Journal 5: 584–586.

34. Lotte, A., O. Wasz-Hockert, N. Poisson, et al. (1988). Second IUATLD study on complications induced by intradermal BCG vaccination. Bulletin of the International Union Against Tuberculosis and Lung Disease 63: 47–59.

35. Maes, R. F. (1999). Tuberculosis II: the failure of the BCG vac-cine. Medical Hypotheses 53: 32–39.

36. Merz, V. W., D. Marth, R. Kraft, et al. (1995). Analysis of early failures after intravesical instillation therapy with bacille Calmette-Guerin for carcinoma in situ of the bladder. British Journal of Urology 75: 180–184.

37. Morales A., D. Eidinger and A. W. Bruce (1976). Intracavitary bacillus Calmette-Guerin in the treatment of superficial bladder tumors. Journal of Urology 116: 180–183.

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38. Morales, A. (1980). Treatment of carcinoma in situ of the bladder with BCG: A phase II trial. Cancer Immunology, Immunotherapy 9: 69–72.

39. Mustafa, A. (1995). Mycobacterium bovis BCG-induced Th 1 type CD4+ suppressor T cells act by suppressing IL-2 produc-tion and IL-2 receptor expression. Nutrition 11: 692–694.

40. Nadler, R. B., W. J. Catalona, M. A. Hudson and T. L. Ratliff (1994). Durability of the tumor-free response for intravesi-cal bacillus Calmette-Guerin therapy. Journal of Urology 152: 367–373.

41. Oates, R. D., M. M. St. Imart, M. C. Freedlund and M. B. Siroky (1988). Granulomatous prostatis following intravesical bacil-lus Calmette-Guerin. Journal of Urology 140: 752–754.

42. Ovesen, H., A. L. Poulsen, A. L., and K. Steven (1993). Intravesical bacillus Calmette-Guerin with the Danish strain for treatment of carcinoma in situ of the bladder. British Journal of Urology 72: 744–748.

43. Ratliff, T. L. (1989). Mechanisms of action of intravesical BCG for bladder cancer. Progress in Clinical and Biological Research 310: 107–122.

44. Samaille, J. (1984). Albert Calmette Commemoration du 50e anniversaire de leur disparition. Bulletin de I’Institut Pasteur 82: 29–39.

45. Schwalb, M. D., H. W. Herr, P. C. Sogani, et al. (1995). Positive urinary cytology following a complete response to intravesical bacillus Calmette-Guerin therapy: Pattern of recurrence. Urologic Oncology 152: 382–387.

46. Smith, F. B. (1993). Tuberculosis and bureaucracy. BCG: its troubled path to acceptance in Britain and Australia. Medical Journal of Australia 159: 408–411.

47. Talic, R. F., T. B. Hargreave, M. C. Bishop, et al. (1994). Intravesical Evans bacille Calmette-Guerin for carcinoma in situ of the urinary bladder. Scottish Urological Oncology Group British Journal of Urology 73: 645–648.

48. Tambryn, K., H. L. Collins and D. G. Russell (1997). IL-6 pro-duced by macrophages infected with Mycobacterium

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species suppresses T cell responses. The Journal of Immunology 158: 330–337.

49. Tardieu, M., C. Truffot-Pernot, J. P. Carriere, Y. Dupic and P. Landrieu (1988). Tuberculosis meningitis due to BCG in two previously health children. Lancet 1: 440–441.

50. Tasaka, S., A. Ishizaka, T. Urano et al. (1995). BCG-priming enhances endotoxin-induced acute lung injury independ-ent of neutrophils. American Journal of Respiratory and Critical Care Medicine 152: 1041–1049.

51. Terashita, M., C. Kudo, T. Yamashita, I. Greeser and F. Sendo (1996). Enhancement of delayed-type hypersensitivity to sheep red blood cells in mice by granulocyte colony- stimulating factor administration at the elicitation phase. The Journal of Immunology 156: 4638–4643.

52. Thurnher, M., R. Ramoner, G. Gastl, et al. (1997). BCG stimu-late human blood dendritic cells. International Journal of Cancer 70: 128–134.

53. Vegt, P. D. J., A. Witjes, W. P. J. Witjes, et al. (1995). A rand-omized study of intravesical mitomycin C, bacillus Calmette-Guerin TICE and bacillus Calmette-Guerin RIVM treatment in pTa-pTI papillary carcinoma and carcinoma in situ of the bladder. The Journal of Urology 153: 929–933.

54. Yersin, A. E. J. (1894). La peste bubonique a Hong Kong. Annales de I’Institut Pasteur 8: 662–667.

55. Yersin, A. E. J., A. Calmette and A. Borrel (1895). La peste bubonique. Second note. Annales de I’Institut Pasteur 9: 589–592.

56. Zhar, B., and W. J. Rapp (1974). Immunotherapy of guinea pig with BCG. Cancer 34: 1532–1540.

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Chapter 27

Charles J. H. Nicolle (1866–1936): Pioneer of Typhus Studies

Source: https://en.wikipedia.org/wiki/Charles_Nicolle(US Public Domain image)

Introduction

Typhus was a terrible disease in Western Europe. It probably killed more humans than any other disease in human history. Charles Nicolle discovered the true cause of typhus. He found that typhus is transmitted by human body louse. His discovery

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was of great importance to both military and civilian medicine. His work made him the recipient of the 1928 Nobel Prize for Physiology or Medicine.

Life of Charles Nicolle

Charles J. H. Nicolle was born on 21 September 1866 in Rouen, France. His father, Eugene Nicolle, was a medical doctor at the municipal hospital and a professor of natural history at the École des Sciences et des Arts. Charles had an older brother, Maurice Nicolle (1862–1932), who became a well-known bacte-riologist and pathologist. Although he was very gifted in litera-ture, he followed the family tradition to study medicine. At the age of 18, for unknown reason, he suffered deafness. His deaf-ness created some problems for his social activities, and also affected his medical practice later on. In 1889, he passed the competitive examinations for a medical residentship.

His brother Maurice encouraged Charles to take a course in bacteriology at the Pasteur Institute which was founded in Paris 1888 by Louis Pasteur (1822–1895). This course was taught by Dr. Emile Roux (1853–1933) and Dr. Élie Metchnikoff (1845–1916), both pioneers in microbiology. Charles studied microbi-ology under them and did research on the bacterium called Ducrey’s bacillus (later called Hemophilus ducreyi) — a causa-tive agent of soft chancre, a type of venereal disease. This research became his doctoral dissertation, and he obtained his medical degree in 1893.

After his obtained his medical degree, Dr. Nicolle returned to his home town of Rouen where he married Alice Avice. They had a happy marriage, with two sons, Marcelle and Pierre. Both of them later became well-known physicians.

In Rouen, he had a staff position in municipal hospital and as an assistant lecture at the local medical school. He was also in charge of a bacteriology laboratory in which he continued his research on Ducrey’s bacillus. He improved techniques for making anti-diphtheria serum.

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Charles intended to develop a major biomedical research center in Rouen. However, he had no support. Dr. Nicolle was an enthusiastic young man with unshakable faith in humanistic ideals. He was very stubborn whenever principles were con-cerned. He often found himself in conflict with an inactive and mediocre bureaucracy.

To his disappointment and also discouraged by the indiffer-ent reception at Rouen, he accepted the post of director of the Institut Pasteur in Tunis, Tunisia in 1902. At that time, Tunis was regarded as poorly developed area. When Nicolle arrived in Tunisia, the Pasteur Institute existed only on paper. Through devoted and hard work, the torn anti-rabies vaccination unit had been successfully transformed into a marvelous institute equipped for large scale manufacturer of vaccine and a center for scientific research. He made the Pasteur Institute in Tunis, a leading center for the study of North Africa and tropical dis-eases. His great medical contributions were all accomplished in Tunis. Nicolle stayed in Tunis for the rest of his life except for an occasional trip back to Paris to give lectures.

In Tunis, he became interested in typhus. He noticed that typhus was common in the general public, but it did not become established in the hospital ward. He discovered that those who collected or laundered the dirty clothes of newly admitted patients typically came down with the disease. He realized that the washing, shaving, and providing of clean clothes to the new patients was possibly the key to the pattern of infection.

An idea suddenly occurred to Nicolle, he suspected that lice, which attached themselves to the bodies and clothes of human beings, transmitted the disease. He infused a chimpanzee with human blood infected with typhus, then transferred the chim-panzee’s blood to a healthy macaque monkey. When the fever and rash of typhus were seen on the infected monkey, he placed 29 human body lice obtained from healthy humans on the skin of the macaque. These lice were later placed on the skin of a number of healthy monkeys. All of these monkeys contracted the disease.

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Nicolle’s experiments, all of them, proved the arthropod-borne nature of typhus. He then established a preventive meas-ure to encounter unsanitary conditions. Nevertheless, the trenches of World War I remained major places for the louse, typhus killed a large number of soldiers. In 1939, Paul Muller (1899–1965) developed the insecticide dichlorodiphenyltrichlo-roethane (DDT) which was a most effective prophylactic against typhus because it killed the lice. Typhus was nearly eradicated among soldiers during World War II.

We now learn that there are two types of typhus: epidemic typhus caused by Rickettsia prowazekii and murine typhus caused by Rickettsia typhi. The epidemic typhus is also called louse-borne typhus because it is spread by body lice Pediculus humanus as stud-ied by Nicolle. The murine typhus is spread by flea, usually rodent flea Xenopsylla cheopis. Both types of typhus can be treated with tetracycline or chloramphenicol. Howard Ricketts (1871–1910) and Stanislaus von Prowazek (1875–1915) pioneered the study of the Rickettsia, which are intracellular parasites. Both of them died of typhus while investigating this deadly disease. Typhus is also called Rickettsiosis.

During his studies of typhus, Nicolle noticed a phenomenon known as “inapparent infection”, which is a state in which a carrier of a disease exhibits no symptoms of the disease, but can transmit the pathogens to others. This is a theoretical discovery which explained how diseases survived from one epidemic to another. It was a great discovery for both bacteriology and medicine.

Along with the typhus studies, he also discovered that injec-tion with serum from a convalescing victim of exanthematous typhus could protect others who had been exposed to the dis-ease. Nicolle applied this finding to other diseases. With the help of his friend E. Conseil, he successful fought against mea-sles. His work became a pioneering work in using gamma globulin in preventive medicine.

Dr. Nicolle with his colleagues also studied African infantile leishmaniasis and differentiated it from kala-azar which was

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found in India. He discovered leishmaniasis in the dogs, which were the major reservoir and vector of this disease. He also developed a culture medium to isolate various Leishmania. With his colleague Louis Herbert Manceaux (1865–1934), he isolated a parasite of Tunisian rodents, Toxoplasma gondii in 1908. Toxoplasmosis was later proved by other investigators to be a human disease. He investigated the role of flies in the transmis-sion of trachoma, a disease which causes blindness. He also found the viral nature of influenzae. He also participated the typhus studies occurred in Mexico in 1931. He published numer-ous scientific papers and wrote five major books. In addition, he was also a literature writer. He published several novels.

Because of his deafness, Dr. Nicolle excluded him from the conversation and dreaded of social gatherings. Therefore, he was introvert. He devoted his time on scientific research, writ-ing, and his family. He was gifted with good imagination and talent of careful observations. He often formulated bold hypotheses, and then scrupulously tested his hypotheses in the light of experimental data and he was also a poet. He was an author of several novels and a collection of stories. He said that scientific creation was similar to poetic inspiration, and was based on personal experiences. He thought that the birth of an idea was comparable to biological mutation.

For many of Dr. Nicolle’s accomplishments, he received the rank of French Commander of the Legion of Honor, and was elected to the French Academy of Medicine. In 1932, he was nominated to the chair of experimental medicine at the College de France. While retaining his post in Tunis, he lectured at the College de France every year from 1932 to 1935. During this period, he began to consider scientific methodology, the major lines of historical evolution of diseases, and human destiny. He had a strong view on the moral responsibility of scientists, and on the biological foundation of creativity. Nicolle died on 28 February 1936 in Tunis.

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Chapter 28

Howard Taylor Ricketts (1871–1910): Pioneer of Rickettsial Diseases Studies

Source: https://en.wikipedia.org/wiki/Howard_Taylor_Ricketts(US Public Domain image)

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Howard Taylor Ricketts made a tremendous contribution to the understanding of a unique kind of microbial disease including Rocky Mountain spotted fever and Mexican typhus. The patho-gens of these diseases are named Rickettsia and diseases caused by these bacteria are generally called rickettsial diseases. This is in memory of Howard T. Ricketts who was the first to discover the bacteria which caused these diseases.

Howard was born on 9 February 1871 on a farm in Findlay, Ohio. His father was Andrew Duncan Ricketts and his mother was Nancy Jane Taylor. They had a modest fortune, and paid a great deal of attention to the education of Howard. Howard grew up on the farm and was a husky and energetic young boy. Howard was motivated by deep Methodist conviction. In 1890, he completed preparatory school and entered Northwestern University. In 1892, he transferred to the University of Nebraska. In 1893, an economic panic occurred to his family which destroyed his family’s modest fortune, and from that time on, Howard worked his way through school. In 1894, he obtained his BA degree and entered Northwestern Medical School, where he was helped by his patron Dr. Walter H. Allport who secured an employment for him in the medical museum. Howard obtained his MD degree in 1897.

For unknown reasons, Howard suffered a nervous break-down in 1897. Fortunately he recovered, and upon his recovery, he became an intern at Cook County Hospital. The next year, he obtained a pathology fellowship at Rush Medical College in Chicago for two years. He married Myra Tubbs on 8 April 1900.

Howard was hungry for knowledge, and always preparing himself for further professional challenges. Therefore, he accepted the suggestion of Dr. Ludvig Hektoen (1863–1951) the head of the pathology department at the University of Chicago, and went to Berlin, Germany, to further his study of medicine. His son, Henry was born in Berlin. Howard later went to the Pasteur Institute in Paris, France. These experiences in Europe, broadened his vision and knowledge. In particular, he

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developed a profound appreciation for microbiology which had been growing in importance in the medical field.

In 1902, Howard was an associate professor of pathology at the University of Chicago. He did some research on blastomyco-sis which resulted in a publication in the journal Infection, Immunity and Serum Therapy. In the late spring of 1906, he traveled to Missoula, Montana, for a vacation, where he discov-ered an unusual disease, and started the medical research career which brought his name into history.

In Montana, he discovered a deadly spotted fever occurred to the inhabitants of the Rockies. He learned that the disease was somewhat geographically restricted and the mortality rate could be as high as 80%–90%. The patients usually had a head-ache, pains in their muscles, and joints, and fever, followed by a hemorrhagic rash. The incubation time for the disease was about 4–8 days.

At that time, the infective agent of spotted fever was not known. It was thought to be caused by Piroplasma and several modes of transmission were suspected. Ricketts spent three years of vigorous investigation and found that certain kinds of ticks transmitted the diseases. Through microscopic examina-tion, he could detect the causative agent was coccobacillus in the blood of people ill with the disease as well as in diseased animals infected with the ticks. He was able to infect guinea pigs by injecting them with the blood of people suffering from the disease and found that the bite of certain kinds of ticks transmitted the disease to the guinea pigs. Later, he proved that only tick Dermacentor andersoni was responsible for trans-mission of the disease. He did not find any Piroplasma associ-ated with the disease. Howard later also proved that ticks acquiring the pathogen by feeding on an infected person or animals transmitted the infection from one generation of ticks to the next. He demonstrated large numbers of pathogens in the eggs of the infected ticks, and verified that transovarian passage explained transmission of the infection from the ticks

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to their offspring. Today, we know that the pathogenesis started by the infection of this pathogen by bite of the tick and organisms multiply at site of bite. The bloodstream is invaded and endothelial cells of blood vessels are infected. Vascular lesions occur and endotoxins are released by the pathogens. This accounts for the pathological changes of the patients and the disease manifested. The laboratory diagnosis was done with fluorescent antibody stain of the skin biopsy, or an Elisa test of patients’ serum for antibodies against rickettsial antigen.

However, whenever there are heroes, there are villains. While Howard Ricketts’ work was shedding a light for fighting the disease, there were oppositions. His work upset several real estate agents who were concerned lest land prices drop. One entomologist from the U.S. Department of Agriculture denied emphatically that the tick was responsible for transmission.

Despite these obstacles, Howard worked diligently to pre-vent the disease. By 1909, he outlined a control program that he hoped would destroy the reservoir of the disease. He based his idea mainly on a successful model of Texas tick fever that had been worked out by Theobald Smith (1859–1934) and oth-ers. His methods of control provided the most practical meas-ures for preventing the spread of the disease at that time.

In 1910, there were outbreaks of typhoid fever and small pox in Montana, and money was diverted to fight these dis-eases. There was no more money left to spend on preventing the spotted fever. Therefore, Howard Ricketts left Montana and accepted an invitation to study the cause of typhus which occurred in the Valley of Mexico. Using the technique similar to his experience with spotted fever, he found that typhus was caused by a bacterium similar to that of Rocky Mountain spot-ted fever, and the pathogen was carried by body lice. He also demonstrated the difference between spotted fever and typhus. He worked closely and efficiently with severely infected patients. Howard and his two associates contracted the disease, and Dr. Ricketts died on 3 May 1910 in Mexico City. Dr. Ricketts’

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body was returned to Illinois, where he was buried in Kirkwood, Illinois.

Dr. Ricketts was a pioneer in using laboratory animals for disease identifications. He worked on the immunity and serol-ogy of laboratory animals. His work became the basis for fur-ther advances in vaccine development for many other diseases. For spotted fever, there is still no vaccine available for spotted fever today. But it can be treated by tetracycline or chloram-phenicol. The best way of prevention is avoidance of tick-infected areas, use of tick repellent, and removal of ticks within 4 h of exposure.

In 1916, a French physician Dr. Henrique da Rocha-Lima (1879–1956) studied the pathogen of Rocky Mountain spotted fever and named the genus Rickettsia in memory of Dr. Howard Ricketts. Dr. Simeon B. Wolbach (1880–1954) made further explo-rations of the genus. The genus Rickettsia now includes all the very small gram-negative bacteria found in arthropods and capa-ble of transmission to some vertebrate. They are obligate para-sites growing in the nucleus and cytoplasm of host cells. In the laboratory, they are usually cultured in embryonated chicken eggs, where they grow within the cells of the yolk sac membrane. They can also be cultured in tissue cultures or in laboratory ani-mals. Laboratory work with the species can be very dangerous because small numbers of cells can cause infections. A number of diseases caused by Rickettsia are listed in the Table 28-1.

We have now also learned that in the eastern United States, R. rickettsia is transmitted by the American dog tick, Dermacator variabilis; in the west, it is transmitted by the wood tick D. andersoni, and in Texas and Louisiana, it is transmitted by the Lone Star tick Amblyomma americanum. Ticks are the primary reservoir of infections, other animal reservoirs of infection include wild rabbits, dogs, sheep, and rodents, in which the disease is perpetuated by tick-borne transmissions.

Dr. Howard Ricketts died in the prime of his career. Dr. Ricketts was survived by his wife, son, Henry, and daughter, Elizabeth. Although Dr. Ricketts lived only 39 years, his

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contribution to humans is tremendous. As Dr. S. B. Wolbach of the Harvard Medical School commented on Ricketts’ contribu-tion, he said: “Ricketts brought facts to light with brilliance and accuracy and indicated by the methods he used, most of the major lines of development subsequently employed in the study of rickettsial diseases”.

Table 28-1: Rickettsial diseases and their pathogens.

Name of Organisms Disease

Rickettsia prowazekii Epidemic (classic, European) typhus

R. prowazekii Brill-Zinsser disease (recrudescent typhus)

R. typhi Endemic (murine) typhus

R. tsutsugamushi Scrub typhus (tsutsugamushi disease)

R. rickettsia Rocky Mountain spotted fever

R. akari Rickettsial pox

Bartonella (Rochalimaea) quintana

Trench fever

B. bacilliformis Bartonellosis

Ehrlichia canis Ehrlichiosis

E. chaffeensis Ehrlichiosis

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Chapter 29

Chaim A. Weizmann (1874–1952): Pioneer of Industrial Microbiology

and First President of Israel

Source: https://en.wikipedia.org/wiki/Chaim_Weizmann(US Public Domain image)

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Introduction

Many people probably remember that Chaim Azriel Weizmann was the first President of the Republic of Israel, which was established in 1948. Chaim Weismann was also a pioneer of organic chemistry and an industrial microbiologist. He pio-neered the study of microbial fermentation, and contributed to the production of acetone and butanol. Butanol and acetone were important fuels before the era of the petroleum industry, and will be important biofuels in the future. His work directly influenced the World War I (1914–1918), past history, and will play an important role in the future civilization.

Life of Chaim Weizmann

Chaim Weizmann was born on 27 November 1874 in Motal near Pinsk in Belarus, Russia. His early religious schooling was in the segregated Jewish area of rural Russia. At the age of 11, he attended the Gymnasium (equivalent to high school) at Pinsk, where he had the opportunity to be exposed to the gentile ways of life and Western European culture, which must have had a great influence on him. He studied hard and was good in almost every discipline. But he was particularly outstanding in chemis-try. His parents encouraged him to pursue his favorite subject, chemistry. He went to the technical institute of Darmstadt, Germany, to study chemistry from 1893 to 1894. From 1895 to 1898, he went to the technical institute of Berlin, where the famous chemist Dr. Carl Libermann (1842–1914) and his student Augustin Bistrzycki (1862–1936) were investigating polycyclic aromatic compounds. The dye manufactures were interested in those chemicals. When Bistrzycki went to the University of Fribourg, Switzerland, Weizmann followed him. In 1899, Weizmann was awarded a PhD sum cum laude with the disserta-tion entitled 1. Elektrolyishche Reduktion von 1-Nitroanthrachinon. Ii. Ueber die Kondensation von Phenanthrenechinon U. 1-Nitroanthrachinon mit einigen phernolen.

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After completing his doctoral degree, Dr. Weizmann contin-ued to do research on the naphthacene quinones. In 1901, he was appointed as assistant lecturer at the University of Geneva, Switzerland. His work won him patents, which were profitably sold to French and German dye companies.

At a young age, Chaim was strongly upset with the anti-Jewish policy of Russia. He joined the Zion Movement. Weizmann only missed the first Zionist conference held in 1897 in Basel, Switzerland, because of a travel problem. But he was very active in this organization ever since. Beginning in 1901, he lob-bied for the founding of a Jewish institution of higher learning in Palestine. Together with Martin Buber (1878–1965) and Berthold Feiwel (1875–1937), they prepared a document empha-sizing the need for more discipline in the fields of science and engineering to present to the Fifth Zionist Conference. The idea was later realized in the foundation of the Technion-Israel Institute of Technology in 1912. He later became a strong leader in the world Zionist movement. He was determined to establish an independent Jewish homeland in Palestine. He was devoted to that cause while he practiced chemistry for a living. He said “Chemistry is my private occupation. It is this activity in which I rest from my social tasks”.

In 1906, he married Vera Chatzmann (1881–1966). The couple had two sons. The younger one, Flight Lt. Michael Oser Weizmann, serving as a pilot in the British No. 602 Squadron RAF, was killed in 1942 when his plane was shot down over the Bay of Biscay.

In 1907, Weizmann first visited Jerusalem, and he helped organize the Palestine Land Development Company as a practi-cal means of pursuing the Zionist dream. He stated, “A state cannot not be created by decree, but by the forces of a people and in the course of generations. Even if all the governments of the world gave us a country, it would only be a gift of words. But if the Jewish people will go build Palestine, the Jewish State will become a reality — a fact”.

In 1917, Weizmann became president of the British Zionist Federation. Sometime before, Arthur Balfour (1848–1930) was

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a member of parliament (MP) of the Conservative party repre-senting the district, as well as Prime Minister. Weizmann and Balfour met during one of Balfour’s electoral campaigns. Balfour supported the concept of Jewish homeland. A very interesting interaction and friendship developed between these two gentlemen. The story goes that Weizmann asked Balfour, “Would you give up London to live in Saskatchewan?” When Balfour replied that the British had always lived in London, Weizmann responded, “Yes, and we lived in Jerusalem when London was still a marsh”.

Weizmann supported grass-roots colonization efforts, by founding so-called Synthetic Zionism, as well as high-level dip-lomatic activity. He was generally associated with the centrist General Zionists. He said, “We have never based the Zionist movement on Jewish suffering in Russia or in any other land. These suffering have never been the mainspring of Zionism. The foundation of Zionism was, and continues to be to this day, the yearning of the Jewish people for its homeland, for national center, and a national life”.

Weizmann’s dream and zest to establish a Jewish country had driven him to do everything possible to facilitate that dream. His research also helped the realization of his dream. Weizmann was a creative industrial microbiologist. Dr. Weizmann became famous for discovering how to use bac-terial fermentation to produce large quantities of desired sub-stances. He was considered to be the father of industrial fermentation. He was the first one to use Clostridium acetobu-tylicum (later called Weizmann organism) to produce acetone (1912). Acetone was used in the manufacture of cordite explo-sive propellants critical to allied war effort. Weizmann trans-ferred the rights to the manufacture of acetone to the Commercial Solvents Corporation in exchange for royalties. Admiralty Winston Churchill (1874–1965) became aware of the possible use of Weizmann’s discovery in 1915, and with Minister of Munitions David Lloyd George (1863–1945) encour-aged Weizmann’s development of the process. Pilot plant

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development of laboratory procedures was completed in 1915. At J & W Nicholson & Co gin factory in Bow, London. Industrial production of acetone could begin in six British distilleries req-uisitioned for purpose in early 1916. A national collection of horse chestnut was required when supplies of maize were inadequate for the quantity of starch needed for fermenta-tion. This effort produced 30,000 tons of acetone during the World War I. The importance of Weizmann’s work to the ongo-ing war effort won his strong support from the Foreign Secretary Arthur Balfour.

Dr. Weizmann’s contribution to Britain was so great that Lord Balfour asked him how he could best be rewarded. He answered that he would like to secure a declaration of British help by establishing an independent country of the Jews. Weizmann worked with Arthur Balfour to obtain the famous Balfour Declaration, which stated that the British government “views with favor the establishment in Palestine of a national home for the Jewish people…it being clearly understood…”.

Also in 1917, indeed Balfour promised that the Great Britain would help the establishment of a Jewish independent govern-ment. On January 1919, he and the Arabic leader Hashemite Prince Faisal (1885–1933) signed the Faisal-Weizmann Agreement attempting to establish favorable relations between Arabs and Jews in the Middle East. At the same month, the Paris Peace Conference decided that Arab provinces of the Ottoman Empire should be wholly separated and the newly conceived mandate system allied to them. Both men made their statements to the conference.

After 1920, Weizmann assumed leadership in the World Zionist movement, serving twice (1920–1931 and 1935–1946) as president of the World Zionist organization. In 1921, Weizmann went along with Albert Einstein (1879–1955) for a fund raiser to establish the Hebrew University in Jerusalem and supported the Technion-Israel institute of Technology.

For some reasons, brewing differences over competing European and American visions of Zionism, and its funding of

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development versus political activities, caused Weizmann to clash with Louis Brandeis (1856–1941), the leader of the precur-sor of the Zionist Organization of America. Brandeis led in fund-raising for Jews in Europe and Palestine during the war time (World War II) Although Weizmann retained Zionist lead-ership, the clash led to the departure from the movement of Brandeis and other prominent leaders.

Weizmann stopped all scientific activity except for promot-ing the growth of the Hebrew University in Jerusalem, and the founding of the Daniel Sieff Research Institute (later called the Weizmann Institute of Science) in Rehovot, Israel, in 1934. Both of these institutions became well known internationally in sci-entific learning.

In 1934, Dr. Weizmann went back to his research at Rehovot and London simultaneously. He studied chemical reactions, which were related to the economy of Palestine. He made preparations for the establishment of his own country. For example, he researched on the commercial synthesis of organic compounds from agricultural products or petroleum. He discov-ered several reaction mechanisms by which petroleum fractions could be reduced by cracking them to ethylene and diene frag-ments, and then recombining them into polynuclear aromatics of the type, which he had used in his dye researches. This was a major progress for industry because such dye intermediates could only be obtained from coal tar previous. Although his researches were based on practical, rather fundamental scien-tific considerations, his work bore considerable technical significance.

In 1936, Weizmann addressed the Peel Commission, set up by Stanley Baldwin (1867–1947), whose job was to consider the working of the British Mandate of Palestine. The Commission published a report that, for the first time, the recommended partition, but the proposal was declared unworkable and for-mally rejected by the government. Weizmann and David Ben-Gurion (1886–1973) accepted the partition and its logic. This was the first official delineation and declaration of Zionist

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vision opting for a new, artificial state with a majority of Jewish population, alongside the existing state with Arab majority. The Arab leaders, headed by Haj Amin al-Husseini (1895–1974), rejected the plan.

Weizmann’s effort to integrate Jews from Palestine in the war against Germany resulted in the creation of the Jewish Brigade, which fought mainly on the Italian front. After the war, Weizmann grew embittered by the rise of violence in Palestine and by the terrorist tendencies among followers of the Revisionist fraction. His influence within the Zionist movement decreased, yet he remained overwhelmingly influ-ential outside Palestine. In his presidential speech at the last Zionist Congress that he attended, he stated: “Massada for all its heroism, was a disaster in our history; It is not our purpose or our right to plunge to destruction in order to bequeath a legend of martyrdom to posterity; Zionism was to mark the end of our glorious death and the beginning of a new path leading life”.

Weizmann met with President Harry Truman (1884–1972) and worked to obtain the support of the United States for the establishment of the State of Israel. Weizmann was unani-mously elected the first President of the Israel Republic. His dream to establish an independent Jewish country was finally realized. In the meantime, his creative scientific career came to an end. He died on 9 November 1952, in Rehovot, Israel. He was buried beside his wife in the garden of his home at Weizmann estate, which is located on the grounds of Weizmann Institute of Science.

Commentary

Weizmann was also a scholar. He was appointed assistant lec-ture at the University of Geneva, Switzerland. In 1904, he moved to Manchester, England. This move was preplanned. It was promoted by a greater professional opportunity and pre-monition that England could do the most for the establishment

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of Jewish State. Initially he enrolled as a student at the University of Manchester. His talent in chemistry was soon dis-covered by Dr. William H. Perkin (1838–1907), the head of the chemistry department. Dr. Perkin appointed him as a research fellow and a reader at the University of Manchester in 1905. In 1906, Dr. Weizmann also secured additional income by serving as a consultant for the local industry and selling patents. In 1907, he was promoted to a senior lecture in biochemistry at the University of Manchester.

Dr. Weizmann used the exceptional scientific facilities at the University of Manchester to conduct much excellent research. He was very effective teacher and attracted many good stu-dents to do research under his direction. He continued to study the chemistry of useful compounds such as alizarin-type dyes and polyhydroxylation of naphthacene quinones. These com-pounds were useful in the dye industry. He continued to make profits on his patents.

About 1909, Weizmann started to do research related to biochemistry. He tried to synthesize various naturally occurring peptides, also studied the biochemical behavior of amino acids, proteins, and ketones. Furthermore, he began to investigate fermentative reactions. He searched for bacteria that would convert the carbohydrates into isoamyl alcohol, which was a precursor of synthetic rubber. In 1912, he discovered a strain of Clostridium acetybutylicum that could break down starch to form alcohol, acetone, and butyl alcohol. This was exciting because these compounds could be used as fuels. During World War I (1914–1918), great quantities of acetone were needed to plasticize the propellant cordite. Dr. Weizmann successfully engineered the massive production of acetone in Great Britain for the Admiralty and Munitions. Plants were also built in India, Canada, and the United States for these purposes. The produc-tion of these fuels continued after World War I. Butanol was the preferred product for use in automobiles. Dr. Weizmann had opened a dimension of microbiology to the production of

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industrial chemicals. He was indeed a pioneer of industrial microbiology. His contribution has been tremendous.

During the World War 1, Weizmann was director of the British Admiralty laboratories from 1916 until 1919. During World War II, he was an honorary adviser to British Ministry of Supply and did resource on synthetic rubber and high-octane gasoline. This is because formerly Allied controlled sources of rubber were largely inaccessible owing to Japanese occupation during World War II.

His also led to the creation of the Daniel Sieff Research Institute in Rehovot, 1934, which was supported by an endow-ment by Israel Sieff (1899–1972) in memory of his late son Daniel Sieff. Weizmann actively conducted research in the labo-ratories of this institute, primarily in the field of organic chem-istry. In 1949, the Sieff Institute was renamed the Weizmann Institute of Science in his honor. Weizmann’s success as a scien-tist and the success of the Institute he founded makes him ani-conic figure in the heritage of the Israeli scientific community today.

Although Weizmann devoted his life to the establishment of the Republic of Israel, he remained as a scholar. Weizmann viewed great promise in science as a means to bring peace and prosperity to his homeland. He said, “I trust and feel sure in my heart that science will bring to this land both peace and a renewal of its youth, creating here the springs of a new spirit-ual and material life. I speak of both sciences for its own sake and science as a means to an end”.

According to those who knew him, they discerned that Weizmann was a brilliant man and with deep warmhearted gen-tleness, exacting toward his coworkers as toward himself. He was a son of the Jewish people and a citizen of the world, a statesman, and a scientist. Everybody who knew him, would never have felt any duality in him — there was in him a oneness of character and purpose, a deep inner peace, which made him accomplish extraordinary tasks both in science and in humanity.

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As we can see, good microbiologists are patriotic. When the country is in danger, microbiologists should always think about the need of their own countries.

Suggested Reading

1. Weizmann, A. (1949). Trial and Error: The Autobiography of Chaim Weizmann. Jewish Publication Society of America, Philadelphia.

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Chapter 30

Oswald Theodore Avery (1877–1955): Microbiological Genetic Transmission

and DNA

“It’s lots of fun to blow bubbles but it’s wiser to prick them yourself before someone else tries to”.

—Oswald T. Avery

Source: https://en.wikipedia.org/wiki/Oswald_Avery(US Public Domain image)

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Introduction

Oswald Avery, before the age of antibiotics, pioneered in the theory and practice of control of virulence in pathogenic organ-isms. He was a professor of the eminent Rockefeller Institute from 1913 to 1948. He work, especially on the discovery of bac-terial genetic transmission, formed the basis for the develop-ment of the famous double-helix structure of the molecular deoxyribonucleic acid (DNA) by James D. Watson (1928 to pre-sent) and Francis H. Crick (1916–2004).

Life of Oswald Avery

Oswald T. Avery was born on 21 October 1877 at Halifa, Nova Scotia, Canada, the second of three sons of Joseph Francis Avery and former Elizabeth Crowdy. They gave him the name of Oswald Theodore. His grandfather, Joseph Henry Avery, was an unusually skilled paper maker of Oxford University. He devel-oped the extra fine thin paper, which took print on both sides and was in the famous Oxford Bibles. His father, also named Joseph, was not satisfied with being a papermaker, even though the paper was used in Bibles. Coming under the influ-ence of a well-known Baptist Evangelist, Charles H. Spurgeon (1834–1892), he left the Church of England and become a Baptist seminarian. He married Elizabeth Crowdy and served as a Baptist pastor in England for several years. Against the advice of Evangelist Spurgeon and friends, he decided to go to Canada, even without a church appointment.

His confidence was rewarded and he became pastor of two churches in Canada over a four-year period. His next call was to New York City, where he served in the Mariner’s Temple. This was a Baptist mission in a part of the city noted for rowdyism and extreme poverty on the lower East Side. The organ of the Temple was broken and there were no funds to repair it. Mrs. Avery employed a young German cornetist to play for the church services. This proved to be great advantage to Oswald.

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He and his older brother Ernest both learned to play the cornet and became so proficient that they were able to obtain scholar-ships in the National Conservatory of Music. Ernest died of tuberculosis earlier in 1892, several months later, Reverend Avery also passed away. But Oswald continued his musical career, even playing Anton Dvorak’s Symphony “From the New World” under the direction of Walter Damrosch. Later, while in the University, he continued to play the cornet and became leader of the band.

Avery attended, first, Colgate Academy and then Colgate University, a British school, but one which encouraged a broad and scientific outlook — usual for the time. He excelled in litera-ture, public speaking, and debate. While at Colgate, he was a classmate of Harry Emerson Fosdick (1878–1969), who would become one of the most notable clergymen in America; it seemed that Avery also intended to enter the ministry. For instance, he and a group of students asked a professor of phi-losophy to give them a class in metaphysics, examining the foun-dations of the Christian religion. In later life, Avery was considered a very cautious scientist, who never took a position unless he felt that he had absolute proof. However, he was so impressed by the course on the scientific background of religion that he once told his colleague: “Fellow, you know there really is a God”.

Avery’s life is one which should be noted by all scientists. He was an excellent student, but not interested in science! The experiences that a microbiologist has in high school and col-lege, or the university, may or may not indicate his adult inter-ests in life and life’s work. He majored in the humanities, taking only those science courses that were required — and his grades were poorest in the science. Avery received a BA in the humani-ties from Colgate in 1900 and this same year entered Columbia University. During his later years at Colgate, he underwent a very deep change of interest, which has never been clearly understood. This change affected his entire life. Instead of con-tinuing in the humanities, where he had gained honors in Oratory, public speaking and debate, he entered the College of

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Physicians and Surgeons. Therefore, his life centered upon the field of medicine. Most of his family had assumed that he would enter the ministry.

Although an excellent student, he made poor grades in bacte-riology and pathology, two fields which later were his main inter-est in life and to which he made monumental contributions.

There was, of course, at this time, no scientific requirement for entrance to medicine, nor was there a single microbiologi-cal course for the aspiring doctor, in spite of later proven fact that a large proportion of diseases are microbial in nature. He completed his medical training in 1904 and joined a group in practice. He remained for general practice for three years, but was distressed to find patients with pulmonary disorders for which he could do nothing. Although his relations with patients was entirely satisfactory, he was neither intellectually nor emo-tionally satisfied with life.

Medicine was changing at this time and research was becoming more important. It was, therefore, not difficult for him to leave practice and shift from clinical to laboratory work. He started working part time for the Board of Health, doing tests on the opsonin indices of patients. In addition, he carried out milk bacteriology for the Sheffield Company. Pasturing of milk was becoming highly important and Avery made bacterial counts of milk before and after pasteurization — for a pay of $50.00 a month.

This work was highly satisfactory to him and was performed so efficiently that he was appointed to the Hoagland labora-tory in Brooklyn, New York in 1907. This was most valuable appointment as it was the first privately endowed bacteriologi-cal research laboratory in the United States which was of crucial importance to his microbiological development. Since the labo-ratory was also associated with a Long Island hospital, Avery’s duties included teaching courses for student nurses. He acquired here his best known and most enduring nickname, “the Professor”, which was often affectionately shortened to “Fess”. Banjamin White, the director, was so pleased with Avery that

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he appointed associate director in 1907 for the then magnificent salary of $1,200.00! His relationship with White was most valu-able, as White was a chemist. Although Avery’s work was in microbiology, he had the advantage of precision and care that came with “the chemical mode of thinking”. This precision was especially beneficial as he developed the methods of microbiol-ogy which enabled him to isolate and characterize the organ-ism of syphilis, Treponema pallidum. He also worked on organisms found in milk and in the fermentation of milk.

In 1910, Avery’s colleague White had a reactivation of tuberculosis, from which he had suffered earlier. Avery went to his sanitarium and spent a great deal of time with him, both as a friend professionally, doing research on tuberculosis. Basically, Avery was a research scientist, more interested in theoretically questions than routine and clinical work. But, for his friend he carried out all the routine work needed. It was during this time that Avery established what his biographer René J. Dubos called the pattern of his career, the “systemic effort to under-stand the biological activities of pathogenic bacteria through a knowledge of their chemical composition”.

This had its reward. His work gained the attention of Dr. Rufus Cole (1872–1966), director of the Rockefeller Institute Hospital. One of Dr. Cole’s goals was to develop a therapeutic serum — like that which had been developed for diphtheria — for pneumonia. Dr. Cole sent a letter to Avery of offering him a position. Since he received no answer, he sent another and more urgent letter. Again there was no response. This time, he came in person and again offered the position at a much higher salary. Dr. Cole had misinterpreted Dr. Avery’s failure to reply as being due to the sal-ary offered; actually at this time in his life, Avery was very poor with regard to any business response. He simply felt too busy to bother with a letter, even though the offer was interesting. Dr. Cole did not learn this for several years. The personal call was effective and Avery joined the Rockefeller staff in 1913.

After having left the medical field his interest in all aspects of infectious disease grew. He studied how microorganisms

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invaded the body tissues, how they grew, the nature of lesions, the responses of the body, and how recovery takes place. These studies grew into the field of microbiology and immunology. It would be 30 years before penicillin, but rational methods of disease prevention and cure were developed by such organiza-tions as Rockefeller Institute. The particular disease, which Avery used as his basis of operation, was lobar pneumonia. At this time, pneumonia was as just as severe and destructive a disease as typhoid fever or tuberculosis.

For the next 35 years, he focused most of his research on a single species of pneumonia-creating bacteria, Diplococcus pneumoniae. He worked with scientists that were widely recog-nized as being among the elite in their fields, including Alphonse R. Dochez (1882–1964), René Jules Dubos (1901–1982), Harriett Ephrussi-Taylor (1918–1968), Michael Heidelberger (1888–1991), Rebecca Craighill Lancefield (1895–1981), Maclyn McCarty (1911–2005), and Colin Munro MacLeod (1909–1972).

Dr. Avery studied the virulence of organisms, which had wide application theoretically to many unrelated species. He found that the virulence of pneumonocci and other organisms was related very often to their ability to produce an ectoplas-mic membrane constituting a cellular external capsule. The bacteria lost their virulence when they failed to be able to secrete the capsule. The capsule gave virulence to organism by providing pneumococci and others with a defense mechanism against the cells of the blood and tissues. He found also that the organisms could be identified by the polysaccharides of the capsules. Although established with pneumococci, the princi-ples were applied broadly to many organisms.

In the early 1940s, Avery and McCarty concentrated on the phenomenon of pneumococcal transformation, in which “R-form” (nonvirulent) peumococcus bacteria changed into the virulent “S-form” after killed S-formed bacteria were added to the cul-ture. The changed bacteria were identical in virulence and type to the killed bacteria, and the changes were permanent and inherit-able. Utilizing that versions of MacLeod’s preparation techniques,

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Avery and McCarty soon isolated active “transforming substance” from samples of pneumococci, and found that substance was DNA. In 1944, Avery, MacLeod, and McCarty published their dis-covery in the Journal of Experimental Medicine. Although some of their peers initially questioned the conclusion of the DNA was the transformation substance, in 1952, Alfred Hershey (1908–1997) and Martha Chase (1927–2003) proved DNA was the heredi-tary material through their work with a bacterial virus. Avery established these principles of immunity and virulence in relation-ship to DNA, which was one of the landmarks of biology, estab-lishing that DNA was a principle carrier of heredity in microbial life and also throughout life itself. He demonstrated that the DNA of other bacteria could be made to enhance the heredity of pneu-mococci — the principle of transformation. This laid down the foundation of work of James Watson and Francis Crick deter-mined the double structure of DNA in 1953. Thus, Avery played an early and critical role in the molecular revolution in biology.

At the time he left Rockefeller in 1948, Avery was recog-nized as one of the greatest of microbiologists. He was com-pletely devoted to his work and never married. But, he was also concerned with his family. A Major reason for leaving Rockefeller was to move to Nashville, Tennessee to be near his brother Roy. Roy was his only living relative. He continued to do some work for the Department of Defense until his death.

Commentary

Soon after starting at the Rockefeller Institute, Avery began to share apartment with Alphonse R. Dochez, who was then col-league in the respiratory disease department at the hospital. The two life-long bachelors were roommates for most of the 35 years. They made complementary housemates and friends, as Avery was somewhat introverted and retiring whereas Dochez was more gregarious and outgoing. They enjoyed dis-cuss science, especially related to microbiology. Dochez said “Dr. Avery was a true scientist with an insatiable curiosity and a

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powerful and unremitting urge to discover the inmost mecha-nisms of the biological facts that came under his observations”. Avery and Dochez used each other as sounding boards for try-ing out new ideas or better defining works in progress.

In the early 1930s, Avery underwent treatment for Graves’ disease. He took a brief leaver from the Hospital in 1934 follow-ing thyroidectomy, but did not fully recover for several years. In 1943, at the mandatory retirement age of 65, Avery became a member emeritus at the Rockefeller Institute; however, he con-tinued his research there until 1948. He then move to Nashville to be closer to the family of his brother, Roy, and quickly became a fixture in the neighborhood. His cousin, Minnie Wandell acted as his housekeeper. While vacationing on Deer Isle last in the summer of 1954, Avery felt terrific pain in the abdomen and was diagnosed with extensive hepatoma, or can-cer of the liver. Oswald Theodore Avery lived to be 78 years of age, dying on 20 February 1955 in Nashville, Tennessee.

Avery was bestowed many honors during his career. He served as president of the American Association of Immunologists — the American Association of Pathologist and Bacteriologists, and the Society of American Bacteriologist (1941). He was elected to the National Academy of Sciences (1933) and Royal Society of London. He received honorary degrees from McGill University, New York University, The University of Chicago, and Rutgers University as well as wards from organizations Royal Society of London (the Copley Medal in 1945), the American College of Physicians (John Philips Memorial award in 1932), and the New York Academy of Medicine, Paul Ehrlich Gold Medal from the University of Frankfurt (1933), Kober Medal from the Association of American Physicians, Lasker Award in basic Medical Research from of American Public Association in 1947, and so on.

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Chapter 31

Frederick Griffith (1879–1941): Discovery of Transformation

Source: https://en.wikipedia.org/wiki/Frederick_Griffith(US Public Domain image)

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Introduction

Frederick Griffith was the first to discover the phenomenon of bacterial transformation. This discovery laid the foundation for the later revealing that deoxyribonucleic acid (DNA) is the genetic material. He also shed light on the virulence of micro-organisms (pneumococci). He is indeed a pioneer of genetics, microbiology, and medicine. However, we really do not know much about him. There was very little literature which described him partly because he lived a very quiet and reclusive life. Another factor is that his work was not fully appreciated until many years after his death. It is difficult to construct his com-plete biography.

Life of Frederick Griffith

It is thought that he was probably born in 1879 (the exact year uncertain) in Hale, Cheshire, England. His exact birthday is not known; neither do we know the names and occupations of his parents. He did have an older brother, A. Stanley Griffith (1875–1941), who was also a microbiologist. He attended Liverpool University and graduated in 1901. He then worked for the Liverpool Royal Infirmary, the Joseph Tie Laboratory (some reference said the Thompson Yates Laboratory), and the Royal Commission on Tuberculosis. In 1910, he was hired by the Local Government Board. During World War I (1914–1918), the laboratory was assumed by the national government and became the Pathological Laboratory of the Ministry of Health. He studied pneumococcus (Streptococcus pneumoniae com-monly called pneumococci) that cause pneumonia and in this capacity, he was sent pneumococci samples taken from patients throughout the country. Pneumonia was a terrible disease which would take the lives of both children and adults. He worked under the supervision of Dr. Arthur Eastwood (1869–1936). An enduring friend William McDonald Scott (1884–1941) worked closely with him.

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Although the facilities at that time were very primitive, he was a diligent worker. The laboratory was sparingly supported by the government, but the laboratory was efficiently used by him and his enduring friend William M. Scott. A friend of him wrote of Griffith in the Lancet: “He could do more with a kero-sene tin and a primus stove than most men could do with a palace”.

His approach to microbiological problems remained very fundamental. The pivotal experiment occurred among many performed by Fred Griffith, in 1920s, as medical officer at the pathological Laboratory of the ministry of Health. Attempting to establish greater and more precise scientific understanding, Griffith tried to clarify the epidemiology of lobar pneumonia. He also tested various pneumococci on mice. He sought improved understanding of the pathology enacted by pneumo-cocci on the individual. He discovered some types of pneumo-coccus, such as Type III (smooth form denoted by S), which could cause the disease more readily than others, such as Type II (rough for denoted by R). He found that the difference in the capability of causing disease is due to the polysaccharide coat-ing. The Type III produced a polysaccharide called capsule out of the cell wall, which protected the bacteria from the host defense system (such as the immune response), and was capa-ble of surviving in the host, therefore, causing disease. Bacterial colonies with capsules were smooth looking and were called “S” colonies; whereas the Type II did not produce a polysaccha-ride capsule. They were rough looking and were called “R” colonies. Types II and III were easily distinguished from each other in culture. The recognition of colony morphology differ-ences and their association with virulence of bacteria was a big discovery for both science and medicine. Yet, Dr. Griffith made a further discovery.

Griffith did an experiment that astonished the world. He injected some mice with Type II pneumococcus, and found that the mice did not get sick. He injected the mice with Type III pneumococcus, and the mice developed pneumonia and died.

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As a control, he injected mice with Type III pneumococcus that had been killed. None of the mice developed pneumonia. All these experimental results were as expected. However, when he injected mice with both live Type II and heat killed Type III pneumococcus, the mice not only developed pneumonia, but live Type III bacteria could be extracted from their blood. Somehow, the Type II bacteria had made protective capsules for themselves. They were transformed into Type III. They had acquired the characteristics from the dead Type III bacteria. This phenomenon was called “transformation”. This interesting dis-covery of transformation was published in the Journal of Hygiene, 27, 113–159, in 1928.

The great discovery of Griffith did not receive much atten-tion. Later, researchers managed to obtain transformed bacte-ria in the test tube instead in live animals. There was very little work done in this area. What were the transforming principles? The basic nature of the transforming materials needed to be answered.

In 1944, Oswald T. Avery (1877–1955), Colin Munro MacLeod (1909–1972), and Maclyn McCarty (1911–2005) took up the Griffith experiment again, and tried to explain what the trans-forming principles were. They extracted the active transform-ing principle from Type III (S) pneumococcus and showed that it was DNA. They showed that DNA was functionally active in determining the biochemical activities and specific characteris-tics of pneumococcal cells.

Other finding of Griffith concerned transformation of sero-logical type, a matter distinct from the presence and absence of a capsule. Type is determined by bacterial antigenicity, Bacteriologist Fred Neufeld (1869–1945) of the Robert Koch (1843–1910) Institute in Germany, had earlier discovered the pneumococcal types, and these were confirmed and expanded by Alphonse Dochez (1882–1964) at Oswald Avery’s laboratory in America at the Hospital of the Rockefeller Institute for Medical Research. Types I, II, and III were each a distinct anti-genic grouping, whereas Type IV was a catchall of varying

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antigenicities not matching any of the other three types. Illustrating S. pneumonia’s plasticity, the abstract of Griffith’s paper reports “The S form of Type 1 has been produced from the R form of Type II, and the R form of Type VI has been trans-formed into the S form of Type II”.

To show that DNA is the genetic material is a milestone of life science. DNA is a long macromolecule made of four bases arranged in varying orders. Scientists were not inclined to sus-pect much of the hereditary role of a molecule of DNA. It seemed too simple for such a complex genetic activity at that time. Later, other researchers used radioactive labeling to show that DNA was indeed the hereditary determinant. Griffith had been the first to demonstrate bacterial transformation and his work laid down the foundation for proving DNA as the heredi-tary material. Griffith’s work was, therefore, seen as pivotal in opening the science of molecular biology!

Griffith’s Further Work

In 1934, Griffith published a lengthy paper carrying his finding on the serological typing of S. pyogenes. S. pyogenes is impli-cated in conditions ranging from the usually minor strep throat, to the sometimes fatal scarlet fever, to the often fatal puerperal fever, to the usually fatal streptococcal sepsis. In the 1930s, streptococci infection was already known as a frequent coinfec-tion complicating recovery from lobar pneumonia by pneumo-cocci infection.

Commentary

As mentioned before, we know very little of his private life, except that he enjoyed skiing, walking, and vacations at his country cottage in Sussex. Dr. Griffith had been described as a shy and reticent man, whose quiet kindly manner, and devotion to his job, made him a lovable personality to those who got to know him. He published little. This was because as W. Hayes

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said “ — must be ascribed to an innate humility and capacity of self-criticism”. But his publications are very valuable.

He was killed in 1941 during the bombing of London by the Germans in World War II (WWII) when a bomb blew up the building in which he was working. The exact date of his death was not for sure. The first Griffith memorial Lecture indicates that Griffith died on the night of 17 April 1941, but the fourth annual lecture indicates that he died in his apartment in February 1941 — alongside friend and colleague William M. Scott amid the air raid during WWII London Blitz. At the time, a few weeks after Scott had become director of the laboratory, which had become Emergency Public Health Laboratory Service with the outbreak of WWII.

Griffith’s finding of pneumococcal transformation had drawn little overall notice from the medical sector. Both dated 3 May 1941, his obituary in the lancet mentioned the historical discovery briefly and his obituary in British Medical Journal does not mention it at all.

In 1966, the Griffith Memorial Lecture, published by the Journal of General Microbiology (now Microbiology) was begun, suggesting the relevance of Griffith’s findings not only to steering the hunt for the specific genetic determinant, thereby contributing to molecular biology and molecular genetics, and also to modern microbiology. Yet, Griffith’s main concern, in conducting research and publication was to improve understanding of the epidemiology and pathology of illness associated with pneumococci.

By 1967, pneumococcal transformation had been shown to occur in vivo naturally. It was indicated that treatment with streptomycin during dual infection by two pneumococcal strains could increase transformation, and increase virulence. It is not only medical important, and its impact on ecology and public health is tremendous.

We mourned his death. Indeed, the Griffith’s findings were enduring relevance to epidemiology, pathology, medicine, and of course, and laid down the foundation for Oswald Avery,

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Colin MacLeod, and Maclyn McCarty’s work on identification of transforming principle. He left the world a legacy that led us into a new era of science — molecular genetics.

Suggested Reading

1. Griffith, F. (1928). The significance of pneumococcal types. The Journal of Hygiene 27: 113–159.

2. (1941). Obituary: Frederick Griffith. Lancet pp. 588–589.3. Avery, O. T., C. M. MacLeod, and M. McCarty (1944). Studies

on the chemical nature of the substance inducing transfor-mation of pneumococcal types: induction of transformation by a deoxyribonucleic acid fraction isolated from Pneumococcus Type III. The Journal of Experimental Medicine 79: 137–157.

4. Hayes, W. (1966). Genetic transformation: a retrospective appreciation. The First Griffith Memorial Lecture. Journal of General Microbiology 45: 385–397.

5. Parsons, J. W. and W. K. Myers (1933). Streptococcic sepsis complicating recovery from pneumococcic pneumonia. Journal of the American Medical Association 100(23): 1857–1859.

6. Collard, P. (1976). The Development of Microbiology. Pp. 97–109. Cambridge University Press, Cambridge.

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Chapter 32

Alexander Fleming (1881–1955): The Discovery of Penicillin

“Professional jealousy can be a most brutal and terrifying phenomenon”.

— Alexander Fleming

Source: https://en.wikipedia.org/wiki/Alexander_Fleming(US Public Domain image)

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Introduction

Molds had been used for therapeutic purposes centuries ago. Ancient Chinese used molds to treat boils and infected wounds, whereas Indian physicians used them to treat dysentery. Ancient Mayan tribes used a cuxum fungus as a therapeutic agent. American Indians dressed war wounds with molds. Hippocrates (460–377 BC), the father of medicine, described the use of mold to treat infected female reproductive organs. Molds were widely used for virtually every disease in the medieval era. They worked! Who cared why they worked?

Antibiotics are secondary metabolites of microorganisms (including molds). Bartolomeo Gosio (1863–1944), in 1896, first discovered an antibiotic which was named mycophenolic acid by Carl Lucas Alsberg (1877–1940) and O. F. Black in 1912. But this antibiotic was not widely used medically. An important medically widely used antibiotic is penicillin. The discovery of penicillin marks the beginning of an important era of microbi-ology and modern medicine, and has a great impact on our civi-lization. It is, therefore, of great interest to know the key person involved in this discovery — Alexander Fleming.

Have you ever wondered what you may have been growing in your refrigerator when you pull out a piece of cheese and find that it has turned into a hairy green piece of fuzz! You might also experience surprise to find that a loaf of bread has dramatically formed itself into another ugly black or green form of life! How in the world could anyone become curious about something so ugly? When you find moldy, transformed pieces of food in your refrigerator, think of what Alexander Fleming was looking at when he walked into his laboratory only to find that he had a couple of culture plates with green fuzzy mold, instead of neat bacterial cultures.

Life of Alexander Fleming

Alexander Fleming was born on 6 August 1881 in Lochfield, Ayrshire, Scotland. His father was Hugh Fleming (1816–1888), a

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Lowland farmer. Hugh Fleming married his first wife Mary Craig and had four children: Jane (born in 1862), Hugh III (born in 1864), Thomas (born in 1868), and Mary (born in 1874). Mary Craig died during childbirth with her fifth child who also died. At the age of 60, Hugh Fleming remarried to Alexander’s mother Grace Morton (1848–1928). In addition to Alexander, Grace Morton gave birth to Grace (1877), John (born in 1879), and Robert (born in 1883). Therefore, he was one of the eight children in this family, and there was no telling then what this young boy who grew up on a farm would become.

We can imagine that Alexander (Alex) and his brothers and sisters explored the valleys and moors there in Scotland during their childhood. He got the chance to observe nature inti-mately. Since he was poor, he enjoyed simple pleasures. At a very young age, he participated in the annual sheep shearing by helping to round up the animals. His father passed away when he was seven. Between the ages of 5 and 10, he attended a tiny moorland school. Then he attended a school of Dorval, a small town, 4 miles from his home, and he had to walk to school every day. He went to school in all kinds of weather. When it was cold, he carried hot potatoes to keep his hands warm, and later he would eat them for lunch. When it rained, he took a change of boots and stockings slung around his neck. When the weather was good, he went to school barefooted. Later, he attended Kilmarnock Academy, which was 12 miles from home. At the academy, he took courses covering inorganic chemistry, physiography, physics, and physiology. He had a very sincere respect for learning, and he always received top grades. Fleming had a solid basic education, a prodigious memory, and a tempered disposition. He was very quiet and kept his personal life to himself.

When just past 13, he and his older brother John went to London to follow his stepbrother Thomas who practiced medi-cine there. His younger brother Robert also joined them a little later. His only sister was to keep house at home.

In London, Alexander attended classes at the Regent Street Polytechnic for 2 years, and then became a clerk in a shipping

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company, the American Line. He did not like the job, but he performed well his assignments. In 1900, he enlisted in the London Scottish Regiment but he did not go overseas as the Boer War (1899–1902) ended. He was good at rifle shooting, enjoyed the military, and stayed attached to this regiment until 1914.

In 1901, at the age of 20, his uncle died, left him a small legacy, and his brother Thomas encouraged him to study medi-cine. He passed the competitive examination and enrolled at St. Mary’s Hospital School from which he received a scholarship. Alexander was a bright student and rewarded for his academic achievements with scholarships and prizes.

While at St. Mary’s, he took biology from Dr. William G. Ridewood, a distinguished zoologist and Director of the Natural History Department of the British Museum. He took chemistry from Sir William Willcox, a well-known toxicologist. Fleming also enjoyed anatomy and became a student demon-strator in 1903, received the senior anatomy prize in 1904. As he became a very keen observer, Alexander had become a naturalist with the habit of noticing everything that went on around him.

During his years in school, Fleming was slow in making friends because of his shyness and reserve. Nevertheless, he belonged to the St. Mary’s Hospital School Amateur Dramatic Society. On one occasion, he played the part of a woman, a sprightly French “widow”. He did a very good job on the play and was highly praised. Also, he was a member of the Medical and Debating Societies. Not forgetting sports, he joined the rifle team and water polo team.

Despite his impenetrable silence and shyness, he enjoyed companionship and liked competition. Fleming would always win in every competitive test that they would enter. He won the competition in chemistry and biology as well as anatomy.

A significant event while a student at St. Mary’s was that he met Sir Almroth E. Wright (1861–1947), a pioneer of the vaccine treatment of disease in 1906. The same year, he obtained the Conjoint Board Diploma. He began to work in the “Inoculation

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Department” which was under the directorship of Sir Wright. He was paid 100 pounds a year. The Department was in a small room and the laboratory equipment was rudimentary. It had an incubator, a sterilizer, some Petri dishes, a few test tubes, and a microscope. The department had 8–9 graduates and many visit-ing doctors.

During this period of time, he spent a lot of time studying phagocytosis. He put a drop of blood containing phagocytes onto a test microbe on a slide with a test microbe and covered it to prevent it from drying. The blood was spread into a thin film, dried, and then stained. In this way, the phagocytes were easily seen, counted, and compared. Another attraction of interest to Fleming was therapeutic vaccination, the treatment of acne. He examined the specific organism’s acne bacillus (Propionibacterium acnes) and made a vaccine from a culture of this organism. He studied the therapeutic effect of this vaccine and published a paper in the Lancet in 1909. In 1908, he gradu-ated MBBS with honors and a gold medal from the University of London. In 1909, he published another paper on “The diag-nosis of acute bacterial infection” in St. Mary’s Gazette, which won him the Cheadle Medal for Clinical Medicine awarded by St. Mary’s Hospital. Starting in 1908, Fleming was also the Casualty House Surgeon while still working in the laboratory of Wright. He gained experience by performing minor surgery and assisted in major surgery. Fleming never truly practiced surgery, but in 1909 he completed the fellowship examinations of the Royal College of Surgeons of England.

From his thesis on bacterial infections, we can picture the line of research that he followed during his life. Fleming sought to find a way to fight the infections that were considered the most dangerous to the human race. He was well equipped for this line of research and was fully conscious of his abilities.

Wright proposed specific immunization against bacterial infections and demonstrated the presence of opsonins and antibodies in the blood. Although Fleming upheld and prac-ticed the principles of Wright, he later questioned “What was

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the point in adding dead microbes (as vaccine) to a body which was already carrying on battle against living ones?” He sug-gested that intravenous administration was not suited to the injection of a vaccine. He once injected himself intravenously with millions of dead staphylococci, and experienced nausea, fever, and headache. He concluded that inoculation of dead bacteria into the blood stream produced the maximum toxic effect with minimum immunization benefits. Fleming also was among the first doctors to treat syphilis with Salvarsan (com-pound 606) discovered by Paul Ehrlich (1854–1915). He invented a very simple apparatus, two glass jars, a syringe, two rubber tubes, and two taps with double nozzles. This apparatus made it possible to treat four patients at the same time. He became known as “Private 606” and had an enormous number of patients.

Although Fleming was busy as a bacteriologist at St. Mary’s, he still made time for some social activities. He joined the Chelsea Arts Club. He went to the club whenever he had time, and he attended the Chelsea Ball on several occasions.

When World War I (1914–1918) broke out, Fleming joined the Royal Army Medical Corps as lieutenant. He served under Colonel Wright in a wound-research laboratory in Boulogne, France. During this period, he made a careful study of new wounds of soldiers. He took swabs from all wounds, examined and identified the bullets, shell fragments, shreds of clothing, dead tissue, and bits of bone removed by the surgeons. He cul-tured and identified the most common infecting organisms. He found that men’s clothing was a major source of infection. These findings resulted in two papers published in the Lancet in 1915.

In 1919, he was demobilized from the Army with the rank of captain, and immediately returned to St. Mary’s to continue working on antibacterial mechanisms. Through sharp observa-tions, pertinacious curiosity, and prepared mind, he observed two important antibacterial effects. In 1921, he noted that nasal mucus dissolved a yellowish colony. This was recorded in

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his notebook labeled XVIII, 16 March 1921. Fleming took three cultures of cocci isolated from his own nose and streaked across a blood agar plate. He then took some of his nasal mucus, diluted with saline and centrifuged it, and placed a drop of the clear fluid (supernatant) on one of the three cultures. After 18 hours of incubation, he noticed that there was no growth in the vicinity of the mucus whereas other plates without mucus had heavy growth. He recorded that “obviously something had diffused from the nasal mucus preventing the germs from growing near the mucus, and beyond this zone killing and dis-solving bacteria already grown”. He continued to study the effect of mucus on bacteria, and found some bacteria were sensitive to it, but some were not. On 21 December 1921, he presented his findings to the Medical Research Club in the hos-pital. However, because of his poor presentation, the audiences were not impressed and left with the feeling of “so what?”

He named the bacteriolytic agent lysozyme and the suscep-tible organism (his nose cocci), Micrococcus lysodeikticus. In collaboration with Dr. V. D. Allison, Fleming detected lysozymes in human blood serum, tears, saliva, milk, leucocytes, egg white, and turnip juice. He also engaged many studies involved with lysozymes. He showed that lysozymes were very useful for bacterial cytology and other studies. Although it is a very sig-nificant finding, the finding did not attract much attention from the scientific community, but in 1922, Wright nominated Fleming for fellowship in the Royal Society.

He continued to work on antibacterial mechanisms. In 1923, he tested the antibacterial activity of more than 20 different antiseptics used by surgeons. He noticed that antiseptics seemed to promote infection instead of fighting infection. He condemned the intravenous administration of chemical anti-septics, and asserting that ideal therapeutical antibacterial agents should arrest the growth of bacterial invaders without affecting the host tissues. Now more than ever, Dr. Fleming was determined to find a bacterial inhibitor not harmful to animal or human tissues.

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One morning in the fall of 1928, Fleming noticed a culture plate displaying a colorful green mold. The culture plate was covered with staphylococci except in the vicinity of the mold, which was near the edge of the plate where there was a trans-parent zone of inhibition. The bacteria that were close to the mold had not grown. Fleming photographed the plate and made it permanent by exposing it to formalin vapor which killed and fixed both the bacteria and the mold. (The original plate is now in the British Museum.) Being a person that thrived on curiosity, he found the culture filtrate of the mold would kill the bacteria. Fleming investigated a list of microorganisms that were inhibited by the mold; among them were streptococci, staphylococci, pneumococci, and meningococci. Fleming identi-fied the mold as a Penicillium that Charles Thom (1872–1956) placed in the species notatum, which is similar some molds grown on bread. Fleming called the active filtrate penicillin. He also discovered that the penicillin was not a protein. He proved that the penicillin was harmless to the leucocyte and would help leucocytes fight against infections. In 1929, he reported that the penicillin was nontoxic to laboratory animals, and might be an efficient antiseptic for application to, or injection into, areas infected with susceptible microbes. He did many experiments with this mold, but he found it hard to isolate the active antibacterial substance even with the help of a biochem-ist H. Raistrick. At that time, no one seemed ready to seek more information on penicillin.

Gerhard Domagk (1895–1964) working for I. G. Farben indus-tries in Germany, was testing new dyes for possible medical applications. In 1932, he found that an orange-red dye, Prontosil, was effective against streptococcal infections in mice but was inactive in vitro. In France, Jacques Trefouel (1897–1977) and coworkers demonstrated that the antibacterial activ-ity of Prontosil was due to part of its molecule sulfanilamide (1935). This opened the door to the synthesis of numerous sul-famides, some of which are still widely used. Fleming also stud-ied the antibacterial properties of sulfamides, although he

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never lost confidence that mold juice could be purified and useful.

It was around 1939 that Dr. Howard Walter Florey (1898–1968) from Oxford University became interested in investigating the Penicillum mold Dr. Fleming had given him. Along with Ernst Boris Chain (1906–1979) who came from Germany and other assistants, Dr. Florey worked diligently on Penicillium notatum.

Florey and his coworkers had little knowledge of how P. notatum worked, but he mixed it with a sugar solution. The mold flourished, forming a mat, and soon golden droplets developed on its surface. The droplets were then dried to a yellowish-brown powder, which was usable form of penicillin. They injected mice with a strain of deadly streptococci. Half of the mice were left untreated and half were injected with peni-cillin at different intervals. All the untreated mice died over-night and all the treated mice survived.

Penicillin was first used on 12 February 1941 on a man that was dying of a blood infection. The drug was used for 5 days and the young man appeared to be feeling much better, but the limited amount of penicillin available did not cure the infection and the man died.

With an increase in supply, Florey’s cases then began to improve. A young girl who was dying of gangrene was cured and so was a man dying from a bone infection.

In the United States, Dr. Wallace E. Herrell (1909–1992) and other doctors were interested in penicillin at the Mayo Clinic in Rochester, Minnesota. Florey visited them and brought 50 mg of penicillin as a gift. Their clinical trials were successful.

The biggest problem was how to produce large quantities of penicillin. American microbiologist Dr. Andrew J. Moyer (1899–1959) devised methods for producing penicillin in large scale. The mounting war casualties entailed securing the high-est priority for its large-scale manufacture, and steps were taken to achieve this in the United States, Britain, and Canada.

In the United States, the mass production of penicillin led by Dr. Chester S. Keefer (1897–1972), chairman of the National

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Research Council’s Committee on Therapeutic Agents and direc-tor of the Evans Memorial Hospital in Boston. Having the reas-surance of the clinical success made by Keefer, the War Production Board took penicillin under its wing in May 1943, and hired Fred Stock to take charge of the mass production of it. In less than a year, more than 300 billion units of penicillin were available.

As supplies of penicillin increased and its efficacy became widely known, Alexander Fleming became more recognized. In 1943, Fleming was elected fellow of the Royal Society. In 1944, he was knighted (KB) and awarded the Moxon Medal by the Royal College of Physicians. Both Florey and Fleming received the Award of Distinction from the American Pharmaceutical Manufacturers Association the same year. In 1945, Alexander Fleming, Ernst Chain, and Howard Florey received the Nobel Prize in Medicine and Physiology. Fleming also was awarded numerous foreign decorations and medals, honorary member-ships in medical and scientific societies, and was decorated by several famous universities. He was elected president of the newly founded Society of General Microbiology in 1945.

It was in August of 1945, that Fleming came to the United States. His purpose was to see the advancements that Americans had made with penicillin. He visited many hospitals and several laboratories. Many people followed this quiet, laconic, and mysterious man, but they were unable to uncover any interest-ing facts about his past life.

Dr. Fleming was married on 23 December 1915 to Sarah Marion McElroy, an Irish farmer’s daughter, who operated a private nursing home. They had a happy marriage despite dis-similar characters. Sarah was more outgoing whereas Alexander was quiet. She often spoke for both of them. Sarah’s twin sister Elizabeth was married to Alexander’s brother John. So there were two bonds between the Flemings and the McElroys. Alexander and Sarah bought a house called Dhoon. It was a Georgian house with a red-tiled roof, built of lath and plaster on a timber frame. It had six bedrooms and three living rooms

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and a 3-acre garden, overlooking the river Lark. The river was big enough for boating and fishing, which Alexander enjoyed and they both enjoyed gardening. The Dhoon was Sarah’s main interest and Alexander’s main relaxation. In a few years, they added a croquet lawn and a tennis court. Fleming enjoyed cro-queting, tennis, fishing, boating, swimming, and playing bil-liards and snooker. They had one son, Robert, born on 17 March 1924 who became a physician. The family spent a lot of time at the Dhoon during the summer along with nephews and nieces. They were often surrounded by children. At Christmas, they had huge family get together. The family remained close. Unfortunately, Alexander’s mother passed away in 1928, and Alexander’s brother John died unexpectedly in 1929. The worst was the death of Sarah in 1949, which was a big blow to Fleming. He became very withdrawn. He devoted himself even more to his laboratory.

Fleming traveled throughout the United States, where he received many awards from several institutions and universities. President Franklin Delano Roosevelt (1882–1945) paid a tribute to this perspicacious scientist.

Even though penicillin was well accepted and used over-seas, the United States was the mass producer of the drug itself. The manufacturers of penicillin in the United States formed the Alexander Fleming Fund. The money went to St. Mary’s Hospital in London for Dr. Fleming to continue his research.

As the years passed, he recovered from the loss of his wife. He was elected rector of Edinburgh University in 1951. He also began to work closely with a female doctor by the name of Amalia Coutsouris-Voureka from Greece. She spent long hours in his laboratory, mastering the science of bacteriology. She decided to continue her studies in Athens, and Dr. Fleming fol-lowed her. They were married in 1953. Friends of Fleming remarked that he was a new man after his marriage. Back in London, Fleming developed a cold, a few days later he died of a heart attack on 11 March 1955. He was buried in St. Paul’s Cathedral.

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Fleming had a long association with the St. Mary’s Hospital. Fleming was assistant director of the Inoculation Department in 1921. The Department merged with the Institute of Pathology and Research in 1933, and it was renamed the Wright-Fleming Institute in 1948. In that year, Fleming retired as professor of bacteriology from the University of London, where he held the chair since 1928. He succeeded the principalship of the Institute in 1946 when Sir Wright retired. He retained the principalship until January of 1955, 2 months before his death.

Commentary

Fleming was a very modest man. He accepted the honors and acclaim diffidently. He once said: “Professional jealousy can be a most brutal and terrifying phenomenon”. Although he became known worldwide, he remained a man of humility who was very quiet and kept his personal life to himself. He enjoyed an evening game of snooker at the Chelsea Arts Club, where he held a long-treasured honorary membership. He had a pawky humor about him, and the basic dedication to the health of mankind. He was the sole or senior author of about 100 scien-tific papers. Among Fleming’s best papers were those prepared as addresses for endowed lectureships. He also published novel techniques or wrote brief biographic memoirs. He was not interested in philosophy or rhetoric. His dedication to science and human health is fully reflected in his writings. A collection of Fleming’s published work and unpublished manuscripts, including letters, diaries, and laboratory notebooks along with other documents are available from the Wright-Fleming Institute.

Penicillin was merely the first of a long list of antibiotics that were proven to have antibacterial qualities. There are problems with penicillin. It is only effective in fighting infec-tions caused by streptococci, staphylococci, gonococci, pneumo-cocci, bacterial endocarditis, and gas gangrene bacilli. It is powerless against malaria, tuberculosis, typhoid fever, and viral

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diseases such as influenza and yellow fever. It caused allergic reactions in certain patients. But it was the first drug in demand that revolutionized the pharmaceutical industry and laid the groundwork for the antibiotic descendants to continue to fight against microbial infections. Fleming’s work inspired many pharmaceutical companies and research institutions to investi-gate the production of antibiotics from different microorgan-isms. Many new antibiotics were later found to be very useful in combating diseases. Antibiotic production today is estimated at a value of more than $16 billion per year. To call Fleming a pioneer for the antibiotics era is very appropriate.

As Andre Maurois (1885–1967) put it, Fleming was gifted with steadfastness and loyalty which created respect and affec-tion. He also possessed the quality of hard and sustained work, a true humility, and a combative spirit which refused to admit defeat. Fleming also had a keen curiosity and perceptiveness, an excellent memory, the technical inventiveness and skill of a highly artistic order, and the mental and physical toughness, that is, characteristic of a great man in many walks of life. Fleming was attentive to things that many other scientists would probably have merely overlooked. Therefore, above all the characteristics mentioned, Fleming kept his eyes and mind open, and he was always prepared for something significant. Success always is given to those who are prepared. Who would care about anything as ugly as a green mold on an apple or a piece of bread? Yet it proved to be the source of life saving antibiotics. The lesson of Fleming is a worthy learning for all microbiologists.

Suggested Reading

1. Maurois, A. (1968). The Life of Sir Alexander Fleming. E. P. Dulton and Co. Inc. Publishers, New York.

2. MacFarlane, G. (1984). Alexander Fleming: The Man and the Myth. Harvard University Press.

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Chapter 33

Albert Jan Kluyver (1888–1956): Unity of Biochemistry and

Pioneer of General Microbiology

Source: https://en.wikipedia.org/wiki/Albert_Kluyver(US Public Domain image)

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Introduction

In the Society of American Bacteriologists at its 1949 National Meeting, there was an “A. J. Kluyver Lecture” entitled “The Dutch School and The Rise of General Microbiology”. In this review, the contribution and profound influence of A. J. Kluyver to General Microbiology was described in detail. Here were reviewed the history of the development of General Microbiology and also was given a glance at this key man, Dr. Albert Jan Kluyver. There may be lessons we can learn from him. Perhaps, we could also appreciate more the growth of microbial knowledge which has resulted from such human endeavors.

Albert Jan Kluyver was born on 3 June 1888 in Breda, the Netherlands. His father was Jan Cornelis Kluyver, an engineer who was later professor of mathematics at Leiden. His mother was Marie Honingh. He entered the Technical University of Delft in 1905, and obtained his first degree of Chemical Engineering with distinction in 1910. He worked as an assistant to Professor Gerrit van Iterson (1878–1972), a well-known bota-nist, from 1909 to 1914 with an interim in 1911 when he spent a year in Professor Hans Molisch’s (1856–1937) Institute in Vienna. In 1914, he obtained the degree of Doctor of Science. His doctoral dissertation was on the development of methods for the quantitative determination of individual sugars in mix-tures, which was published in Biochemische Suikerbepalingen. Because of the outbreak of the First World War, this young doc-tor was inducted into the Dutch Army, where he spent more than a year. As soon as he was released from military duty, he resumed his work in Dr. Van Iterson’s laboratory until 1916, when he accepted a position as a consultant for the Dutch East Indies to the Department of Agriculture, Industry, and Commerce. In the same year, he married Miss Helena Johanna Van Lutsenburg Mass, whom he met at the Delft School.

While working in the Dutch East Indies, he was given a special assignment to make a detailed study of the copra fiber industry

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in Ceylon and Malabar, which resulted in a 300-page report, pub-lished in 1923, “Klaervezel- en Klappergarennijverheid”. In 1920, Kluyver became Director of the research laboratory of a large East Indian Concern. The authority there planned to appoint him to a scientific position at the Dutch “Colonial Institute”, but this never materialized. In 1921, his career changed and he was offered a professorship at the Delft Technical University to suc-ceed Dr. Martinus W. Beijerinck (1851–1931) who retired that year because of age. This was a big surprise and also caused him considerable hesitation because he feared that his own limited training in microbiology might make it impossible for him to do the job, especially as Dr. M. Beijerinck was known as an interna-tional scientist. Dr. van Iterson was deeply impressed with Dr. Kluyver’s scientific ability and personal characteristics, and strongly recommended him to take the post and he finally accepted the position. This started his eminent career as a teacher and founder of general microbiology for his own generation and generations to come. He held this position until his death on 13 May 1956.

The contribution of Kluyver to microbiology might be per-ceived in his inauguration speech on 18 January 1922 at Delft School. In this speech, he predicted that the human race would be facing a serious problem resulting from the depletion of the limited supply of fossil fuels, the main source of energy for the world’s rapidly expanding industry. He foresaw that human beings might someday be in a position to utilize atomic energy. He also pointed out that the application of microorganisms would be a great service in this respect. He mentioned the manufacturing of alcohol by fermentation of carbohydrates as a potential means of supplying fuel for internal combustion engines. Many products such as glycerol, butanol, acetone, fats, and proteins, could be produced by microorganisms from cheap and readily available sources of agriculture were raw materials that would also serve as energy sources. Others such as large scale production of lactic acid, citric and fumaric acids with the aid of microbes were further possibilities.

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His vision in 1922 was largely realized by the development of human civilization over the last 75 years. In this regard, the Delft laboratory under his direction was busily engaged in detailed studies of several types of biochemical transformation by various types of microorganisms with result that more than 200 publications came from his laboratory. His approaches resulted in paramount significance. One example was that he made the first investigation of Acetobacter suboxydans and recognized its importance in the production of sorbose, an intermediate stage in the commercial manufacture of ascorbic acid. He developed the ties between theoretical and applied microbiology. He also had evolved a concept of “unity in bio-chemistry”, in which he interpreted that biochemical processes as the net result of more or less extended chains of simple and chemically intelligible step reactions, each one representing an (enzymatic) transfer of hydrogen from a donor to an acceptor. In due course of implications of the “unity concept” were envis-aged, yielding equally important principles of “comparative biochemistry”, which permit inferences concerning special bio-chemical events to be drawn from studies on processes which at first sight appeared totally unrelated. These two ideas guided his research and had inspired many impressive investigations, worldwide, during the Kluyver’s life time.

Kluyver’s concepts gave a great impetus to biochemical research. The sound valuation of their scope can be seen in a small book entitled The Chemical Activities of Microorganisms, published by the London University Press, and also in the first two chapters of The Microbes’s Contribution to Biology (Harvard University Press, 1956).

Another important direct contribution of Kluyver in science is his introduction of the submerged culture technique. In 1932, Kluyver collaborated with L. H. C. Perguin on the submerged cultivation of molds. This has produced many other publica-tions on this subject. This technique was not only applied to the study of mold biochemistry but later was applied to the manu-facturing of riboflavin and penicillin, and still other antibiotics,

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from cultures of molds and actinomycetes. Kluyver’s study cov-ered a wide range of subjects in the field of biochemistry, tax-onomy, microbiology of foods, brines, rumen, and other specialized environments. His many series of monographs on yeasts represented an exhaustive survey of the literature and a meticulous comparative study of the prodigious collection of yeast cultures. The collection of yeast cultures at the Delft Laboratory in collaboration with the “Center Bureau of Fungus Culture” were probably the best culture collection of yeast in the world at his time. He was also interested in problems con-cerning the morphology and development of microorganisms. During World War II, he used electron microscopical techniques to study the microbial morphology. He was also interested in classification and he was the member of the International Commission for Nomenclature of the International Congress for Microbiology.

In addition to this, Kluyver was an excellent teacher. His lec-tures were stimulating and inspirational; he educated many good investigators. An excellent student, Dr. Cornelis B. van Niel (1897–1985), later migrated to the United States and brought the Delft School of Microbiology with him to this fertile land, which resulted in the blossoming of General Microbiology in this century. Furthermore, he was a man of noble wisdom. His knowledge of the problems and the freedom of science and his deep conviction of the supreme importance of the uniqueness of the individual, are truly noble assets to science and humanity.

He worked hard and was devoted, but his last 15 years of life (1941–1956) were not happy ones. This was because the war had left wounds that never quite healed, his health was pre-carious, and his wife died in 1952 which was a big blow to him since she was his deep, understanding, devoted companion, and friend for 36 years. He often stated “This is a great and ter-rible world”. He became more pessimistic and more fearful for the failure of humanity. Therefore, he took little interest in his health although he was advised by his medical doctor. He suf-fered from angina pectoris. He worked strenuously seven days

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a week from 9 a.m. to midnight or later; when he died of heart attack on 13 May 1956, he had two sons and three daughters.

Kluyver received many honors. He was elected member of the Royal Netherlands Academy of Sciences in 1926. He served as president of Natural Science section of this organization from 1947 to 1954. He was a member of the executive council of the Netherlands Reactor Center. He was also active in Biophysical Research Groups Delft-Utrecht — an organization dedicates to investigate biophysical problems. He was one of important figures responsible for the progress of science in Holland.

As van Niel put it: “Kluyver has erected a monument to the power of intelligence, guided by great compassion, and cou-pled with undeviating dedication to his work; and who in con-sequence has engendered more inspiration and devotion than falls to the lot of most people in a comparable situation”. He has much for us to learn, which he helped not only in microbiol-ogy, but humanity as well.

Suggested Reading

1. Kluyver, A. J. (1961). Unity and diversity in the metabolism of microorganisms. In T. D. Brock. Milestones in Microbiology. American Society for Microbiology, Washington, D.C.

2. Van Niel, C. B. (1956). In Memoriam: Prof. Dr. A J. Kluyver. Antonie Van Leeuwenhoek J. Microbiol. Serol 22: 209–217.

3. Van Niel, C. B. (1949). The Delft School and the rise of gen-eral microbiology. Bacteriology Review 13: 161–174.

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Chapter 34

Gerhardt J. Domagk (1895–1964): Pioneer of Sulfur Drug Chemotherapy

“I am convinced that before long we shall dispose of effective agents against cancer — I hope I shall live to see the victory over cancer”.

“I made all the decisive tests myself until I was quite certain as what was right”.

— Gerhardt J. Domagk

Source: https://en.wikipedia.org/wiki/Gerhard_Domagk(US Public Domain image)

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Introduction

The above statement by Gerhardt J. Domagk was not realized, but Domagk was known for his discovery of the chemothera-peutical action of Prontosil (a sulfur drug) in various infectious diseases. He left the legacy to the world and he opened a new era of medical therapy. For this work, he was bestowed with the Nobel Laureate list of Medicine or Physiology in 1939, although was never received the prize. His discoveries have saved the lives of countless people and contributed greatly to the world of microbiology and medicine.

Life of Gerhardt J. Domagk

Gerhardt Johannes Paul Domagk was born on 30 October 1895, in a small town called Lagow in Brandenburg, which was then part of Germany, and is now in Poland. His father was a teacher called Paul Domagk, and his mother was named Martha Reimer. They lived in a schoolhouse in Sommerfeld, a small town, which existed primarily because of textile industry, where his father Paul was assistant headmaster of a school in Sommerfeld. Gerhardt Domagk attended this school, which specialized in science instruction, until he turned 14. Then he transferred to the secondary education school at Liegnitz in Silesia. In 1914, he commenced his study in medicine at the University of Kiel. The outbreak of the World War I (1914–1918) interrupted his education. He volunteered with the Leibgrenadier regiment of Frankfurt on the Oder. Later, he went to Flanders and saw action in the battle of Langemarck. In December 1914, he was transferred to the Eastern front of the conflict and was wounded in battle. After, he joined the Sanitary Service, serving as a medical officer throughout the remainder of the war. He worked in the cholera hospital of Russia, and assigned to the medical corps Verdun, France until the end of war. He experiences the powerless of medical treat-ment of bacterial infections.

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After the war, he resumed his study of medicine and received his doctorate in 1921. For a while, Domagk stayed at Kiel with doctors Hoppe-Seyler and Emmerich, and assisted them in studies on different organs and tissues, which played a part in the manifestation of different diseases. He also worked at the Pathological Institute in Greifswald. In 1924, at the age of 28, Domagk was qualified as “Privatdozent“ (University lec-ture) for general pathology and anatomy at the University of Greifswald. He gave a dissertation titled “Investigation of the significance of the reticulo-endothelial system for the destruc-tion of infecting microorganisms and the formation of amy-loid”. For this title, we can see that he was already interested in the sterilization of tissues with bacteria. However, he sought after the principal factor as being in the body’s defense mecha-nisms at this time. He was also interested in the effect of certain types of radiation upon kidney ailments and some forms of cancer. Next year (1925), he moved to the University of Munster, which appointed him to the same position. Also in 1925, he married Gertrude Strube, and together, the couple eventually had four children — three sons and one daughter.

At the University of Munster, Domagk done his studies of cancer, but these studies never resulted in any major break-through. From 1927 to 1929, Domagk, took leave of absence to conduct research at I. G. Farbenindustrie (Bayer), in Wuppertal-Elberfeld. In 1928, he was promoted to professor of General Pathology and pathological Anatomy by the University of Munster. He held this position until his death in 1964.

While still in leave of absence, in 1929, the I.G. Farbenindustrie established a new research institute for pathological and bacte-riology, and appointed Domagk as its director of research in experimental anatomy and bacteriology. Motivated by his war time inability to treat bacterial infections, Domagk focused his investigation on finding antibacterial agents, first in vitro, or in test tubes, and then in vivo, or in living organisms, such as mice and rabbits. He researched different dyes for antibacterial activity. Domagk was neither a chemist nor a pharmacologist;

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he did not synthesize any compounds, but he was untiringly in his search for active substances that would kill bacteria. One of the great achievement of Domagk was the discovery of the dye now known as Prontosil, which exhibits a strong antibacterial activity. The compounds were submitted to him for testing by two chemists, Dr. Fritz Mietzsch (1896–1958) and Dr. Joseph Klarer (1898–1953), who were working with him. This was sev-eral days before Christmas in 1932, Domagk brought the impor-tance to the world attention shortly afterward.

Prontosil Rubrum (Prontsil Rubruym was the original name, it was later abbreviated to Prontosil [C12H13N502S]) was a red coal-tar dye used on leather, had already been known for 20 years and could be produced in the laboratory. Domagk discov-ered that Prontosil exhibited effect against bacteria in test tubes and proved nontoxic to mice. He conducted an experi-ment whereby he injected 26 mice with a hemolytic streptococ-cal bacteria culture, then injected 12 mice with a single dose of Prontosil Rubrum an hour and a half later. The 14 control mice, which did not receive the potential antibacterial agent, all died within four hours as expected. The 12 treated mice, on the other hand, all survived. Further, Domagk discovered that Prontosil Rubrum affects streptococci in vivo, but not in vitro. For reasons unknown, Domagk waited three years before his findings. During this period, a very dramatic (but not published story) was that Domagk tested this compound on his four-year-old daughter. In December of 1933, Domagk’s four-year-old daughter (Hildegarde) contracted a streptococcal infection at his laboratory when she was accidently pricked with a needle; after making 14 incisions, the surgeons could find no other solution than to recommend to the parents that they have the arm amputated. At this moment, Domagk himself intervened in the treatment. Domagk injected her with a dose of Protonsil Rubrum, and she recovered. He finally published his results in a paper titled “Ein Beitrag zur Chemotherapie der bakte-riellen Infektionen” in 15 February 1935 of the German journal Deutscghe Medizinische Wochenschrift (but not mentioning

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anything about his daughter.) The news spread, and many other researchers began to experiment with Prontosil. Later in 1936, French scientist Thérèse Tréfouël (1892–1978) discovered that Frontosil was metabolized into sulfanilamide (para- aminophenylsulfonamide) and identified the sulphonamide group as the active ingredient in Protonsil. Ensuing studies dis-covered that this treatment does not, in fact, kill bacteria, but rather prevents the bacteria from reproducing by blocking metabolism. Sulfanilamide derivatives proved effective against pneumonia, meningitis, blood poisoning, and gonorrhea. Domagk’s discovery thus transformed the medical fields, empowering the medical doctors to treat bacterial infection. Because the active gradient sulfanilamide contains sulfur in the structure, sulfur drugs thus gain its name, and became the major antibacterial agent in that era.

In 1939, Domagk published a book titled Chemotherapire bakterialer Infektionen, which summed the results of much of the studies concerning antibacterial drugs up to that time. Also in 1939, the Royal Swedish Academy of Sciences decided to award the Nobel Prize for Medicine or Physiology to him after realizing the importance of the effects of sulfanilamide, but Adolf Hitler (1889–1945) had forbidden German citizens from receiving the prize. In fact, the Nazi government arrested Domagk and he was confined for over a week in prison when he informed it of his honor. Not until 1947, after World War II (1939–1945) had ended. During the World War II, Domagk’s mother died of hunger in refugee camp in 1945. Domagk did receive the medal, but the prize money had reverted to the Nobel Foundation.

At the same time, Domagk also worked to Vitamin A. Later, Domagk turned his attention to a disease, which had been little affected by sulphonamide; this disease was tuberculosis. In 1940, Domagk reported the discovery of a compound sulfathia-zole, which has antitubercular effects. In the next few years, he isolated other compounds that were effective in destroying tuberculosis bacteria, even those strains of bacteria, which are resistant to antibiotics streptomycin. Domagk published another

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book titled Chemotherapie der Tuberculosis mit den Thiosemikarbonazonen in 1950. In this book, he covered much information about tuberculosis. In 1952, he discovered another drug, Neoteben, which is effective in fighting tuberculosis.

The later years of Domagk were devoted to seeking a chem-otherapeutical agent against cancer. Although he was a confi-dent that he would be able to find a weapon against cancer as he said “I am convinced that before long we shall dispose of effect agents against cancer — I hope I shall live to see the vic-tory over cancer”. In this hope, Domagk was disappointed, because he retired in 1958, and died on 24th April 1964 without having discovered the desired remedy. He left that work for us to accomplish.

Domagk lived a rich life with his wife Gertrude. They appar-ently had a happy family. Domagk received many honors dur-ing his life time. Beside the Nobel Prize, he received numerous other honors. He was awarded the Emil Fischer Memorial Plaque by the German Chemical Society in 1937. The Cameron Prize was awarded to him by the University of Edinburgh in 1938, and the Klebelsberg Prize by the University of Szeged, Hungary, also in 1938. He was knighted in the Order of Merit in 1952 and was awarded the Grand Cross of the Civil Order of Health of Spain in 1955. The University of Frankfurt granted him its Paul Ehrlich Gold Medal and Prize in 1956. The Royal Society of London and the British Academy of Science inducted him into their fellowships in 1959, and the Japanese govern-ment bestowed on him its Order of Merit of the Rising Sun in 1960. He received honorary degrees from the University of Munster, the University of Bologna, and the University of Limas, Cordoba, and Buenos Aires.

Commentary

The Prontosil is a kind of azo dyes, which are widely used in industry. A large amount of these dyes are discharged into the environment. Some of these compounds may accumulate into

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food chains and eventually reach the human body through ingestion. One of the authors, K. T. Chung demonstrated that intestinal microbiota and to a lesser extent, the liver enzymes (mainly azo reductase), are responsible for the cleavage of azo dyes into aromatic amines just the same way as Prontosil is cleaved into sulfanilamide. This explains why that Prontosil is not effective in vitro, but is effective in vivo. Many aromatic amines are biological active such as carcinogenic and/or muta-genic. We therefore, have illustrated the significance of azo reduction is a metabolic activation of the carcinogenic and/or mutagenic properties of this group compound. It is interested to learn that Domagk in about 90 years ago illustrated the metabolic activation of the antibacterial property of the Prontosil. Of course, in his era, carcinogenic potential of many sulfanilamides were not major concerned. Neither the role of intestinal microbiota was known. Domagk worked extremely hard. He worked persistently and tirelessly over his entire life. Although colleagues made chemical preparations available to him, he always worked quite alone when testing them for their curative properties. In his words, “I made all the decisive tests myself until I was quite certain as what was right”. He was very critical and precise himself. This was why the reports of others in regard to the contrary left him unshaken regarding his own work. What he had discovered by experiment to be correct was for him “incontrovertible”. We live in an age of fighting against cancers and AIDS, the example of Domagk is much of an inspi-ration and worth remembering today.

Suggested Reading

1. Armin, H. and K. Jurgen (1968). German Nobel Prize Winners. Pp. 67 and 68. Heinz Moos Verlagsgesellschaft, Munich, Germany.

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Chapter 35

Paul Henry de Kruif (1890–1971): Gas Gangrene Research and Historian

of Microbiology

“— the relief of suffering and prevention of dying cannot be best served for all, so long as there remains any money consideration between the people and the fighters for their lives”.

— Paul de Kruif

Source: http://www.findagrave.com/cgi-bin/fg.cgi?page=pv&GRid=93482178&PIpi=88557392

(US Public Domain image)

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35 Paul Henry de Kruif

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Introduction

Most of us would remember the fascinating book called The Microbe Hunters, written by Paul Henry de Kruif in 1926. The book dramatized the battle between the unseen “germ” and the “microbe hunters”, microbiologists whose brief biogra-phies are in the book. At the same time, it gave a good descrip-tion of how microbiology started in the beginning. This book was not only a bestseller for a length of period after publica-tion, it remained high on the list of recommended for science and has been an inspiration for many physicians, scientists, especially microbiologists. This book inspired many talents invest their life into the field of microbiology and medicine. Some of them became notorious figures including Nobel Prize winners in the field of medicine and Physiology. Even today, this book is still very powerful in inspiring the young minds to devote to the big enterprise of microbiology. However, Paul Henry de Kruif (or called Paul de Kruif) was an excellent micro-biologist himself and also contributed greatly to the develop-ment of microbiology. He is a part of history.

Life of Paul de Kruif

Of Dutch ancestry on both sides, Paul was born on 2 March 1890 in Zeland, Michigan. His mother was Hendrika Kremer and his father was named Hendrik de Kruif. Hendrik was a hard-working farmer with only second grade education, but he was a natural mathematician and pointed his son toward the uni-versity. A magazine article on the famous German microbiolo-gist Paul Ehrlich (1854–1915), which he read as a freshman, stimulated Paul’s choice of bacteriology as a profession. After completing his BS in 1912 at the University of Michigan, de Kruif worked in the bacteriology department pursing his PhD degree in microbiology. He obtained his PhD in 1916. He imme-diately entered service as a Private in Mexico on the Pancho

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Villa Expedition and afterwards served as a Lieutenant and later, Captain in World War I (1914–1918) in France.

Because of his service in the Sanitary Corps, he had occa-sional contacts with leading French microbiologists of the period. Bacteriology was new science, especially to the Army, and de Kruif did his main work on the bacillus of gas gangrene on the European front. Gas gangrene was a serious malady dur-ing the extensive trench warfare of World War I. Captain de Kruif was with the 5th Division of the American Army and car-ried out the first prophylactic use of gas gangrene serum with American troops. While in France, de Kruif also did research at the Pasteur Institute and the military’s General Medical Department Laboratory at Dijon.

Upon returning to the United States, he left the academic field and he worked as a bacteriologist at the Rockefeller Institute in New York City. (Ironically, this Institute became a university long after he had left.) From the experience of de Kruif, one learns that life is not always a smooth path. In addi-tion to his research in bacteriology, he began writing on micro-biology and medicine. His writing brought almost immediate fame in the years between 1920 and 1922. His first major book was Our Medicine Men (1922). Some reviewers accused him of “sensationalism” and being too critical of the medical profes-sion; however, the truth of his accusations was generally accepted.

While at the Rockefeller Institute, de Kruif became acquainted with a laboratory assistant named Rhea Barbarin; they were married in 1922 and collaborated for the rest of life. De Kruif is the major example of a first-rate microbiologist who uses his expertise for the general good on the printed page, rather than at the laboratory bench. In spite of the worldwide recognition of his writing on microbiology, some of his writings created problems for him. Because of the “sensationalism” of his articles on medicine and microbiology, some essays written while working for the Rockefeller Institute led to his dismissal. He learned the “science for science’s sake” did not always

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pay — de Kruif, as a microbiologist, was deeply concerned with the lack of knowledge of his field by many doctors.

De Kruif collaborated with Harry Sinclair Lewis (1885–1951) with his Pulitzer winning novel Arrowsmith (1925), by provid-ing the scientific and medical information required by the plot, along with the sketches of characters. Even though de Kruif’s contribution was very significant, Lewis was listed as the sole author. In the preface of the book, Lewis acknowledged that de Kruif “not only furnished most of the bacteriological and medi-cal material in this tale, but equally (he should be credited with) — the planning of the fable itself — for his realization of the characters as living people, for his philosophy as a scien-tist”. De Kruif received 25% of the royalties.

In 1926, de Kruif published his next book titled The Microbe Hunters, which was perhaps his most famous book. The Microbe Hunters sold more than one million copies in a few years and inspired great interest. Ronald Ross (1857–1932), one of the scientists featured in The Microbe Hunters, took exception to how he was described, so the British edition deleted that chap-ter to avoid a libel suit.

Almost as popular was his next major work, Hunger Fighters. The American Library Association published it in its list of “Forty Notable Books in 1928”. Besides his books, Paul de Kruif was a staff writer for such magazines as Ladies Home Journal, Country Gentleman, Fortune, The Nation, The New Republic, and the Readers Digest. He also contributed articles on Science and Medicine. Many of his articles were considered “too emo-tional” for a scientist. In fact, de Kruif was far ahead of his time in calling for medical care: “— the relief of suffering and pre-vention of dying cannot be best served for all, so long as there remains any money consideration between the people and the fighters for their lives”.

From this fundamental knowledge of microbiology, de Kruif took up the battle in the public interest regarding such diseases as maternal mortality, infantile paralysis, syphilis, and gonorrhea. He proposed systems of national care but was not

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opposed to the American Medical Association. He actually always attempted to win the support of medical organization. In 1940, he published Health is Wealth, presuming a national program of medical care. He considered this book a national program for human conservation. His plans did not envision a change in the relationship of doctors and patients, nor would it disturb doctor’s “ancient rights”. The support for his writing with his knowledge of microbiology prompted the Saturday Review of Literature to state that he wrote with “Parresting facts marshaled in militant words”.

In the beginning of World War II (1939–1945), de Kruif again brought his knowledge of microbiology to bear on the war effort. His chapter in a national symposium titled “America Organizes to Win the War” called for improvement in national health to strengthen America’s war effort. He insisted that as we became more heavily engaged in the War, “more and more cracks are going to show in the pleasant façade of our essentially healthy America if something isn’t done now”. He also testified before Senate Subcommittee in November of 1942 on the neces-sity of the compulsory allocation of physicians in the United States. Dr. de Kruif recommended Federal supervision of the allo-cation of all members of medical profession as a war measure.

He was appointed secretary of the General Advisory Committee of the National Foundation for Infantile Paralysis and promoted research into Infantile Paralysis. In his later years, he also took a great deal of interest in vitamins. He loved vigorous exercise, such as chopping wood and “swimming against strong current”. Another deep interest of de Kruif was music. Many times he listened for hours and even days to the music of Beethoven. He had reported listening to the Third and Ninth Symphonies all night long. His second marriage was highly successful and he dedicated most of his books to his wife, whom he considered “indispensable”. He passed away in February 1971 in Holland, Michigan.

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Commentary

De Kruif was a first-rate bacteriologist who lived before the true flowering of science in America. He realized that he could have had a pleasant life as a microbiologist teaching in a uni-versity, but he chose the more difficult path, instead, of using words to interpret microbiology for both the public and scientists.

De Kruif published many books. His first published book was Our Medicine Men in 1922. Then he published Microbe Hunters in 1926. Many books published in sequences were Hunger Fighters (1928), Men against Death (1932), Why Keep Them Alive (1937), The Fight for Life (1938), Health is Wealth (1940), Life Among the Doctors (1940), Kaiser Wakes the Doctors (1940), The Male Hormone (1945), A Man Against Insanity (1957), and The Sweeping Wind (1962). Among the books, de Kruif’s most celebrated and influential 1926 book Microbe Hunters consists of chapters on the following medi-cine’s “heroic Age”:

1. Antonie van Leeuwenhoek (1632–1723), invention of a sim-ple microscope and the discovery of microorganisms.

2. Lazzaro Spallanzani (1729–1799), biogenesis. 3. Robert Koch (1943–1910), identification of pathogens. 4. Louis Pasteur (1822–1895), bacteria, biogenesis. 5. Emile Roux (1853–1933) and Emile von Behring (1854–

1917), diphtheria. 6. Élie Metchnikoff (1845–1916), phagocytosis. 7. Theobald Smith (1859–1934), animal vectors and ticks. 8. David Bruce (1855–1931), tsetse fly and sleeping sickness. 9. Ronald Ross (1857–1932) and Battista Grassi (1854–1925),

malaria.10. Walter Reed (1851–1902), yellow fever.11. Paul Ehrlich (1854–1915), the magic-bullet concept, syphilis.

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This book was not only widely selling, but also inspired tre-mendous young mind flooded into the emerging microbiology. This book is about most influential promoter in the develop-ment of microbiology. He also wrote many influential articles; for example, the How we can help feed Europe (in Reader’s Digest, September 1945, pp. 50–52) also greatly affected the public policy and humanity. The authors have the courage to write this book, partly owing to the inspiration of de Kruif.

Paul de Kruif is a good human being; he left a valuable legacy to let us enjoy today.

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Chapter 36

William C. Frazier (1894–1991): Pioneer of Dairy and Food Microbiologist

Food Microbiology (McGraw-Hill, 1958). The book has been widely used and is now in its fifth edition. It was originally written by William C. Frazier, a pioneer dairy and food microbi-ologist. During his career at The University of Wisconsin, he played an important part in the development and growth of

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the Bacteriology Department and the education of many dairy and food microbiologists. His former students have made great contributions to the development of dairy and food industries in this country and abroad as well as in other areas of microbiology.

The Frazier family had origins in Wisconsin when William C. Frazier’s grandparents, William and Plurna Frazier, migrated from Ohio to the new state in 1855 and began farming in northwestern Wisconsin in what eventually became Vernon County. William C. Frazier’s parents William S. Frazier and Eva Palmer were married in Astoria, Illinois in 1891. Following their marriage, they resided at a home on Francis St., Madison, Wisconsin, while his father continued his studies leading to a law degree granted on 19 June 1898. At this home, William C. Frazier was born on 26th September 1894 or 1895. The official year is given as 1895 on a birth certificate issued many years later, but, found in a paper of his father, was a letter from a dear friend, Jasper Dexter, dated 2 October 1894, congratulat-ing him on the birth of a son with the following comment: “Will S. Frazier Dear Friend: Your letter with the good news received accept congratulations. Say you needn’t feel so big you ain’t the only fellow that has a boy. I do hope the boy will be stouter than his dad — if he ain’t, he won’t be much for stout”.

After his father received his law degree, the family which now also included his sister Mildred, moved to 961 Island Avenue (renamed Palmer Ave.), Milwaukee, Wisconsin, where his father was admitted to the Bar of Milwaukee County and practiced law.

The family remained in Milwaukee, where William C. Frazier completed his primary and high school education, graduating from North Division High School in 1912 in the “Science Course”. His primary education was rather interesting as several of the classes, in the then predominantly German Milwaukee, were taught in German in which by necessity he became quite proficient. While in high school, William C. Frazier was greatly

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36 William C. Frazier

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influenced by both his English and Chemistry teachers of whom he spoke kindly of in his later years. A surviving report card from that period shows that he received excellent marks in all subjects. Both teachers encouraged him to apply himself to those subjects when he entered college. During his formative years, summers were spent with family either on a farm of one of his many aunts or uncles, or at Camp Cleghorn Assembly on the Chain O’Lakes near Waupaca, Wisconsin. The latter was a favorite family place where he developed his great love of fish-ing and the outdoors. As a boy he worked there doing odd jobs and he never tired of telling of his adventures, such as canoeing down the Crystal Creek, catching big fish or working on such odd jobs as tarring the ice house roof. He was a very creative person, as a youth, enjoying writing and art, examples of those still survive. He tended to love the humorous side of life as illus-trated by the following limerick written sometime during his high school.

There was a small boy in Quebec, Who was buried in snow to his neck,

When they said “Are you frizz?” He replied, “Yes I is”

But we don’t call this cold in Quebec

He also enjoyed the game of basketball and played on a YMCA team, the Monarchs, since the public schools then didn’t support the game. When he entered college, he tried out for basketball and would have made the team but for the fact the coach said he was too light for the game as it was then played. When he entered the army in 1917, he was 6 feet tall and weighed some 130 pounds.

When Frazier entered the University of Wisconsin, the entire family immediately moved from Milwaukee to Madison, as was the custom of many families in those days when the oldest son entered college. He continued to live at home while

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studying for his degree in Agricultural Bacteriology. To earn money for his college expenses, he spent summers working at Dugan’s Dairy on a farm near Beloit, Wisconsin, driving a milk wagon, milking cows, and working in the processing plant or working in a canning factory in Cambria, Wisconsin. While at Dugan’s, he met his friend, Ellsworth Hay, who was to be his pal during service in the 32nd Division in World War I and who was a chemistry major at the University. His summer employment at a dairy and at a canning factory was early practical experience in areas which continued to interest him throughout his entire professional career.

With the Declaration of War in 1917, his senior year in col-lege was cut short. Students were encouraged to help the war effort by assisting in food production and he returned in April 1917 to Cambria, Wisconsin to work in the canning factory. He also applied for a position in Washington, DC, at the Bureau of Dairy Industry under Dr. Lore A. Rogers. However, as interested as he was in this position, he was at this time of his life more interested in becoming directly involved in the war effort. To this end, he enlisted as a private in the Wisconsin National Guard, The 32nd “Red Arrow Division” on 2 September 1917.

The 32nd Division saw active combat duty in France and Frazier rose to the rank of Sergeant, Headquarters Company 127th Infantry, seeing actions in all battles in which this famous division was involved. He and his friend Ellsworth Hay were offered the rank of 2nd Lieutenant twice while in combat, but both turned it down for reasons only known to them but one might guess the life expectancy was short for a 2nd Lieutenant. His duties at that time were in the Intelligence Section and he was awarded the Silver Star for bravery in action. During this period, he kept in contact by mail, when he could, with both the Bureau of Dairy Industry in Washington and his professor, Edwin G. Hastings (1872–1953), at the University of Wisconsin, looking forward to future days. Both gave him encouragement for postwar opportunities. After months of combat, both he

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and Ellsworth began efforts to obtain transfers out of the infantry to the Medical Corps and Chemical Corps respectively, where their college training could be put to better use. He said in a letter written on 11 November 1918 (Armistice Day): “— I guess I’ve never mentioned that I have applied for a transfer to the A.E.F. Bacteriological Laboratories and expect to leave this outfit almost any day. If the war had not ended I would prob-ably received a commission, but I suppose I’ll not now. Ellsworth goes to the Chemical Lab. We prefer to be in our real profession & doing something during the time which will necessarily be spent in France before we get back. We’ll have a good oppor-tunity to study up and get in swing again, while in the infantry we’d either be doing nothing or bossing some natural labor like handling rations, cleaning up battle fields, etc”.

Sergeant Frazier’s transfer to The Army Medical Corps occurred while he was in Germany with the 32nd Division marching towards the Rhine after the armistice. He reported to Central Medical Department Laboratories, Dijon, France, on 6 December 1918. His duties there are best described by part of a letter written by him to his mother on 3 April 1919.

“I am a medical Bacteriologist. I do the bacteriological work of all kinds that come in (except the few anaerobes which are taken care of by the Wound Bacteriology Department). I may have feces, urine, or blood come in for examination for typhoid, paratyphoid, or dysenteries. There may be blood for strepto-cocci, pneumococci, etc., pleural fluid for tuberculosis, and so on. These are cultures of various sorts for identification, diph-theria, influenza, glanders, typhoid, and all those I have men-tioned. And then there are smears, direct from the subject or sent in, to be stained for: Vincent’s Angina (decay of gums in mouth), TB (septum for tuberculosis), B diphtheria (swabs of throat or tonsils for diphtheria). Then, there are examinations for venereal disease. Stains of urethral smears are made for gonococcus (the causal organism of gonorrhea) where the ‘bugs’ show up within the pus cells. In the examination for

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syphilis, serum from the edge of a sore is placed on a slide under a special microscope and rays of light from a strong elec-tric arc light are thrown up through the liquid, so that the bac-teria in the fluid refract the light and show up as a bright spot of light, while the body of the liquid is black. By this means is shown the causal organism of syphilis: Treponema pallidum, a spirochaete (animal — not bacterium), a cork screw shaped affairs with 8 to 12 turns in it, stiff moving by means of flagella at the ends”.

Immediately after his discharge from the Army, Frazier returned to the University of Wisconsin to work on his PhD in Agricultural Bacteriology under the direction of Professor Edwin G. Hastings. For his minor, he chose Physical Chemistry with Professor Louis Kahlenberg (1870–1941). Even at that time, Wisconsin’s interest in dairy helped shape the activities and careers of both Professor Hastings and his students.

Frazier served as an instructor in the Department until he finished his doctorate in 1924; then he joined the US Department of Agriculture’s Bureau of Dairy Industry to inves-tigate a variety of problems involving dairy products. During his 10 years at the Bureau, Dr. Frazier worked with a group of eminent dairy scientists under the overall direction of Dr. Lore A. Rogers (1875–1975) (ASM President, 1922). Among his major achievements was unraveling the microbiology of Swiss cheese, which is one of the most complicated natural fermentations in the entire food supply.

They published a series of papers that took much of the “luck” and mysticism out of Swiss cheese manufacture. He was active in the then Society of American Bacteriologists (to become the American Society for Microbiology) and again showed his sense of humor in a series of spoofs published in a booklet at one of their meetings in Washington, DC in 1929. Any student of Bacteriology knows Bergey’s Manual as the authority on bacterial classification and Dr. Frazier had a good time writing several verses in the booklet entitled “A New

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Genus Omitted From Bergey’s Book”. The verse about Bergey follows:

Index in bacterium bergi Every time we see that guy

We wonder how we classify. He throws us each into our genus

And knows the differences between us. Indeed, I’ve often heard it said,

He’s classed the hairs upon his head, Black ones, gray ones, those just started,

Even those long since departed.

Dr. Frazier returned to the University of Wisconsin in 1934 to become Professor of Agricultural Bacteriology and to start a new program in Food Microbiology. He was a popular teacher and his program flourished. In 1943, he was appointed Chairman of the Department, a post he held for 10 years. World War II decimated the Bacteriology faculty, as one professor after another took wartime assignments away from Madison. For a while the Department had only three faculty members at the University: Dr. Frazier, Dr. Elizabeth McCoy (1903–1978), and Dr. Wayne Umbreit. These three shared the teaching load while carrying on active research programs on problems important to the war effort (production of penicillin, butyl alcohol, etha-nol from wheat, food yeasts, etc.) As World War II neared its end, Dr. Frazier took the lead in rebuilding the Department’s faculty. W. B. Sales, P. W. Wilson, and I. L. Baldwin returned from wartime service, though Dr. Baldwin soon moved to higher administrative posts in the University. O. N. Allen, J. B. Wilson, S. G. Knight, K. B. Raper, and Edwin Michael Foster (1917–2013) were added to the faculty soon after the war ended. These men became world renowned microbiologists in their own fields. At the same time, Dr. Frazier accelerated efforts to obtain a new building to house the Department. At

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this time, the department occupied most of the space in the west half of Agricultural Hall. With funds became available in the late 40s, Frazier was ready with a detailed analysis of departmental needs and ideas for a building that was com-pleted in 1955. It was during Frazier’s tenure as chairman that the departmental name was changed from Agricultural Bacteriology to Bacteriology to accommodate the broadening interest of departmental faculty. These years demonstrated Dr. Frazier’s skill as an administrator, just as a prior decade had revealed his excellence as teacher and researcher.

But tragedy struck in 1953, when he suffered a heart attack. After recovering his health, Dr. Frazier returned to full-time teaching and research, a role he filled until his retirement in 1966, at age 70. His last 10 years on the faculty were among the most productive of his academic career. During his 32 years as Professor of Bacteriology, Dr. Frazier served as the major professor and adviser of 30 candidates for the PhD and 36 can-didates for the Master degree. One-third of these were gradu-ated during the 10 years after his illness. Another notable achievement of this period was the completion of an excellent text book in food microbiology (McGraw-Hill, 1958) previously mentioned. He also was a coauthor of a general text in micro-biology “Microbiology, General and Applied” and of several laboratory manuals concerned with food and dairy microbiol-ogy. He was a productive scientist but, first and foremost, he never forgot who he worked for. The people of Wisconsin paid his salary, and the people of Wisconsin deserved his service. He didn’t do research just to have fun or to satisfy his personal curiosity; he did research to solve problems that needed to be solved. Among some of his 90 scientific papers were studies concerned with the microbiology of market milk, concentrated milk, brick cheese, cheddar cheese, and alfalfa silage. He did pioneering work on heat resistance and moisture requirements of bacteria important in foods, besides on a host of other prob-lems important to society.

Dr. Frazier received many honors during his career. He joined the Society of American Bacteriologists (now American Society

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for Microbiology) (ASM) in 1920. He served the Society as coun-cillor, secretary-treasurer (1943–45), program chairman for four years, associate editor of Bacteriological Reviews for 14 years, and editor of Journal of Bacteriology for seven years. He also held offices in The North Central Division of the ASM. He was named Honorary Member of the ASM after he retired. Dr. Frazier was a life member of the American Dairy Science Association, a charter member of the Institute of Food Technologists, an honorary life member of the International Association of Milk, Food, and Environmental Sanitarians, a fellow of the American Association for the Advancement of Science, and a member of Wisconsin Academy of Arts, Letters and Sciences, as well as the Washington, DC, Academy of Science.

Dr. Edwin Michael Foster (1917–2013) was one of Dr. Frazier’s early graduate students; he wrote his own personal experiences and feeling regarding the man on the occasion of Dr. Frazier’s 91st birthday in 1986. It gives one an insight as to what kind of person he was during his career at the University of Wisconsin.

“As a student in his class: We first met in the fall of 1937 when I showed up in Madison, a green kid of 20, after a 48-hour bus ride from my home in Texas. I warmed up to him immediately. His lecture was solid and businesslike — he was not there to entertain, but to teach us. Almost 50 years later I can conclude that while taking Dr. Frazier’s course a student learned facts; but when the course was over, he had knowl-edge. Dr. Frazier taught us to reason, to generalize, to draw logical conclusion from specific data.

As a graduate student in his lab: Dr. Frazier did not ‘direct’ his students’ research. He asked questions, made suggestions, proposed hypothetical situations. He made us think clearly and logically without seeming to try to do so. But when it was over we saw the picture and we knew how to write English. Dr. Frazier would not tolerate sloppy writing.

As a junior colleague: Dr. Frazier was Chairman of the Department when I joined the Wisconsin Faculty. I was supposed to replace Professor E. G. Hastings, who taught dairy bacteriology,

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but Dr. Frazier proposed that we share the instruction in food and dairy bacteriology. For 21 years he taught both courses one year and I taught them the next. Thus we kept up with the field and our students benefited accordingly. This was not a senior-junior relationship between us, but a true division of responsi-bility. During those early years, Dr. Frazier repeatedly steered research projects and outside financial support in my direction when he could just as well have kept them for himself. He seemed to enjoy helping young professors get established, and he went out for his way to give us credit for ideas and sugges-tions, even though they probably originated with him in the first place.

As a senior colleague: Friendly respect is the way I would characterize the relations between Dr. Frazier’s colleagues and himself. Our department was a remarkably pleasant and friendly group, both during and after his chairmanship. Much of this happy situation can be credited to Dr. Frazier’s skill in getting along with people and getting them to work together.

As a lifelong friend: Dr. Frazier somehow managed to get the best out of his students. As one of them, I responded immediately to his warm, friendly smile; his helpful suggestions; his stimulat-ing questions. We used to go fishing together, visit cheese facto-ries together, attend meetings together, drink beer together. It was a stimulating relationship that led to a lifelong friendship. Yet I never once called him Bill, and I don’t to this day. He would not have objected, but I preferred it the way it was. It is my way of showing the enormous respect I have for the man”.

Dr. Frazier died in Madison, Wisconsin on 21 August 1991, just 5 weeks short of his 97th birthday, survived by his wife Hildegarde; a son, William R. Frazier; a sister, Mildred Bruff; three grandchildren; and five great-grandchildren. Mr. William R. Frazier was also a microbiologist. He recently retired from E. R. Scripps Company.

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Chapter 37

Ira Lawrence Baldwin (1895–1999): Pioneer of Agricultural

Microbiology and Education

“The seeing eye, the inquiring mind, and the innovating spirit”.

— Ira L. Baldwin

Source: http://www.wisconsinacademy.org/sites/default/files/ WiscAcad_AnnualReport 2014_2.pdf

(US Public Domain image)

Introduction

Ira Lawrence Baldwin’s remarkable life experience with soil microbiology represents an era of the development of agricultural

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microbiology. He contributed not only to the understanding of the role of microorganisms in the soil but also to their agricul-tural applications. He was also an eminent educator. He exerted a great influence on the growth of microbiology in the United States and in some areas around the world.


Ira Baldwin was born on 20 August 1895, on a farm near Oxford, Indiana. His father was Thomas Atkinson Baldwin (1849–1933) and his mother was Eva Mock Baldwin (1854–1924). Baldwin was the youngest son and the sixth child of the family of seven children. They lived on an isolated farm with no dwellings within at least half mile in any direction. They worked in the farm and reading magazines or newspapers was their only entertainment, so Baldwin developed very good reading habits starting at a young age.

At the age of 6 (1901), he attended a public elementary school in Oxford, Indiana, which was built in 1866 and covered grades from 1 to 12. Baldwin stayed in that school for eleven years and then transferred to a new school, which offered classes in agriculture. Baldwin liked this curriculum and devel-oped an interest in becoming an agricultural scientist. In 1912, he was admitted to Purdue University in Lafayette, Indiana, not far from his home, and majored in agriculture. During his sophomore year, he took a course in bacteriology in Purdue’s medical school, which was taught by Charles A. Behrens (1885–1950). Bacteriology inspired Baldwin and he took an advanced course in bacteriology during his junior year.

During his college years, Baldwin joined the Reserve Officers’ Training Corps (ROTC) training program for two years; he was not excited by military drills and dogma and abandoned ROTC for other pursuits. However, in 1917, when World War I loomed on the horizon, Baldwin volunteered for officer’s training and left Purdue to join a unit at Camp Sherman in Chillicothe, Ohio. He was commissioned as a 2nd Lieutenant in December 1917.

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From Camp Sherman, Baldwin was sent to Camp Jackson in Columbia, South Carolina, where he was assigned to teach alge-bra to the new recruits. Later, he was sent to Fort Sill, Oklahoma, for training in light artillery and then to the 72nd Field Artillery Range in Camp Taylor in Louisville, Kentucky. While at Camp Taylor, there was an epidemic of flu. Lt. Baldwin’s responsibility was to bury the victims of the flu. He buried as many as two to eight casualties per day during the crisis of the pandemic in 1918. (Pandemic influenza flu occurred in 1918–1919; more than 20 million people worldwide died. There is uncertainty concerning the origin of this pandemic. The best reliable reports place the first well-documented cases among U.S. Army recruits at Camp Funston, Kansas, in March 1918.)

When World War 1 ended in December 1918, Baldwin was immediately discharged from the army and returned to Purdue. As a veteran and a more mature man, he devoted his full energy to his studies. He earned his Bachelor’s degree in Agriculture Chemistry in 1919 and was employed as an instructor. He was in charge of laboratory instruction and assisted in teaching a general bacteriology course. On 29 December 1920, he married Mary Mock Lesh (1892–1952) who was from Delphi County of Indiana. They had a wonderful marriage for 32 years until her death in 1952. They had two daughters and one son. Baldwin married Ineva Reilly Meyer (1904–2000), from Cook County in Illinois, on 17 April 1954. They also had a wonderful marriage; Ineva was also an educator and accompanied Baldwin in travels.

Baldwin decided to pursue graduate work at Purdue University partly because he was interested in teaching, which he did during his senior undergraduate year. He obtained his MS degree in Agricultural Chemistry in 1921 and was offered a chance to teach a course in soil bacteriology and in glass blow-ing. The third year, he was asked to teach a graduate course in dairy bacteriology. He did that for two years. In 1924, he was promoted to Assistant Professor. He was also employed as an Associate Physiologist for the Agricultural Experiment Station in 1926.

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Beginning in 1922, Baldwin spent the summer at the University of Wisconsin, Madison, to conduct research and to earn some extra money. During this time, he met Edwin G. Hastings (1872–1953) of the Department of Bacteriology. This encounter led Baldwin to decide to do further graduate work at the University of Wisconsin. He spent the summers of 1923 and 1924 there, as well as the entire school year of 1925–1926, and completed his doctoral research project under the direction of Edwin B. Fred (1887–1981). His PhD dissertation was on the identification and differentiation of Rhizobium species in the laboratory. He obtained his PhD in 1926 and was offered an Assistant Professor position, which he started in 1927.

Achievement

At the University of Wisconsin, Baldwin continued to study Rhizobium and also researched new leguminous crops, such as peas and soybeans. He was asked by Dean Christensen of the College of Agriculture to chair a committee to determine the need of personnel training for the food industry, which was booming in Wisconsin. The committee studied the statewide need and recommended that training opportunities should be provided. Baldwin set up a program to educate farmers on techniques for inoculating their legumes with Rhizobium spe-cies and demonstrated “field plots” across the state to facilitate the process. Consequently, Baldwin became very acquainted with farmers, county agents, and canners.

He also studied and solved problems such as contamination of commercial yeast and penicillin production facilities encoun-tered by these extension activities, which were very practical to agricultural industries. These problems were broad in scope but intimately relevant to agribusiness.

Within the Department of Bacteriology, Baldwin developed a course called Bacterial Physiology with a laboratory, which was first taught in the fall semester of 1928. He was promoted to Associate Professor in 1929 and to Professor in 1932. He

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collaborated with many faculty members, including Edwin G. Hastings who was chairman of the department. Later, he worked again with Fred. After that, he collaborated with William Stovall (1887–1971), Director of the State Laboratory of Hygiene and with Paul Clarke of the Medical School. Baldwin also collabo-rated and published papers with Albert J. Riker (1894–1982) and William H. Peterson (1880–1960) as well as Edmond Joseph Delwiche (1874–1950) and Laurence Graber (1887–1977). He also had frequent collaboration with Marvin Johnson (1906–1982) of the Department of Biochemistry on projects in yeast cell pro-duction, citric acid fermentation, and improved penicillin yield under optimized culture conditions.

Together with Fred and Elizabeth McCoy (1903–1978), Baldwin wrote a textbook on the root nodule bacteria titled Root Nodule Bacteria and Leguminous Plants (University of Wisconsin Press, 1932, p. 343). It was regarded for many years as the authoritative text in the field. Other people he had col-laborated with included Perry Wilson (1902–1981), Oscar N. Allen (1905–1976), and Phillip Gerhardt (1921–2008).

Baldwin was made chairman of the curriculum committee. He did an excellent job on this assignment, which resulted in his being promoted to Assistant Dean of the College of Agriculture (1932–1941). In this capacity, he was responsible for managing the entire instructional program of the college. In 1941, he was appointed Chairman of the Department of Bacteriology (1941–1944), as well as serving as the Assistant Dean (for one more year). In 1942, he was called to government service due to the need for scientific researches created by World War II. He moved to Fort Detrick, Frederick, Maryland, for top secret work on biological warfare (BW) and became the technical director of the Army’s BW program (1942–1945). He still held the pro-fessorship and chairmanship at the University of Wisconsin but traveled frequently between Madison and Fort Detrick. In the fall of 1944, he was also appointed Dean of the Graduate School, a position he held only briefly. He left the post at Fort Detrick in April of 1945 and returned full time to Madison.

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In September of 1945, Baldwin became Dean and Director of the College of Agriculture, a position he held until 1948.

Baldwin now carried a huge responsibility. He was respon-sible for the instruction throughout the college, research throughout the experiment station, and service throughout the Agricultural Home Economics Extension Service. The College of Agriculture is a large college within the University of Wisconsin. The notable departments within the college include agricultural economics, agricultural engineering, agricultural journalism, agronomy, biochemistry, bacteriology, dairy sciences, dairy and animal husbandry, entomology, genetics, horticulture, plant pathology, rural sociology, soil science, veterinary sciences, and wildlife management. The scope of the responsibilities was broad and complex, yet Baldwin did an excellent job.

In 1948, Baldwin was appointed Vice President for Academic Affairs and he held this position until 1958. This was a rapid expansion period since the veterans returned to the campus. As many as 40 university centers were created across Wisconsin to offer freshman and sophomore-level courses to veterans. Enrollments escalated rapidly. Many dormitories were con-structed and local armed forces training facilities were con-verted to veteran housing. Research became increasingly important. The National Science Foundation and the National Institutes of Health were created in Washington to provide research funding. Dr. Baldwin was chiefly responsible for many of these changes. The University of Wisconsin became a top university in the nation and the world.

In 1958, Baldwin stepped down from the vice presidency and became Special Assistant to the President of the University until 1966, when he retired as Vice President Emeritus and Professor Emeritus. During this period, he devoted the major portion of his time to the theory and practice of building insti-tutions of higher education both in the United States and throughout the world.

After retirement, Dr. Baldwin and his wife Ineva lived most of their time in Madison. They would retreat to their condominium

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apartment in Key West, Florida, in winter months. They moved to Tucson, Arizona, in December of 1996 because of the warm weather and also they could be closer to one of their daughters. Dr. Baldwin passed away on 7 August 1999, 11 days prior to his 104th birthday. He had seven grandchildren and numerous great grandchildren. His wife passed away on 2 October 2000, at the age of 96. They had donated a total of 21.7 million dollars to the University of Wisconsin Foundation.

Services and Recognitions

Baldwin’s service to education not only included the state of Wisconsin, but also the nation and the world. He served the North Central Association of Colleges and Secondary Schools in various capacities in its educational building activities for over 20 years. He also was the University of Wisconsin’s representa-tive on the Joint Staff of the State Coordinating Committee for Higher Education from its inception in 1956–1963, playing a key role in its studies of Wisconsin’s educational resources.

During the 1960s, Baldwin devoted the major portion of his time to the problems of building educational and research insti-tutions in developing countries. On behalf of the Association of State Universities and Land-Grant Colleges, he established the Washington Office for International Rural Development and served as its director from February 1963 to June 1964. From 1964 to 1968, Baldwin served as director of a nine-university Agency for International Development (AID) — Committee on Institutional Cooperation (CIC) research project entitled “Rural Development Research Project — A study of University-AID Technical Assistance Projects in Building Instructions to Serve Agriculture”. From 1968 to 1971, Dr. Baldwin served as project administrator of an AID-MUCIA (Midwest Universities Consortium for International Activities) — Indonesia technical assistance project in building institutions to serve agricultural education, research, and public service. In additional to many trips to Indonesia, Baldwin’s interest in building institutions to serve

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agriculture in developing countries took him — literally — all over the world both for research and service. His contribution to these countries is beyond description. He left large footprints wherever he trod.

Scientifically, Baldwin studied the valuable industrial and soil microbiology and contributed greatly to the understanding of how microorganisms work in the soil. He was the leader in exploring the frontier of BW during World War II. He then served continuously on various federal government advisory boards and committees, serving as the chairman of a Department of Defense Research and Development Board committee and the Army Chemical Corps Advisory Council.

Baldwin also maintained a keen interest in the conservation of natural resources and served as the university representative on the Wisconsin Natural Resources Committee of State Agencies from its establishment in 1951 to his retirement in 1966. During 1960–1963, he served as chairman of a National Academy of Sciences — National Research Council Committee on Pest Control and Wildlife Relationships.

Baldwin was a man who belonged to many organizations. He became a member of the American Society for Microbiology (ASM) in 1920 and served as the Secretary-Treasurer from 1935 to 1942, Vice President in 1943, and President in 1944. He was a charter member of the American Academy of Microbiology and served as member of the Board of Governors from 1956 to 1961 (Chairman from 1957 to 1960) and also 1962 to 1968. He was member of many societies and clubs including the American History Society; the American Association for the Advancement of Science, the American Forestry Association, the American Institute of Biological Sciences, the American Phytopathological Society, the American Society of Agronomy, the American Society of Plant Physiology, the National Educational Association, the Society for Experimental Biology and Medicine, the Society for International Development, the Soil Conservation Society of America, the Royal Society of Arts (England), and many other societies in Wisconsin and Indiana. He was devoted whole heartily to the progress of each society.

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Baldwin also received honorary doctoral degrees from his alma maters Purdue University in 1945 and the University of Wisconsin, Madison, in 1972. His name is listed in many bio-graphic directories including Who’s Who in America, Who’s Who in American Education, Who’s Who in the Midwest, Who’s Who in the Central States, American Men of Sciences, Leaders in America Science, and Leaders in Education.

Personal Caliber

Dr. Baldwin had an amiable personality. He was well liked by everybody associated with him. His superb personality and administrative skills won him the trust of his superiors, the love of his associates, and the support of his students and fellow workers. Due to this, he made great accomplishments. “He was an exceptional microbiologist who helped discover, for exam-ple, which bacteria improve crop yields. … His skill as an admin-istrator and the respect others had for him brought him a series of administrative promotions and tasks in the 1940s, 1950s, and 1960s”, commented by the former Dean of Agriculture Neal Jorgenson who presented him the College of Agriculture’s 1997 Distinguished Service award. Former University of Wisconsin President Conrad Elvehjem (1901–1962) once said, “It is impos-sible to measure the impact of a quiet, self-effacing man like Dr. Baldwin for he has always worked as part of the team and stepped back when credit was given for progress made. He was, for instance, the chief architect in laying the groundwork for the formation of University of Wisconsin-Milwaukee”. This is why many microbiologists still hold Baldwin in deep memory.

Commentary

Baldwin was a man with a world view. His concerns were not just in Wisconsin but the whole world. His charm and concern for the living conditions of people around the world was impressive and infectious. He said, “…Over 30 years ago, a series of short biog-raphies of early agricultural research workers of the United

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States appeared in a trade magazine. One was entitled, ‘The Seeing Eye and the Inquiring Mind’. This is an excellent capsule description of the qualities of a research worker. Over the years, I have added another phrase, making the capsule description read, ‘The Seeing Eye, the Inquiring Mind, and the Innovating Spirit’”. He also said “Progress in any field is due to the actions of individuals, but social, economic, and political institutions and conditions are prime determinants of the actions of men”. That is essentially what he had been doing during his life. He contributed not only to the knowledge of agricultural microbi-ology in the field of nitrogen fixation, but also instituted the conditions in the world scale that facilitated the progress in this type of research. Many students, including those from the devel-oping worlds that may now be scientists in different scientific fields, have benefited from his generous efforts.

I am not quite sure about the wording in this last sentence — or if I have helped it.

While we are enjoying the marvelous progress of biotech-nology, let us not forget the work of Ira Lawrence Baldwin, one of the founders of the agricultural microbiology, a field that is a cornerstone of modern biotechnology.

Suggested Reading

1. Acker, R. F. (1999). In memoriam: Ira Lawrence Baldwin (1895–1999). SIM News 49: 355–358.

2. Baldwin, I. (1977). Soil Bacteriology: reflections on the past; directions for the future. Speech delivered in October, 1977, to the Agricultural Faculties of the National Taiwan University, Taipei.

3. University of Wisconsin-Milwaukee. Oral History Project. Records, 1981–1990. UWM Archival Collection 16. University Archives, UWM Libraries, University of Wisconsin-Milwaukee.

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Chapter 38

Cornelis B. van Niel (1897–1985): Educator and Pioneer of Bacterial

Photosynthesis and General Microbiology

Source: http://www.ranker.com/pics/N775750/cornelius-van-niel-writers-photo-1?ref=image_1072524

(GNU Free Documentation License)

In December of 1928, there arrived a young PhD, Dr. C. B. van Niel at the Jacquez Loeb Laboratory of the Hopkins Marine Station belonging to Stanford University and located on the Monterey Peninsula, California. Many of us remembered Dr. van Niel for his

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work on photosynthesis. He developed a revolutionary concept of the chemistry of photosynthesis that was to influence research on the topic for many years. He also was the first in the United States to teach “General Microbiology”. His teaching resulted in the great blossoming of microbiology in this century. Many distin-guished microbiologists including Nobel Laureates, were influ-enced by him directly or indirectly. Van Niel was the third important figure of the Delft School of microbiology — Martinus W. Beijerinck (1851–1931), Albert J. Kluyver (1888–1956), and Cornelis B. van Niel (1897–1985). He brought the rich inheritance of the Delft School to the United States. To call him a “pioneer of General Microbiology” of this country is very appropriate.

Van Niel was born in Haarlem, The Netherlands on November 4, 1897. His father, Jan Hendrik van Niel, died when he was seven years old. His mother was Geertien Gesiena Hagen. He was raised by his mother with his uncles. They affected his education at a great deal. At the age of 15, when his family spent their summer vacation as guests of a friend on a large estate in northern Holland devoted to various agricultural activities, he was impressed by the host who introduced him to methods of agricultural research. He learned that one could raise a question and obtain a more or less definitive answer to it as a result of an experiment. This greatly influenced van Niel’s interest and guided him in the exper-imental approach of solving scientific problems. In high school, he was greatly influenced by his chemistry teacher Dr. H. van Erp, and later, he developed a strong interest in chemistry and would set up a small laboratory at home where he analyzed samples of fertilizers in his spare time. Of course, good grades in high school would lead him to enter the Chemistry Division of the Technical University, a prestigious school in the Netherlands, in Delft in 1916. But after only 3 months, he was inducted into the Dutch Army, in which he stayed until the end of 1918.

In the Army, he was very much disturbed by the rough and impersonal life of the military training. In his memory, he said he was “utterly unaware of the many problems to which man is exposed and with which he must learn to cope”. Yet he was

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influenced by one of his good friends, Jacques de Kadt (1897–1988), later a famous Dutch sculptor, who introduced van Niel to a new world of literature, art, and philosophy. Under Jacques’ influence, he read the works of Emile Zola (1840–1902), Anatole France (1844–1924), Henrik Ibsen (1828–1906), August Strindberg (1849–1912), George Bernard Shaw (1856–1950), Friedrich W. Nietzsche (1844–1900), and so on. These might have greatly influenced on van Niel’s philosophical thinking during which he called himself “a rebel”. But this period might have helped him to become a superlative teacher later on.

After quitting the Army, he returned to school and contin-ued his education in chemistry. He took many courses, including Gerrit van Iterson’s (1878–1972) course in genetics and plant anatomy and chemistry, and also M. W. Beijerinck’s course in general and applied microbiology. Most important of all, he decided to major in Microbiology after hearing the inaugural lecture of A. J. Kluyver, who succeeded Beijerinck in 1921. Here he began his career as a microbiologist.

In 1923, he received Chemistry Engineering degree, and also accepted a position as an assistant to Kluyver. He helped taking care of a large pure culture collection of bacteria, yeasts, and fungi, and preparing demonstration in Kluyver’s courses. In addition, van Niel also isolated pure cultures of photosynthetic Chromatium species and Thiosarcina rosea, and demonstrated that oxygen is not produced. (For the purple bacteria, H2S is required for their growth.)

Van Niel expected to continue his study of purple bacteria for his PhD dissertation. In the meantime, van Niel developed an effective method for isolating propionic acid bacteria from Swiss cheese as a side project. But Kluyver suggested that he work on propionic acid bacteria for his PhD dissertation and he agreed. His PhD dissertation was entitled The Propionic Acid Bacteria and was published in 1928. In this dissertation, he dis-covered that diacetyl was the compound responsible for the characteristic aroma of high quality butter.

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Van Niel in the United States

Van Niel came to the United States in 1928 by the invitation of Lourens G.M Baas-Becking (1895–1963) professor of physiology of Stanford University. Van Niel never intended to migrate to the United States because he did not like the materialism of American society. However, when van Niel arrived in Carmel, California, he was immediately impressed by the charm of the town, and the beautiful site of the Jacques Loeb Laboratory. He also liked the freedom from outside pressure that Hopkins Marine Station provided. Van Niel loved his job, continued his study, and never intended to leave even later when he was offered the Chair of the Kluyver professorship of his alma mater.

Scientific Contribution

Van Niel continued his study of purple and green bacteria. In his study, he demonstrated that photosynthesis is essentially a light-dependent reaction in which hydrogen from a suitable oxidizable compound reduces carbon dioxide to cellular materi-als. This can be expressed by:

2H2 A + CO2 → 2A + CH2O + H2O

According to this formula, H2O is the hydrogen donor in green plant photosynthesis and is oxidized to O2. When H2S or another “oxidizable” sulfur compound is the hydrogen donor for purple and green sulfur bacteria, the “oxidation” product is sulfur or sulfate depending on the organism. In this fashion, the evolution of O2 in the green plant is coming from H2O, not from carbon dioxide. This was later proved by using radioisotopic techniques. This certainly was a “milestone contribution” to the understanding of photosynthesis in general. Van Niel also stud-ied the culture, morphology, and physiology of purple and sulfur bacteria, and also the nonsulfur purple and brown bacteria. He classified 150 strains isolated from natural sources into six species and two genera, Rhodopseudomonas and Rhodospirillum.

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One other great contribution of van Niel on photosynthesis is how radiant energy participates in photosynthesis. Many investi-gators considered that radiant energy could be used to activate either carbon dioxide and/or the hydrogen donor. Through extensive study, van Niel concluded that radiant energy activates the hydrogen donors instead of carbon dioxide. He also con-cluded that both plant and bacterial photosynthetic reactions have a common reaction; the photolysis of water to a strong reducing agent and a strong oxidizing agent. He postulated that the reducing agent was used through a series of enzymatic reac-tions to convert carbon dioxide to cellular constituents, whereas the oxidizing agent was used to generate oxygen in green plant photosynthesis or to oxidize the hydrogen donor in bacterial photosynthesis. This interpretation of the photochemical event laid down the foundation for the current understanding of photosynthesis. The current concept of photosynthesis is that a special type of chlorophyll (rather than water) is considered the source of the light-generated oxidizing and reducing agent.

Van Niel also made many other contributions to photosyn-thesis. For example, he did a series of studies of the pigment of the purple and green bacteria, which help us understand the role of pigment in photosynthesis. He also did many cultural works, in physiology, ecology, biochemistry, enzymatic investigation, etc. He often directed, gave encouragement, advice, and inspired many of his postdoctoral students, associates, or even graduate students to work on different aspects of photosynthesis.

Van Niel’s studies of photosynthetic bacteria led him to con-sider other processes in which carbon dioxide utilization might occur. One of these is methane formation. He postulated that methane formation from organic compounds by anaerobic bac-teria was the result of carbon dioxide reduction.

From methanogenesis, van Niel also discovered that CO2 was utilized by other bacteria, fungi, and protozoa. He concluded that carbon dioxide fixation generally occurs by carboxylation reaction and that carbon dioxide is required to counteract the decarboxy-lation of oxaloacetate, which “constitutes a leak through which

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certain essential cell constituents are drained off”. Van Niel also did some work on the chemistry of denitrification. He postulated that nitramide, H2N – NO2 was a possible intermediate of denitri-fication. He showed that NO3

− could serve as an additional elec-tron acceptor in the nitrate adapted Chlorella in photosynthesis. Van Niel’s interest in microbial taxonomy started from his PhD dissertation work. He had reviewed the main features of bacterial taxonomy and proposed a possible sequence for the evolution of various morphological types of bacteria. He and A. J. Kluyver formulated a taxonomic system dividing bacteria into four mor-phological types defined by cell shape, type of flagella, and sporu-lation. They were subdivided into 63 genera. In 1941, he and Roger Y. Stanier (1916–1982) undertook an analysis of the prob-lem of classification of the larger taxonomic units among micro-organisms. He considered that morphological characteristics should be given priority over physiological characteristics. In 1961, van Niel and Stanier defined the prokaryotes as cells in which the nuclear material (nucleoplasm) is not surrounded by a nuclear membrane. This definition of prokaryotes is still used today.

Teaching

Perhaps the greatest contribution of van Niel toward microbiol-ogy is his teaching. His unusual technique of conveying the scientific message and extraordinary high skills of inspiration attracted the top brains in this country to devote their careers in the development of General Microbiology and Comparative Biochemistry for several decades. His lecture techniques were partly from his own teachers, such as Beijerinck and Kluyver, and of his own caliber. His lectures often lasted for several hours and were presented with such clarity and histrionic skill as to capture the complete attention and stimulate the enthu-siasm of his students. These sounded as though he delivered universal truth and required the whole attention and no one ever felt tired. He gave you the impression that you were par-ticipating in the most significant part of scientific progress.

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Every one of his students was so highly inspired and excited that they were willing to devote their whole career to the endeavor of microbiological research. This kind of inspiration and his per-sonal charisma is beyond imagination. For instance, this author listened to his lecture only once, but changed his study concept and decided to choose microbiology as his subject. Some nota-ble scientists would give up their original disciplines and choose to devote themselves to aspects of microbiology for research. Many of his students and postdoctorals became the major pio-neers of different fields of microbiology. His influence on the development of microbiology in the United States, particularly on the West Coast, has been tremendous. He is surely the major pioneer in the development of microbiology and comparative biochemistry of our time.

As more microbiological knowledge developed, his lectures expended from three afternoons a week to 3 days a week, with class hours often extending from eight in the morning to well into the evening, with time out only for lunch and tea or coffee breaks.

He discussed the metabolism of each group of microorgan-isms, emphasizing the most recent findings regarding interme-diatory metabolism, similarities, and differences in degradative pathways, and the chemical and enzymatic relationships between degradative metabolism and synthesis of cellular components. He examined the structures of bacterial cells, aspects of bacterial genetics, variation and adaptation, bacterial and yeast taxon-omy and also the philosophy of science.

According to Dr. Robert E. Hungate (1906–2004), van Niel’s first graduate student, van Niel’s lectures were also accompa-nied with a series of simple experiments. He was always in the laboratory guiding the work and commenting on each student’s observation and results. He would stimulate students to make judgements about the meaning of their observations. Sometimes, he intentionally led them to incorrect conclusions so that in a later experiment, already planned, he would reveal the error. After the experiment, he would make a detailed presentation

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of its historical background, usually starting with the primitive ideas and progressing to the latest development. He always emphasized possible alternate interpretations of the available information at each phase of scientific investigation. He also lectured on the frequently slow and difficult process of moving from clearly erroneous to more nearly correct, but never immu-table conclusion.

During the early years, only a few students attended. But as van Niel’s reputation as a teacher spread, his classes expanded. Initially there were only undergraduate and graduate students from Stanford; later they came from many institutions. There were also auditors of the discussions and lectures, who did not do the experiments, mostly postdoctoral fellows or established scientists who wished to extent their background in general microbiology. The list of students and auditors who attended van Niel’s lectures between 1938 and 1962 read like a Who’s Who of the biological scientists in the United States with sev-eral other countries. Many of his students became pioneers of some field of microbiology and several were Nobel Prize laure-ates. Both directly and indirectly through his students, van Niel exerted a powerful influence on teaching and research on gen-eral microbiology for at least a generation.

One of the great merits of van Niel’s teaching is that he was interested in students. He did not believe in simply directing research. Instead, he encouraged his students or associates to follow their own interests, which were mostly stimulated by him. He would do everything possible to help the research. The range of research in his laboratory was exceedingly wide, which included the culture and physiology of blue-green algae and diatoms, nutritional and taxonomic studies of plant-path-ogenic bacteria, biological methane formation, pteridine and carbohydrate metabolism of protozoa, germination of mold spores, biology of Caulobacteria, cultivation of free-living spi-rochetes, induction of fruiting bodies in myxobacteria, decom-position of cellulose, the role of microorganisms in the food cycle of aquatic environments, adaptation of bacteria to high

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salt concentrations, cultivation of spirilla and colorless sulfur bacteria, bacterial fermentations, thermophilic bacteria, deni-trification, pyrimidine metabolism, and thermodynamics of liv-ing systems. To all students, van Niel gave, freely of his time, advice and enthusiasm, drawing on his own extraordinary knowledge of the literature. His memory of literature was so powerful that it was like a living dictionary. He often pointed out the most relevant literature of each subject of his interest to the right page number of different journals. But he was seldom willing to become a co-author of the final publication of his students. Therefore, many of his scientific contributions are sim-ply embedded in the publications of his students and associates.

Van Niel retired from the Marine Station in 1962, and worked as a visiting professor at the University of California, Santa Cruz from 1964 to 1968. One of the authors, K. T. Chung was fortunate to meet him at this period and establish a memorable friendship since then. Through his influence, this author became a doctoral student of Dr. Robert E. Hungate. Most memorable was that this author had the honor to be given by Dr. van Niel in person a copy of his PhD dissertation The Propionic Acid Bacteria and also a copy of book by his master A. J. Kluyver entitled The Chemical Activities of Microorganisms.

In 1972, van Niel gave up teaching and research entirely and lived quietly with his wife Christina van Hemert in Carmel, California. Van Niel married his wife on 17 August 1923 in Netherlands after eight years of engagement. They raised three children: Ester, Ruth, and Jan. They had a wonderful marriage. He died on 10 March 1985, after a life as a great teacher. His success in teaching is a result of his charm of personality, the breadth of his understanding and the comprehensiveness of his memory. Those who knew him were fully aware that he was a fine man. His warm, kind wisdom and inspiration impressed each mind profoundly and never faded away.

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Chapter 39

Max Theiler (1899–1972): Yellow Fever Vaccine Developer

“It looks as if yellow jacks get me the jackpot”.

—Max Theiler

Source: https://en.wikipedia.org/wiki/Max_Theiler(US Public Domain image)

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Max Theiler was the leader in the development of the yellow fever vaccine. For his work on the yellow fever vaccine, he was awarded the Nobel Prize in Medicine or Physiology in 1951.

Max was born on 30 January 1899 in a farm village called Daspoort near Pretoria, South Africa. His father was Sir Arnold Theiler (1867–1936) and his mother Emma Jegge. Arnold Theiler and Emma Jegge were both from Switzerland, Arnold’s father was a school master in Switzerland. Arnold inherited the char-acters of caution and obstinacy of his Swiss peasant forebears, and also had a religious reverence for hard work. He also had a rebellious character. Arnold was excited with Charles Darwin’s (1809–1882) Voyage of the Beagle and David Livingstone’s (1813–1873) accounts of his travels in Africa. He devoted him-self to prepare for a career as a veterinarian. He graduated from the Veterinary School at Zurich and served as an assistant to the new but primitive Bacteriological Institute attached to the University there and performed his compulsory service in the Swiss Army. All these activities did not satisfy his curiosity and ambition. He fell in love with his schoolmate Emma Sophie Jegge, but received a strong disapproval from his father. With his rebellious character, he decided to leave Switzerland and look for a new life in South Africa in 1891. Arnold had an extremely hard time in the beginning. He lost one of his arms in an accident. Emma Jegge who had gone to England for some education, joined him in Africa, and they were married in 1892.

In Africa, Arnold worked as a veterinary doctor. Initially it was not easy for them to make a living. They had four children. Hans was born in 1894, Margaret born in 1897, Gertrude in 1898, and Max in 1899. Max was the youngest of their four children. The first year of young Max’s life was a crisis. Arnold Theiler was away to attend an international veterinary congress in Germany in most of the spring and summer. The Dutch-descendent Boers were fighting against the British Empire for independence of South Africa. The Theilers were in the Boers’ side. Arnold was assigned to guard the horse of the Boer’s artillery against the devastating disease that menaced the animals of South Africa.

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The Theiler family survived the war. Arnold Theiler devoted his effort fighting against animal diseases and protected cattle from outbreaks that regularly wiped out whole herds. Through hard work and cleverness, Arnold became the director of South Africa’s Veterinary services. He had contributed tremendously to the animal industry of South Africa and became Sir Arnold Theiler.

Max worried his parents as he grew to adolescence. He was very short for his age and too fragile to excel in sports. He attended Pretoria Boy’s High School for one year when he was in Basle, Switzerland. His grades were not very good. But he contributed nobly to the family’s project of natural history. His father Arnold encouraged and dictated the project. The children wrote and edited The Field-Naturalists’ Journal. Each issue was a systematically indexed and handwritten notebook with drawing of the local flora and fauna such as grasshoppers, butterflies, leaves, worms, birds or tools of naturalists. Max served as vice president of the Field-Naturalists Society, which was exclusively Theilers. This journal was terminated when the elder Theiler siblings went off to boarding school. Max’s talents in writing, editing, and drawing were clearly reflected in this journal at a very young age.

At the age of 13, Max contracted schistosomiasis which caused a serious abdominal pain. This may be the reason that he remained small. But his spirit was vital and his joy for living was great. In 1916, he went to Cape Town to attend a two-year premedical program at the Cape of Good Hope University (some called University of Cape Town Medical School). Max was glad to leave home because he could thrive away from the day-to-day scrutiny of his attentive parents. In 1918, the epidemic of Spanish influenza struck Cape Town. Max joined the team to do what they could in the teeming slums of Cape Town.

In 1919, at the end of World War I, Max decided to sail to England to enter a full-fledged medical school. At this stage of life, he was 20, but stood barely 5 feet 2 inches. He enjoyed his self-image and was glad to be out of the shadow of his father. Like his father, he was somewhat rebellious toward his parents.

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Things were not as smooth as he imagined. The University of London refused to accept his two years training at Cape Town as the equivalent of a pre-med course. To meet the entrance requirement of an English medical school, he would have to take once again all the basic courses he had completed in South Africa. Max firmly refused to do that. Instead, he attended the four-year training program of one of England’s oldest and busiest hospital — St. Thomas. It would not give him a medical degree, but once certified as having taken the hospi-tal courses, he could pass an examination, that would enable him to practice medicine.

To his further disappointment was that in the St. Thomas hospital, he walked the wards filled with patients who had tuberculosis, nephritis, typhoid fever, and so on, yet there were no cures for these diseases. He said once “To me all this pill-giving was hogwash”.

He did not do anything more than it was necessary to get at St. Thomas. He sought relief from life onward in the bright, postwar London theater scene. He also read, went to art galler-ies, and enjoyed the cosmopolitan delights of London. When he completed St. Thomas’s curriculum, he took the licensing examination and was duly qualified to practice medicine.

At this time, Max decided to further his clinical training with a four-month course at the London School of Hygiene and Medicine. This course gave him hope and confidence. It gave him the inspiration that answers to the cures of some seemingly incurable diseases might come out of hard work in the labora-tory research. He was further inspired by reading Infection and Resistance by Hans Zinsser (1878–1940), the American bacteri-ologist for whom Max had a great respect. Zinsser’s book made him realize that research was an alternative to clinical practice. That is the way he would pursue it. One fellow student of Zinsser, Oscar Teague had been asked to look for a keen young student at the London school who might like to work as an assis-tant for Andrew Watson Sellards. Max applied for the position

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and got the job as an assistant in the Department of Tropical Medicine at Harvard Medical School in Cambridge, Massachusetts in 1922.

At Harvard, Max learned the methods, the protocol, the language, and the politics of scientific research from Sellards and his colleagues. He made friends with Zinsser who became not only a model, but also a friend and mentor. Max’s abdom-inal pain reoccurred in his first year at Harvard. It was quickly diagnosed. Surgery excised the scar tissue, he never again had the abdominal problem that had bothered him since boyhood.

He met a medical technician Lillian Graham and fell in love with her. They were married in 1928. He did not inform his par-ents of his marriage. It is probably because he anticipated that his parent would not approve his marriage with a non-Swiss, non-South African, and even non-European. They had a good marriage. Lillian was a great help in his career. They had a son Arnold Theiler II (Noldi) in 1929. Unfortunately, Noldi was killed in a car accident in front of his home in Hastings in New York in 1937. They had a daughter Elizabeth born in 1939.

At Harvard, Max was assigned to work on amoebic dysen-tery and rat-bite fever. He intended to devise ways to induce a mild form of that disease as a means of prophylaxis. Gradually, he developed an interest in yellow fever. An interest he had started from his school years in England.

Yellow fever is a serious viral disease with a high rate of death. The first known outbreak of yellow fever occurred in 1648 in Mexico. The last major outbreak in the United States was in 1905 which claimed 435 lives in New Orleans. A Cuban physi-cian, Carlo Finlay (1833–1915), speculated that mosquitoes were the carriers of this disease in 1881, but his claim was ignored by the medical community. In 1900, Walter Reed (1851–1902) led a team of investigators to study the cause of yellow fever, and concluded that yellow fever was caused by a blood-borne virus carried by mosquitoes. In 1916, the Rockefeller Foundation

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launched a worldwide program to control and eventually eradi-cate yellow fever, but the program was not successful.

By the 1920s, the strategy of control of yellow fever was to find a vaccine to prevent the spread of the disease. In 1928, researchers discovered that the Rhesus monkey could contract yellow fever and could be used for experimentation. But the Rhesus monkey was most a difficult laboratory animal. At 1928 prices of $7 a head, it was too expensive to get.

By now, Max Theiler was a full instructor with a salary of $3000 per year. He tried to use mice as the experimental animal for yellow fever although he realized it had been tried before, but failed. He injected a preparation of infected monkey into the brain of a Swiss mouse, the mouse developed severe enceph-alitis, which was different from the normal symptoms of human when infected with yellow fever. He proved that the yellow fever virus was neurotropic. When he injected the extract from the brain of one of the sacrificed mice into the monkey, the monkey promptly suffered a fatal attack of yellow fever.

In 1929, Max Theiler accidentally contracted yellow fever from one of his mice, which caused him to have a slight fever and weakness. This kept Theiler out of his lab and in bed for nearly a week. Theiler was very lucky, this small bout of yellow fever gave him an immunity to the disease. He was in fact the first recipient of a yellow fever vaccine.

In 1930, encouraged by Zinsser and helped by his wife Lillian, Theiler published his findings on the effectiveness of using mice for yellow fever research in Science. The article drew sharp disapproval from his immediate supervisor Sellards, who still pursued the theory of a bacterial origin of yellow fever. He also received negative responses from his Harvard col-leagues. Theiler was so disappointed that he felt rejected. However, he undauntedly continued his work. Fortunately, his work was noticed by Dr. Wilbur Sawyer (1879–1951), head of the Yellow Fever Laboratory at the Rockefeller Foundation, International Health Division in New York. Dr. Sawyer visited him and collaborated with him some experiments. Sawyer then

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sent Max samples of blood taken from laboratory workers, who had been immunized by mild attacks of yellow fever. Using his own immunized blood along with what Dr. Sawyer sent, Max was able to protect his mice from encephalitis even after injec-tion of the deadliest strain of yellow fever virus. The experi-ment proved surely that the mouse encephalitis was indeed caused by the yellow fever agent. He also had a test of immu-nization that could reveal the presence of yellow fever antibod-ies in any population.

Dr. Sawyer offered him a job at the Rockefeller Foundation in New York City with double his present salary. With the encour-agement of Dr. Zinsser, Max accepted and moved to Hastings-on-Hudson, New York. He commuted by train to his new laboratory in Manhattan at the Rockefeller Foundation on the grounds of Rockefeller Institute. He was supported by the Rockefeller Foundation, and continued experiments using mouse as the research animal of choice.

About this time, Dr. Wilbur A. Sawyer at the Rockefeller Foundation used Theiler’s mouse-virus strain, a combination of yellow fever virus and immune serum as a human vaccine. He inoculated the mouse-virus strain into 10 volunteer workers in the Rockefeller laboratories. It proved to be effective. Because of this success, Sawyer was often credited with discovering the first human yellow fever vaccine. The fact was that Dr. Sawyer simply transferred Theiler’s work from the mouse to humans. However, there were problems with this vaccine which might lead to encephalitis and in some incidences caused side effects such as headache or nausea.

This mouse-virus strain was subsequently used by the French government to immunize French colonials in West Africa. Later a “Scratch” vaccine was developed. The “Scratch” vaccine was a combination of infected mouse brain tissue and cowpox virus. The “Scratch” vaccine could be quickly administered by scratch-ing the vaccine into the skin. This was used throughout Africa for nearly 25 years and led to near total eradication of yellow fever in the major Africa cities.

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At the Rockefeller Foundation, Max was assigned to work directly with Dr. Eugene Haagen (1898–1972), a German bacteri-ologist. Together they succeeded in culturing yellow fever virus in chick embryo cells which made possible an unlimited supply of virus for experimentation. Max was in strong disagreement with the political views of Haagen, who was pro-Nazis. When Haagen returned to Germany, Max began working with Dr. Wray Lloyd.

Max was not satisfied with the mouse virus strain as a vaccine. He was confident that a better attenuated vaccine, one weak enough to cause no harm to humans, yet strong enough to generate immunity could be developed. He used a new strain of yellow fever virus called “Asibi”, which was harvested from a 28-year-old West African named Asibi. It was highly virulent and was both neurotropic and viscerotropic. It would kill a monkey immediately when injected under the skin. Theiler, W. Lloyd, and the technician Nelda Ricci worked dili-gently to culture this virus. The virus would not grow on their first attempt. The 17th culture was a culture of minced mouse embryo which supported the growth of this virus. They devised methods to subculture every five days to avoid contamination. During this period, W. Lloyd died in Rio de Janeiro where he had married a Uruguayan girl, and fell from an apartment win-dow. W. Lloyd was replaced in the laboratory by Hugh Smith. They continued transferring this virus in tissue culture, passing it from chicken embryo to embryo until they reached strain number 176. They tested the strain on monkeys, and the mon-keys that were tested survived and acquired the expected immunity. They continued to work on this culture number 176. The culture 17D was tried over and over again on monkeys with very favorable results — no harmful clinical manifestation was observed and a good amount of antibodies was obtained.

In March of 1937, Max tested this strain of vaccine on him-self and others. Again, it proved successful. Then he announced that he had developed a new, safer, attenuated vaccine called the 17D strain. (Theiler called it that out of whimsy because there had been no 17A, B, or C.) This new strain was easier to

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produce and cheaper and caused very mild side effects. The 17D strain is the vaccine used today.

The vaccine from number 17D strain vaccine had been extensively used. From 1940 to 1947, more than 28 million 17D-strain vaccines were produced inexpensively. These vaccines were supported by the Rockefeller Foundation. The vaccines were distributed freely to people in tropical countries and the United States. The vaccine was so effective that the health lead-ers thought the yellow fever had been totally eradicated world-wide. As a result the Rockefeller Foundations decided to end the yellow fever program with the perception that yellow fever was no longer a problem. Thereafter, very few people studied the cure of the disease. Unfortunately, yellow fever is still around particularly for people in tropical climates who live outside of the major urban centers. A major outbreak in Ethiopia in 1960–1962 claimed about 30,000 lives. The World Health Organization (WHO) still uses Theiler’s 17D vaccine and is attempting to inoculate people who live in remote areas.

The work of the 17D vaccine had made Max Theiler famous. He was awarded the Chalmer’s Medal of the Royal Society of Tropical Medicine and Hygiene in 1939. Harvard. University also recognized his contribution and awarded him the Flattery Medal of Harvard University in 1945. He also received the Lasker Award of the American Public Health Association in 1949. In 1951, Max Theiler received the highest honor, the Nobel Prize in Medicine or Physiology for his discoveries concerning yellow fever and how to combat it.

However, there was a shadow of the Nobel Prize for Theiler. Wilbur Sawyer was very disappointed because he thought the award should be given to him instead of Theiler. He had served as head of the Yellow Fever Laboratory at the Rockefeller Foundation. A yellow fever vaccine was his life dream. He had hired Theiler and had taken over as director of the International Division, opening Theiler’s way to head the laboratories. Sawyer retired in 1946, but never ceased to regard the victory over yel-low fever in large part his own. He had been involved in a lot

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of research although his job was mainly administrative. He surely had contribution in the development of yellow fever vaccine. He was dying when Theiler was awarded the Nobel Prize. Mrs. Sawyer declared in 1964: “The award of the Nobel Prize to Max Theiler killed my husband” in 1964.

Theiler did not stop his research, he continued to do research on other viral diseases including the Bwamba fever and Rift val-ley fever which were unusual and rare diseases. He also studied polio and discovered polio-like infections in mice known as encephalomyelitis or Theiler’s disease. In 1964, he retired from Rockefeller’s Foundation after achieving the rank of associate director for medical and natural science and the director of the Virus Laboratory. In the same year he accepted the position of professor of epidemiology and microbiology at Yale University in New Haven, Connecticut and retired there in 1967.

He was interested in philosophy and history which occupied his leisure time. He never liked fictional literature. He was a baseball fan, he particularly beloved the Brooklyn Dodgers. When he received the Nobel Prize in 1951, he was asked what he would do with the money, he said, “buy a case of Scotch, and watch the Brooklyn Dodgers”. According to Lillian, he never bought the Scotch, he did buy tickets and watched many games of Brooklyn Dodgers.

Dr. Theiler had written three books. One is entitled Viral and Rickettsial Infections of Man, published in 1948; another is “Yellow Fever”, published in 1971. In 1973, he and Dr. Wilbur G. Downs (1913–1991) published another book titled “Arthropod-Borne Viruses of Vertebrates”. Besides, he pub-lished numerous research papers.

Theiler came to America, in 1923 and lived in this country till his death. However, he never applied for a citizenship. Therefore, he probably never voted at any political elections. He consid-ered himself as a citizen of the world. However, he did have a strong love to the United States. As Elizabeth, his daughter recalled, when they heard of the death of President Franklin Delano Roosevelt (1882–1945) on the radio, they were so

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smitten with grief that she held her breath so long that her family panicked. He died on 11 August 1972 at the age of 73. Elizabeth said that her father knew very well that life has its ridiculous components.

Brief Biographies of Important Persons Mentioned in the Text

Darwin, Charles Robert (1809–1882). English naturalist. Father of modern evolutionary theory.

Downs, Wilbur George (1913–1991). American epidemiologist. Research and publication on epidemiology of malaria, insect transmitted virus diseases, use of naturalists methods, and insecticides in malaria control.

Finlay, Carlos Juan (1833–1915). Cuban physician. Author of numerous works on tuberculosis, exophthalmic goiter, fila-riasis, trichinosis, leprosy, cholera, and tetanus. Contribution to the etiology and pathology of Yellow Fever. Founder of doctrine that Yellow Fever was a mosquito-borne disease.

Livingstone, David (1813–1873). Explorer, physician. Author of Missionary Travels and Research in South Africa, 1857; The Zambezi and Its Tributaries, 1865. Described relapsing fever often resulting from tick bite, also tsetse fly and cattle dis-ease caused by its bite. First to use arsenic in treatment of nagana (horse and cattle diseases in Africa).

Reed, Walter (1851–1902). American physician. Proved that Yellow Fever was transmitted by mosquito.

Sawyer, W (1879–1951). American hygienist. Contribution to Yellow Fever research and international health.

Zinsser, Hans (1878–1940). American bacteriologist. Author of Text Book of Bacteriology, Infection and Resistance, Resistance to Infectious Disease; Rats, Lice and History. Developed immunization against typhus fever; Research on cholera and other bacterial diseases.

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Chapter 40

Rene• Jules Dubos (1901–1982): Pioneer of Bacterial Antibiotics and

Environmental Microbiology

Source: http//www.ranker.com/pics/N1892921/ren-dubos-writers-photo-1?ref=image_621393

(Freely licensed under the CC-BY license)

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Earlier Years of René J. Dubos

René Jules Dubos was born in Saint-Brice-sous-Forêt, France, on 20 February 1901. His father was named George Alexandre Dubos and his mother was named Adeline (de Bloedt) Dubos. They lived in several small agricultural villages north of Paris. When ten years old, he contracted rheumatic fever and for seven years was unable to have vigorous exercise. This was a blessing in disguise as he formed the habit of reading. One book, which had a lasting effect was Essay on the Eafble of La Fontaines (1860) by Hippolyte Taines (1828–1893). As a boy of 15, he ruminated on Taine’s concept that spirit of the fables was caused by the nature of the landscape in the story. All his life, René Dubos was to be aware that the environment had a strong influence on an individual’s development.

In World War I (WWI) (1914–1918), René father bought a butcher shop in Paris, but was almost immediately called into the military service. His mother and René ran the butcher shop during the war. They were very successful. The neighborhood of his upbringing, was, however, quite diverse; he lived in rather drab surroundings, but not far away in his part of Paris there were better homes. He completed his high school train-ing at College Chaptal in 1919. At this time his father died of wound received in the war; he was needed to support his mother and could not go to the university as he would like. However, he did manage to enroll in the nearby Institut National Agronomique and won scholarships, which enabled him to major in science — rather than history, which he would have preferred. A microbiologist might learn from Dubos’ life that sometimes such a situation turns out satisfactory.

After receiving his BS in 1921, he was drafted into the Army as an officer trainee. A mild recurrence of rheumatic fever caused his discharge, and he went to Rome, Italy to be assistant editor of a science magazine. This was published by the International Institute of Agriculture and he served for seven years in this posi-tion. An article by a Russian microbiologist, Sergei Winogradsky

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(1856–1953) changed his life. This article taught that soil bacteria should be studied in their own environment, not merely in labo-ratory cultures. He later stated that this is where his intellectual life began. After reading this article he decided to become a microbiologist.

He decided further that he would have the best opportuni-ties to learn microbiology in the United States. To earn money for the trip he translated books and guided tourists around Rome. Finally, with enough money he boarded ship to New York. By good fortune, one of the passengers he met was Dr. Selman A. Waksman (1888–1973), who was director of the soil microbiology laboratory at the New Jersey State Agricultural Station of Rutgers University. Waksman helped him get a posi-tion as bacteriologist at Rutgers and at the same time work his PhD as a research assistant for Waksman. His PhD research was on the way different organisms in the soil decompose cellulose; this research revealed to him the wide range of microbiological activity that existed in natural environments. This observation was one, which he never forgot; his later experiences related to it strongly. After he earned his PhD in 1927, Dubos joined the staff of the Rockefeller Institute for Medical Research as a fellow.

Dubos continued his research here and in 1965, this institu-tion became Rockefeller University. During this period, Dubos moved up the scientific ladder from assistant, in 1928 to profes-sor in 1956. He held this position until 1971. A great hero to Dubos was Louis Pasteur (1822–1895) and one idea that took hold was the power of microbes to use energy and break and decompose any organic substances. With that idea firmly in mind, Dubos began to study soil, and especially swamp soil, try-ing to find organisms, which could break down the protective coat surrounding human pneumonia bacteria. He did isolate a swamp bacterium, which produced an enzyme, tyrothricin, which enabled the human body to attack the pneumonia germ directly. Later, he isolated bacteria, such as Bacillus brevis, which could consume deadly germs. Other microbiologists were stirred

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by Alexander Fleming’s (1881–1955) discovery of penicillin and Dubos discover of tyrothricin to make still other discover-ies of broad spectrum antibiotics.


Pasteur, Louis (1822–1895). French chemist. Father of microbiol-ogy. Studied anthrax, fermentation, pebrine, rabies chicken cholera, and many other diseases. Disproved the fallacy of spontaneous generation.

Taine, Hippolyte Adolphe (1828–1893). French critic and historian. Chief theoretical influence of French naturalism, a major proponent of sociological positivism.

Waksman, Selman A (1888–1973). American soil microbiologist. Pioneered antibiotics research. Involved in the discovery of streptomycin and many other antibiotics. Nobel laureaut in Medicine or Physiology, 1952.

Winogradsky, Sergei Nikolaievich (1856–1953). Russian pioneer microbiologist. Studied soil microbiology, bacteriology of cellulose decomposition and autotrophic bacteria; established existence of microorganisms that transform ammonia into nitrous acid.

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Chapter 41

Barbara McClintock (1902–1992): Pioneer of Microcellular

Directed Genetics

Source: https://en.wikipedia.org/wiki/Barbara_McClintock(US Public Domain image)

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Barbara McClintock never worked directly with bacteria or viruses, but she was a great microbiologist. Her pioneering directly affected the genetics of the microbial world. When we use the word “microbiologist”, we usually think of virolo-gists or bacteriologists. But, we need to remember that many helminths (and we think of them as the “parasites” even though bacteria and viruses are also parasites!) are microscopic. Considering life cycles, starting with the egg and sperm, all “life” is microscopic, at some stage. All living things are at any one time, divided into the microscopic or the macroscopic. The microscopic, the world of invisible, is as vast and surprising as the macroscopic universe, with its enormous solar systems and spiral nebulae. Barbara McClintock was a true pioneer of the microscopic world.

Barbara McClintock worked with cellular and subcellular life and its denouement in the adult plant. She was able to view chromosomes and even their components and determine their effect upon the adult corn plant, which allowed her to visualize genes before the name “gene” was known. McClintock pub-lished her work 30 years before it was generally acknowledged and accepted. Today, biologists can learn from her life the need for courage and the ability to “go at it alone” when necessary.

McClintock was born on 16 June 1902 in Hartford, Connecticut. She was the youngest of the three sisters. She grew up first in rural Massachusetts and later in Brooklyn, New York. She was active in sports, but also enjoyed just “thinking” and especially a wide range of subjects reading. After graduating from high school in 1918, McClintock enrolled in Cornell University as a botany major, a field in which she remained for her entire life. While in college McClintock was president of her Freshman class and very socially active, playing the banjo in the school band. She earned her BS degree in 1923 and immediately began grad-uate work. One of her first advances was in identifying individ-ual maize (corn) chromosomes under microscope. In 1925, she completed the MA degree and started work on her PhD, which she completed in the field of plant genetics in 1927. In addition,

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she also received an honorary Doctor of Science degree from Rochester University.

The new Dr. McClintock took a position as an instructor in botany at Cornell, where she stayed until 1931. She also served brief periods at the Western College for Women, Smith College, and Williams College. During all of her years as a teacher of botany, McClintock also did research on the genetics of the maize plant. One important paper published in 1931 was on the exchange of genetic information in meiosis, a form of cell division; this paper was in the Proceedings of The National Academy of Science. She also received a National Research Council Fellowship for further work on maize. In this year, she also was appointed a research fellow at the California Institute of Technology. In 1933, she received a Guggenheim Fellowship and did research in Germany. Her work on maize continued.

In 1936, she was appointed Assistant Professor of Botany at the University of Missouri where she taught and did her research until in 1941. The following year, she accepted a research posi-tion at the Carnegie Institute’s Department of Genetics at Cold Spring Harbor, Long Island, New York. While here, she made her home in Cold Spring Harbor. She continued her research at the Institute until her death on 2 September 1992.

The life of Barbara McClintock is one that every science student and teacher needs to study! In the time in which she did her work, science was largely a male institution. Her pio-neering was done over a 30-year period when geneticists did not understand the work she was doing and institutions were not prepared to accept it. As stated before, she was also a woman working in a predominantly male field. In addition to these big obstacles, she was an intensely individualistic person. She did not need the approval of others, but enjoyed her own observations and her own private experiences. To some extent, she was like Gregor Johann Mendel (1822–1884), the monk who worked with peas to initiate the science of genetics. His work was done in the garden of a monastery in Brunn, Moravia, part of Austrian empire. Monks were not supposed to be

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learning or teaching biology and science itself was considered an enemy of the church. Mendel selected such contrasting char-acters in his peas as tall/short, wrinkled/smooth, and green/yel-low. He planted the peas, pollinated accurately, and kept track of the percentage of the plants; he continued this work for 8 years. From his work, he deduced that hereditary characteristics were controlled by factors. These factors were in pairs, one from each parent. The factor pairs separated during the forma-tion of the sex cells, with the egg receiving one factor and the sperm the opposite but related factors, such as tall/short. He found that in crossing the plants, one factor may prevent the expression of the other; one factor was dominant and the other recessive. Like McClintock later, his results were not recognized. Although he published his results and the journals were widely received, science was not yet ready for his findings.

The great emphasis in science at the time was on spontane-ous generation; the scientific world was shocked by Louis Pasteur’s (1822–1895) refutation of this idea; in addition, Pasteur startled the scientific world with his demonstration of microbial disease and the saving of animals and humans from death by such diseases. Mendel, living in the same general period, was not able to show his factors under a microscope, but instead, had to offer mathematical proof. Pasteur was recognized as a devout man as well as a scientist; Mendel as a monk was not regarded as a scientist, but as a peripheral person and somewhat of an “enemy” to the church. Mendel’s work, like McClintock’s, was rediscovered many years later at the turn of the century. Among the discoveries of science, which helped in the rediscovery of Mendel (and also McClintock) was that of nuclear material which took stain more deeply; it was called chromatin. The chro-matin went through a cycle; at cell division chromatin threads split lengthwise, then consolidated and shortened to become definite bodies called chromosomes. Barbara McClintock became an expert in the study of these chromosomes under the micro-scope. Her history was very much like Mendel’s, whom she updated and whose work she modernized.

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Barbara McClintock was recognized as a brilliant scientist and an impeccable worker, but she was also considered acerbic, with a sharp tongue, too specialized in her interest in corn and a bit eccentric. But, one is quick to raise the question: are not these typical of many professors? The answer is yes — but at that time, only male professors! Such characteristics were con-sidered a detriment in a woman! Cornell University did not grant a full professorship to women until 1947 — and that was in home economics! Barbara grew her own com, and with mini-mal support through fellowship with the National Research Council and later, the Carnegie Institution. In the 1940s, she carried out experiments which ultimately led to the 1983 Nobel Prize. Both careful observation of the macroscopic plants and the chromosomes under the microscopic were required, plus some unusual sharp insight regarding unusual happenings. The kernels of some plants showed a purple color and others were colorless. But some kernels which should have been colorless were purple in color! She was astute enough not to bypass the unusual. After many trials, she noticed a regularity to the pattern. This was the basis for her discovery of transposition, for which she received the Nobel Prize. She discovered not only the major genes (she called them “controlling elements” as this was long before the name “gene” was given), but also other factors which caused the genes to be expressed or not expressed. These factors were on the 10 chromosomes of the corn plant, which she studied extensively as a true “microbiologist” and microscopist.

She discovered also that the chromosomes can cross over during the formation of sex cells, sperm, and ova. The cross-over point was called chiasmata; these resulted in an actual exchange of genetic material between chromosomes.

There are “band-wagons” in science, but Barbara was not one to get on them! Barbara McClintock stuck with her corn plants for the study of genetics. Corn takes a year to grow. The mice reveal genetic characteristics in months; fruit-flies respond in weeks; bacteria reveal them in days and viruses may do so in

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hours! Most geneticists, therefore, used the more rapidly grow-ing species for their genetic experiments. Nevertheless, chroma-tin and genes are characteristic of all living matter and the findings of Barbara on corn were applicable to microbes. The findings of James Watson (1928–), Francis Crick (1916–2004), and Maurice Wilkins (1916–2004) regarding DNA and RNA applied to all genetics; but we will find there are differences, also. The organisms microbiologists worked with, largely bacteria and viruses, seldom had chromosomes (some microscopic parasites, as well as blue-green bacteria do have chromosomes); genetic mate-rial was found throughout the organism and genes were formed at reproduction. All living things were first divided into two “kingdoms” — plant and animal. Then the “kingdoms” became the “karyote” and “prokaryote” kingdoms, since this seemed to be a more general division. Microscopic organisms, mostly, were placed in the prokaryote kingdom-living forms without nuclei. Nucleated organisms were placed in the “ karyote” kingdom.

Recently, a third kingdom has been proposed: the “archae-bacteria”. These bacteria are often found around ocean vents and live on the sulfurous fumes which come from the interplay of the magma of the earth and the ocean. Giardia lamblia, a parasite, is related to these, and by some biologists is considered a “missing link”. One of the characteristics of the archaebacteria is a double nucleus with two sets of chromosomes which remain through life (this name for the third kingdom will probably be changed, since more than bacteria are found in it).

The scientific world was highly excited about the work of Watson and Crick, unlike the response to Barbara McClintock. They published their findings in Nature (1953) and became instant heroes. One reason was the clarity of the model which they developed. This was the basic model of DNA with the double helix and the repeating subunits, the nucleotide with phosphate, sugar, and hydrogen bonds.

Ten years later, they shared with Maruice Wilkins (who had done photographic analyses of DNA) the Nobel Prize in Medicine and Physiology. Barbara McClintock’s work helped form the

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“central dogma” of DNA, but she remained outside the core group during the development of the concepts. The central dogma was far more rigid than the concepts fostered by McClintock and her “jumping genes”, which moved about and affected other genes. She also called attention to the differ-ences between the smaller and larger organisms in regard to genetics. Jacques Monod (1910–1976), 1965 winner of the Nobel Prize in Physiology and Medicine once said: “What is true for Escherichia coli (an intestinal bacterium) is true for the ele-phant”. There are elements of truth to this, but McClintock pointed out that in the larger animals the cells were more com-plex and in the embryonic stage actually directed the develop-ment of an entire large organism, which might become an elephants, a whale or a gigantic redwood tree. The cell, she said, had to make “wise decision” in these cases. Bacteria and viruses had genes and DNA, but were not destined to become enormous multi-celled beings, whose development was guided by the origi-nal embryonic cells. These were exceedingly formidable concepts, well ahead of their time. Monod’s concepts were a holdover from the nonliving world of physics, but McClintock knew better! An organism consisting of cells in corn stalk tassels and kernels which is starting a corn plant has a great many more decisions to make than a single-celled E. coli bacterium! The largest chromosome of a Drosophila fruit-fly is 19 times the size of E. coli and the human genome is a 1000 times its size. McClintock stated that molecular biologists had no “feel” for what these biological cells did. This was heresy when molecular geneticists with a physics background were at the top of the scientific world.

It is well for the new microbiologist and scientist — and his professor — to see that the advances in sciences often come not from the “central dogma”, but from the peripheral areas, of which Barbara McClintock was an ideal example. Since 1945 on, she showed that genes were not immutable and that they moved — opposite to many ideas in classical Mendelian genet-ics, which she, herself, had helped modernize. New discoveries often refute the “central dogma”, as the examples of Columbus

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and Galileo reveal. Even when her work was available, Jacques Monod and François Jacob (1920–2013), two French geneticists who corroborated her work, failed to read and cite her find-ings, because there was no settled terminology and they may have been difficult to find. A scientist, therefore, needs to search diligently and be aware that terms a little different from his may be in use.

One scientist has stated that after 60 years of work, geneti-cists are back to the original questions: “How does the egg from the organism?” Many scientists are uncomfortable with Barbara McClintock’s answer. Intuition! Speaking of chromosomes and genes, she said that the more she worked with them, the bigger and bigger they got. Finally, as she looked through her micro-scope as all microbiologists must, she began to “feel” that she was there with the genes! She said “you forget yourself — it surprised me that I actually felt that — these were my friends. As I looked at the genes they became part of me — The main thing about it is that you forget yourself”.

As you try to find the field in science which most appeals to you, can you apply Barbara McClintock’s way?

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Chapter 42

George W. Beadle (1903–1989): Pioneer of Biochemical Genetics

“Our knowledge of living things at the molecular level has continued to increase exponentially. In a real sense genetics has come to be recognized as an integral and basic part of all biology, of biochemistry, biophysics, immunology, virology, physiology, the behavioral sciences, plant and animal breeding, and all the rest. Largely as a result of its advances, the opportunities and challenges have never been greater in the areas of biology. Nor have the intellectual reward to those adequately prepared and sufficiently motivated”.

“The separation between the sciences and the humanities is a fallacy that is annoying to me. Science is not opposed to culture any more than culture is opposed to science. Intelligent people seek balance”.

—George W. Beadle

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Source: https://en.wikipedia.org/wiki/George_Wells_Beadle(US Public Domain image)

Introduction

George Wells Beadle shared the Nobel Prize for Medicine or Physiology in 1958 with Joshua Lederberg (1925–2008) and Edward Lawrie Tatum (1909–1975). He investigated the biochemical genetics of Indian corn, fruit fly, and Neurospora. He pioneered the approach of identifying many genes for individual biochemical steps leading to biosynthesis of vitamins, amino acids, or other important metabolic products. His work helped prove the one gene/one enzyme or one polypeptide concept, which was the foundation of modern molecular genetics.


George Wells Beadle was born on 22 October 1903, on a farm near the small town of Wahoo, Nebraska. The Beadles owned

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and operated a small farm of about 40 acres. Their farming was very diversified with field crops such as alfalfa, potatoes, and corn; truck crops included asparagus, strawberries for market; plus cattle, horses, hogs, and chickens. The income from these crops were supplemented by selling produce such as out-of-state apples and potatoes purchased in carload lots. Life on the farm was difficult but never dull because they kept rabbits, ferrets, bees, cats, dogs, and, for a time, a pet coyote. They also did hunting, fishing, and trapping, which were enjoyable pastimes for the two brothers. Great sorrow came to the Beadle family with the passing of Beadle’s older brother. Beadle assumed tacitly that he would eventually take over the family farm, but education changed his life course.

Beadle attended a genuine little red, one-teacher, wooden schoolhouse in Wahoo and was influenced by one of his teachers; Ms. Bess MacDonald, who taught him chemistry and physics, and encouraged him to go to college; although Beadle’s father was not very keen about this idea. Instead, he expected him to inherit the farm and thought that higher education was not necessary for farmers.

Beadle did manage to attend the Agricultural College, University of Nebraska, Lincoln, with the full intention of returning to the farm. However, as recorded in his recollections, Beadle said that he doubted he would have attended college had it not been tuition free and had he not had an opportunity to work.

In the first year of college, Beadle was interested in English, and he intended to follow on that subject. But in the second year, he was given a summer job to help classify genetic traits in a wheat hybrid population for Professor Franklin D. Keim (1885–1956) of the Agronomy Department. After reading about genetics, Beadle realized he had a strong interest in the subject. He was also given other assignments including laboratory instruction in an agricultural high school program offered by the college. In addition, he grew various exotic plants, collected, and mounted representative weed seeds,

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filled orders, mailed them out, and kept records. In his senior year, he worked on projects related to root development, the survival of fall-seeded grasses of economic importance, and the development of a key for the identification of local native grasses by using vegetative characters. Beadle obtained his BS degree in 1926 and Master’s degree in 1927, both in agronomy. He seemed destined to be an agricultural scientist.

Professor Keim had an uncanny ability to evaluate students and encourage them to pursue the right career. He strongly recommended that Beadle pursue a PhD degree, and in fact Keim arranged for Beadle to have a teaching assistantship at Cornell University researching the ecology of the pasture grasses of New York State. However, when Beadle met Professor Rollins A. Emerson (1873–1947) of Cornell University, he resigned his teaching assistantship and started working in genetics and cytology with Professor Emerson, who gave him a part-time research assistantship.

Professor Emerson was the leading maize geneticist in the United States. The group of graduate students who worked with him at that time, included George F. Sprague (1902–1998), Marcus Rhoades (1903–1991), Barbara McClintock (1902–1992), Hsien-Wen Li (1902–1976), and others. All of them were known worldwide in the field of genetics. Emerson’s work on kernel and plant color inheritance in maize was ground breaking and, he was responsible for many contributions to the basic science of genetics, particularly the genetics of maize. Emerson’s remarkable work in basic genetics significantly elevated the art and science of plant breeding. For example, by genetically transferring resistance to the disease anthracnose to commercially desirable dry bean, the important bean industry of New York State was saved from collapse. Emerson also succeeded in transferring disease resistance to commercially grown cantaloupes.

For his PhD dissertation, Beadle worked on the cytogenetics of maize. He studied the genetical control of meiosis using corn lines, in which the chromosomal behavior was genetically modified. He proved that heritable defects in pollen production

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were caused by the abnormal behavior of the chromosome during meiosis and that this behavior was in turn determined by genes. In 1931, he completed his dissertation and obtained his PhD degree. During this time, Beadle audited courses in physical chemistry and biochemistry which later influenced him to develop the new discipline of biochemical genetics.

Achievement

Although Beadle intended to continue his corn cytogenetics work at Cornell with the award of a National Research Council Fellowship, he was persuaded by the wise chairman of the Fellowship Board, Dr. Charles E. Allen of the University of Wisconsin, to go to California Institute of Technology (Caltech). At Caltech, Beadle worked with Dr. Thomas Hunt Morgan (1866–1945), the renowned geneticist, who had just moved from Columbia University to California. He immediately found that he was working with distinguished American geneticists such as Alfred H. Sturtevant (1891–1970), Jack Schultz (1904–1971), Calvin Bridges (1889–1938), Theodosius Dobzhansky (1900–1975), Ernest G. Anderson (1891–1973), Sterling Emerson (1900–1988), Karl J. Belar (1895–1931), and Carl Lindegren (1896–1986), and also with scientists of international renown such as C. D. Darlington, John B. S. Haldane, and George D. Karpechenko. In the beginning, Beadle concentrated on his cytogenetic program but soon became actively interested in the fruit fly, Drosophila. Beadle shifted his work to genetic recombination of Drosophila collaborating with Dobzhansky, Emerson, and Sturtevant at various times. He focused on the problem of crossing-over in Drosophila. He found that various aspects of crossing-over and recombination of characters occurred at random when two cells united. He also investigated homozygous and heterozygous translocation using Drosophila.

Beadle served as an instructor from 1933 to 1935 at Caltech. During that time, Boris Ephrussi (1901–1979) arrived at Caltech from Paris. From his association with Ephrussi, Beadle became interested in tissue culture because he liked to bring together

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genetics and embryology. As a result of this interaction, he completed a successful tenure with Ephrussi’s laboratory in Paris (1935–1936) and worked on embryonic bud transplantation.

At Caltech, general enthusiasm in research was at a high level and Linus Pauling (1901–1994) took a personal interest in the study of genetic crossing-over. However, this was also the time period of the Great Depression and Caltech was in a serious financial difficulty at that time. Beadle’s salary of $1500.00 during his time in Paris was actually paid by Dr. Morgan. Beadle was not aware of this situation at the time and learned about it later. Dr. Morgan was a scientific visionary and was always generous in supporting promising young scientists.

Beadle and Ephrussi experimented with transplanted ovaries of Drosophila and showed that “functional connections of the implanted ovary with a host oviduct may be established”. They also worked on how genes controlled the expression of vermilion eye of Drosophila by using mutants. With the help of transplantation techniques, they successfully demonstrated that two genes (Cinnabar and Vermilion) are in direct control of the two postulated chemical reactions responsible for the biosynthesis of the vermilion color of the eye. From this work, they originated the one gene/one enzyme concept.

The next logical step was the identification of the two brown pigment precursors. Ephrussi continued to work on this problem in Paris, whereas Beadle brought this project back to the United States.

Upon returning to the United Stated in 1936, Beadle was appointed assistant professor at Harvard University, working with Dr. Kenneth V. Thimann (1904–1997). In 1937, he was appointed professor at Stanford University, Palo Alto, California. There, Beadle hired Dr. Edward L. Tatum and Dr. Clarence Clancy as research associates to continue to work on the genetic control of the biosynthesis of the vermilion color of the eye of the Drosophila. Tatum demonstrated a functional relationship between one of the precursors of tryptophan. Tatum in collaborating with Arie J. Haagen-Smit (1900–1977)

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at Caltech attempted to identify the precursor without success. This substance was later identified as kynurenine by Adolf Butenandt, W. Weidel, and E. Becker in Germany.

At Stanford University, Beadle found an easy way of identifying genes with known chemical reactions. This occurred to him while he audited a course on comparative biochemistry offered by E. Tatum. Since they were convinced that all enzymatically catalyzed reactions were gene controlled in a one-to-one basis, it would be easy to discover additional such relationships by finding mutant organisms, which had lost the ability to carry out specific chemical reactions already known or postulated. In order to facilitate this approach, Beadle used Neurospora because the cytogenetics of that organism had already been worked out by mycologists Bernard O. Dodge (1872–1960) and Carl Lindegren. So why not determine the minimal nutritional requirements of Neurospora, produce mutant types by X-ray or ultraviolet irradiation and then test these mutants for loss of ability to synthesize one or more components of the minimal medium?

Combining genetical and biochemical techniques, Beadle and his colleagues worked out the pathways for the biosynthesis of vitamin B6 and the genes controlling each biosynthetic step of this vitamin. They also identified many genes for many other individual biochemical steps leading to the biosynthesis of many other vitamins and amino acids.

Beadle’s research group expanded over the years. Herschel K. Mitchell, Norman H. Horowitz, David M. Bonner, Francis Ryan, Mary Houlahan, and others joined his team. He also trained many graduate students including Adrian M. Srb, August Doermann, David Regnery, Frank C. Hungate, Taine T. Bell, and Verna Coonradt; all of whom were successful in their endeavors.

Beadle’s genetical findings were also applied to the manufacturing of penicillin, which resulted in a four-fold increase in production, and in the development of new ways of assaying vitamins and amino acids in food and tissues. These achievements, thus, helped the war effort during the World War II.

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Beadle’s most significant contribution was the identification of many genes with specific chemical reactions. Around 1941–1942, John B. Haldane (1892–1964) and Sewall G. Wright (1889–1988) had rediscovered the early observation of inborn errors of metabolism by Archibald E. Garrod (1857–1936) who had demonstrated that the human disease alcaptonuria was a simple Mendelian recessive trait characterized by the inability to degrade 2,5-dihydroxyphenyl acetic acid (alcapton or homogentisic acid). The normal healthy person would degrade alcapton; whereas, the alcaptonurics would excrete it in the urine where, upon exposure to air, it would be oxidized to a blackish compound. This observation implied the one gene/one enzyme concept. However, this concept wasn’t appreciated nor even mentioned in Garrod’s time. Beadle’s work reconfirmed the concept that one gene specifies the sequence of one enzyme (or polypeptide chain). But this concept was received with reluctance and skepticism by the scientific community. This reluctance of acceptance continued until the double helix structure of DNA was described by James Watson (1928–), Francis H. Crick (1916–2004), and Maurice Wilkins (1916–2004). With the understanding of how DNA replicates and the role of DNA in protein synthesis, the difficulty in accepting the concept of one gene/one enzyme theory finally disappeared, but today, with the discovery of RNA exon splicing, the one gene/one enzyme or polypeptide concept is no longer an accurate statement. Nevertheless, this concept served as a good guiding light for many molecular biological investigations.

In 1946, Beadle returned to Caltech and succeeded Morgan as chairman of the division of biology. In December of that year, he was elected president of the American Association for the Advancement of Sciences.

From 1959 to 1960, Beadle was an Eastman Visiting Professor of Oxford University in London. In 1961, he was appointed chancellor of the University of Chicago. He then served as the president of that university from 1962 to 1968. In 1968, he accepted the directorship of the Institute for Biomedical

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Research of the American Medical Association and moved it to the Chicago campus, where he continued to teach and do research until 1978. From 1969 to 1975, he carried the title of William E. Wrather, Professor of Biology. Concurrently, he served as a trustee member of the Caltech (1970–1978) and an honorary trustee of the University of Chicago (1971).

Organizational and Administrative Activities

Beadle was active in many scientific organizations. He was a member of the National Academy of Sciences and was chairman of the committee on genetic effects of atomic radiation for a number of years. He was a member of American Philosophical Society, American Society of Zoologists, American Society of Naturalists, Botanical Society of America, and Genetical Society of America and numerous societies. Beadle also served on various boards including the advisory committee of biology and medicine of the Atomic Energy Commission from 1948 to 1950, the divisional committee for biological sciences of the National Science Foundation, and worked with the Los Angeles county branch of the medical and scientific advisory board of the American Cancer Society. He was also on the board of directors of the Los Angeles County Medical Association Research Foundation.

Beadle was president of the University of Chicago during the Vietnam War era. It was a period of turbulence and intense changes for universities across the country. However, Beadle strongly believed that the role of the university was to advance knowledge, and he held steady to its traditional values of research and intellectual excellence. He increased the faculty number from 860 to 1080 and full professors from 345 to 433. The average salary increased 50%, and total campus expenditures doubled. Under his leadership, new facilities were built including the Joseph Regenstein Library, the high energy physics and astrophysics laboratories, the children’s hospital, and the School of Social Service Administration.

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Honors and Awards

Beadle received many awards in his life. He was the recipient of the Lasker Award of the American Public Health Association in 1950, the Dyer Lectureship Award in 1951, the Emil Christian Hansen Prize of Denmark in 1953, Albert Einstein Commemorative award in science in 1958, the Nobel Prize in 1958, American Cancer Society award in 1959, Kimber Genetics award in 1960, and Donald Forsha Jones Medal in 1972. More than 31 universities and colleges from the United States and foreign countries have conferred him honorary degrees.

In addition to many research papers, Beadle coauthored with Dr. A. H. Sturtevant on a book titled An Introduction to Genetics (Saunders, 1939). He wrote a book titled Genetics and Modern Biology (American Philosophical Society, Philadelphia, Pennsylvania) in 1963. In 1966, he coauthored with his second wife, Muriel Burnett Beadle, another book The Language of Life: An Introduction to the Science of Genetics (Doubleday and Company, Inc., New York, New York). Later in 1976, he also wrote a book titled Thomas Hunt Morgan: Pioneer of Genetics with his colleagues Ian B. Shine and Sylvia Wrobel.

Personal Calibers

Beadle married Marion Cecil Hill on 22 August 1928, and had a son, David. But the couple was later separated. On 12 August 1953, he married Muriel McClure Burnet. He had a stepson Raymond James Burnet. In 1982, he and his wife Muriel moved to a retirement village in Pomona, California, and lived there until his death on 9 June 1989.

According to Adrian M. Srb (1917–1997), Beadle was an attractive and interesting person. He had a good sense of human and he was tickled by the amusing name of his native town. He enjoyed gardening, mountain climbing, and tennis playing. He liked activities that involved physical exertion; on all these he showed his basic competitiveness.

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Commentary

Beadle was a brilliant cytogeneticist by training, yet he was able to bring other disciplines into his research work. Initially, he added biochemistry to his study of genetics. Later, he tried to integrate embryology into the already marvelous genetical work of Drosophila and Neurospora. Bringing different but interrelated disciplines together in a revealing way will often broaden the scope of research and result with surprising reward. Naturally, his vision and penetrating observations, in addition to his persistent endeavors, helped lead to the great discovery of one gene/one enzyme or polypeptide concept. One should be open-minded to adding new disciplines to one’s research. Beadle’s success story is a vivid example of the success of such an approach.

Despite the great accomplishments in Beadle’s life, he remained a farmer at heart. He pioneered the new field of genetics, which revolutionized the plant breeding and the entire understanding of biological reproduction. He further established the relationships between genes and enzymes in the organisms he studied. Yet, he kept his initial childhood warm hearted kind, perseverant farmer’s spirit. He grew corn behind his house and in other plots in campus wheas president of the University of Chicago and was often mistaken for a university gardener. After retirement, gardening was still his favorite activity.


Allen, Charles Elmer (1872–1954). American botanist. Author of A Textbook of Botany (with Edward M. Gibert in 1917); A Textbook of General Botany (with Gibert M. Smith and others in 1924). Researched in cytology of meiosis, heredity.

Anderson, Ernest Gustav (1891–1973). American geneticist. Researched on genetics of maize; mechanisms of crossing

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over in Drosophila; hereditary effects of radiation; applica-tion of cytogenetics to plant breeding.

Bonner, David Mahlon (1916–1964). American biochemist. Researched on leaf growth factors; relation of chemical structure to physiological activity of plant growth hormones; chemistry of flower hormone; growth factors for plants and microorganisms; biochemistry of genetically controlled reac-tions in Neurospora; genetic control of enzyme formation.

Bridges, Calvin Blackman (1889–1938). American geneticist. Contributed to the research on genetics of Drosophila, for-mulation of concepts of modern genetics.

Butenandt, Adolf Friedrich Johann (1903–1995). German bio-chemist. Researched on plant insecticides, hormones. Nobel laureate in Chemistry in 1939.

Clancy, Clarence William (b.1905). American geneticist. Researched on genetics of Drosophila melanogaster; development of eye colors and gene action; intraspecific sterility.

Crick, Francis Harry Campton (1916–2004). British biologist. Nobel laureate in Medicine or Physiology (with J. D. Watson and Maurice H. F. Wilkins) in 1962; proposed model for double-helix structure of DNA.

Dobzhansky, Theodosius (1900–1975). Russian geneticist. Researched on heredity and cultural evolution of Drosophila.

Dodge, Bernard Ogilvie (1872–1960). American mycologist. Research on reproduction of fungi, fungal parasites of fruit and trees, development of plant disease control.

Emerson, Rollins Adams (1873–1947). American geneticist. Investigated problems of orchard management including insect and disease control, winter hardness, effect of envi-ronmental factors in seed production in potatoes, heredity studies on common bean, nature of somatic mutations, and compiled genetic analysis of corn.

Emerson, Sterling Howard (1900–1988). American geneticist. Researched on cytogenetics and self-sterility in Oenothera; crossing-over in Drosophila; genetics and physiology of adaptive changes in Neurospora.

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Ephussi, Boris (1901–1979). Russian geneticist. Researched on cell differentiation, genetic mechanisms, somatic cell varia-tion, and cell cultures in vitro.

Garrod, Sir Archibald Edward (1857–1936). English physician. Author of Inborn Errors of Metabolism (1923); The Inborn Factors in Disease (1931). Researched on genetically deter-mined human diseases, cryptonuria, alcaptonuria, fructosuria, and albinism.

Haagen-Smit, Arie Jan (1900–1977). American biochemist. Researched on chemistry of natural products, plant hormones, essential oils, alkaloids; physiologically active substances.

Haldane, John Burdon Sanderson (1892–1964). British geneticist. Applied mathematics to genetical studies. Researched on evo-lution, human physiology, gene linkage, and so on. Author of many books.

Horowitz, Norman Harold (1915–2005). American biologist. Researched and numerous publications on biochemistry, natural inheritance, evolutionary biochemistry, amino acid synthesis in living cells, regulation of enzyme synthesis, strategy and tactics search for extraterrestrial life.

Keim, Franklin David (1886–1956). American agronomist. Researched on pasture improvement; forage crop; grass identification; and wee control.

Lederberg, Joshua (1925–2008). American geneticist. Discovered bacterial conjugation and numerous other contributions. Nobel laureate in Physiology and Medicine (with George W. Beadle and Edward L. Tatum) in 1958.

Li, Hsien-Wen (1902–1976). Chinese geneticist and plant breeder. Researched on cytogenetics of Italian millet, wheat, sugar cane and rice. Improved crop varieties in wheat, rice, Irish potatoes, and sugar canes; eretoides in rice by use of atomic energy and mutagenic chemicals.

Lindegrens, Carl Clarence (1896–1986). American geneticist. Researched on genetics of yeasts, bacteria, and fungi.

McClintock, Barbara (1902–1992). American geneticist. Discovered transposon. Nobel laureate in Physiology and Medicine in 1984.

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Mitchell, Herschel Kenworthy (1913–2000). American geneti-cist. Researched on growth factors for microorganisms; bio-chemical genetics in Neurospora; synthesis of compounds of biological significance.

Morgan, Thomas Hunt (1866–1945). American zoologist and geneticist. One of the founders of modern genetics using Drosophila. Demonstrated physical basis of heredity and importance of gene. Nobel laureate in Medicine or Physiology in 1933.

Pauling, Linus (1901–1994). American chemist. Nobel laureate in Chemistry in 1954 and in Peace in 1963.

Rhoades, Marcus Morton (1903–1991). American geneticist. Researched on a wide variety topics in maize cytogenetics, including crossing over and basic cytogenetic principles; cytoplasmic male sterility; centromeric misdivision; the first transposon-type mutator system; a nuclear gene, iojap, that affects the chloroplast genome; meiotic mutations, includ-ing ameiotic 1; meiotic drive by abnormal chromosome 10; properties of heterochromatin; and the effect of B chromo-some on heterochromatin.

Ryan, Francis Joseph (1916–1963). American geneticist. Researched on general zoology and microbial genetics.

Schultz, Jack. (1904–1971). American geneticist. Researched on nature and function of the gene; chemical techniques of Drosophila as studies by genetics, cytochemical and nutri-tional techniques; pattern of human chromosomes.

Sprague, George Frederick (1902–1998). American geneticist. Pioneered in the inbred-hybrid concept for developing superior corn hybrids. Leader in the science of corn genetics and breeding.

Sturtevant, Alfred Henry (1891–1970). American geneticist. Researched on fruit flies; discovered gene position effect; developed methods of mapping chromosome; proved cross-over inhibitors in the fruit fly resulted from chromosome inversion, sex determination, and so on.

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Tatum, Edward Lawrie (1909–1975). American biochemical geneticist. Discovered one gene-one enzyme theory. Nobel laureate (with George W. Beadle and Joshua Lederberg) in Physiology and Medicine in 1958.

Thimann, Kenneth V. (1904–1997). American biologist. Co-discovered plant hormone auxin and its functions. Researched on plant biochemistry and effects of light and gravity in higher and lower plants.

Watson, James D. (b.1928). American biologist. Nobel laureate in Medicine or Physiology (with Francis H. C. Crick and Maurice Wilkins) in 1962; proposed model for double-helix structure of DNA.

Wilkins, Maurice, H. F. (1916–2004). New Zealand biochemist. Nobel laureate in Medicine or Physiology (with Francis H. C. Crick and James D. Watson) in 1962; proposed model for double-helix structure of DNA.

Wright, Sewall Green (1889–1988). American geneticist. One of the primary founders of population genetics which led to the modern evolutionary synthesis.

Additional Resources

Abbot, D. (1984). Biologists. In The Biographical Dictionary of Scientists. New York: Peter Bedrick Books.

Beadle, GW. (1974). Recollections. Annual Review of Genetics, 8, 1–13.

Berg, P. and M. Singer (2003). George Beadle, an Uncommon Farmer. The Emergence of Genetics in the 20th Century. New York: Cold Spring Harbor Laboratory Press.

California Institute of Technology Oral History Project. (1981). Interviews with James Bonner, Sterling Emerson, Norman Horowitz and Donald Poulson by Judith Goodstein, Harriet Lyle, and Mary Terrall. Pasadena, CA: Caltech Archives.

Candee, MD (1956). Current Biography Year Book, pp. 37–39. New York: H. W. Wilson Company.

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Suggested Reading

1. Candee, M. D. (1956). Current Biography Year Book, 1956. pp. 37–39. H. W. Wilson Company, New York.

2. Beadle, G. W. (1974). Recollections. Annual Review of Genetics. 8: 1–13.

3. Abbot, D. (1984). Biologists. The Biographical Dictionary of Scientists. Peter Bedrick Books, New York.

4. California Institute of Technology Oral History Project. 1981. Interviews with James Bonner, Sterling Emerson, Norman Horowitz and Donald Poulson by Judith Goodstein, Harriet Lyle, and Mary Terrall, Pasadena, California, Caltech Archives.

5. Berg, P. and M. Singer (2003). George Beadle, An Uncommon Farmer: The Emergence of Genetics in The 20th Century. Cold Spring Harbor Laboratory Press, New York.

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Chapter 43

Edward Lawrie Tatum (1909–1975): Pioneer of Molecular Genetics

Source: https://en.wikipedia.org/wiki/Edward_Lawrie_Tatum(US Public Domain image)

Edward Lawrie Tatum was well known for his pioneering work on the mutational analysis of biochemical pathway, which laid foundation for the discovery of the gene control of the biosyn-thesis of proteins. He was a pioneer in postulating one gene-one

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enzyme theory. Edward Tatum together with George W. Beadle (1903–1989) and Joshua Lederberg (1925–2008) were awarded with Nobel Prize in Physiology and Medicine in 1958.

Edward was born in Boulder, Colorado on 14 December 1909. His father was Arthur L. Tatum (1884–1955), and his mother Mabel Webb Tatum. He had a twin brother Edwood, but Edwood died shortly after birth. Edward had another brother, Howard, and a sister, Besse. Edward’s grandfather, Lawrie Tatum, was a Quaker, who had settled in the Iowa Territory, and had been an Indian agent after the American Civil War (1861–1865). Lawrie Tatum had written a book Our Red Brothers. The Tatum’s family migrated several times in rapid succession as many fron-tiers did in those days. They moved to Madison, Wisconsin; Chicago, Illinois; Philadelphia, Pennsylvania; Vermillion, South Dakota, and in 1918 back to Chicago.

When Edward was born, Arthur Tatum was an instructor in chemistry at the University of Colorado at Boulder, Colorado, where Mabel Webb’s father had been the Superintendent of School. Arthur Tatum held a succession of teaching positions while earning a PhD degree in Physiology and Pharmacology from the University of Chicago, and a MD degree from Rush Medical College. By 1925, Arthur Tatum was settled at the University of Wisconsin, Madison as professor of pharmacology in a department that was a major center for the training of professors of pharmacology. Arthur Tatum was a scholar, and had done many good researches. Among the significant contri-butions were the introduction of picrotoxin as an antidote for barbiturate poisoning and the validation of arsenoxide (mapharsen) for chemotherapy of syphilis.

Edward Tatum was influenced by his remarkable family background as mentioned above which helped nurture him to be a good scientist. He was also benefitted from the Laboratory School at the University of Chicago he attended while his father was in Chicago. He continued his education at the University of Wisconsin when his family moved to Madison. He earned a bachelor’s degree in 1931. For the bachelor’s degree, he worked

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on the effect of associated growth of bacterial species Lactobacillus and Clostridium septicum giving rise to racemic acid (Biochem. J, 1932, 26, pp. 846–852). He demonstrated that C. septicum racemized the D-lactic acid produced by the lactic acid bacteria. He continued his graduate work along the same line under Dr. Edwin B. Fred (1887–1981) and William H. Peterson (1880–1960), and obtained his MS degree in 1932 and completed his PhD dissertation entitled Studies on the biochemistry of microorganisms in 1934. The same year he married June Alton.

In his dissertation research, Dr. Tatum found in potatoes a chemical compound, later identified as asparagine, to be a growth factor for C. septicum. He continued this research in collaboration with Harland G. Wood (1907–1991) and Esmond E. Snell (1914–2003) in a series of pioneering studies on the role of growth factors, vitamins, in bacterial nutrition. For example, they demonstrated that thiamine was a growth factor for pro-pionic acid bacteria in 1936. His work won him a General Education Board postdoctoral fellowship to spend a year at Utrecht, The Netherlands. In Netherlands, he worked with Fritz Kogl (1897–1959) and met many friends such as Nils T. E. Fries (1912–1994), another research fellow from Uppsala, Sweden, Albert J. Kluyver (1888–1956), B. C. J. G. Knight, and P. Fildes. These are leading investigators of bacterial chemistry and nutri-tion at that time. (Dr. J. H. Mueller in Harvard and Dr. Andre M. Lwoff (1902–1994) in Paris were also leading figures of bacterial nutrition research.) Dr. Tatum was in the leading edge on the research of bacterial nutrition.

After returning to Wisconsin, a big career change occurred for Edward, which significantly affected the future role of Tatum in embracing the birth of modern molecular biology. Only a man with great courage and wisdom could do that. George Beadle at the Stanford University was looking for a research associate to work on “hormone-like substances that are concerned with eye pigments in Drosophila”. Edward Tatum wrote to him to accept his offer and work on Drosophila. This was a big challenge for Tatum. Dr. Tatum’s work was related to

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the chemical microbiology of butter, which is of economic importance in the state of Wisconsin. Therefore, Dr. Tatum’s academic future would be bright if he chose to stay in Wisconsin where funding for butter-related bacterial nutrition research would likely be more adequate. Nevertheless, Tatum accepted Beadle’s offer and the multiple challenges of comparative bio-chemistry that went with it.

Between 1937 and 1941, Tatum engaged with the arduous task of extracting pigment precursors from Drosophila larvae. Boris Ephrussi (1901–1979) and G. Beadle had previously dem-onstrated that a diffusible substance produced by the wild type flies was critically lacking in the mutant strains. Ephrussi and Chevais had also reported that normal eye color of Drosophila could be restored in culture media supplemented with trypto-phan. Tatum, however, found that the above phenomenon could happen only with Drosophila culture medium carrying a bacterial contaminants. Tatum cultured the contaminant and proved that the bacterium, a Bacillus species, was the source of the elusive hormone. It was due to this bacterial product that Drosophila was able to synthesize the pigment. Tatum and Arie J. Haagen-Smit (1900–1977) also identified that the V+ hormone was kynurenine, a metabolite of tryptophan. These were the frontier work of the interchangeability of growth factors for bacteria and animals. This work also bolstered the theory that many microbes synthesized vitamins required by other species.

In the meantime, Tatum volunteered to develop and teach an unprecedental comparative biochemistry course for biology and chemistry graduate students at the Stanford University.

In the course, Dr. Tatum described the yeast and fungi, some of which exhibited well-defined block in vitamin biosynthesis. Dr. Beadle also attended these lectures and recalled the earlier elegant work of Bernard Ogilvie Dodge (1872–1960), which was followed up by Carl C. Lindegren (1896–1986) in Caltech. After learning to culture Neurospora in a well-defined medium, Tatum and Beadle X-rayed the Neurospora and sought for mutants with biochemical defects marked by a nutritional

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deficiency. This was a painstaking work because they had to examine one by one the single spore culture isolated from the irradiated parent strain. They had examined thousands of cul-tures and found 299 proved to be the first recognizable mutant required pyridoxine for growth. The trait (gene) was segre-gated according to simple Mendelian principles, and could be mapped onto a specific chromosome of the fungus. After extensive study of the nutritional mutants of Neurospora, Drs. Beadle and Tatum then shifted the main stream of genetic study from Drosophila to Neurospora. From these extensive studies with the genetic mutants of Neurospora, they sug-gested that a direct and simple role of genes is the control of enzymes. They hypothesized, therefore, that enzymes were the primary products of genes. This was later becoming the one gene-one enzyme theory. This was before the genetic material was identified as DNA by Oswald T. Avery (1877–1955), Colin Munro MacLeod (1909–1972), and Maclyn McCarty (1911–2005) in 1944.

In 1942–1945, Tatum was an associate professor of Stanford Biology Department. He developed an increasingly independ-ent research program exploiting the use of Neurospora mutants for the exploration of biochemical pathways. His laboratory rapidly engendered a library of mutants blocked in almost any anabolic pathway. The growth of these mutants would require extra nutrients. They also discovered many distinct genes for controlling metabolic pathway in the Neurospora.

It is worth mentioning that the one gene-one enzyme theory was actually the results of hard work of primarily Beadle and Tatum. One might think that Archibald E. Garrod (1857–1936) who published his study of what was then called “inborn errors of metabolism” including alcaptonuria in man in 1908, was ahead of Tatum in formulating the one gene-one enzyme theory. The Garrod’s basic findings on alcaptonuria might be in parallel with the metabolic blocks in Neurospora. However, many geneticists who specialized in maize and Drosophila were not aware of Garrod’s work. The Garrod’s findings received

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little comments from geneticists until after Neurospora was launched in 1941. Garrod himself did not recognize enzyme is the direct product of gene in the normal function, only referred to mutational anomalies as freaks or aberrations to be com-pared with the effects of infections or intoxication. He had no concept of the one gene-one enzyme theory. It was the concep-tional and experimental methodology of both Beadle and Tatum using nutritional mutants thus provided the break-through during 1937–1941, which led to the birth of the one gene-one enzyme theory.

Tatum also studied the tryptophan metabolism of Escherichia coli. He and his group demonstrated that many biochemical mutants were similar to those of Neurospora in basic principle.

While all these good research were going on and both Beadle and Tatum became world famous, Tatum’s career in Stanford was in jeopardy. This was partly due to leadership trou-ble at the top university level, and the role of a chemist in a department of biology was particularly controversial. Despite of the unequivocal support of Cornelis B. van Niel (1897–1985), Tatum was not granted promotion. In 1945, Tatum moved to Washington University, St. Louis, Missouri, where Carl Lindegren hoped to find a niche for him. (Dr. Beadle also left Stanford en bloc the next year and moved to Caltech.) But only staying there for one semester, Tatum was invited as an associate professor of botany (1945–1946), and later (1946–1948) a professor of micro-biology at Yale University, New Haven. Dr. Tatum was charged with development of a biochemically-oriented microbiology program in the Department of Botany. At Yale, Dr. Tatum was again disappointed in the University’s commitment to biochem-ically-oriented research in a department still heavily dominated by morphological-systematic tradition. In 1948, Dr. Tatum returned to Stanford as a full professor persuaded by Dr. Douglas Whitaker who took over the leadership of biological research at Stanford.

Nevertheless, a great fortunate thing happened while Tatum was at Yale. A young student named Joshua Lederberg with the

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strong recommendation of Francis J. Ryan (1916–1963), came to the laboratory of Dr. Tatum in the summer of 1946. The collabo-ration between Tatum and Lederberg yielded a fantastic break-through of molecular genetics — bacterial conjugation. A milestone paper of bacterial recombination was published with Lederberg in 1947. At Yale, Dr. Tatum also recruited David Bonner among others to join research on the biosynthesis of tryptophan and bolster the academic program in microbiology.

From 1948 to 1956 at Stanford as a professor of biology and 1956–1957 as a professor of biochemistry and head the bio-chemistry department, Tatum pursued and supervised research projects that reconciled a variety of interests introduced by his students and colleagues with his particular branch of biochemi-cal insights. He became increasingly interested in the analogy between mutagenesis and carcinogenesis, and foresaw the upcoming now famous Bruce Ames (1928 to present) Salmonella typhimurium/microsome mutagenicity screen test. He nurtured many students to work on the biochemical genetics of Escherichia coli, which was considered technically superior to Neurospora. Tatum encouraged his students or intellectual heirs to work on E. coli so they obtained an ultimost leeway for their own devel-opment. He credited his students and associates even though he participated actively intellectually. He took pride of having cultivated them as gifted investigators.

He was also involved with many activities including as an energetic spokesman for the rapidly emerging disciplines of biochemistry. He was an influential member of the National Science Board, strongly supported the predoctoral and postdoc-toral fellowships for creative talents in the new field. He was also a strong advocate of the international cooperation among scientists and helped to set up a joint program with Japan. He gave a strong support for the new science-oriented curriculum in medical education at Stanford University.

Overshadowed his other plans, Dr. Tatum’s marriage was not a happy one. He and June Alton separated. In 1956, he mar-ried Viola Kantor who was a staff employee at the National

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Foundation/March of Dimes, where Tatum spent a great deal of time as a scientific adviser. He left Stanford in 1957, and moved to Rockefeller Institute (now Rockefeller University) as a profes-sor and remained there until his death in 1975. In New York, he rebuilt his personal life. However, due to Viola’s illness and her death from cancer in 1974, the personal life of Dr. Tatum was surely not a happy one.

At Rockefeller, Tatum was also concerned himself with insti-tutional affairs and many others. He served on the National Science Board and strengthened fellowship programs and other measures that would bolster supports for young people entering scientific work. He was also chairman of the board of the Cold Spring Harbor Biological Laboratory.

Of course, the great honor Nobel Prize came in 1958. The wisdom, vision, and human compassion of Tatum could be clearly discerned from his Prize lecture, he not only recalled the history of biochemical genetics in his and Dr. Beadle’s hands, he saw cancer as a genetic change subject to natural selections. He looked forward to “the complete conquering of many of man’s ills, including hereditary defects in metabolism and the momentarily more obscure conditions such as cancer and the other degenerative diseases — This may permit the improvement of all living organisms by processes that we might call biological engineering”. Today we see biological engineer-ing (genetic engineering or biotechnology) is such an industri-ous enterprise; Dr. Tatum’s vision is fully realized.

Dr. Tatum continued to work on Neurospora as a model for genetic control of development. Numerous research subjects, such as inositol deprivation, effect of sorbose on the morphol-ogy of the fungus, mycelial branching, surface versus aerial hyphae, and formation of peritheciae, micro- and macroconidia, were investigated by him and his students and/or associates in his New York laboratory.

Dr. Tatum’s health was never robust. At the time of Viola’s death, Dr. Tatum’s health was already failing. The last stage of his personal life was quite a misfortune. He died on 7 November

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1975 from heart failure complicated by progressive chronic emphysema. He was survived by two daughters from his first marriage: Margaret Carol (Mrs. John Easter) and Barbara Ann. His brother Howard worked for many years with the Population Council doing research on contraception, and his late sister Besse was married to Dr. A. Frederick Rasmussen, professor of microbiology at UCLA.

Dr. Tatum received many honors in addition to Nobel Prize in 1958. In 1952, Tatum was elected to Fellow of National Academy of Science (NAS). In 1953, he received the Remsen Award of the American Chemical Society and was elected to the American Philosophical Society. He was the president of the Harvey Society (1964–1965) and the recipient of at least seven honorary degrees. He also served on many committee and organizations. He served on the NAS Carty Fund Committee from 1956 to 1961. He was a member of the Advisory Committee on the Biological Effects of Ionizing Radiation from 1970 to 1973 for the National Research Council. He also did service on advisory committees for the National Institutes of Health, American Cancer Society, and The National Foundation (March of Dimes). He was the chairman of the Scientists’ Institute for Public Information and an advisor to the City of Hope Medical Center, Rutgers University Institute of Microbiology, and Sloan-Kettering Institute for Cancer Research, and a consultant in microbiology for Merck and Co. He was also editors for many scientific publications including Annual Reviews, Science, Biochemica et Biophysica Acta, Genetics, and the Journal of Biological Chemistry.

Tatum started from bacterial nutrition to biochemical muta-tions of Drosophila and later Neurospora, and ultimately led to the formulation of fundamental concept of one gene-one enzyme theory which has been the central paradigm of experi-mental biology since 1941. Now we take the concept for granted. What a contribution a scientist can make for a life time? His success was partly, as Dr. Lederberg described it, that he was ingrained with the ability of balance critical scientific objectivity,

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personal ambition and interdependence on others. His combi-nation of generosity of spirit and scientific vision marked the serendipity of his career. He also embraced the joy of having so many of his fellow scientists who he joyfully nurtured and looked to him warmly as to a father or brother. Dr. Tatum’s concept of science and education can be seen from his testi-mony to a Congressional Committee on behalf of the National Science Foundation in 1959. He said, “The general philosophy is concentration on excellence — making it possible for (the scien-tists) to use his capacities, both for research and for training the next generation — whether it is a particular research program in a given area, whether it may or may not be immediately practicable in its application — freedom to develop the intel-lectual curiosity and abilities of the individual”. He gave us an immense inheritance, inheritance of wisdom, generosity, and human compassion.


Ames, Bruce Nathan (1928 to present). American biochemist and geneticist. Researched on intermediates and enzymes in the pathway of histidine biosynthesis, and operon regula-tion. Invented Salmonella typhimurium/microsome muta-genicity assay.

Beadle, George Wells (1903–1989). American geneticist. Nobel Prize in Physiology and Medicine (with Edward L Tatum and Joshua Lederberg) in 1958. Researched on genetics of Indian corn and cross-over in fruit fly. Codiscovered one gene-one enzyme concept. Pioneer of biochemical genetics.

Ephrussi, Boris (1901–1979). Russian geneticist. Researched on cell differentiation, genetic mechanism, somatic cell variation, and cell culture in vitro.

Fred, Edwin Broun (1887–1981). American bacteriologist. Researched on root nodule bacteria and leguminous plants. President of the University of Wisconsin, Madison (1945–1958).

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Fries, Nils Thorsten Elias (1912–1994). Swedish plant physiolo-gist. Researched on physiology of fungi, vitamin require-ment, multiple sexuality, spore germination, and mutations, vitamins and metabolites in seedling development. Invented filtration technique.

Garrod, Sir Archibald Edward (1857–1936). English geneticist. Researched on genetically determined human diseases, cryptonuria, alcaptouria, fructosuria, and albinism. Author of Inborn Errors of Metabolism, 1923; The Inborn Factors in Disease, 1931.

Kluyver, Albert J. (1888–1956). Deutsche yeast biologist, micro-biologist. Well known for his unity of biochemistry theory.

Lederberg, Joshua (1925–2008). American biochemist, geneti-cist. Nobel Prize in Physiology and Medicine (with George W. Beadle and Edward L. Tatum) in 1958. Discovered bacte-rial conjugation. Numerous other contributions.

Lwoff, Andre Michael (1902–1994). French microbiologist and virologist. Nobel Prize in physiology and medicine (with Francois Jacob and Jacob Mono) in 1965. Researched on morphogenesis in Ciliates, kinetosome development, repro-duction and evolution, nature and function of growth factors, physiology of viruses, enzyme regulation, and latent bacteria viral induction.

Snell, Esmond Emerson (1914–2003). American biochemist. Researched on nutritional requirement of bacteria, vitamins assay and functions.

Van Niel, Cornelis Bernardus (1897–1985). American microbiolo-gist. Well known for his research on photosynthesis and microbial education.

Wood, Harland Goff (1907–1991). American biochemist. Researched on carbohydrate metabolism and discovered the utilization of CO2 by heterotrophic organisms.

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Chapter 44

Horace A. Barker (1907–2000): Pioneer of Anaerobic Metabolism

Source: https://history.nih.gov/exhibits/stadtman/popup_htm/web-3–7.htm(US Public Domain image)

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Horace Albert Barker is well known for his work on various aspects of bacterial metabolism, including synthesis and oxida-tion of fatty acids, fermentation of amino acids and purines, carbohydrate transformations, methane formation, and isola-tion, structure and function determination of some bacterial enzymes and coenzymes. He elucidated many anaerobic meta-bolic pathways which are reported in many microbiology text-books today.

H. A. Barker was born on 29 November 1907 in Oakland, California. His father, Albert C. Barker, came from Maine during the big western migration period. Mr. A. C. Barker was a high school teacher, and had been a high school principal and a pub-lic school administrator in several cities, including Oakland and Palo Alto, where Horace grew up. His mother, Nettie Hindry Barker, came from Denver, Colorado. She had a AB degree in Classical Literature and a MA degree in Latin. Horace had an elder brother who later became a professor of English. Therefore, we can see that H. A. Barker came from a well-educated family, although he mentioned that there was nothing in his family background that predisposed him to science.

As a child, Horace liked music, fishing, hiking, camping in the Sierra area, and outdoor activities, most of which he recalled were solitary activities. He was interested in music primarily because his good friend, Robert Vetlesen, was a talented pianist. But, Barker did not feel that he was good enough to pursue a career in music. After he graduated from high school in 1924, Horace spent a year (1924–1925) in Europe with his parents. They stayed most of the time in Dresden where he studied piano, learned German, read classical German literature, and went to innumerable operas and concerts of every kind, includ-ing a musical festival in honor of Richard Strauss’s (1864–1949) 60th birthday.

In 1925, right after coming back from Europe, Barker, still undecided on a career, entered Stanford University. He took a course in systematic botany taught by Professor LeRoy Abrams

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(1874–1956) who inspired him to take another course in plant ecology. That course included a 7-week field trip throughout the southwest of the United States. During that period, over 5000 plants were collected which are still in the Stanford her-barium. In 1929, Barker graduated with an AB degree in Physical Sciences, which provided him with a good background in mathematics, physics, chemistry, and geology.

The same year, Barker entered the graduate school of Stanford. He was interested in biology but had no definite idea as to an area of specialization. He was interested in protozool-ogy taught by Charles V. Taylor (1895–1946), whom he liked, and for whom he worked as an assistant. In the spring of 1930, Barker moved to the Hopkins Marine Station on the Monterey Peninsula, where he met Robert E. Hungate (1906–2004), a graduate student of Professor Cornelis B. van Niel (1897–1985), who was a new staff member from Holland. Hungate advised him to take van Niel’s course of general microbiology during the summer. From then on, Barker was convinced that microbi-ology was the most exciting subject. He was also impressed by the developing knowledge of the biochemistry of yeast and bacterial fermentations, but he felt that his knowledge in chemistry was insufficient, and changed his major from biology to chemistry.

In the summer of 1931, Barker was a research assistant of Dr. C. V. Taylor working on some experiments on the develop-ment of starfish eggs. Dr. Taylor was committed to spend the following academic year as a visiting professor of Zoology at the University of Chicago, and Barker followed him to Chicago. There, Barker worked on the effects of the environment on the encystment of the protozoon Colpoda cucullus. He also studied the effect of moisture on the survival of Colpoda cysts exposed to high temperature. He, thus, developed an interest in the relationship between moisture and heat resistance of living organisms. With the help of Dr. Taylor, he was able to work with Dr. James W. McBain (1882–1953) of the Chemistry Department

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(Stanford University) for a PhD degree. Just before he started graduate work in chemistry, he spent another summer at the Hopkins Marine Station as an assistant to Dr. James P. Baumberger (1892–1973) of the Physiology Department.

Barker devoted his time from 1931 to 1933 to his PhD research studying the relationship between relative humidity and heat denaturation of egg protein. When he completed his PhD dissertation, he secured a National Research Council Fellowship, which allowed him to extend his training at the Hopkins Marine Station for two more years (1933–1935) doing microbiological research. First, he isolated two species of diatom and three spe-cies of small photosynthetic dinoflagellates. He also maintained several other species of dinoflagellates in species-pure culture. He studied the environmental conditions affecting the growth of these microorganisms. Later, he studied the utilization of organic substrates by a colorless alga, Prototheca zopfii, which was brought back by van Niel from Delft, The Netherlands.

Barker then obtained a fellowship from the General Education Board of the Rockefeller Foundation and spent a year in the Delft Microbiology Laboratory with Professor Albert J. Kluyver (1888–1956). Just before he left for Holland, Barker started to investigate the degradation of glutamate and the biological formation of methane. Van Niel had done no experi-mental work on biological methane formation, but he had developed an ingenious hypothesis for the origin of methane based mainly on the earlier experiments of Nicolas Louis Sohngen (1878–1934). Van Niel proposed that CO2 was reduced by molecular hydrogen, which is the basis of the CO2 reduction hypothesis. When Barker reached Delft in 1935, he first started the work on the research subjects that Dr. Kluyver was inter-ested in tartrate fermentation by Aerobacter aerogenes, and later glutamate degradation by Clostridium tetanomorphum. While working on these problems, he also started an enrich-ment culture for methane-producing bacteria using inoculum from sewage sludge. Soon, he obtained crude cultures that

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would rapidly convert ethanol to methane according to the following equation:

2CH3CH2OH + CO2 → 2CH3COOH + CH4

This was rather exciting. All the experimental data seem to support the CO2 reduction theory of Van Niel. Meanwhile, he also noticed the formation of C4 (butyrate) and C6 (caproate) fatty acids in high yields from ethanol in an anaerobic environ-ment. All these discoveries greatly influenced his future research.

In 1936, Barker returned from Delft to the University of California, Berkeley, Division of Plant Nutrition of the Agricultural Experimental Station, as an Instructor in Soil Microbiology and Junior Microbiologist. He assisted Dr. Charles B. Lipman (1883–1944) in teaching a laboratory and lecturing in a course of soil microbiology. Later, he assumed the whole responsibility of teaching this course. Some years later, he collaborated with Dr. Michael Doudoroff (1911–1975) of the Biochemistry Department, Dr. Reese H. Vaughn (1908–1988), and Dr. Maynard Alexander Joslyn (1904–1984) of the Food Technology Department to design a new course, “Microbial Metabolism” in the Bacteriology Department. Later, Dr. Roger Y. Stanier (1916–1982) and Edward A. Adelberg (1920–2009) also participated in teaching this course. This course attracted many graduate students from several areas of biol-ogy, and Berkeley’s school of microbiology began to blossom.

At that time in California, there were some diseases of fruit plants and other plants, particularly a disease called “little leaf”. Upon the request of Dr. Dennis R. Hoagland (1884–1949), Barker did some research on the subject, but he got nowhere. He abandoned research on bacteria-plant interactions and devoted all his efforts to investigate simple microbial system.

During this period, Barker began to isolate various kinds of interesting anaerobic bacteria, including Methanobacterium omelianskii (later discovered to be a mixed culture), the organ-ism responsible for the conversion of ethanol and carbon

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dioxide to acetate and methane; Clostridium kluyveri, which is responsible for the formation of butyric and caproic acids from ethanol; C. acidi-urici and C. cylindrosporum, which decompose uric acid and other purines; Streptococcus allantoicus, which degrades allantoin anaerobically; C. tetanomorphum and C. cochlearium, which ferment glutamate; C. propionicum and Diplococcus glycinophilus, which utilize alanine and glycine, respectively; and Butyribacterium rettgeri and C. lactoacetophi-lum, which ferment lactate in different ways. These organisms provided many of the biochemical problems which Barker later investigated. Many of these bacteria were isolated from rather simple enrichment cultures. But, some of them, for example, C. kluyveri, were isolated with great difficulty. From the isola-tion process, he discovered that C. kluyveri derived energy from the conversion of ethanol and acetate to butyrate, caproate, and hydrogen. The nutritional requirements include acetate, CO2, biotin, and para-aminobenzoic acid.

Barker was among the pioneers in applying radioactive carbon in biological investigations. In those days, 11C was the initial avail-able radioactive carbon used in the study of photosynthesis and dark CO2 fixation by higher plants and algae. The use of 11C has many limitations. The half-life for 11C is 21 min, so there was only a very short period of time to prepare the radioactive CO2 and set up the experiment. In 1939, with the help of Dr. Sam Ruben (1913–1943) of the Chemistry Department and Dr. Martin D. Kamen (1913–2002) of the Radiation Laboratory, Barker used 11C to study the incorporation of CO2 into acetate during the fermentation of purines by C. acidi-urici. Similarly, he found that CO2 was incorpo-rated into the carboxyl groups of propionic and succinic acids dur-ing the fermentation by propionic acid bacteria. Later, when 14C became available, Barker in collaboration with Marin D. Kamen, discovered that CO2 was also incorporated to produce acetate in C. thermoaceticum. They also discovered that B. rettgeri could convert CO2 to acetic acid and butyric acid during anaerobic deg-radation of lactate. Ethanol and acetate were also found to be converted to butyrate and caproate by C. kluyveri.

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In 1941, Barker was eligible for a sabbatical leave. He obtained a fellowship from the Guggenheim Foundation and spent 2 months in Dr. L. F. Rettger’s laboratory at Yale University to study the fermentation of B. rettgeri, and another 2 months in Dr. W. H. Peterson’s (1880–1960) laboratory, at the University of Wisconsin, to learn methods for investigating bacterial nutrients and assaying for growth factors using microbiologi-cal methods. He worked the rest of the year with Dr. Fritz A. Lipmann (1899–1986) at the Surgical Laboratories of the Massachusetts General Hospital, primarily on the pyruvate oxi-dation of Lactobacillus delbrueckii.

On returning to Berkeley, Barker continued to study various bacterial fermentations. Together with Dr. Michael Doudoroff and Dr. William Z. Hassid (1899–1974), they studied sucrose degradation by Pseudomonas saccharophila and found the synthesis of disaccharide by glucosyl transfer from sucrose in the absence of phosphate. They established sucrose phosphorylase as a glucosyl-transferring enzyme.

At this time, Barker also studied factors influencing the deteriorations of dried apricots with the help of Earl R. Stadtman and later Victoria Haas. This work has had a beneficial effect on the quality of commercial dried fruits.

After World War II, Barker and his graduate student Earl Stadtman engaged in the study of the oxidation and synthesis of fatty acid by C. kluyveri. Stadtman found that acetylphos-phate is a product of both ethanol and butyrate in a phosphate buffer. Acetylphosphate is also an essential substrate for the synthesis of butyrate when hydrogen is used as a reducing agent. Stadtman also discovered the acetyl-transferring enzyme (phosphotransacetylase), (later he and Lipmann showed an enzymatic system for using acetylphosphate to activate other fatty acids, and requiring CoA to catalyze the formation of acetyl-CoA compounds).

In 1951, Barker was invited to give a lecture at the first sym-posium on phosphorus metabolism at John Hopkins University. Barker reviewed the literature and his own experimental data,

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and he concluded that acetyl-CoA compounds are not only the primary oxidation products of pyruvate and acetaldehyde, but also the primary substrate in the synthesis of acetoacetate and citrate. They must also be the intermediates in the oxidation and synthesis of butyrate. This conclusion was later proved to be correct.

Barker and colleagues also did some very interesting stud-ies on amino acid biosynthesis in bacteria. They noted that C. kluyveri used ethanol, acetate, and CO2 as the only carbon sources. They found that 25% of the cellular carbon was derived from CO2 and 75% from acetate. Barker and his stu-dent Neil Tomlinson proved that amino acid carboxyl groups come from CO2, whereas a-carbon atoms are derived from the carboxyl carbon of acetate. Tomlinson further discovered that the a-carboxyl and b-carbon atoms are derived mainly from the carboxyl carbon of acetate and that g-carboxyl car-bon atoms are derived mainly from CO2. This was later explained by Gerhard Gottschalk (1935-) who found that C. kluyveri contains a (R)-citrate synthase rather than the (S)-citrate synthase. The (R)-citrate synthase occurs in only a few anaerobic bacteria.

Another important contribution of Barker was that he and his student Thressa Stadtman discovered that the methyl carbon of methane (formed by Methanobacterium barkeri) is derived from the methyl group of acetate. This is contradictory to van Niel’s CO2 reduction hypothesis in which the methyl carbon was believed to come from CO2. With further study, Barker proposed a general pathway for the formation of meth-ane from either acetate, methanol, or carbon dioxide, all are known to be used as the precursor by some methane-forming bacteria. This conceptional scheme proved to be valuable for later studies, particularly after Ralph Wolfe (1921–) had made a detailed study of the pathways and discovered various unique coenzymes (cofactors).

Barker was initially hired as a soil microbiologist in the division of plant nutrition of the agricultural experimental

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station of the University of California, Berkeley. In 1950, Barker together with William Z. Hassid, Paul Stumpf, Eric E. Conn, and Constant C. Delwiche formed a new department of Agricultural Biochemistry in the College of Agriculture. But soon (maybe in 1951), Barker transferred to the Department of Biochemistry in the School of Medicine whereas the rest of the group moved to the Davis campus of the University of California.

From 1936 to 1948, Barker’s students obtained advanced degrees in the graduate curricula of Bacteriology, Microbiology, or Agricultural Chemistry. In 1948, Barker organized the group of Comparative Biochemistry to offer advanced degrees to stu-dents working on biochemical problems who were not in the Medical School. From 1948 to 1958, there were a total of 75 students who obtained their PhD degree in Comparative Biochemistry. All of them had made tremendous contributions to the progress of biochemistry in the last 50 years.

At about this time (1950), Barker and his student, Leo Kline, discovered that lipoate was required for the metabolism of B. rettgeri. They demonstrated that lipoate functions as an elec-tron carrier in the oxidation of lactate to pyruvate.

From 1937 to 1957, Barker studied the degradation of uric acid and other purines by clostridia isolated from chicken drop-pings. These bacteria, C. acidi-urici and C. cylindrosporum, decompose uric acid, xanthine and guanine readily, and hypox-anthine more slowly, with the formation of acetate, CO2 and ammonia as the major products. C. cylindrosporum also pro-duces glycine. Together with his graduate students, Jay Beck, Norman Radi, Jesse C. Rabinowitz, J. Karlsson, and William Bradshaw, they illustrated the detailed enzymatic steps in purine degradation.

In 1951, Arthur Kornberg (1918–2007) spent a few months in Dr. Barker’s laboratory learning how to handle anaerobic bacteria. The following year, Barker spent 6 months in Kornberg’s laboratory at NIH to learn purification of enzymes.

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Barker also studied glutamate fermentation by C. tetano-morphum. He discovered that the degradation of this amino acid was through a unique b-methylaspartate rearrangement using mesconate as the intermediate. He and his associates isolated B12 coenzymes and demonstrated the role of corrinoid coenzymes in many biochemical reactions. The students and associates involved in these studies, included Joseph Wachsman, Agnete Munch-Peterson, Herbert Weissbach, Harry Hogenkamp, John Toohey, Benjamin Volcani, Jeff Ladd, David Perlman, Axel Lezius, Roscal Brady, Robert Smyth, Arthur Iodice, P. G. Lenhert, Fujio Suzuki, Robert L. Switzer, and Raymond Blakley.

Another great contribution of Barker was his studies of lysine degradation by clostridia. He and his students and asso-ciates, including Olga Rochovansky, Thressa Stadtman, Ernest A. Rimerman, Ralph B. Costilow, Thomas P. Chipch, Eugene E. Dekker, John J. Baker, Su-Cheng L. Hong, Ing-Ming Jeng, Takamitsu Yorifuji, Henry N. Edmunds, Gerhard Bozler, John M. Robertson, and Mesahiro Ohsugi, illustrated the detailed b-lysine decomposing pathways and the enzymes involved. It is worth mentioning that Thressa Stadtman later indepen-dently made a tremendous progress in the understanding of the lysine metabolism by clostridia.

In Dr. Barker’s life, he received many honors and awards, which include the Gowland Hopkins Medals from the Biochemical Society, London in 1967 and the National Medal of Sciences in 1968. He was also a member of the National Academy of Science, American Academy of Art and Sciences, and many other professional societies. He wrote a book entitled “Bacterial Fermentation”, which was published in 1956. He had published a total of more than 230 research papers. He also served on the editorial boards of various scientific journals and contributed his generous services to many organizations. He retired in 1975.

In brief summary, Barker spent his whole life to illustrate the anaerobic bacterial metabolism. Through his life efforts, we understand many biochemical pathways today, which are

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essential for the development of biotechnology for the tomor-row. He is a pioneer microbial biochemist indeed.

Horace married Margaret D. McDowell on 29 August 1933. They are happily married and have one son, Robert, and two daughters, Barbara and Elizabeth.

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Chapter 45

Deam Hunter Ferris (1912–1993): Pioneer of Epizoonotic Studies

”...there will always be an extra room for you!” “Infectious microbiology is parasitism”. ”As a microbiologist, don’t get in a rut, but keep your eyes open”.

—Deam H. Ferris

Courtesy of Dr. Deam Hunter Ferris

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Deam H. Ferris had done much work on the infectious microbiol-ogy. His book Illustrated Manual for the Recognition and Diagnosis of Foreign Animal Disease has been translated into more than eight languages and used worldwide. He was a pio-neer in the study of diseases common to animals and humans — zoonosis. He had done major work on vesicular stomatitis (VS); developed transmissible gastroenteritis (TGE) vaccine for swine. He was the first to isolate Micronema deletrix in horse and dem-onstrated the defective infection of rabies in animals. His work benefits the human welfare tremendously.

Deam Hunter Ferris was born on 8 July 1912 in Mankato, Minnesota. His father was called Joseph and his mother, Ruby. His first name is the family name of an editor that his parents admired. His second name was well chosen as he truly became a hunter, both of microbes and of their hosts. His family moved to St. Joseph, Missouri, about 1916, where his father was an insurance executive. Deam’s early interests were in natural things: fields, forests, streams, and lakes around St. Joseph, Missouri — and the great “Big Muddy”, the Missouri River, on whose banks the old city of St. Joseph was founded by Joseph Robidoux, a French fur trader.

Deam attended Missouri Western College at St. Joseph, Missouri for two years (1930–1932), and then continued at Drake University in Des Moines, Iowa, where he obtained his Bachelor degree in Zoology in 1934, and his MA in parasitology in 1938.

Deam’s first hunting of microbes and their hosts concerned certain parasites of snails. When he collected living snails in jars or jelly glasses, he kept them alive with a piece of lettuce in their water. He soon noticed that with some snails the water became milky. Looking at a drop under the microscope, he found that the milky appearance came from thousands of wrig-gling microbial forms with a body and tail. Some had forked tails and some with single tail. These were Cercaria and they arose from a spore cyst engendered by a still smaller form in the snail called a Miracidium. The Miracidium arose in the snail

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from the egg (or ovum) of a macroscopic flatworm (Platyhelminthes), deposited in the water by some parasitized animal. Deam did a survey of the snails in the vicinity of Des Moines, Iowa, and found a wide range of these microscopic parasites which caused “swimmer’s itch”, a much feared malady in human beings. Actually, people, in this case, were “dead end” hosts, even though these parasites caused a sever itch, but died for their trouble. In a proper animal, however, such as fish, in some cases, the hosts was eaten by large animals and the ingested Cercarium grew in the gut to become a flatworm, repeating the cycle. In each case, the snail was an “intermedi-ate” host, home to microscopic forms: “Microbes”. These find-ings were Deam’s Master’s thesis at Drake University, but were not published in the literature at that time.

During World War II (1939–1945), Deam first served in the infantry, but because of his MA degree in parasitology, he was pulled out of combat roles to help build the Army’s new bio-logical station known as Ft. Detrick, Frederick, Maryland. Deam actually helped draw up the first plans and served there during the later years of the war. He worked on many bacteria, includ-ing the microbes of anthrax and brucellosis. The purpose of the post was to develop both a capability for biological warfare (BW) and mainly, defense against it. It was known that both Germany and Japan had a BW capability. Germany, in World War I had used glanders, a zoonotic disease of both horses and men, against the United States to prevent the shipment of horses and mules, for military use, to France.

At Ft. Detrick (about 1945), the Army BW station, Deam did his first research resulting in a paper. There were many infec-tions at Ft. Detrick. Deam himself was hospitalized for brucel-losis, caused by Brucella abortus. He was convinced that these infections were largely the result of aerosols produced by care-less handling of laboratory equipment, especially the pipette. The pipette was, and still is, a major instrument for transferring liquid cultures of organisms. Deam and his colleagues took cul-tures around people working in the laboratory and revealed

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airborne contamination. However, this was often denied, and Deam had to show the germs right in the air to demonstrate the particulate nature of aerosols.

Electronic flash, so common today, was not available at that time, but Deam and a colleague were able to get plans for one and built an electronic flash which could, when used with a highly sensitive film in a camera, actually recorded an other-wise invisible aerosol. By this means, he demonstrated that the last drop blown from the pipette, particularly, had a “cloud” around the tip, composed of thousands of germs — many of which reached the nose and mouth of the pipetter. Even the platinum loop, used to transfer solid cultural media was shown to emit tiny unwanted droplets, if carelessly used.

The resulting paper (published in 1946) was widely acclaimed by laboratory workers and especially the pharmaceutical indus-try. Blowing the last drop from a pipette, formerly a regular practice, was universally banned because of this research. Much greater care was taken to prevent laboratory aerosols. Mouth pipetting was also prohibited in the laboratory.

Deam became skilled in scientific photography, and this skill helped him later to work as an assistant and, later, an instructor which supported his graduate school expenses.

After the war ended, Deam served as an instructor in auto-tutorials and photography at the University of Wisconsin at Wisconsin (1946–1947), and then he was a general science and biology teacher in Des Moines, Iowa public school systems (1947–1948). From 1948 to 1957, Deam was a professor of biol-ogy and natural history at the Graceland College, Lamoni, Iowa.

Deam decided to pursue a PhD education in the University of Wisconsin while he still holds the position at the Graceland College. He was interested in parasitology. Unfortunately, the only parasitologist in the Department of Veterinary Science died. The only other professor available in the department was Dr. Robert P. Hanson (1918–1987) who became his major profes-sor. Deam wondered if he should change his field from “parasitol-ogy” to “microbiology”. After doing some hard thinking, he

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suddenly realized that he had been working all this time with microbial forms. He was really already in “microbiology”. He also suddenly realized that viruses were the ultimate parasites. They could not exist long outside of a host. In fact, it became very clear to Deam at this time that “all infection is parasitism”. No matter whether a germ or a worm or a virus particle, the infectious agent is the parasite of a host! He was not leaving his beloved “parasi-tology” to work on such microbial forms as bacteria and viruses.

At that time, one of the mysteries of science was a disease called “Vesicular Stomatitis”. This disease had struck dairy farms in northern Wisconsin, horses in western states, and swine in southern states, appearing usually in wooded areas without warning and then disappearing after a year or so. It had almost exactly the same signs and symptoms as the dreaded “foot-and-mouth” disease (FMD). The only method of differentiating it from FMD was to inoculate a horse, a cow, and a pig (from another region) with material from the blisters of an infected animal. If only the cow came down, it was FMD; if all three came down, it was VS. In Georgia, particularly, but also in several southern states, all three animals were involved with the disease. But the US Department of Agriculture also had to determine if there might also be FMD present. An outbreak of VS was bad, but FMD would cost millions of dollars to control. Deam’s department at the University of Wisconsin was a major site for study of VS, since it appeared in dairy cows of that state frequently. Deam had been studying it in northern Wisconsin, but was sent to Georgia to determine if this might not be trans-mitted by arthropods, with a reservoir in wild animals. A proto-type disease he also studied was equine encephalomyelitis, with both Eastern and Western forms of virus. This virus was carried by mosquitoes from a variety of birds and small mammals and caused a paralytic disease in horses and human beings.

Deam used embryonated eggs to grow the VS virus. He also used both young and suckling mice to isolate the virus. Tissue culture methods were not employed then. He collected the rafts of mosquito eggs of a various kind and reared them in the

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laboratory. Mosquitoes were induced to suck infectious egg fluids through the egg shell membrane and these were used to infect cattle to demonstrate the feasibility of the virus to be carried by arthropods. In addition, the stable flies, horse and deer flies, ticks, and other biting arthropods were studied for their propensity for carrying the VS virus. He found that horse flies, deer flies, and mosquitoes were potential carriers.

Deam studied the major wild fauna in the natural foci of VS. Antibodies against VS virus were found in the blood of deer and other species. The virus was also isolated from cattle, swine, and horses in the South. It was also later isolated from the mosquitoes and birds. All these findings were Deam’s dis-sertation work. Deam was on leave from the Grace College 1952–1953 and completed his PhD dissertation in 1953 at the University of Wisconsin.

After obtaining the doctoral degree from the University of Wisconsin, Deam initially worked as a Research Associate at the Department of Veterinary Science, University of Wisconsin at Madison, and later as director of field laboratory in Glenville, Georgia under Dr. Francis Mulhern and Professor Robert Hanson on VS outbreaks and Eastern equine encephalomyelitis (EEE) in the summer from 1953 to 1958. In 1957, he secured a faculty position at the College of Veterinary Medicine, the University of Illinois at Urbana-Champaign. He made contributions par-ticularly in the field of zoonotic diseases — leptospirosis and porcine TGE. He was the first to isolate a leptospire from an eastern hog snake (Leptospira ballum). He was among the first in the United States to demonstrate capability of wildlife to maintain foci of leptospires endangering domestic animals and men. Such foci included deer, skunks and opossums, the feral cat, marsupials, rodents, and snakes.

TGE was a very serious killer of baby pigs. Farmers would often lose their entire crop of baby pig. It was a costly disease among the worldwide farmers. Deam began to use a culture of porcine kidney cells to grow the TGE virus. He was the first to patent a vaccine for this disease, assisted by Dr. Angel Arambulo, professor of pharmacy, who helped him prepare a coating for

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the attenuated virus. The coating would allow the vaccine to survive the acidity of the stomach and reach the target cells in the small intestine. This vaccine did not really function as a stimulating the formation of antibody as normal vaccine, but instead, prioritized the target cells against invasion by the viru-lent field TGE virus. Since the kidney cell culture were efficient, and large amounts of the attenuated virus could be produced and coated with the anti-acid layer at relatively low cost. This vaccine was fed by farmers or veterinarians to both sows and newly born pigs during winter TGE months and became a very effective measure to prevent the TGE. His method has been patented in many countries and is believed to be the basis on which other patents were later devised. Deam’s work has sig-nificantly benefitted to the hog-raising industry.

On one occasion, Deam was invited to work for the United Nations for two years in Egypt (1963–1965). He set up a virology laboratory in Cairo to study Near East Equine Encephaolmyelitis, a disease which resembles Eastern, Western, and Venezuelan equine encephalomyelitis (EEE, WEE, and VEE). Horses, don-keys, and mules in Egypt and other countries of the Near East frequently died with a paralytic disease. The disease, like EEE, WEE, VEE, and VS, was a sporadic one, appearing suddenly in a variety of Egyptian villages and farms. Deam and his collabora-tor Dr. Livio Badiali, made numerous isolations of a virus which would kill mice, giving them first paralytic signs. The major test for rabies at that time was to stain for Negri bodies. Deam sent all samples to a government rabies laboratory and neither labo-ratories could demonstrate Negri bodies. In Dr. Badiali’s absence, Deam inoculated donkeys with infected mouse brain, having read of abortive rabies (with the absence of Negri bodies) in American skunks. The donkeys came down with paralytic signs as had the mice. To the surprise of both Drs. Ferris and Badiali (on his return), the donkeys survived, but with antibodies against rabies virus.

However, his complacence and that of Dr. Badiali was shaken when they found that few scientists were willing to accept the concept of abortive rabies. At that time scientists

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believed that rabies was invariably fatal. The papers were finally published, and later, in their laboratories in both Italy and the United States. Deam and Livio proved conclusively that they had isolated a rabies virus. This was confirmed by the Center for Disease Control (CDC) at Atlanta, Georgia.

The major problem in Egypt was the source of the virus. Dogs were ruled out as the Egyptian farmers lived very near their animals and always knew of canine-derived rabies. In each village, there were posters showing rabid dogs and urging that their heads be sent to the rabies laboratory. Bats had been found in the United States to carry rabies, but the virus was not isolated from bats in Egypt. There were numerous rodents on all these farms; Deam theorized that they might be responsible, but was not be able to demonstrate this. Also, at this time, no scientist believed that rabies was rodent disease. Later, how-ever, rabies was demonstrated abundantly in rodents; abortive types were demonstrated by molecular biologists at Rockefeller Institute. Some human beings who survived rabies were even found. Drs. Ferris and Badiali were far ahead in demonstrating abortive rabies. They were indeed pioneers in the study of abortive rabies.

Another finding in Egypt startled the microbiologists. Many of encephalitis were caused by moldy corn, a common malady of cattle and horses on American farms! It took a great deal of experimental work to prove this to the satisfaction of the Egyptian authorities and farmers. It was found that cattle and horses were often fed in fields where the standing corn was moldy. They were also fed corn known to be moldy and unfit for human consumption.

Deam made an early retirement from the University of Illinois in 1973, but continued his work as a Research Microbiologist at the US Department of Agriculture, Animal and Plant Inspection Service (APHIS), National Veterinary Service Laboratories (NVSL), and Foreign Animal Disease Diagnostic Laboratories, Plum Island, New York (1973–1985). Deam did many significant scientific works there. There were

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more than 160 scientific papers and numerous books pub-lished. One of the notable books is the Illustrated Manual for the Recognition and Diagnosis of Foreign Animal Disease of which, Dr. Ferris was the chief editor and originator of the book. This book had been translated into more than eight lan-guages including French, German, Spanish, Arabic, Chinese, Japanese, and so on; adapted by many countries as a standard manual for animal diseases control.

Dr. Ferris in collaboration with Dr. Badiali isolated the Micronema deletrix, a microscopic nematode from cases of equine encephalomyelitis in horses in Egypt. This was the sec-ond isolation made and first in the Near East. His work extended the understanding of etiological agents of diseases. The horses had all the signs of viral equine encephalomyelitis, but the etio-logical agent is a nematode.

Dr. Ferris also demonstrated a large scale, but low-grade infections of Coxiella burnetii in dairy herds and men, which went unrecognized in the Mideast. He studied plasmodial babesial and related hematropic diseases of domestic and labo-ratory animals.

Dr. Ferris has combined research with advanced photo-graphic cinematrophic and autotutorial methods to advance autotutorial learning in veterinary medicine. He also partici-pated in developing diagnostic methods for the Food and Drug Administration (FDA) such as Cytauxzoonosis in domestic cats and others. He developed a new fluorescent antibody (FA) test for wildebeest-derived malignant catarrhal fever virus (WMCFV). He developed a diagnostic test system for detection of Trypanosoma vivax, which was a major cause of loss in cattle, sheep, goats, and equidae in Africa and Latin America.

Dr. Ferris work covers a various aspects of microbiology including virology, bacteriology, parasitology, immunology, serol-ogy, host-parasite biology, and application to epizootiobiology.

He has contributed to the better understanding and methods of prevention and treatment of a number of microbial diseases including African sleeping fever (ASF), duck plague, malignant

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catarrhal fever (MCF), FMD, rinderpest in deer, protozoan dis-eases such as T. vivax, T. congolense, and feline Cytauxzoon. Few scientists did so much research on these animal diseases. These diseases eventually affect human health. If Dr. Ferris’s work were on human disease, he would be much more well known and recognized. He is in the same gentry as Louis Pasteur (1822–1895) and Robert Koch (1843–1910). Over years of hard work, Deam discovered that “infectious disease is parasitism” — a belief had been held much earlier by Dr. Theobald Smith (1859–1934). This has become his working moto.

The pioneering experience of Dr. Ferris taught us a lesson. Dr. Ferris found that bacteria, fungi, virus, and/or parasites, can cause diseases which could not be distinguished by signs and symptoms. Even toxic agents could cause diseases which were similar to the parasitic and/or infectious ones. As a microbiolo-gist, don’t get in a rut, but keep your eyes open!

After retiring from the government post in Plum Island, Deam continued his microbiology teaching and research at the International University Osaka Learning Center from 1985 to 1986. He also taught microbiology at the Department of Microbiology, Soochow University in Taipei, Taiwan 1986–1988 when the author of this book, King-Thom Chung, was the chairman of that department.

Since then, Deam and King-Thom became good colleagues and friends. Deam also worked as a consultant on Foreign Animal Diseases to the Council of Agriculture and visited and gave demonstrations and lectures at all the Animal Health Laboratories in Taiwan. In 1988, Deam returned to the United States and resided at Independence, Missouri to enjoy his retirement. However, Deam is still a microbe hunter and prac-ticing microbiology, in addition to serving as a Chaplain in the local hospital in Independence, Missouri.

Around 1990, when the author began to write the biogra-phies of pioneer microbiologists, Deam gave a tremendous help to this endeavor. He suggested the author to publish a book; helped and edited many of his writings and coauthored some

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of the articles. This was a learning and enjoyable experience. Unfortunately, he passed away before seeing anything pub-lished on this subject. This article is an example of this unfin-ished task.

Deam was a firm “believer” as the great microbiologist Louis Pasteur did. Deam felt that he should do everything pos-sible for humanity with a true altruism. He appreciated having had the opportunity to travel around the world and demon-strated his affection for mankind by his high microbiological skill and knowledge.

In 1935, Deam married Merle Wildey in Des Moines, Iowa. Merle is also a teacher. She had a Master’s degree in English from the University of Illinois. When they were in Taiwan, Merle also taught English at the Soochow University. Both of them nurtured many good students all over the world. They enjoyed entertaining students from different part of the world. His famous words were: “there will always be an extra room for you!” Even at their old age, their house was always flooded with international visitors including colleagues, friends, and students of different ages.

Deam and Merle had a wonderful marriage. They raised four children: Sara Jo, Tary Jeanne (now Dr. Tary), Karen, and Deborah Joan. They had at least 13 grandchildren and many great-grandchildren. They are scattered “everywhere” in the world and are doing well.

The author K. T. Chung had the honor of knowing Deam personally and felt that he was not only a great scientist who contributed greatly to the understanding of both human and animal diseases, he was also a great human who practiced com-plete altruism. Deam passed away on 8 December 1993, in Harvest Cluster, Missouri.

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Chapter 46

Herman J. Phaff (1913–2001): Pioneer of Yeast Biology

Source: https://en.wikipedia.org/wiki/Herman_Phaff(US Public Domain image)

When we enjoy beverages at a party, we may be aware that some of them are products of the life processes of yeasts. Yeasts have made numerous contributions to human welfare but have also added to the scope of human suffering. How much do we

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know about the yeasts and how are they different from bacte-ria or higher fungi? Dr. Herman J. Phaff has dedicated his career to the study of the biology and biochemistry of yeasts. His work covers classical physiology, ecology, taxonomy, and modern molecular systematics and biology. Among the greatest micro-biologists of all time, Phaff stands out as the most important contributor of fundamental knowledge of yeast biology. He is truly a pioneer microbiologist.

Humble Beginnings

Herman Phaff was born on 30 May 1913 in the small city of Winschoten in the province of Groningen, The Netherlands. His father and uncle operated a winery. Both his parents died while he was still quite young, and he and his brother were adopted by his uncle Jacob. As a boy, Herman started learning to per-form those regular chores of crushing, pressing, and observing fermentation operations of the winery. He learned how to use a microscope to observe the budding and growing of yeast cells long before he received formal training in microbiology. This early childhood had a profound influence on his development as a yeast scientist.

In 1932, Phaff attended the well-known Technical University of Delft, where he met and was greatly influenced by the mas-ter of microbiology, Dr. Albert J. Kluyver (1888–1956), professor of general and applied microbiology. At Delft, Phaff specialized in technical microbiology and worked on extracellular pectin-hydrolyzing enzymes by fungi as an undergraduate thesis. As Dr. Phaff recalled, he also was privileged to meet the world renowned scientists Dr. Charles Clifton (1904–1976), Robert Lyman Starkey (1899–1991), and Benjamin E. Volcani (1915–1999), who were guests in Dr. Kluyver’s laboratory.

In 1939, after earning his degree in chemical engineering and working on the taxonomy of yeasts, Herman Phaff came to the University of California, Berkeley. He met professors William V. Cruess (1886–1968), Emil M. Mrak (1901–1987), and Maynard

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Alexander Joslyn (1904–1984) in the Division of Fruit Products (later known as the Department of Food Technology). Initially, Phaff worked as a research assistant to Dr. Emil Mrak and helped in the identification of yeasts responsible for the spoilage of figs and dates in the orchards of central and southern California. At the same time, he pursued his PhD under Dr. Maynard Joslyn on the subject of pectic enzymes of Penicillium chrysogenum. When Dr. Joslyn was called to war duty, however, Phaff pursued his studies under the mentorship of Professor Horace A. Barker (1907–2000). In his PhD dissertation, Phaff illustrated the inducibility of the hydrolytic enzymes required for the break-down of pectin, pectin esterase (PE), and polygalacturonase (PG). The enzyme inducibility was a grand new concept long before the lactose operon was proposed. His PhD was com-pleted in 1943 and he was offered a faculty position at Berkeley in the Department of Food Technology by department chairman Dr. Cruess.

As Phaff recalled his graduate years, one of the highlights was the annual spring seminar held at the home of Professor Cornelis B. van Niel (1897–1985) in Carmel, California, where microbiology students from the Berkeley campus met with van Niel’s students from the Hopkins Marine Station in Pacific Grove. During this occasion, Herman met Dr. Michael Doudoroff (1911–1975), who became one of his best friends.

Academic Career

From 1943 to 1953, Dr. Phaff taught an upper level course related to taxonomy, ecology, and physiology of yeasts with Dr. Emil Mrak at Berkeley. This was one of the few, or perhaps the only one, of its kind in the United States. He also conducted numerous research projects on the dehydration of fruits and vegetables, including developing a process for sterilizing dried prunes, figs, and dates by treatment with ethylene oxide. In 1948, he married Marinka Boratynski and in 1951 returned to Delft on sabbatical.

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In 1952, the Department of Food Technology was moved from Berkeley to the University of California, Davis campus. At first, Dr. Phaff was reluctant to move because Davis was a small place with few cultural activities. Dr. Phaff commuted to Davis three times weekly to fulfill his teaching obligations and still continued research in his old Berkeley departmental labo-ratory for a year. Eventually, however, Dr. Phaff moved to his newly built home in Davis.

At Davis, Phaff devoted himself to the study of a broad area of yeasts. He was assisted by three graduate students brought from Berkeley, Bor Shiun Luh (1916–2001), Arnold Demain (1927–), and Moshe Shifrine (1928–). Dr. Phaff continued the study of a hydrolytic enzyme of polygalacturonic acid produced by the yeast Saccharomyces fragilis (later renamed kluyveromy-ces fragilis). He also studied, with graduate student Harry Snyder, the degradation of a fructose polymer, inulin, by the same yeast. Later, the research expanded to the breakdown of pectin and pectic acid by a new class of enzymes, called lyases, produced by the bacterium Clostridium multifermentans, and a fungus, Aspergillus fonsecaeus. These research projects were accomplished with a group of graduate students including Bor Shiun Luh, Arnold Demain, D. Patel, James Macmillan, and Ron Estrom.

Also during this period, Dr. Phaff isolated a number of yeasts and elucidated their taxonomy and natural habitats. Some of these yeasts were Candida diddensiae (originally described as Trichosporon diddensii) from marine shrimp, Pichia haplophila, Torulopsis nitratophila, and Candida silvicola (later renamed Hansenula holstii) from bark beetles of the genera Ips and Dendroctonus. One of the important pioneering works was the isolation of a CO2 requiring yeast Saccharomycopsis guttu-lata (now renamed Cyniclomyces guttulatus) from the stomach content of domestic rabbits by graduate student Moshe Shifrine. The effect of CO2 on the growth, life cycle, physiology, and ecology of this particular yeast was extensively illustrated by graduate student Ed Bucher. This was a big advancement at

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that time since the CO2 requirement for growth was a new and exciting finding for the biology of yeast as well as many other microorganisms.

Dr. Phaff turned his interest in yeast ecology to species of Drosophila that require a yeast source for their life cycle. He traveled to the Sierra Nevada mountains of California and iso-lated a number of yew yeast species from wild species of Drosophila in that area. These included Saccharomyces dros-ophilarum, S. dobzhanskii, and S. phaseolosporus (these three species were later transferred to the genus Kluyveromyces). The predominant yeasts isolated from Drosophila of the southern California Sierras were Pichia pastoris (then known as Saccharomyces pastori ), Pichia fluxuum (initially described as Debaryomyces fluxorum), and Pichia silvestris (later considered synonymous with P. membranifaciens).

He later turned his interest to yeasts in the Yosemite area of central California and identified 240 yeast isolates. The predomi-nant yeasts were Kluyveromyces veronae (syn. K. thennotoler-ans), K. drosophilarum, and K. dobzhanskii. Several new species including K. wickerhamii, S. kluyveri, and Trichosporon aculea-tum (syn. Aciculoconidium aculeatum) were also described. A number of graduate students participated in these studies. They includes El Tabey Shehata, Moshe Shifrine, and Martin Miller. Dr. Elisa Knapp, a visiting scientist from Brazil, also joined in these efforts.

Around 1960, Dr. Phaff’s research interests shifted from pec-tolytic enzymes to those that were capable of hydrolyzing the cell walls of yeast, since a better understanding of the composi-tion of the cell envelope of different yeasts by their susceptibility to specific hydrolytic enzymes might give a better understanding of the relationships among species and genera. With the help of a number of graduate students including Hirosato Tanaka, Graham Fleet, Melvin Meyer, Ahmed Abd-El-Al, and postdoc-toral visitors, Dr. Frank Rombouts from The Netherlands and Dr. Thomas Villa from Spain, Dr. Phaff made tremendous pro-gress on exploring the enzymology that affects the integrity of

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the yeast cell envelope. They demonstrated several bacterial and endogenous glucanases, both endo- and exoforms, and studied their activity in detail.

In the early 1960s, in collaboration with the visiting scholar Dr. Minoru Yoneyama (1924–) from Hiroshima University of Japan, Dr. Phaff studied the yeast flora associated with bark beetles of the genus Scolytus which attack species of fir (Abies) and Douglas fir (Pseudotsuga). They isolated Endomycopsis sco-lyti (later reclassified as Pichia scolyti) and studied the genetics of its mating system.

Also, in collaboration with another visiting scholar, Dr. Lidia do Carmo Sousa from Portugal. Dr. Phaff isolated four new spe-cies of yeast from bark beetle frass of the coastal hemlock Tsuga heterophylla. These four species were Cryptococcus skinneri, Candida oregonensis, Bullera tsugae, and Sporobolomyces sin-gularis. They also demonstrated the quantitative trans-glycosylation of sugars by S. singularis, a phenomenon unique to biosynthe-sis. With the help of visiting scholars Dr. J.F.T. (Frank) Spencer from the Prairie Regional Laboratory in Canada and Dr. Phil Gorin from the Saskatoon laboratory, they demonstrated the biosynthesis of unusual disaccharides, trisaccharides, and tetra-saccharides by this yeast. This process of biosynthesis is quite unique since chemical synthesis of such saccharides would be extremely difficult and laborious. At about the same time, Dr. Phaff, Frank Spencer and Martin Miller identified and described two new yeast species, namely Pichia trehalophila from exu-dates of cottonwood trees and P. salictaria from insect frass in willows.

Around 1967, Drs. Phaff and Martin Miller (then a faculty member at the University of California, Davis) applied to the National Science Foundation for a grant under the US-Japan Cooperative Science Program to do a broad study of yeasts in tree exudates on all of the major Japanese islands on the west coast of North America. With the cooperation of Japanese counterparts Drs. Minoru Yoneyama and Masumi Soneda, Drs. Phaff and Miller crisscrossed the Japanese islands for about

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two months by train and locally by jeep, mainly in forested mountain areas, staying in forest lodgings, Japanese inns, and occasionally in university dormitories. They carried out the yeast isolations in improvised field laboratories or occasionally in university laboratories. In 1968, Drs. Phaff and Miller traveled to Anchorage, Alaska to meet their Japanese colleagues and collected samples of tree exudates along Alaskan roads and the Alcan Highway, with various side trips through the Yukon Territory, British Columbia and the states of Washington, Oregon, and California. During this trip, the samples were streaked and analyzed in the field, usually in forest campsites and occasionally, in a motel.

In this capacity, they collected and identified about 400 yeast strains in Japan and more in North America. They discovered that because of the widely separated geographic areas and the very different tree species in Japan and the Pacific Northwest, some profound differences in yeast biota were noted. For example, Dipodascus aggregatus and Nadsonia elongata were extremely common in Japan, but were not found in the Pacific Northwest; conversely, P. pastoris and two novel species of Torulopsis were common in the Pacific Northwest but were not found in Japan. Noteworthy is that they isolated a number of fermenting, carotenoid-producing yeasts. The principal carotenoid pigment was identified as astaxanthin. Drs. Miller, Yoneyama and Soneda created a new genus for the fermenting red yeast and named it Phaffia. Some of the Phaffia rhodozyma strains have industrial potential because of the pigments they produce.

Toward the end of the 1960s, Dr. Phaff began applying modem molecular biological techniques, including DNA base composition [guanine plus cytosine (G +C) content] and DNA-DNA hybridization for the classification of yeasts. With the help of graduate students Sally Meyer, Chet Price, and visiting scholars Dr. Leda Mendonca-Hagler from Brazil and Dr. Allessandro Martini from the University of Perugia in Italy, Dr. Phaff worked

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extensively on the systematics of yeasts. Also, with the help of another graduate student, Andre Lachance of Canada, they applied the immunological enzyme technique to compare the relatedness of different species, particularly in the genus Kluyveromyces. They found an excellent correlation between DNA relatedness data and the immunological enzyme comparison.

Beginning in 1970, Dr. Phaff began collaborating with Dr. William B. Heed of the University of Arizona, Tucson on exploration of another yeast habitat, the rotten cactus tissue in the Sonoran desert of the southwestern United States. Dr. Heed spent a sabbatical year in 1971 at Davis with Dr. Phaff, and together, they identified more than 300 yeast isolates and found the most common was P. membranifaciens. They later found that some isolates belonged to a new species, P. c actophila, which is among the most cosmopolitan species of cactophilic yeasts. Another similar species, P. heedii, was found in only two species of cactus, Carnegiea gigantea (saguaro) and Pachycereus schottii (senita). Later, they also discovered Candida sonorensis and C. cereanus in cactus necroses in the state of Sonora and northern Baja California, Mexico. C. cereanus was later placed in a new genus, Sporopachydermia.

The study of the yeast-cactus-Drosophila ecosystem was con-tinued with the participation of Dr. Tom Starmer, who obtained his PhD with Dr. Heed. Collecting areas were greatly expanded to include Baja California, southern Mexico, northern Venezuela, 12 islands in the Caribbean, Texas, Hawaii, and eastern Australia. Many new species of yeasts were identified and their ecophysi-ological information described. For example, Don Holzschu, Dr. Phaff’s last PhD student at Davis, found that P. pseudocact-ophila is different from P. cactophila, not only in DNA related-ness, but also in the electrophoretic mobility of four major metabolic enzymes. Much new biochemical, ecophysiological, genetic, and evolutionary information about yeast was obtained in Dr. Phaff’s laboratory. The yeast-cactus-Drosophila ecosystem

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probably provided the most valuable subject for Dr. Phaff’s career research. Phaff and his coworkers have described close to 50 new yeast species. This research is still continuing today in Dr. Phaff’s laboratory on the Davis campus, although he officially retired in 1983.

Administration and Honors

In addition to serving on the faculty in the Department of Food Science and Technology of the University of California in Berkeley and Davis, Dr. Phaff was appointed chairman and pro-fessor of the Department of Bacteriology in 1970 and stayed at this administrative position for five years. His principal teaching appointment from 1970 was in the bacteriology department.

In 1954, Dr. Phaff became the editor of the Yeast Newsletter, a journal Dr. Mrak had started in 1950. The Yeast Newsletter later became the official organ of the “International Commission on Yeast and Yeast like Organisms” of the International Union of Microbiological Societies (IUMS). Dr. Andre Lachance took over the editorship in 1987. Dr. Phaff was also an associate edi-tor of the International Journal of Systematic Bacteriology for more than 20 years, as well as several other journals.

Dr. Phaff received many professional honors. In 1969, he was awarded the Faculty Research Lectureship on the Davis campus. His lecture was entitled “Changing Aspects in Yeast Systematics”. He was the annual lecturer of the Mycological Society of America in 1976, recipient of the J. Roger Porter Award from the American Society for Microbiology in 1984 and the James F. Guymon lecturer of the American Society of Enology and Viticulture in 1986. Also in 1986, Dr. Phaff was named the John L. Etchells, Memorial Lecturer at North Carolina State University. A special issue of the Journal of Industrial Microbiology was published in 1995 with articles by his former students and coworkers. In October 1996, the administration of the Davis campus officially dedicated the “Herman J. Phaff

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Culture Collection of Yeasts and Yeast-like Microorganisms” consisting of thousands of strains collected over the years by Phaff and his coworkers. The dedication was accompanied by a minisymposium of papers presented by a number of his former associates from around the world.

“The yeast-cactus-Drosophila ecosystem probably provided the most valuable subject for Dr. Phaff’s career research. Phaff and his coworkers have described close to 50 new yeast species”.

He has been active in many scientific organizations including the American Chemical Society, American Society for Microbiology, Mycological Society of America, Institute of Food Technology, a charter member of the American Academy of Microbiology, New York Academy of Science, Canadian Society of Microbiology, Society of General Microbiology, United Kingdom, British Biochemical Society, and the Dutch Microbiology Society.

In 1966 Dr. Phaff, together with Drs. Mrak and Miller, pub-lished a book on The Life of Yeast (Harvard University Press). In 1978, it was followed by the extensively revised second edition. This is a very good book for the study of yeast and is the only one of its kind. A Japanese translation is also available, prepared by Dr. Phaff’s friend Dr. Susumu Nagai.

One of the authors had the honor to take some of Dr. Phaff’s courses back at Davis in the late 1960s and found him an honest and enthusiastic teacher, always straightforward and direct. I found him to be kind, gentle, and helpful. He was well liked by his colleagues, friends, and students; including Ellen Barker, Gayle Fuson, Mary Miranda, Heather Presley, Joanne Tredick, John Blue, and Jim Haudenshield who highly spoke of his personality and devotion.

Herman Phaff is an excellent cellist and at Davis became chairman of the Committee for Arts and Lectures, helping to develop the arts program on the campus. He has played an active role in the UC-Davis Symphony Orchestra and in local chamber music activities.

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Herman Phaff has spent his life studying the biology of yeasts and has laid down the most fundamental knowledge for our understanding of yeasts today. His contribution is comparable to that of Louis Pasteur and Robert Koch. He has also greatly influ-enced the sciences of microbiology and food science as well as other scientific disciplines.

As a microbiologist, while enjoying the benefits of yeasts have contributed to human civilization, we must always acknowl-edge Dr. Phaff’s contributions.

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Chapter 47

Harold Boyd Woodruff (1917-2017): Antibiotics Hunter and Distinguished

Soil Microbiologist

“One tenet of psychology is that childhood experiences shape the adult”.

“One must learn to accept the uninteresting while appreciating the good”.

“After the easy success possible at my relatively small school, it took a failure to make me recognize my deficiencies”.

“I have found that even the most basic scientist enjoys discussing the practical so long as his actual working time is not diverted. Occasional gems of wisdom may shine through in such discussions, but more often the specialist serves to draw out submerged ideas of the practical laboratory scientist, striking a spark of enthusiasm that will raise a project from the routine to creative”.

“Even the most dedicated basic scientist enjoys discussing practical objectives, and as the applied scientist becomes the recipient of the challenges presented, his cup runneth over with pleasure”.

“… soil is not simply dirt but a teeming mass of microbial life, who could have envisioned the challenges, the frustrations, the thrill of accomplishments that have been the lot of this industrial microbiologist”.

—Harold Boyd Woodruff

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Source: http://www.nasonline.org/member-directory/members/3005853.html(US Public Domain image)

Introduction

Dr. Harold Boyd Woodruff is a soil microbiologist with the desir-able characteristics of an applied approach to research. He holds 16 patents and has over 100 publications and presentations. He was credited for the discovery and production of several antibiot-ics including actinomycin, streptothricin, 19A (thermophyl), bacil-lin, 2′-ethoxygriseofulvin, histidomycin, mycosubtilin, streptavidin, streptin, antibacillin, and hadacidin. He was also chiefly responsi-ble for the production of a number of useful natural products including vitamin B12, glutamic acid, 3′-deoxyadenosine, tenua-zonic acid, coenzyme Q, bottromycin, penicillin acid, nicarbazin, and so on. All these findings are extremely beneficial to humans. For example, a member of the actinomycin complex was later found to have an antineoplastic effect in non-toxic doses to fight against Wilm’s disease, a childhood cancer. This drug has con-verted a 100% fatal disease to more than 90% survival and is still the best means to cure this disease.

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Dr. Woodruff achieves personal satisfaction by working toward practical objectives. He seizes opportunities of service to society and to microbiology. He has contributed significantly to the development of natural products important to human and animal health and to agriculture. His life experiences are worth learning about.


Harold Boyd Woodruff was born on 22 July 1917 in Bridgeton, New Jersey. His father was Harold E. Woodruff and his mother Velma Smith Woodruff. They were originally farmers in the state of Washington. They later moved to the state of New Jersey and lived in a farm community. Both of his parents respected education although they could not achieve educa-tion themselves because of financial limitations. Therefore, they were very instrumental in bringing a consolidated school system to their New Jersey farm community to replace a group of single-room schools, which were common in those days. Boyd attended elementary school in Bridgeton. He was impressed with the eighth grade teacher who stimulated stu-dents to educate themselves instead of formal instruction in the classroom. They were taught to conduct research and write reports. One of the valuable lessons he learned in that stage of life was as he said, “We in the United States are not the center of the world, that we are in fact just one of the many varied cultures vying for position on the earth”.

In high school, Boyd was interested in experimental sciences. Boyd recalled vividly the excitement of certain laboratory expe-riences, particularly in the course of freshman agriculture. He also enjoyed the chemistry that provided free experimentation such as coating a mirror, generating a source of heat to convert water to working steam, and making pigments to dye cloth, which were useful objectives.

Boyd was a teenager during the Depression. Farmers had food to eat but no money to spend. They often worried about

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unexpected hospitalization, unpaid fertilizer bills, and the necessity to move from one sharecrop farm to another when unpaid bills became overwhelming. Nevertheless, these prob-lems meant little to a little boy. Boyd had fun at local swimming holes, grange parties, marshland fishing, crabbing in salt water ponds, and Sunday school socials. Occasionally, a magazine route provided a little spending money, and a temporary job at the school cafeteria provided free lunches. Those were happy days for a country boy.

After graduation from high school in 1935, Boyd marched directly to Rutgers University with numerous financial aids. He won a scholarship established by a local farmer for a country resident planning to major in some aspect of agriculture. Rutgers University provided a free room. He was also helped by the director of resident instruction at the Agricultural College, Dr. Frank Helyer convinced the chairman of the Poultry Department to release ten unused poultry houses for student use. He was given 125 white Leghorn hens, whose eggs pro-vided a source of income via a local egg route. The eggs also provided a source of food, together with the inexpensive col-lege farm milk and vegetables and supplemented by canned foods provided in abundance by his family.

Boyd took many interesting courses including Geology, Zoology, Botany, and Chemistry in all aspects, Agronomy, Plant Physiology, and Soil Pedology. He also took some uninteresting liberal arts courses such as Educational Psychology and Economy in order to insure Phi Beta Kappa election, which was recom-mended by the university advisors. He said “… one must learn to accept the uninteresting while appreciating the good”.

College experiences were exciting, overwhelming, and broadening. He met many interesting people and did many important things. He attended concerts for the first time and tried out for track. He met Miss Jeanette I. Whitner, the Woman’s College student, and fell in love with her and got married on 25 July 1942. Another important thing that hap-pened to him was that he was appointed the Danforth Fellow

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for the state of New Jersey to attend a comprehensive training program in business practice at the Ralston Purina Company in the summer of 1937. He had a chance to work for a short period on the company’s research farm during which time he participated in the ongoing research projects. He was exposed to the significance of fully controlled experimentation, which gave him valuable experience in his later career development in clinical evaluation of antibiotics.

Another significant influence that happened to him was that he took a course called “soil microbiology” taught by Professor Selman A. Waksman (1888–1973) with laboratory instruction by visiting instructor Wayne Umbreit (1913–2007). Through this course, Boyd was very fortunate to have frequent contacts with truly inspirational teachers, who had a lasting influence on his life.

Boyd did well in his undergraduate studies in soil chemistry major, which was the usual ploy at that time to obtain a chem-ist’s education at the lower tuition rate of the state supported agriculture school. One little disappointment was that he was nominated for a Rhodes scholarship but failed the interview session because he had no plan for the future, did not know what he wanted to do with his life, and did not understand his need for future supplemental education. This instance made him realize that, “after the easy successes possible at my rela-tive small school, it took a failure to make me recognize my deficiencies”.

Boyd obtained his BS degree with soil chemistry major in 1939, and he immediately pursued graduate studies. He was neither prepared for nor did he expect to receive an offer from Dr. Waksman to join his laboratory group on a University fellowship permitting full-time research at the then unusual stipend of $900 per year. With hesitation and consultation with Dr. Jacob Lipman (1874–1939), dean of the Agricultural School, and others, Boyd accepted this offer with pleasure and began a career of an applied microbiologist. He learned directly by examples and by discussion with two distinguished senior

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scientists, Dr. Waksman and also Dr. Robert Lyman Starkey (1899–1991), another member of the departmental staff. He also had the opportunity to work in the lab with international students and research topics of extreme diversity and interest.

After his brief probes at defining the steps of sulfur oxida-tion in soil, Dr. Waksman called his attention to Alexander Fleming’s (1881–1955) work on penicillin. He immediately initi-ated the first search for antibiotics from actinomycetes on which Dr. Waksman was an expert. The work of searching for antibiot-ics was very successful. He gained the experiences of reisolating the old bacterial product, pyocyanase, and soon isolated a new antibiotic actinomycin. Actinomycin was highly active in killing microorganisms, but it is very toxic. It took a while later to find the usefulness of the actinomycin in treatment of a rare form of cancer, Wilm’s disease.

Research with Antibiotics

Actinomycin provided Dr. Woodruff’s first introduction to Merck & Co., Inc. which became his eventual employment. He worked with Dr. Jack L. Stokes, a former Waksman student, who offered to provide facilities for large-scale production of actinomycin. He also worked with Dr. Max Tishler (1906–1989), who later became president of the Merck Sharp & Dohme Research Laboratories. Dr. Tishler joined with Dr. Waksman in bringing actinomycin to the final stage of purity and investigation of its chemical structure.

The successful isolation of actinomycin changed the research direction of Dr. Waksman’s laboratory. The whole department specialized in antibiotics and talked of antibacterial substances, but nothing else. At one lunchtime discussion, Dr. Waksman coined the word “antibiotics” and stated its definition. “Antibiotics” became a scientific word at that time. Incoming graduate students were assigned to antibiotic research. A cul-ture isolated by Dr. Walter Kocholaty was assigned to him, and from the culture he isolated streptothricin, the first of the basic water soluble antibiotics. Streptothricin was shown to produce

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a significant cure of experimental infections including conta-gious abortion caused by Brucella abortus.

Wartime came to the United States, and Dr. Waksman turned full attention to antibiotics as a cure for diseases. Dr. Waksman gave encouragement to the budding interest of Merck & Co., Inc. in microbial processes. Merck was already known for its success in isolation of natural products and their chemical synthesis such as L-phenyl-1-acetylcarbinol, pre-cursor of L-ephedrine and sorbose, and precursor of vitamin C. Dr. Waksman convinced Merck & Co., Inc. to build a pilot plant for citric acid production using the new isolated fungus Aspergillus wentii and kept the Merck research directors informed of his work on antibiotics from streptomycetes. He strongly suggested the initiation of developmental studies at Merck with tyrothricin, an antibiotic isolated by Dr. René J. Dubos (1901–1982). Most importantly, Dr. Waksman sup-ported the Merck staff’s interest in the Oxford’s (England) stud-ies on penicillin.

Penicillin Studies

Merck & Co., Inc. warmly welcomed Drs. Howard Walter Florey (1898–1968) and Norman Heatley (1911–2004) from England to work on the production of penicillin, and H. Woodruff was asked by Dr. Waksman to participate in the program as a part of his graduate training. He had the chance to work with Dr. N. Heatley at the same laboratory bench to learn the penicil-lin fermentation and isolation process. This was exciting and challenging to him. They successfully worked out the labora-tory methodology of fermentation of Penicillium notatum in surface culture and later in submerged culture. With the coop-eration of 17 companies in the penicillin program, penicillin was finally produced in industrial amounts to supply the war-time need in 1941.

Woodruff’s PhD dissertation was not on the production of penicillin but on streptothricin. He was able to isolate strepto-thricin in pure form with excellent yield and developed a

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method of quantitative assay. He also discovered that strepto-thricin was effective against the growth of both Gram (+) and Gram (−) pathogens and even mycobacteria. Infected mice were cured with streptothricin. Clinical trials were under way, and the factory for the production by fermentation was designed. It seemed that streptothricin would be another use-ful antibiotic. However, extreme renal toxicity was found with streptothricin. The program was stopped instantly. What a disappointment to Woodruff! Nevertheless, he obtained his PhD in 1942 and was employed by Merck & Co., Inc. where he stayed for more than 40 years.

About this time, streptomycin was described by graduate students Albert Schatz (1920–2005) and Elizabeth Bugie (1921–2001) in Waksman’s laboratory. There was great excitement about the usefulness of streptomycin. As an important discov-erer of the first successful chemotherapeutic drug for tubercu-losis, Dr. Waksman received much adulation. Awards including the Nobel Prize were bestowed overwhelmly upon Dr. Waksman. A distressing instance with the allocation of the credit for this discovery occurred. Although the episode was also an interest-ing story, this author will not cover it in detail in this article. Nevertheless, the allocation of credit provides a good model for succeeding cases that followed.

Dr. Woodruff’s career at the Merck & Co., Inc. was rather smooth. He was the chief assistant to Dr. Jackson W. Foster (1914–1966), laboratory director of antibiotics studies in Merck. Dr. Foster had introduced penicillin production in postwar Japan. Drs. Woodruff and Foster coauthored a very influential paper, “Microbiological aspect of penicillin IV. Production of penicillin in submerged cultures of Penicillium notatum” (J. Bacteriol, 1946, 51, pp. 465–478.) This paper had opened doors of many research institutions in Japan to him. When Dr. Foster left Merck to accept a position at the University of Texas, Dr. Woodruff was promoted to head of the Research Section (1948) of the department. In 1949, he was assistant direc-tor of the Microbiology Department. In 1952, he was promoted

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to director of the same department. He was also responsible for microbial nutrition, including the vitamin and amino acid assay laboratories. In 1957, he became director of the Microbiology and Natural Product Research Department (1957–1969), and, finally, executive director for Biological Sciences (1969–1973).

Studies with Vitamin B12

There were many other studies that Dr. Woodruff was involved in at Merck & Co., Inc. They isolated the antipernicious anemia factor, cynocobalamin (Vitmain B12) from the liver, Streptomyces griseus fermentation broths, and eventually from other micro-organisms as well. From this research, Dr. Woodruff also was led to become an amateur expert on patent law as applied to fer-mentation because he experienced firsthand the stimulatory effect of the patent system on research activities. He appreci-ated the economic values derived from the patent system, which provides a means of continued support of industrial research, the flow from royalties often supports fundamental research in academic institutions as well.

Development of Bioengineering

In order to produce fermentation products on a large scale, a new engineering process was needed. With experiences with small laboratory production of penicillin, streptothricin, and eventually streptomycin, scientists at Merck & Co., Inc. marched into a new era of science, which was the birth of bioengineer-ing. This new science was developed under the leadership of Dr. Woodruff, Dr. Edward O. Karow, Waksman-trained microbi-ologist; Dr. William H. Bartholomew, petroleum engineer; and Mr. Michael R. Sfat (b. 1921), Cornell MS graduate also partici-pated in this effort. A theoretical chemical engineer Richard Wilhelm of Princeton University also loaned a big hand to this endeavor. Dr. Per K. Frǿlich (1899–1977), director of the research laboratories of the Merck, also gave a big encouragement to this project.

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Amino Acids

The next endeavor of Dr. Woodruff was the industrial fermenta-tion of amino acids. Dr. Woodruff participated in the American development of fermentation of two amino acids, lysine and glutamic acid, using processes invented by Dr. S. Kinoshita and his associates at the Kyowa Hakko organization. This experience also led Dr. Woodruff to propose an approach based on base-pair ratios and recovery of related microorganisms from nature for defining the range of microorganisms encompassed in a microbiological process patent disclosure.

Dr. Woodruff was involved in many types of research, which gave him excitement, practical accomplishments, publications, and invitations to speak at seminars at universities. His research included new antibiotics such as antibiotic 19A (thermophyl), bacillin, 2′-ethoxygrisreofulvin, histidomycin, mycosubtilin, streptavidin, and streptin; antagonist including antibacillin; and herbicide hadacidin. He also investigated antitumor activi-ties of 3′-deoxyadnosine for KB cells, tenuazonic acid for human adenocarcinoma-1, tenuazonic acid and hadacidin in human tumor-egg host system. In addition, he participated in the stud-ies of fermentation processes for coenzyme Q10, antimyco-plasma activity of bottromycin, penicillic acid production by molds, combination of nicarbazin and antibiotic in animal feeds. Because of his high productivity, his reputation grew, and he was overwhelmed with requests to write chapters, reviews, encyclopedia descriptions, and so on. He was most proud of one thing that he was invited to edit a book in recog-nition of Dr. Waksman’s 80th birthday. In this book, he had the opportunity to express his profound personal feeling toward Dr. Waksman.

Developmental Research Problems

Merck & Co., Inc. continuously manufactured penicillin for more than 35 years up to 1976. Improvement was made each year in either cost reduction or fermentation yield raised, but

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to exist as a research-based organization, Merck & Co., Inc. needed to have new products in order to provide greater finan-cial return and support continued research. While solving peni-cillin problems for factory operation, basic and new products research was neglected. For a time, Merck & Co., Inc. was left behind in the antibiotic field. Fortunately, other products such as vitamin B12, the amino acids, and the steroids provided targets for application of research creativity, but they were con-stantly oscillating from a developmental to a more basic research orientation. In order to survive one has to apply the same basic level approach to two areas: one is to apply the full knowledge of biochemical pathways for synthesis and their control as an approach to increase yields for the existing prod-ucts; and the other is to apply knowledge of the biochemical nature of the medicinal problems to be solved in order to devise screens for new products discovery. This new recognition was actually led through a new employee Dr. Arnold Demain (1927 to present), who approached developmental microbiol-ogy as applied to penicillin from a fundamental viewpoint while working in their fermentation factory in Danville, Pennsylvania. Dr. Demain attacked penicillin research success-fully through a biochemical approach. He later was advanced to the directorship of the Fermentation Research Department in the headquarters’ laboratory, which held the leading posi-tion for the research and development of Merck & Co., Inc. Dr. Demain later became a professor at the Massachusetts Institute of Technology, Boston, Massachusetts.

It is appropriate to mention that the Fermentation Research Department of Merck & Co., Inc. nurtured a good number of scientists who later became successful academicians and con-tributed greatly to the development of applied microbiology and technologies. To name a few, Drs. Jackson Foster, Jacob L. Stokes, and David Perlman, became leaders in research at the University of Texas, Washington State University, and the University of Wisconsin respectively. Dr. Roger Stanier (1916–1982), who was also a former Merck Co., Inc. laboratory staff

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member, became a professor at the University of California, Berkeley. Dr. Stanier introduced their penicillin fermentation process to the Merck Canadian laboratory. Dr. Stanley Carson became deputy director of the Biology Division, Oak Ridge National Laboratories. Dr. Lloyd McDaniel joined the Waksman Institute for Microbiology. Dr. Wayne Umbreit became chair-man of the Department of Bacteriology at Rutgers University. Dr. Paul F. Smith, expert in the field of Mycoplasma spp., became department chairman at the University of South Dakota. Dr. Walter Konetzka became professor at the University of Indiana. Dr. Robert Acker became an executive director at the American Society for Microbiology (ASM). Dr. Norman Somerson became professor at Ohio State University. Dr. George E. Schaiberger (1920–2005) became professor in the School of Medicine at the University of Miami. Dr. Lemuel D. Wright (1913–1995) became professor of Nutrition at Cornell University. Dr. Willard Verwey (1913–1979) became a depart-mental head at the University of Texas at Galveston. Dr. Robert Finn became founding chairman of the Bioengineering School at Cornell. Dr. Richard Shope (1901–1966), a world famous virologist, joined Merck Research and later became Professor of the Rockefeller Institute in New York. Under the leadership of Dr. Woodruff, Merck & Co., Inc. became one of the cradles for nurturing many pioneer microbiologists in the United States.

During the long stay in Merck Co., Inc. Dr. Woodruff also found time to do some research of his personal interest. He investigated natural products that act as antiviral agents. He was also interested in the study of aspects of RNA accumulation in Semliki Forest virus-infected tissue culture cells.

Further Antibiotics Research

Although Dr. Woodruff was involved in many executive and other activities, he never stopped his research. His laboratory continued to isolate and discover new antibiotics including

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cycloserine, novobiocin, and cephamycin C. He also developed a close association in research with foreign companies. For exam-ple, he helped to establish a cooperative new antibiotics pro-gram with the Compania Espanola de la Penicilina y Antibiotics in Spain. From this cooperation program, he and his group discovered fosfomycin, which is now marketed in Spain, Portugal, South America, and Japan. Cefoxitin was also devel-oped through this cooperative program. In addition, antibiotic thienamycin and combo-antibacterial MK-641/MK-642 were also developed through this stage of investigation.

New Fields of Challenge

Dr. Woodruff’s research was not limited to antibiotics; he explored other research directions. At that time, the National Institute for Dental Research (NIDR) had developed excellent models for diseases employing laboratory animals and had elu-cidated the bacterial species involved in the cariolytic process. Merck & Co., Inc. groups had an earlier research program that had been carried out in association with the Northern Regional Research Laboratory involving the use of enzymes to degrade dextran to produce a polymer with potential as a blood plasma extender. Dr. Woodruff and Dr. Thomas Stoudt (b. 1922) recog-nized the potentiality of utilizing similar enzymes to destroy microbial plaque attached to teeth, thereby reducing the ten-dency for caries formation. They developed a patent for this possibility in 1972. As a result, they manufactured dextranase under license from Merck & Co., Inc. and introduced it as a com-ponent of tooth paste by a company in Japan. Dr. Woodruff also looked into the excellent research ideas of Dr. Michael Heidelberger (1888–1991) on the antigenic properties of bacte-rial polysaccharides and recently pursued by Dr. Robert Austrian (1916–2007) to practical fruition. Eventually, Dr. Woodruff and his group successfully developed a new type of submerged fer-mentation pilot plant to produce these products, which resulted in the marketing of a 14-valent pneumococcal vaccine as well

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as group A and group C meningococcal vaccine by Merck & Co., Inc. They also continued extensive research on Haemophilus and gonococcal vaccines.

Foreign Diplomacy

Merck Sharp & Dohme Research Laboratory (MSDRL) decided to expand their research and development through interna-tional collaboration. One of the countries chosen by MSDRL was Japan. Dr. Woodruff was assigned half-time residence in Tokyo with the opportunity to meet with and discuss issues with Japanese microbiologists. Another half-time was spent in the Unites States to ensure that Japanese discoveries were given the credence they deserved in laboratory study. For about seven years the Merck research laboratories had been given the opportunity to share in research and evaluation of products originally discovered in Japanese laboratories. Dr. P. Roy Vagelos (1929 to present), the Research Laboratories president of that time introduced a system that provided equal developmental opportunity to products originating from out-side laboratories and those from the basic section of the Merck Sharp & Dohme Research Laboratories. Dr. Woodruff devel-oped a longtime friendly association with Dr. Toju Hata (1908–2004), president of the Kitasato Institute, and later with Dr. Satoshi Ōmura (1935 to present), successor of Dr. T. Hata. The collaborative Merck/Kitasato Institute research program was significantly strengthened. Starting with an examination of growth- promoting antibiotics and moving to a study of enzyme inhibi-tors, the program extended to an evaluation of production of antiparasitic agents by microorganisms. For example, a new class of natural products, the avermectins, was isolated from Streptomyces avermitilis. Avermectins are now under develop-ment worldwide as broad spectrum anti-helmintic agents of exceptional potency. This was a very enjoyable period of Dr. Woodruff’s career.

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Returning to Soil Microbiology

For the last 50 years, his life journey brought him from the ini-tial introduction to problems in soil microbiology to the many opportunities available in applied industrial research at Merck Co., Inc. and finally returning to soil microbiology. He became aware, as he said that “… soil is not simply dirt but a teaming mass of microbial life, who could have envisioned the chal-lenges, the frustrations, and the thrill of accomplishments that have been the lot of this industrial microbiologist”.

Dr. Woodruff’s life effort was in screening the production of biological active chemicals from soil actinomycetes, which was the most favorite subject of Dr. Waksman. He believed the viewpoint of Waksman who regarded that the actinomycetes are active members of the soil population and are important. But in obtaining firm proof for this idea, they were often disap-pointed. In collaboration with Dr. Robert L. Starkey, they found the response of soil microorganisms including the soil proto-zoa, the soil bacteria, and soil fungi multiplied quickly follow-ing partial treatment of soil with toluene. However, based on total counts, the responses of actinomycetes were often slug-gish. Soil microbiologists at other institutions, who did not have Waksman’s confidence, concluded that the actinomycetes seen on soil plates have little significance in the soil. The com-mon conclusion of many workers at the time was that actino-mycetes can be ignored as important soil components. Much disappointment to both Waksman and Woodruff was that a discovery was made that actinomycetes played little role in the soil. This conclusion was contradictory to Drs. Wasksman and Woodruff’s expectations. But there was no solid evidence to dispute this conclusion.

However, an opportunity arose; Dr. Woodruff had an occa-sion to visit Australia in association with his responsibility for the veterinary research laboratory in Sidney and to various research laboratories throughout that country in search for new research opportunities. He took the opportunity to collaborate

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with Dr. David Keast of the Department of Microbiology of the Medical School at the University of Western Australia in Perth. They collected hundreds of actinomycetes isolated from unique soil types in Western Australia, and they did not measure the total number of actinomycetes present in soil by direct count. Instead, they enumerated the number of distinct morphological types present on soil dilution plates. They were also assisted by Dr. John W. Tukey (1915–2000), professor of statistics at Princeton University, who devised a mathematical expression for the degree in which two soils differ in types of actinomy-cetes present. They did a lot of laboratory work such as growing cultures under standard conditions observing soluble pigments, spore formation, and characters that enter into actinomycetes classification. Furthermore, when they compared the type of actinomycetes at the end of a dry period to samples 5 days after rain and again after 13 days, a major change in the actinomy-cetes types had occurred at the 5-day sampling and even greater changes in 13 days. No great change was seen in the total actinomycetes counts, so the effect would have been missed by simply obtaining the total count. The revised technique of studying the types present showed clearly that the population of actinomycetes in the soil is dynamic. Soil actinomycetes respond to physical factors and associated nutritional factors, and they do so with great speed. Soil diversity is important and the actinomycetes population of the soil is dynamic. Actinomycetes have significance beyond their abilities to pro-duce antibiotics and are significant members of the diverse soil population. Actinomycetes respond, change, and multiply in soil with rapidity. One actinomycete population can replace another. Actinomycetes do not differ from other members of the soil biota. They truly are important. Although, with credit of isolating many antibiotics and other biological chemicals that are beneficial to human welfare, Dr. Woodruff regarded the experiment described above was the most satisfying experiment in his career.

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Endeavor After Retirement

In 1982, Dr. Woodruff formerly retired from the Merck Sharp and Dohme Research Laboratories in Japan, but he did not stop research in soil microbiology and antibiotics. He established Microbiology Associates, Inc., and as president and director of research, he continued his active study. His wife was his work-ing partner. Since many natural products of potential useful-ness had been isolated from soil, he realized that new approaches had to be adapted in order to have a better chance to get new antibiotics and to avoid the redundancy of isolating the same products. This could be achieved through two approaches: first, the widest range of microorganisms possible should be screened, and second, the most predictive screening procedures possible to detect useful products must be employed. The experience of his previous collaboration with the University of Western Australia provided him valuable lessons. From that experience, had applied that approach to characterize all actin-omycetes possible in the soil samples to screen for the produc-tion of new antibiotics. He also found that soils of many unusual, especially stressed environmental niches should be sampled. Employing these two approaches, he and his wife, col-lected approximately 5500 actinomycete cultures isolated from highly stressed conditions, and several new natural products were obtained and the chemical structures established. For example a compound named chloropeptin was isolated from Streptomyces sp. WK-3419, which was obtained from marsh-land adjacent to San Padro Island, Texas. Chloropeptin is a strong inhibitor of the HIV virus, which causes AIDS. Pure chlo-ropeptin proves 100% effective as an HIV binding inhibitor. Louisianin was obtained from Streptomyces sp. WK-4028, which was isolated from a bayou in Louisiana. Louisianin was proved to be able to inhibit testerone-responsive prostate cancer cells at 0.6 microgram per ml. Norma cells from other sources were not harmed by 2000-fold greater concentration. Diolmycin was isolated from Streptomyces sp. WK-2955, which was obtained

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from a plant rhizosphere taken at the Red Cross Hospital in Tokyo. Diolmycin was proved to have a strong anti-Eimeria tenella coccidian property. Similarly, a new product structurally similar to virginiamycin was isolated from a streptomycete, which was isolated from a soil sample collected in Pimpri, India. The new virginiamycin analog can inhibit the binding gastrin to receptors at very low concentration and very specific in the intestine. Excessive binding of gastrin to receptors in the intes-tine has been suggested to be involved in intestinal ulcers. Another bioactive substance called hexadepsipeptide was isolated from an active actinomycete, which was obtained from the rhizosphere of a plant present in a Japanese garden. Hexadepsipeptide is an inhibitor of anaphylatoxin C5, which is present in the blood serum during the complement fixation and believed to serve as a mediator of inflammation.

In collaboration with Kitasato Institute in Japan, Woodruff also isolated phenazinomycin from Streptomyces sp. WK-2057, which was isolated from the rhizosphere of a fireweed plant growing along the Tiekal River in Alaska. Phenazinomycin is a new antitumor agent. It can prolong the surviving time of mice infected intraperitoneally with sarcoma 180 cells. Again in col-laboration with Japanese workers, antifungi antibiotic phtho-ramycin was isolated from Streptomyces spp. WK-1875, which was obtained from the shore of Tokyo Bay in Japan. Phthoramycin was found to inhibit the growth of Pyricularia, Mucor, and Phytophthora at low concentrations. It was later found that mechanism of action of phthoramycin is the inhibition of the cellulose synthesis in resting cells of Acetobacter xylinum. A new group of protease inhibitors called ahpatinins was obtained at the Kitasato Institute from actinomycetes isolated from a bayberry plant growing in the Izu Peninsula, Japan, and another inhibitor identified as a phenyl derivative of pepstatin was found at Merck from a culture, which was isolated from an unusual black organic rich soil of the isolated island of Shikinejima in the Pacific Ocean. These compounds were shown

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to be able to inhibit the rennin, which is a factor causing high blood pressure. An actinomycete culture isolated from a forest floor in Pimpri, India was found to produce oxytocin antago-nists. Oxytocin is a physiological inhibitor for labor in birth. It is believed to be involved in premature labor, which is associated with neonatal morbidity and mortality.

There may be more new products useful in research and potentially useful for disease control that were isolated during this stage of Dr. Woodruff’s retirement. The crucial point is to employ more innovative approaches to reduce the redundancy in the organisms supplied for screening. Dr. Woodruff strongly believes that the natural product research remains an exciting, challenging, and productive endeavor. He said, “if young students chose microbial natural products as their research objective, they can be guaranteed fulfilling and exciting lives”.

Academic Societies Services

Through his work, he was provided with opportunities of encountering many scientists worldwide, both basic and applied researchers. He had relationships with applied research scien-tists in pharmaceutical companies located in France, England, Spain, Italy, Sweden, Brazil India, Germany, and Japan. He recalled that he had visited 37 state universities in the continen-tal United States and 26 foreign countries for exchange of ideas, which enriched his research and life. As he said, “Even the most dedicated basic scientist enjoys discussing practical objec-tives, and as the applied scientist becomes the recipient of the challenges presented, his cup runneth over with pleasure”.

He also enjoyed the participation in the Merck Foreign Fellowship program. He spent the winter term 1953–1954 in attendance at lectures at Cambridge University, in the Sub-Department of Chemical Microbiology headed by Dr. Ernst Gale (1914–2005). He took the opportunity to enhance his knowledge of biochemistry, particularly the biochemistry of

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protozoa and helminthes, which was important for his later responsibilities to include parasites as well as more common forms of microorganisms as targets for drug development. He also established a lifetime relationship with Dr. Gale, whom he believed was instrumental to his participation as a speaker in three of the Annual Symposia of the Society for General Microbiology of England.

Dr. Woodruff was also active in academic society’s activities. He first attended the annual meeting of ASM in Baltimore in 1941 and almost never missed it since then. He was the found-ing member of the Journal Applied Microbiology (now Applied and Environmental Microbiology) and the first editor-in-chief of that journal (1953–1962). During his tenure, he won respect for both the journal and himself. He was later elected Society treasurer for six years (1964–1970). He is a loyal member of ASM and continued to serve on the Board of Directors for ASM Foundation (1972–1974) and served as president in 1972. He was also active in the Society for Industrial Microbiology (SIM) and was elected president (1954–1956). In addition, Dr. Woodruff was president of the Theobald Smith Society (1949–1950); member of the US National Committee on the International Union of Biological Sciences (1963–1967); member of the Scientific Advisory Committee Charles F. Kettering Research Laboratory (1972–1975); member of Board of Trustees Biological Abstracts (1972–1977) and its treasurer (1974–1977). He also served on the Executive Board of the US Federation for Culture Collections (1973–1976) and Board of Trustees, American Type Culture Collection (1981–1987). He was also chairman of the Scientific Advisory Council of Waksman Institute for Microbiology (1982–1987).

In Dr. Woodruff’s professional life, he was a member of sev-eral academic societies including Alpha Zeta, American Academy of Microbiology, American Association for the advancement of Science, America Chemical society, ASM, National Academy of Sciences of the United States, Sigma Xi, Society for Actinomycetes, Japan, Society for General Microbiology, S IM, Theobald Smith

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Society, and US Federation for Culture Collection. To each soci-ety, he dedicated his totally unselfish services.

Honors

Numerous honors were bestowed upon Dr. Woordruff during his lifetime. As an undergraduate at Rutgers University, he was an Alpha Zeta member, State of New Jersey appointee Danforth Summer Fellowship, and also a Rutgers University nominee for Rhodes Fellowship in 1937. In 1938, he was elected member of Phi Beta Kappa. He received a sabbatical award to attend Cambridge University in 1953. In 1953, he was awarded with the Charles Thom award, SIM. In 1982, he was elected honorary member for ASM and also the Kitasato Institute, Japan. In 1983, he was elected Fellow of SIM. In 1995, he was honorary member of the Society for Acetinomycetes, Japan. The highest honor was the election to be a member of the National Academy of Sciences in 1998. At the Rutgers, he was recipient of the George Hammell Cook Distinguished Alumni Award in 2002 and inducted into the Rutgers University Hall of Distinguished Alumni in 2004. Both Dr. Woodruff and his wife Jeanette Woodruff were the recipi-ents of the Rutgers University Medal for Philanthropic Excellence.

Recently, Dr. Woodruff was selected by the Theobald Smith Society, the New Jersey Branch of the ASM, for their Waksman award on 3 May 2007. This award was presented to a scientist with a distinguished career in microbiology. As stated by Douglas Eveleigh, Eveleigh and Fenton Chair in Applied Microbiology at Rutgers School of Environmental and Biological Sciences, “Boyd Woodruff has introduced major new directions in the pharma-ceutical industry, stimulated development in what was at the time a fledgling fermentation engineering industry and in bringing practical antibiotics to fruition, clearly revolutionized the practice of international medicine. Woodruff was one of the driving forces who established the United States as a world leader in the discovery of antibiotics and inspired a revolution in medicine and agriculture”.

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Personal Life

H. Boyd Woodruff married Jeanette Whitner, who was a gradu-ate of the Women’s College at Rutgers; Jeanette became his coworker in their joint household and career. They had a wonderful marriage and raised two children. Brian earned a PhD in statistics; and Hugh a doctorate in computer applica-tions to chemistry. Dr. Woodruff is a wonderful kind person as you can feel his honesty and sincerity. For so many years, he has been capable of associating with many competitive profession-als and maintaining such a warm association. Everyone associ-ates with him would like to be his friend. His high respect and love to his mentor Dr. Waksman can always be discerned when-ever he talked or wrote about him. His sincerity is infectious to everyone. This author has the honor to have met him at a spe-cial occasion at one of the ASM meetings in 2006, and requested bluntly to write his life story. His response was happily “yes”. It is indeed an honor to able to tell his story and share his valu-able life experiences, which represents an era of antibiotics. The progress of this world is made by those endless hard and sincere workers like Dr. Woodruff, who make all of us live better.

Dr. Woodruff was also very interested in nurturing the younger generation, particularly in his native state of New Jersey. He set up The H. Boyd and Jeanette Woodruff Microbiology Fellowship, a graduate scholarship fund in soil/environmental microbiology awarded annually to outstanding Rutgers School of Environmental and Biological Sciences ( formerly Cook College) students and to which the Woodruffs contributed one million dollars. The Woodruffs also donated monies for the H. Boyd Woodruff Undergraduate Scholarship Fund to aid scholars from Cape May, Cumberland, or Salem Counties of the New Jersey State and gave funds for the devel-opment of the Waksman Soil Microbiology Laboratory Museum, Martin Hall of the Rutgers University.

Dr. Woodruff contributes so much to society and remains humble and kind. He always wants to do more for the benefit

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of others. He is an altruist indeed. He represents a generation of American who had humble beginning but willing to work hard and have a successful life and share the credit with others. He said, “If students do choose microbial natural products as their research objective, they can be guaranteed fulfilling and exciting lives”. His life is a shining example for students who are willing to work hard and persistently pursue their goals with love.

Suggested Reading

1. Woodruff, H. B. (1981). A soil microbiologist’s odyssey. Annual Review of Microbiology 35: 1–28.

2. Keast, D., P. Rowe, B. Bowra, L. Sanfelieu, E. O. Stapley and H. B. Woodruff (1984). Studies on the ecology of west Australian actinomycetes: factors which influence the diver-sity and types of actinomycetes in Australian soils. Microbial Ecology 10: 123–136.

3. Woodruff, H. B. (1996). Impact of microbial diversity on anti-biotic discovery, a personal history. Journal of Industrial Microbiology 17: 323–327.

4. Woodruff, H. B. (2000). Natural products from microorganisms and odyssey revisited. Actinomycetologica 13(2): 58–67.

5. Kamiyama, K., S. Takamatsu, Y.-P. Kim, A. Matsumoto, Y. Takahashi, M. Yayashi, H. B. Woodruff and S. Omura (1995). Louisianins A, B, C, and D: Non-steroidal growth inhibitors of testosterone-responsive SC 115 cells. 1. Taxonomy, isolation and biological characteristics. The Journal of Antibiotics 48: 1086–1089.

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Chapter 48

Ralph S. Wolfe (1921 to Present): Pioneer of

Biochemistry of Methanogenesis

“The degree of success depends on attention to details. Assume nothing!”

“The thrill of discovery on one’s own is the best motivating force”.

“Appreciate the importance of hard data in nailing down a concept as well as the importance of freedom in exploring and making discoveries”.

“As an independent investigator, each should realize the importance of choosing a problem that is not moving and move it”.

“To get back in the laboratory is a necessary component of scientific survival”.

—Ralph S. Wolfe

Introduction

Ralph Stoner Wolfe (b. 1921) is well known for his work on the biochemical studies of methane formation (methanogenesis). He has devoted his research career to work on the metabolism and physiology of bacteria, particularly the methanogens, which reduce CO2 with molecular hydrogen to produce meth-ane. Methane is a basic component of natural gas — a biofuel.

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However, how methanogens reduce CO2 to form methane has puzzled biochemists and microbiologists for a long time. Dr. Wolfe’s patient and meticulous work has put in place numer-ous pieces of the puzzle which illustrate one of nature’s most important pathways. This in turn has given us a new apprecia-tion for the complexity of the microbial world. His work dem-onstrates the amazing capabilities of microbes in the natural environment. This information has had a great influence on the development of biotechnology which we are facing today.

Ralph was born on 18 July 1921, in New Windsor, Maryland; however, in 1937 his family moved to Bridgewater, Virginia. His father was a professor of religion and philosophy at Bridgewater College. During his youth he was fascinated with animal fossils. One might speculate that he would be a paleontologist and work in a museum. This aspiration quickly faded away after he visited the paleontology research area of the Academy of Natural Sciences, which gave him a sobering experience. His dream was to be a small college professor like his father. This would permit him to enjoy the freedom of the academic life and a three month summer vacation each year. However, his dream never came true. He never reached a small college and never had a true three month summer vacation. Instead, he was associated with big universities and worked laboriously during the summer months.

He attended Bridgewater College majoring in Biology, and graduated in 1942. During his undergraduate years, he received some sound advice from one of his professor Dr. Harry G. Jopson (1911–2012), who persuaded him to take organic chemistry and minor in chemistry. This advice had a great influence on him, and this became the foundation for working on biochemical prob-lems of microbes. Ralph taught in high school for a brief period and later went to the University of Pennsylvania hoping to get a Master’s degree. Professor Schramn advised him to take a course in “General Bacteriology” taught by Dr. Wesley G. Hutchinson. While taking the course, he also worked part-time in a herbar-ium, where he mounted pressed flowers on sheets of papers.

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Dr. Hutchinson stimulated his interest in bacteriology. Later he became a research assistant for Dr. Hutchinson. In the second year of his graduate study, Ralph became a teaching assistant for a new assistant professor, Dr. Daniel J. O’Kane (1919–2007). Dr. O’Kane accepted Ralph as a Master’s degree student for a thesis project he worked on “Hippicurase from Streptococcus”. As a teaching assistant, Ralph learned some valuable lessons: “The degree of success depends on attention to details; every-thing must be checked; the cultures, the media, the glassware, the incubator. Assume nothing!” He also learned that “the thrill of discovery on one’s own is the best motivating force”.

From 1947 to 1949, Ralph was an Assistant Instructor of Microbiology in the Department of Botany at the University of Pennsylvania, with a stipend of $950 for nine months. In 1949, Ralph finished his MS degree. At this time he wanted to see what a nonacademic life was like and wanted to improve his financial condition. He accepted a position as a technician in Ruth Patrick’s laboratory at the Academy of Natural Sciences in Philadelphia. He helped Dr. Patrick work on methods to analyze the toxicity of stream pollutants. He began to appreciate the potential application of microbiology. During this period, he fell in love with Gretka Young, who worked for the American Friends Service Committee in Philadelphia. They were married in September, 1950.

Ralph went back to Dr. O’Kane’s laboratory and obtained his PhD degree from the University of Pennsylvania in 1953. His dissertation research was concerned with the oxidation of pyru-vate by Clostridium butyricum. He discovered that diphospho-thiamine, coenzyme A, and ferrous ions were required for the oxidation of pyruvate. The lesson that he learned from working in Dr. O’Kane’s laboratory was “I owe much to O’Kane, he made me appreciate the importance of hard data in nailing down a concept as well as the importance of freedom in exploring and making discoveries”.

In 1953, Ralph was hired as an Instructor in the Department of Bacteriology at the University of Illinois, Urbana-Champaign.

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He became a colleague with internationally known microbiolo-gists such as Halvor O. Halvorson (1897–1975). Solomon Spiegleman (1914–1983), Salvador E. Luria (1912–1991), and Irwin C. Gunsalus (1912–2008). In the beginning of his appoint-ment, Ralph was determined to write a manuscript from his dis-sertation for publication, so he closed his door whenever he had a chance, to work on the manuscript. One day there was a knock on his office door, Professor Halvorson entered, sat down, and in a very concerned manner said “I just want to tell you one thing — you are paid to teach; you get promotions for doing research”. This was an unforgettable memory as Ralph recalled. This statement is still true today.

In the summer of 1954, Ralph attended a summer course taught by Dr. Cornelis B. van Niel (1897–1985) at the Hopkins Marine Station, Stanford University, in California. Van Niel’s course introduced him to a microbial world of unfamiliar micro-organisms. Since then Ralph always regarded himself a disciple of the Delft School of Microbiology. Upon returning to the University of Illinois, with assistance from Gunsalus, Luria, and Sherman; Ralph organized a lecture-laboratory course (Microbiology 309 and 409) that emphasized the biological properties and fascinating diversity in the major groups of microorganisms. He taught this course for the next 30 years. At the same time, he found a unique niche at the University of Illinois. He set out to study unusual microorganisms and began an in-depth investigation into the physiology and biochemistry of methanogens.

One of his initial scientific contributions at the University of Illinois came from his studies, which explained the biochemical significance of ferredoxin, an electron carrier of many anaer-obes. This was accomplished chiefly by Ralph’s PhD student, Raymond Valentine, and Dr. Leonard E. Mortenson (b. 1928) who was working at the DuPont Company in Wilmington, Delaware. In 1960, Ralph began to work on Methanobacterium omelinskii, a bacterial culture obtained from Dr. Horace A. Barker (1907–2000) from the University of California, Berkeley. This organism

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was thought to grow by oxidizing ethanol to acetate using CO2 as the electron sink with the formation of methane. With the collaboration of Dr. Meyer (Mike) J. Wolin (b. 1930) and others, he was able to grow M. omelianskii in large quantities and demonstrate the formation of methane by cell-free extracts of this organism from various substrates including methylcobala-min, cobamide derivatives, methyl-Factor B, and methyl-Factor III. While these discoveries were exciting, it was soon found in col-laboration with Dr. Marvin P. Bryant (1925–2000) that M. omeli-anskii was not a pure culture. M. omelianskii was a mixture of Methanobacterium M.O.H., the true methanogen, and another organism called the “S” organism. The “S” organism, isolated by Bryant, produced hydrogen from the oxidation of ethanol to acetate. Methanobacterium M.O.H consumed the hydrogen with the formation of methane from CO2. This caused a temporary set back of Ralph’s work on methanogenesis, but led to the discovery of the “interspecies hydrogen transfer”, which was a milestone in understanding interactions between anaerobic microorganisms. Ralph’s laboratory soon isolated coenzyme M (CoM), the terminal methyl carrier from Methanobacterium M.O.H. They also deter-mined that the chemical structure of CoM was 2-mercaptoethane-sulfonic acid and began to study its functions. At this time, because large quantities of methanogens were required, they modified Hungate’s roll tube method of culturing methanogens by using a pressurized atmosphere in standard serum bottles sealed with a solid rubber stopper.

The discovery of CoM and the elucidation of its structure led Ralph to document the distribution of a new vitamin-coenzyme relationship with a classical growth-dependent assay. Thus, he and his associates examined whether or not CoM was present in other bacteria, plants or animals. This work established that CoM was unique to methanogens. Moreover, it was the first of several unusual coenzymes that were found to be required for methanogenesis.

Further studies eventually led Ralph to a collaboration with Dr. Carl R. Woese (1928–2012) to sequence the 16s RNA of

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methanogens. Again a surprise, the sequence of 16s RNA was found to be totally different from any other microorganisms. Dr. Woese proposed that “these things (methanogens) are not even bacteria”.

With the collaboration of Dr. Otto Kandler (1920–) in Munich, Germany, they discovered that methanogens did not have the regular bacterial cell wall component peptidoglycan. Instead they have pseudomurein. That is uniquely different from bacteria, plants or animals. Collectively, these data led Dr. Carl Woese to propose that methanogens (along with halo-philes and certain thermoacidophiles) belong to a distant phy-logeny — the Archaebacteria, now known as the Archaea. The work defining the properties of the Archaebacteria is one of the greatest contributions in microbiology in the past two dec-ades. Dr. Wolfe is the center figure for this discovery.

Further studies by Ralph and his associates led to new discov-eries of a variety of coenzymes (cofactors) involved in the bio-chemistry of methanogens. The significant ones are: Coenzyme F420, a deazaflavin, which is an important electron carrier; F342, a methanopterin or coenzyme of methyl group transfer enzyme; coenzyme F430, the first nickel natural tetrapyrrole, or coenzyme for methylreductase; methanofuran, a coenzyme for formyla-tion or CO2 reduction and 7-mercaptoheptanoylthreonine phosphate, which is the coenzyme that donates electrons to methylreductase.

Discoveries of these coenzymes led to studies involving the enzymology of methane formation from CO2 and H2. However, this was an exhausting effort that spanned about 15 years. Eventually, Ralph and his associates were able to put all the cofactors together in the biochemical pathways they had pro-posed for methanogenesis from CO2 and H2. This is truely one of the unique biochemical pathways in the microbial world. This is indeed a great achievement. Although Ralph was well known for his work on methanogenesis, his interest and studies were much broader than one group of microorganisms. As a disciple of the Delft School, Ralph is interested in a great variety of

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microorganisms. Microbial diversity is also his specialty. For example, with his students he worked on iron bacteria, Gallionella species, isolated from rusty patches along the Rocky Hollow trail in Turkey Run State Park, Indiana. He worked on Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, R. palustris, and purple nonsulphur photosynthetic bacteria. Other organisms included in his studies were Beggiatoa (a glid-ing trichome), Sarcina ventriculi, Streptococcus allantoicus, Sphaerotilus natans, Arthrobacter crystallopoietes, Myxobacter, C. tetanomorphum, Geodermatophilus species, M. thermoauto-trophicum, Methanogenium cariacii, M. marisnigri, Methanococcus voltae, M. jannaschii, M. thermophilum, Acetobacterium woodii, Acetogenium kuvui, Methanospirillum hungatei. Ralph also studied many important microbial ecologi-cal phenomena such as synthrophic cultures of A. woodii and M. barkeri, Spirillum 5157 and Chlorobium, and enrichment cul-tures of magnetotactic bacteria.

Dr. Wolfe always appreciates the association with his colleagues, friends, and students. As he said “I was fortunate to have a series of talented graduate and postdoctoral students to work with”. Dr. Ralph was a humble and hardworking scientist, he was always in the laboratory to conduct experiments himself or directing students. Students enjoy working with him. Yet he never dictated to them. As he said: “In operating my laboratory, my purpose has not been to play the role of the brilliant intel-lectual leader, but rather to stay in the background and try to create an atmosphere in which students could develop into inde-pendent investigators”. His humble demeanor attracted many bright students to work with him. He also said: “To get back in the laboratory is a necessary component of scientific survival”. He led by example. He represents a great microbiologist who is dedicated, enthusiastic, and contributes greatly to the progress of science, yet he would rather remain anonymous.

Dr. Wolfe’s awards and honors were numerous. Most recently he received the Abbott Lifetime Achievement (1996) and the Selman Waksman Award (1995). Other awards include

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the Alexander von Humboldt Senior Award (1984), Pasteur Award (1974), and the Carski Foundation Distinguished Teaching Award (1971). He was elected to a honorary membership in the American Society for Microbiology (1995), elected to the National Academy of Sciences (1981), and to the American Academy of Arts and Science (1981). Dr. Wolfe received a Distinguished Alumnus Award, Bridgewater College (1978), has given numerous distinguished lecturer presentations, was a Foundation Lecturer for the Amaerican Society for Microbiology (1972) and was a Guggenheim Fellow (1960–1975). He is actively involved in numerous study sections for granting agencies and participates in numerous organizational activities.

In conclusion and as a tribute of respect to Dr. Wolfe, one of us (VV) has known him for 25 years, and has discussed his accomplishments on numerous occasions with students, post-doctoral candidates and colleagues. Everyone has had positive comments to offer, and not once during that time have I heard anyone ever speak negative comment about Ralph. He has had a positive impact, scientifically and personally, on all who have become acquainted with him.

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Chapter 49

Esther Miriam (Zimmer) Lederberg (1922–2006): Transduction and

Replica Plating

Source: https://en.wikipedia.org/wiki/Esther_Lederberg(US Public Domain image)

Esther M. (Zimmer) Lederberg contributed greatly to the birth of modern bacterial genetics. With the collaboration of her husband, Joshua Lederberg (1925–2008), she contributed to the elucidation of lyogenicity, bacterial recombination, transformation, DNA repair, phase variation of flagellic antigens in Salmonella, drug

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resistance, and transduction, which is the transfer of bacterial genes from one organism to another through bacteriophage. She invented the “Replica Plating” method, which has been widely used in bacterial genetic research. She discovered lambda phage and made a big contribution to the understanding of plasmid biology. Despite her contributions, Joshua Lederberg [along with George W. Beadle (1903–1989) and Edward Lawrie Tatum (1909–1975)] received the Nobel Prize in medicine or physiology in 1958. Esther was left out.

Esther was born on 22 December 1922 in New York City. Her father was David Zimmer and her mother was Pauline Geller. She was a smart student and she obtained her bachelor’s degree in genetics from Hunter College in 1942 and her master’s degree from Stanford University in 1946. The same year Esther worked as an assistant to Dr. Tatum at Columbia University where she met Joshua Lederberg. They quickly fell in love and got married. In 1947, Joshua accepted an appointment as assistant professor at the University of Wisconsin where Esther was a National Cancer Institute predoctoral fellow. She completed her PhD degree from the University of Wisconsin in 1950.

While in Wisconsin, Esther worked as an associate in genetics. She continued to collaborate with her husband to work on the genetics of bacteria. She demonstrated the mutability of several lactose mutants of Escherichia coli and lysogenicity of E. coli K-12. She did many recombination analyses of bacterial strains. This was a very exciting period. She participated in many impor-tant works that promoted the understanding of how genetical information is transmitted. She and her colleagues demonstrated that a fertility factor was required for bacteria to undergo sexual conjugation and the exchange of genetic materials.

The transfer of many different bacterial colonies from plate to plate was very tedious and time consuming. Esther developed a technique of replica plating. In this technique, the bacteria from a liquid culture were evenly spread on a master agar plate and allowed to grow for a period of time. A sterile velveteen pad was then gently pressed against the surface of the master

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plate to pick up organisms from each colony. The tiny fibers of velveteen act like hundreds of tiny inoculating needles. The pad was carefully kept in the same orientation and used to inoculate another agar plate containing the medium composition desired for selecting the organism. Replica plating is a very effective means of isolating mutants that require one or more new growth factors. Any mutant microorganisms having a nutri-tional requirement, that is, absent in the parent is known as an auxotroph. The parent would be called a prototroph. This tech-nique also demonstrates the spontaneity of mutations.

Replica plating becomes a powerful tool for mutant selections. It speeds up the research of bacterial genetics. It is still widely used to study changes in the characteristics of many bacteria.

In 1951, Esther discovered a lambda phage in E. coli. She isolated the phage and proved that it could be transmitted to other bacterial cells via sexual conjugation and recombination. This also led to the discovery of transduction, transfer of bacte-rial genes from one organism to another via phage, which was published in 1956. The lambda phage is also the first plasmid that has ever been discovered. Esther’s findings tremendously helped the development of plasmid biology, which leads to the rapid progress of recombinant DNA technology. Esther had made a mark in the development of science.

In 1956, Esther and Joshua were jointly awarded the Pasteur Award of the Illinois Society for Microbiology. In 1957, Esther was also awarded a Fullbright Fellowship in bacteriology to spend a year at Melbourne University in Australia. In 1958, Joshua Lederberg alone without Esther was awarded with the Nobel Prize for the work in bacterial genetics, but much of the work had been carried out in collaboration with Esther. Was this an instance of sexual discrimination of the Nobel Prize Committee?

In 1959, Dr. J. Lederberg accepted the chairmanship of the Department of Genetics at Stanford University Medical school, while Esther was a research geneticist in the department. They worked closely on bacterial genetics problems. However, their

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marriage was at risk and they were divorced in 1966. After that Esther became a research associate in the Department of Medical Microbiology, where she also held an American Cancer Society Senior Dernham Fellowship. She was promoted to senior scientist in 1971 and research professor in 1974. In 1976, she was appointed as director of the Plasmid Reference Center where she supervised the collection and cataloging of bacterial plasmids, which were the most important tools in molecular biol-ogy and recombinant DNA technology. She also did a lot research on bacterial transformation, recombination, and DNA repair mechanisms. She held that position until 1986. A year before, Esther retired from active administrative duty and remained as an Emeritus Professor in Microbiology and Immunology at Stanford University.

Esther was a fellow of the American Association for the Advancement of Science and a number of other scientific organizations. She forfeited under the shadow of Dr. Joshua Lederberg. Her contributions were equally as significant as Dr. Joshua Lederberg, but unfortunately were largely ignored because of her gender.

Suggested Reading

1. Lederberg, E. M. (1948). The mutability of several lac-mutants of Escherichia coli. Genetics 33: 617.

2. Lederberg, E. M. (1951). Lysogenicity in Escherichia coli K-12. Genetics 36: 560.

3. Lederberg, E. M., J. Lederberg, N. D. Zinder and E. R. Lively (1951). Recombination analysis of bacteria heredity. Cold Spring Harbor Symposia on quantitative Biology 16: 413–443.

4. Lederberg, E. M. (1952). Allelic relationships and reverse mutation in Escherichia coli. Genetics 37: 469–483.

5. Lederberg, E. M and J. Lederberg (1952). Replica plating and indirect selection of bacterial mutants. Journal of Bacteriology 63: 399–406.

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6. Lederberg, E. M., L. L. Cavalli and J. Lederberg (1952). Sex compatibility in Escherichia coli. Genetics 37: 720–730.

7. Lederberg, E. M. and J. Lederberg (1953). Genetic studies of lysogenicity in Escherichia coli. Genetics 38: 51–64.

8. Lederberg, E. M., M. L. Morse and J. Lederberg (1956). Transduction in Escherichia coli K-12. Genetics 41: 142–156.

9. Lederberg, E. M. and S. N. Cohen (1974). Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. Journal of Bacteriology 119: 1072–1074.

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Chapter 50

Marvin P. Bryant (1925–2000): Bacteria in Methanogenic Ecosystems

Marvin P. Bryant is one of the most outstanding rumen microbi-ologists who extended the study of the rumen ecosystem initi-ated by Robert E. Hungate (1906–2004) in the United States. Dr. Bryant gives the most detailed and accurate descriptions and analyses of the types and kinds of bacteria in the rumen, and discloses many important biochemical pathways and metabolic interactions which affect the development of other branches of biology such as biochemistry, animal nutrition, ecology, and genetics and phylogenetics. He has made a very significant con-tribution. He is an example of the self-learning and hardwork-ing contemporary American Scientist, who help to greatly expand our knowledge of microbiology.

Marvin was born on 4 July 1925 on the edge of the foothills in Boise, Idaho. He spent his childhood in this countryside often with summer and fall excursions to the family ranch on the edge of the middlefork of the Salmon River, where he developed a strong love for his country. Marvin attended Boise Junior College for one quarter before he joined the Army Air Corps (January 1944–November 1945). Immediately after his discharge from the Air Corps in 1945, he returned to Boise Junior College to con-tinue his education. He studied Forestry and Soil Science and obtained his diploma in June 1947. In Boise, he worked as a common laborer for the summers or part-time engineering aid because he needed the money to support the family. His favorite

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professor, Dr. Donald Obee advised him to move into bacteriol-ogy. He traveled to Washington State University at Pullman, where he obtained his bachelor’s degree with honors in 1949 and a master’s degree in 1950.

At Washington State University, Marvin first met Robert E. Hungate, the pioneer of rumen microbiology. He worked as a research microbiology technician to help Hungate and his PhD student, R. H. McBee. Due to poor scientific support, Hungate, McBee, and Marvin had to make all of their glass apparatus by hand. For example, they were able to “manufacture” a complete Warburg apparatus, micromodification of the Newcommer-Haldane constant pressure, volumetric gas analyzer, all condens-ers, and units for the determination of lactic and volatile fatty acids, which was very important for their work. These experi-ences helped Marvin to develop a strong interest in anaerobic microbiology, and taught him how to gain new knowledge through experiments.

Determination of fermentation end products and fermenta-tion balances of various anaerobic bacterial species including the cellulolytic Clostridium thermocellum, Ruminococcus albus, and Butyrivibrio fibrisolvens was some of Marvin’s initial work. Marvin was the first to isolate a small spirochaete of the genus Treponema from the rumen. This included the first determina-tion of fermentation products, for example, succinate, of an anaerobic spirochaete and the demonstration of its metabolic interaction with cellulolytic bacteria in using sugar products of cellulose.

While Marvin wanted to continue with a PhD degree at Washington State University, Hungate went on a sabbatical leave to Cornell University. Hungate arranged to continue Marvin’s assistantship through Washington State University for a period of eight months at Cornell University. In this capacity, Marvin took excellent courses from distinguished professors at Cornell University. These include Professors Jack Loosli, Gene Delwich, and Knaysi. He also associated with Professor Dukes and James Sherman, and undergraduate student Meyer J. Wolin.

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Drs. Bryant and Hungate did the first detailed microbial study on acute indigestion (lactic acidosis) by implicating Streptococcus bovis in sheep at Professor Robert Dougherty’s laboratory and anaerobic lactobacilli, while at Cornell University.

Although Marvin wanted to complete his PhD degree at Washington State University, a rumen research bacteriology position became available at the Bureau of Dairy Industry at Beltsville Agricultural Research Center. Marvin was the only qualified person at that time. Hungate strongly persuaded Marvin to take the position, and so he did. This was in February 1951.

While at Beltsville as a full-time research bacteriologist, Marvin registered as a part-time graduate student under Professor Raymond Doetch at the University of Maryland, and completed his PhD degree in 1955.

At Beltsville, Marvin was chiefly responsible for the advance-ment of the basic knowledge of rumen bacteria. Initially, he was devoted to systematics with emphasis on ecologically important metabolic features of bacteria and identifying their unusual growth factors in rumen fluid. The isolation and identification of rumen bacteria became possible because of the development of Hungate’s anaerobic roll tube technique and habitat-simulating media. Bryant modified the anaerobic roll tube method of Hungate and isolated numerous important rumen bacteria and studied them in detail. These include Fibrobacter succino-genes, Prevotella ruminicola, Ruminococcus albus, R. flavefa-ciens, Eubacterium cellulosolvens, E. ruminantium, Selenomonas ruminantium, Lachnospira multipara, Succinivibrio dextrinosol-vens, and Succinimonas amylolytica. The important microflora of young calves which are different from the mature rumen, include Fusobacterium necrophorum, Clostridium clostridio-forme, Lactobacillus vitulinus, Megasphaera elsdenii, Eubacterium limosum, and so on. He illustrated their physiological functions, biochemical properties, and nutritional requirements, which are not only important for the understanding of the animal nutri-tion and metabolism, but also important for the study of other

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anaerobic microbial habitats such as human gastrointestinal tracts which are important for human health. Dr. Bryant also generously provided the anaerobic cultures to other investiga-tors around the world for more than 30 years, before the American Type Culture Collection began to handle anaerobic bacteria which are difficult to grow. His studies with students demonstrated straight- and branched-chained fatty acids and heme requirements of rumen bacteria and discovery of plasm-alogens in bacteria, the reductive carboxylation of fatty acids to amino acids and the presence of cytochromes in anaerobic fermentative bacteria.

In Beltsville, Dr. Bryant worked closely with his professional colleagues, Dr. Milton Allison, his first PhD student, and Prof. Mark Keeney and Ira Katz of the University of Maryland, Dr. Howard Bladen, Dr. Daniel Caldwell, and his assistant Mr. Isadore M. (Ike) Robinson, as well as Dr. David C. White at Rockefeller Institute in New York (Ike Robinson later became the world expert on anaerobic mycoplasmas). He described many of the rumen bacterial species and discovered many important parameters involved in cellulose degradation, lipid metabolism, ammonia nutrition, amino acid, and peptide assimilation, which are essential to rumen fermentation.

In 1963, Dr. Bryant’s career began to change. He was invited to deliver a paper at the Third Rudolf’s Conference at Rutgers University. Dr. Bryant met Dr. Ralph Wolfe (1921–) of the University of Illinois, Dr. Perry McCarty of Stanford University, and other influential scientists. The University of Illinois began a campaign to recruit Dr. Bryant. Because of the advantages of being more closely associated with basic microbiological research at the University of Illinois, and his ability to grow many anaerobic bacteria, needed by new colleagues, Wolfe and Wolin, Dr. Bryant accepted the offer and moved to Illinois in 1964 as an Associate Professor in the Department of Dairy Science. Two years later, he was promoted to Professor and became a faculty member of the combined Department of Animal Sciences and the Department of Microbiology.

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At the University of Illinois, Dr. Bryant continued his work on rumen microbiology. Along with Dr. Meyer J. (Mike) Wolin, probably one of Dr. Bryant’s greatest scientific contributions to microbiology, was that he illustrated the “interspecies hydro-gen transfer” mechanism. This mechanism was significantly influenced the experiments being conducted at the time and when the dust settled, the knowledge gained from these experiments formed a new chapter in microbiology textbooks.

Dr. Bryant’s interest in nutrition of methanogens started in Beltsville and continued at the University of Illinois. He initially worked on the nutritional requirements of the rumen methano-gen, Methanobrevibacter ruminantium. In collaboration with other scientists and graduate students, he discovered coenzyme M and some functions of cofactor F420, which are crucial for the study of the biochemistry of methanogenesis, research project in collaboration with Dr. Ralph Wolfe’s laboratory.

The discovery of “interspecies hydrogen transfer” mechanism stemmed from the suspicion of the impurity of Methanobacillus omelianskii which was isolated from San Francisco bay mud in the 1930s by H. A. Barker. Dr. Bryant and his colleagues discovered that there was an “S” organism which converted ethanol to ace-tate and hydrogen, and a methanogen which converted hydro-gen and CO2 to methane. Together they converted ethanol to acetate and methane. The “S” organism can only utilize a small amount of ethanol while producing acetate and hydrogen because the accumulation of a very small amount of hydrogen would stop the ethanol-utilizing reaction needed for growth. This was the first demonstration of a syntrophic metabolic association of two microbial species. “Interspecies hydrogen transfer” was a term first used by Dr. Mike Wolin, who was once Marvin’s closest colleague at the University of Illinois. This phenomenon is now known to be of great importance in anaerobic digestion in the rumen. It is considered even more important in many methano-genic ecosystems where more complete anaerobic degradation occurs. Now methanogens have been discovered in many habitats and their ecological significance illustrated. This also led to the

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discovery of the phylogenetics of Archaebacteria — a milestone of microbial systematics — by Drs. Carl Woese (1928–2012) and Ralph Wolfe in the Microbiology Department of the University of Illinois.

Many examples of interspecies hydrogen transfer were illus-trated which shed a great deal of light on rumen fermentation. Dr. Bryant and his students also studied fatty acid and benzoate degradation. This involves hydrogen transfer between fatty acid and benzoate degraders and sulfate-reducing bacteria, which have greater affinity for hydrogen than methanogens under the slow growth rate necessary for their degradation. The new fatty acid and benzoate-degrading bacteria were isolated in co-culture with the H2 users and characterized. Biological potential for methanogenesis of cattle wastes was also thoroughly investigated. Methane is a biofuel. Much research work on the generation of biofuel from animal wastes were conducted in the last two decades, and much of this knowledge was derived from the understanding of interspecies hydrogen transfer in which methanogenesis plays an important role. This aspect of work will continue to be important in the future when biomass energy may eventually be an important energy source.

Many other aspects of rumen microbiology were investi-gated by Dr. Bryant and his colleagues during his tenure at the University of Illinois. Growth yields of rumen bacteria, improve-ment of rumen microbial protein synthesis available to the ruminant animals, regulation of ruminal microbial ammonia assimilation, demonstration of pathways of anaerobic metabo-lism of gallic acid, pyrogallol, and phloroglucinol via the previ-ous unknown 1,2,3,5-tetrahydroxybenzene and the first acyclic intermediate, 3-hydroxy-5-oxohexanoate; study of acetogens that used CO, H2-CO2, or methanol to produce acetate, and urea hydrolysis are just a few examples.

Over the years, Dr. Bryant has directed many graduate students. Students who obtained their doctoral degree from Dr. Bryant include Milton J. Allison, Howard Bladen, Kenneth Pittman, C. A. Reddy, Sin-Fu Tzeng, Dennis Short, Ronald

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Isaacson, Daniel Schaefer, C. J. Smith, M.J. McInerney, Barbara Genthner, Vincent Varel, Lee Krumholz, Joel Dore, and Hongxue Zhao. Those who obtained master’s degree from Dr. Bryant are Daniel R. Caldwell, C. A. Reddy, Andrew John, Mary Ann Wozny, Robert Leedle, Sin-Fu Tzeng, M. J. McInerney, Janice Herbeck, William Brulla, Holly Whetstone, Rogelio Gomes-Alarcon, Vincent Varel, Bill Lorowitz, and Lee Krumholz. Those who worked with Dr. Bryant at one time or another included Dr. J. M. Gawthome, Dr. Roderick Mackie, Dr. Won Maeng, Dr. M. P. Sharma, Dr. Douglas Mountfort, Dr. Olga Nal- bandov, Dr. Alan E. Joyner, Jr., Dr. Arthur Wellinger, Dr. James Wohlt, Dr. David Boone, Dr. Russell Frobish, Dr. Leonard Slyter, Dr. Sudhakar Barik, Dr. Hongxue Zhao, Ms. Karen Robins, Ms. Ann Denholm, Dr. Joe Salanitro, Dr. Robert Gardner, and Mr. William Brulla. Drs. Robert Hespell and Bryant White are also close colleagues.

Dr. Bryant received many honors during his professional career, most notably, he was elected as a member of the National Academy of Sciences in 1987. That is one of the highest honors for scientists in the United States. He is a member of Phi Beta Kappa and Phi Kappa Phi (1949) and was also awarded the Dairy Science Borden Award and the Fisher Award from the American Society of Microbiology. He also served on Bergey’s Trust as the vice chairman and as the associate editor of the famous Bergey’s Manual of Systematic Bacteriology, Vol. 3. He was active in many professional societies such as American Dairy Science Association; the American Society for Microbiology, in which he served many functions including being a member of the Editorial Board, Publication Board, Council, founding Editor-in-chief of Applied Environmental Microbiology, and various other functions; Society for General Microbiology; American Association of Advancement Science; Society for Intestinal Microbial Ecology, and Disease; and so on. He also served in many professional organizations as a speaker, program organizer, reviewer, con-sultant, board of trustees, and so on, and advised professional activities in many countries. His unselfish service to science and professional societies are indeed impressive.

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Dr. Bryant is a great man. At one occasion, he commented that his scientific success was due to his innate competence in growing anaerobic bacteria and in having been at the right places at the right time to be associated with many outstand-ing, creative, and unselfish colleagues. But most important of all is that he is honest, sincere, and compassionate which attracted those who happily associated with him. Many people who have associated with Marvin once, would immediately choose to be his friend for life. He also has great respect and love for his mentor, Dr. Robert E. Hungate, and his spouse, Alice.

Marvin was happily married to his teenage love Margaret A. Bryant in 1945. They have five children, Dr. Peggy Bryant, Mrs. Susan Bryant (Kruidenier), Mrs. Katherine Smith, Dr. Robert Bryant, and Mr. Steven Bryant who are a testimony to their wonderful marriage. Their children are now spread around the country, from Illinois and Minnesota to California and Washington State with success stories of their own.

Marvin worked day and night in the laboratory and traveled abroad frequently. Encephalitis of unknown etiology seriously affected Marvin’s health in 1979. However, he was still active in science. In 1988, Dr. Bryant took disability from full faculty duty at the University of Illinois. This would allow him to rest more and have more time to retreat to Idaho for some of his first hobbies, high mountain buckarooing, and wild animal and bird watching. I have had the honor to visit him several times in his laboratory. Through his work, the knowledge of microbiology has progressed by at least one more chapter.

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Chapter 51

Joshua Lederberg (1925–2008): Pioneer of Microbial Genetics

“Life is a hobby. Science has an intrinsic excitement to it that is much more important than the competition for wealth and glory that we hear too much about”.

“Try hard to find out what you are good at, and what your passions are, and where the two converge, and build your life around that. Make deliberate choices, do not just wait for things to happen”.

—Joshua Lederberg

Source: https://en.wikipedia.org/wiki/Joshua_Lederberg(US Public Domain image)

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Joshua Lederberg was awarded the 1958 Nobel Prize in Physiology or medicine for his discovery of “sexual recombina-tion” in bacteria (bacterial conjugation). This award was shared with Drs. George Wells Beadle (1903–1989) and Edward Lawrie Tatum (1909–1975). Conjugation is mediated by the presence of the P factor (fertility factor or plasmid) and requires cell-to-cell contact. The plasmid can reside inside the donor bacterium as an extrachromosomal element with its own origin of replication or it can be integrated into the bacterial chromo-some. When integrated in the chromosome, replicative transfer begins within the F plasmid region at the oriT region and con-tinues into the chromosomal region. Thus, the chromosomal genes as well as F plasmid genes are transferred to the recipient cell during conjugation. Via a process of recombination (Hfr — high frequency recombination), the newly acquired deoxyribonucleic acid (DNA) is incorporated into the recipient’s chromosome and the recipient cell in turn becomes a donor cell, provided an intact F plasmid is present. When the plasmid DNA is maintained as an extrachromosomal element, the F plas-mid is transferred at a high frequency. Dr. Lederberg made the distinction between Hfr and F+ and coined the term plasmid in 1950 to describe extrachromosomal genetic elements. However, the term was not widely accepted until the 1970s when bacterial drug resistance was shown to be mediated by self-transmissible plasmids and became a major medical problem.

Joshua Lederberg was born on 23 May 1925 in Montclair, New Jersey. His father was called Zwi H. Lederberg, an orthodox Jewish rabbi, and his mother Esther Goldenbaum, a housewife; both of them immigrated from Israel in 1924. They moved to New York City when Joshua was only 6 months old and they settled in the Washington Heights neighborhood. Joshua spoke proudly of his parents; he considered his father an idealistic per-son and his mother “a heroic soul” who had to work extremely hard to help keep the family together during his father’s pro-longed illness. Joshua has two brothers. One of them, Seymour, was a professor of biology at Brown University. The other

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brother, 16 years younger, lived in Jerusalem and was with the Lubavitcher rabbi’s group of the Hasidim.

The young Joshua was a “precocious youngster” who devel-oped a keen interest in science as early as 6 or 7 years of age. This interest was inspired as a result of “sublimation of his father’s and family traditions, religious impulses, but within a secular framework” as described by Dr. Lederberg at a later day. Young Joshua was an extremely intelligent, active, inquisitive, and optimistic person with a strong desire to learn. Later, he developed an analytical mind that gave him the power to look at subject matter with an unusual insight and an amazing capa-bility for innovative thought. This could be seen during his early school days when he constantly confronted his teachers with questions that they could not answer. This was so annoying to some of his teachers that they made a deal with him: “if you (Joshua) do not ask too many disruptive questions, you can sit and do your own work in the back of the classroom”. He began to read Bodansky’s Introduction to Physiological Chemistry at the age of 11, which impacted his scientific development greatly.

He attended Stuyvesant High School (a high school famous for budding Nobelists) and graduated with top grades in 1941. Young Joshua was an avid reader of science books during his high school years. Among the books he read were Eddington and Jeans’ Physics, Jaffe’s Crucibles in Chemistry, and Wells, Huxley & Wells encyclopedia The Science of Life. The popular culture in those days that idealized medical scientists with novels and movies like Arrow-Smith, The Magic Bullet, The Life of Louis Pasteur, and The Symphony of Six Million also inspired this young man’s desire for a career in medical research.

Joshua then enrolled at the nearby Columbia College as a student of the premedical curriculum, the main reason for enrollment being that he had obtained a scholarship. He worked as a laboratory assistant to Professor Francis J. Ryan (1916–1963) of the Zoology Department and helped to conduct experiments on the mutation and adaptation of the bread mold, Neurospora.

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Professor Ryan had a great influence on Lederberg and went far beyond his duty to encourage Joshua. Ryan became his lifelong mentor. Joshua obtained his bachelor’s degree with honors in 1944. He continued his education at the College of Physicians and Surgeons at Columbia University while working for Professor Ryan. In 1944, he read papers published by Oswald T. Avery (1877–1955). Colin Munro MacLeod (1909–1972) and Maclyn McCarty (1911–2005) learned that DNA was the genetic material. These papers inspired Joshua greatly. In 1946, with Professor Ryan’s encouragement, he was able to work for 3 months with Dr. Edward L. Tatum at Yale University. His research with Dr. Tatum was so interesting that he never went back to Columbia. Instead, he continued his graduate studies with Dr. Tatum.

At Yale University, Joshua Lederberg and Dr. Tatum studied the reproduction of Escherichia coli and discovered that certain strains of E. coli possessed the mechanism for transfer of genetic material with subsequent recombination in the recipient host. Lederberg showed that genetic material was exchanged by con-jugation, a process by which cell-to-cell contact is required and which may result in the transfer of an entire complement of the bacterial chromosome.

In 1947, Joshua received his PhD degree in Microbiology from Yale University and accepted an appointment as an Assistant Professor of Genetics at the University of Wisconsin. In 1950, he was promoted to Associate Professor and in 1954, to the rank of full professor at the age of 29. In 1957, Joshua organized the department of medical genetics and served as its first chairman. In the same year, Dr. Lederberg was a Fulbright Visiting Professor of Bacteriology in the laboratory of the emi-nent immunologist Sir Frank MacFarlane Burnet (1899–1985), at Melbourne University in Australia.

Upon his return to the University of Wisconsin, Dr. Lederberg was involved in research projects involving bacterial genetics. He had two great associates; one was his wife, Esther Marilyn Lederberg (1922–2006). They had met and married in 1946

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when both of them served as assistants to Dr. Tatum. Esther had obtained a bachelor’s degree from Hunter College, a master’s degree from Stanford University, and her PhD from the University of Wisconsin where she had also received a United States Public Health Service Fellowship for research. In 1956, Joshua and Esther Lederberg were named joint recipients of the Pasteur Award of the Society of Illinois Bacteriologists.

Esther Lederberg helped in the development of the tech-nique of replica plating in 1952. This technique was essential in the study of bacterial genetics, more specifically in the selection of mutants from among hundreds and hundreds of bacterial colonies on a plate. Joshua and Esther devised sterile velveteen pads, which facilitate picking up a few bacteria from each colony. In their original replica plating studies, organisms from a liquid culture were evenly spread on an agar plate and allowed to grow for 4–5 h. Then sterile velveteen pads were gently pressed against the surface of the plate to pick up organ-isms from each colony. The tiny fibers of velveteen act like hun-dreds of tiny inoculating needles. The pad was carefully kept in the same orientation and used to inoculate a series of agar plates containing different media supplemented with essential nutrients, such as amino acids and vitamins. Esther and Joshua used this technique as an indirect selective method to prove the spontaneous origin of mutants with adaptive advantages. This is a significant contribution to the progress of microbial genetics. The technique of replica plating is still widely used in genetic laboratories today.

In 1953, Esther provided another contribution to the Lederberg laboratory; it was her discovery of lambda phage (λ phage) in E. coli, and her initial observation of the F factor, at a time during which Joshua and Esther exemplified the importance of team work.

Joshua Lederberg’s other great assistant at the University of Wisconsin was Norton D. Zinder (1928–2012), then a doctoral candidate. Dr. Zinder, later at the Rockefeller University, also became a great bacterial geneticist. Between 1951 and 1952,

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Joshua Lederberg and Zinder together discovered that bacterial viruses could carry hereditary material from one cell to another by transduction. This is accomplished as follows. After a virus has adsorbed to a host cell and caused lysis of a bacterium with the release of some 100 copies of phage particles, occasionally a bacterial gene (s) is packaged into the head of the viral parti-cle. When this “defective” phage particle starts the infection cycle over again, the bacterial gene is transferred into the new host bacterium with subsequent incorporation into the chromosome via recombination.

In 1959, the Lederbergs moved to Stanford University where Joshua assumed the chairmanship of a newly created depart-ment of genetics in the medical school. Unfortunately, Joshua and Esther’s marriage did not last. They were separated in 1966.

Joshua remarried in 1968 to Marguerite Stein Kirsh, who was an attending psychiatrist at the hospital. She was born in Paris and was hidden as a child during World War II in Southern France. She came to the United States after the war. Dr. Lederberg has one daughter born in 1974. Marguerite also has a son David Kirsch who was born in 1964 from her first marriage.

Dr. Lederberg enjoyed working in the environment of the Stanford medical school because he could relate genetics to a wider context of human health and biology, particularly neuro-biology and mental illness. He oversaw a large and diverse research group and helped institute a human biology curricu-lum for undergraduates. He was also interested in computer sciences. He formed a collaboration with Dr. Edward Feigenbaum, chairman of computer science at Stanford, Professor Bruce Buchnan (1936–), computer scientist, and Dr. Carl Djerassi (1923–2015), professor of chemistry. Together, they created DENDRAL, a computer program to generate structures of organic molecules and to explore how molecules exist in nature. In 1974, with support from the National Institutes of Health, they established Stanford University Medical Experimental Computer (SUMEX) to provide hardware for research projects all over the country.

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Additionally, he was interested in extracurricular activities. He served as a panel member of the President’s Science Advisory Committee. He served on President John F. Kennedy’s (1917–1963) Panel on Mental Retardation and directed research on the genet-ics, development, and neurobiology of retardation at the Kennedy Laboratories for Molecular Genetics at Stanford University.

Since 1957, Dr. Lederberg has also been concerned with the contamination of the moon by lunar rockets. He and a biophysicist Dr. Dean B. Cowie (1913–1977), from the Carnegie Institute of Washington, were seriously concerned about the use of earth satellites for moon missions. They were particularly concerned that non-sterile rockets reaching the moon could destroy or distort the picture of potential biochemical evolution of life on the moon. As a consequence, Dr. Lederberg served on the National Academy of Science’s committee for the space biol-ogy program from 1958 to 1977, and on the National Aeronautics and Space Administration’s (NASA) lunar and planetary mission’s boards involved with the Mariner and Viking missions from 1960 to 1971.

Dr. Lederberg was a great advocate for having a public well informed about science issues. In 1966, he initiated and wrote a weekly column for The Washington Post over a period of six years. He commented on everything from gene cloning to manipulating weather, science ethics, education, the envi-ronment, the history of medicine, and the state of science reporting itself.

In 1978, Lederberg was appointed President of The Rockefeller University, New York City. This brought him back to the place where he grew up. During his administration, he built many new laboratories and renovated old ones. Research projects conducted at most of these laboratories concentrate on biomedical investigations and lean heavily on the insight and methods of molecular biology. He also established the Howard Hughes Medical Institute. He added new university apartment buildings and residences for scholars from around the world.

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The university’s thrust was expanded to include research in such areas as heart disease, cancer, mental and neurological illnesses, and infectious diseases, including diseases of the Third World.

In 1989, Dr. Lederberg retired from administration, but he still keeps an active research program and remains involved in public affairs. His current research interest is to find out how mutagenesis varies with the conformational changes that DNA undergoes during the cell cycle or under conditions of transcription. As a member of the National Academy of Science Committee on International Security and Arms Control, he maintains his participation in public activities. He is deeply con-cerned about the threats to humanity from naturally emerging viruses. In addition, as a cochairman of the Carnegie Corporation’s blue-ribbon commission, he advises federal and state govern-ments on issues of science and technology. He is also an adjunct professor at his alma mater, Columbia University, and serves as a mentor to undergraduate students.

Dr. Lederberg has been the recipient of many awards, most notably, the Eli Lilly award in 1953 for being an outstanding young bacteriologist at the annual meeting of the Society of American Bacteriologists (now called American Society for Microbiology) in San Francisco; the Nobel Prize in Physiology or Medicine in 1958; and in 1989, he received the National Medal of Science, the nation’s highest scientific award.

As a multitalented man of wisdom, Dr. Lederberg is consid-ered the father of microbial genetics. His insight, compassion, motivation, integrity, and scores of other wonderful qualities make him a shining star and role model for many scientists.

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Chapter 52

Hubert A. Lechevalier (1926–2015): Antibiotics Hunter and Actinomycetologist

The discovery of the antibiotic penicillin in 1928 by Alexander Fleming (1881–1955) did not attract much attention until 1940, when Howard Florey (1898–1968), Ernst Chain (1906–1979), and associates published a paper in Lancet on the therapeutic effect of this compound. At that time, René Jules Dubos (1901–1982) and collaborators demonstrated that bacilli produced bacteri-cidal substances. Following these leads, many microbiologists became interested in hunting for antibiotics. Of these, a group led by the soil microbiologist Selman A. Waksman (1888–1973) at the College of Agriculture of Rutgers University, the State University of New Jersey, was probably the best known. Hubert A. Lechevalier was a member of that group and discovered neo-mycin (1949) and candicidin (1953), thus contributing to the warfare that mankind wages against infection. A few more antibiotics were later isolated in his laboratory but did not find practical application. Later, Dr. H. A. Lechevalier, with his wife Mary, investigated, mainly the taxonomy of mold-like bacteria, the actinomycetes. They made a useful contribution by develop-ing a system of classification of actinomycetes based on mor-phology and chemical composition (1965). Also, one of their associates, Dr. Nancy N. Gerber (1929–1985), extended the range of their studies to the chemistry of pigments and volatile sub-stances produced by actinomycetes. In addition, Lechevalier developed an interest in the history of microbiology and the

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book that he wrote in collaboration with Morris Solotorovsky (1913–1992); Three Centuries of Microbiology is one of the best on the subject.

Hubert Arthur Lechevalier was born on 12 May 1926 in Tours, France. His father was Jean Gaston Lechevalier, the son of a building contractor, who after surviving the first World War (1914–1918) as an officer in the heavy artillery, attended the letters section the prestigious École Normale Supérieure, where Louis Pasteur (1822–1895) had previously been Director of Scientific Studies. His mother was Marie Emilie Delorme, the daughter of the controller of an insurance company. Both parents were born in Paris, but the Lechevaliers came from Normandy and the Delormes from Burgundy. When Jean Lechevalier was in high school, his best friend was Pierre Allorge. Both boys shared a passion for dead languages and botany, and explored together the flora of the surroundings of Paris, which were so dear to the impressionist painters. Pierre Allorge (1891–1944) became an algologist and eventually the director of the Museum of Natural History in Paris, whereas Jean Lechevalier became a professor of Latin.

In 1932, when Hubert was only 6 years old, the family moved to Canada as Jean Lechevalier was appointed to the Chair of Latin at Laval University in Quebec City. Under the prodding of his father, Hubert learned to recognize common plants around Quebec. Some of the plants that he and his sister Genevieve collected are still in the herbarium of Laval University as they are the first reports of their occurrence in the Quebec region. Later, Hubert’s interest in natural history, under the guidance of an entomologist, Brother Joseph Quellet (1869–1952), broadened to include insects, mainly coleoptera, and, under the influence of one of Laval’s professors, Dr. Franco Rasetti (1901–2001), to dabble with fossils of invertebrates, mainly trilobites and graptolites.

Professor Jean Lechevalier was an educator who decided to homeschool his children. However, his methods were unorthodox and by the time Hubert was 9 years old, results were not

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impressive. Professor Lechevalier, at the urging of his wife, decided to send Hubert to a private school and eventually to the Jesuit High School of Quebec City. In 1944, Hubert entered the Faculty of Sciences of Laval University to study botany, zoology, and geology. He received a Licenses Sciences Naturelles (summa cum laude) in 1947.

While an undergraduate at Laval, Hubert spent his spare time in the laboratory of Dr. Rene Pomerleau (1904–1993), a mycologist who was the director of the section of forest pathology of the Ministry of Land and Forest of the Province of Quebec. Dr. Pomerleau also taught mycology and plant pathol-ogy at Laval University. Dr. Pomerleau had studied in France where he had been a student of Pierre Dangeard (1895–1970), the discoverer of sexuality in higher fungi. Dr. Pomerleau was then building a collection of mushrooms in view of preparing a monumental flora of the mushrooms of Quebec, which was eventually published in 1980. During the academic year, Hubert would learn some mycology by helping Dr. Pomerleau organize his collections; during the summers, he had a paid job in Dr. Pomerleau’s laboratory. His most important phytopathological function was in the diagnosis of Dutch elm disease.

Athletic college students, under the supervision of forest inspectors, would travel throughout the part of the Province of Quebec where elms were growing and would climb elm trees with wilted branches to collect samples that were sent to the laboratory in Quebec City. There, more college students would prepare culture plates that were inoculated with chips of wood aseptically collected from the samples of branches which had been received. Hubert’s contribution to this effort at the eradication of the disease from the Province of Quebec was to examine the plates and determine if the pathogen, then called Ceratostomella ulmi, was present. He noted that, in some plates, there were bacteria growing that inhibited the growth of C. ulmi. Dr. Pomerleau suggested that Hubert should isolate these bacteria, identify them, make crude extracts of the active principles, and inject them in elms. Hubert received a bursary

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from the Research Council of Canada and carried out this study, which was a pioneering effort at chemotherapy of trees for which he received a MS degree from the Laval University in 1948.

The main conclusion that Hubert drew from his master’s study was that he should learn more about antibiotics and, as he wanted to continue his graduate studies in that field, Dr. Selman A. Waksman’s laboratory was a logical choice since this was where actinomycin, streptothricin, clavacin, fumigacin, streptomycin, chetomin, micromonosporin, and grisein had been isolated. Dr. Waksman accepted Hubert as graduate student and offered him a fellowship.

In 1948, Dr. Waksman was the head of the Department of Soil Microbiology of the College of Agriculture of Rutgers University. Dr. Waksman’s range of knowledge was impressive but from a taxonomic point of view, his favored microorgan-isms were the actinomycetes. The staff of his department was small. There were another outstanding soil microbiologist, Dr. Robert Lyman Starkey (1899–1991), a chemist, Dr. E. Augustus Swart, five technicians, two secretaries and an assort-ment of graduate students, and postdoctoral fellows. Part of the laboratory training was done by having the more advanced graduate students help the new arrivals.

When Hubert arrived, eager to look for new antibiotics, Dr. Waksman naturally suggested that actinomycetes should be screened for antibiotic activities and, since he was departing for an extended trip, he assigned to Dorris Hutchison, a student who was finishing her PhD thesis, the task of teaching Hubert how to recognize and isolate actinomycetes. Having mastered this task, Hubert proceeded with the isolation of about 200 actinomycetes that he subjected to two screening programs; one, directed by Dr. Waksman against mycobacteria which yielded a strain of Streptomyces fradiae producing neomycin and the other, directed by a mycologist, Dr. Conrad M. Haenseler, against fungi that resulted in the selection of a strain of Streptomyces griseus producing candicidin. Both neomycin

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and candicidin proved to be useful antibiotics and are still used today.

In 1949, it was Hubert’s turn to help a new student get started in Dr. Waksman’s laboratory. The new student was Mary-Jean (Midge) Pfeil who had just graduated from Mt. Holyoke College where she had received a special award for being both a good student and a good athlete. One year later, Midge became Mrs. Lechevalier. Another year later, as Midge received an MS degree and Hubert a PhD degree, a son Marc was born (1951). The family was completed with the birth of another son, Paul, in 1953.

Midge was not only a sweet wife, but also an outstanding coworker since she was a professional biologist-biochemist. For a few years, Midge searched for novel actinomycetes at home and isolated a few new actinomycetes with unique morpholo-gies. Midge went to work in the industry, but a few years later, after a stay in Paris at the Institute Pasteur, she decided to stay in Hubert’s laboratory and became an expert on the chemical taxonomy of actinomycetes.

In 1951, Hubert was employed as an assistant professor of microbiology first at the College of Agriculture of Rutgers University, then at the Waksman Institute of Microbiology of the same university. For a while, he continued his antibiotic hunting. A heptaenic antibiotic candicidin was isolated in 1953 and proved to be very effective against fungal infections caused by Candida albicans. Many aspects of microbiology, such as antifungal properties of the actinomycetes, nutritional require-ments for the production of antibiotics, biochemistry, and physiology of the actinomycetes were studied in Dr. Lechevalier’s laboratory. He and his associates also developed methods for the detection of specific types of antibiotics such as those active against intracellular bacteria. He also did some work on cross resistance between antibiotic and antiseptics (Argyrol). He was promoted to an associate professor in 1956.

Hubert and his wife Midge were both interested in taxon-omy and classification of actinomycetes. Jointly, they described a

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new genus of actinomycetales, Waksmania in 1957. In 1961, he and his colleagues described another new genus Micropolyspora. Another new genus of actinomycetales, Microellobospira, was also described by Hubert, his wife, and Tom Cross in 1963.

With the increase in scientific contributions, Dr. Lechevalier became well known. He was selected as an exchange scientist with the Academy of Sciences of the USSR, and spent a winter in Moscow (1958–1959). He and his wife also spent a summer as visiting investigators at the Czechoslovak Academy of Sciences, Prague in 1961. The same year, he obtained a special fellowship from the US Public Health Service and spent a sabbatical year in the mycology laboratories of the Pasteur Institute in Paris. Midge was also engaged an active study on rare actinomycetes at the Pasteur Institute.

Upon returning from Paris (1962), Hubert’s laboratory was busier than ever before. He and his collaborators studied numerous subjects, for example, the antitumor activity of sulfur- containing antibiotics and 3′-amino-3′-deoxyadenosine produced by a strain of Helminthosporium, electron microscopy of spores and other structures of actinomycetes, classification and taxon-omy of actinomycetes, secondary metabolites produced by actinomycetes, nematode-trapping fungi, and so on. He was promoted to full professor in 1966. He remained at the Rutgers University until his retirement in 1991. Between 1980 and 1988, he was also the associate director of the Waksman Institute of Microbiology.

The Lechevaliers’ laboratory became quite popular with investigators in the field of actinomycetes and by the time the laboratory was closed in 1991, Hubert noted that postdoctoral fellows from 25 different countries had spent time there. Midge and Dr. Nancy Gerber (1929–1985) became expert at understanding foreign accents.

Over a period of 40 years, Hubert also taught a number of courses at Rutgers including “Antibiotics”, “Actinomycetes”, “History of Microbiology”, “Comparative Morphology and Physiology of Microorganisms”, “Practical Microscopy”, and

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“General Microbiology”. He emphasized microbial diversity when he taught general microbiology. All these courses are at the graduate level. From time to time, the Lechevaliers gave workshops on the taxonomy of actinomycetes.

The research from Dr. Lechevalier’s laboratory covers a wide spectrum of microbiological problems usually related to actino-mycetes. New compounds were isolated and, thanks to Nancy Gerber, their structures were determined. Those include cell sugars, phenazines, polyacetylenic compounds (peniophoryns), and odorous metabolites (geosmin, methylisoborneol, 2-isopropyl- 3-methoxypyrazine).

He and his wife with the help of associates and graduate students, reported many new genera of actinomycetes including Waksmania, Actinosynnema, Micropolyspora, Sporichthya, Microcellobosporia, Actinomadura, Saccharothrix, Oerskovia, and Glycomyces; described many new species including Nocardia amarac, Actinoplanes rectilineatus, A. minutisporangius, Chainia kunmingensis, and Actinomadura yumaensis. Taxonomy of actinomycetes and the role of actinomycetes in various process, such as sewage treatment and nitrogen fixation were the main topics of study in Lechevaliers’ laboratory. In the study of the morphology of actinomycetes, the Lechevaliers used long work-ing distance condensers and objectives which were available for metallurgical studies to examine the structures formed by actino-mycetes and observe the mode of formation of these structures in situ. These methods had not been used by microbiologists who used biological optics designed to be used with coverslips. The advantage of these methods was that the morphology of the cultures were not ruined. Through these studies, he and his wife first proposed the classification of actinomycetes according to their chemical composition and morphology in 1965. He and his wife were among the world leading authorities in the studies of actinomycetes.

Hubert also studied how to recover precious metals from spent waters from smelters using killed microorganisms and their components.

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He published 143 research or review papers and coau-thored or coedited 10 books. The more notable books include: Neomycin — Nature, Formation, Isolation and Practical Application (published by Rutgers University Press), 1953; A Guide to the Actinomycetes and Their Antibiotics (Williams and Wilkins), 1953; Neomycin — Its Nature and Practical Application (The Williams and Wilkins, Co., Baltimore), 1958; Three Centuries of Microbiology (McGraw-Hill Book Co., New York), 1965; The Microbes (Lippincott, Philadelphia), 1971. Hubert was coeditor with Allen I. Laskin (1928) of the big volume: Handbook of Microbiology (first edition volume I to IV, 1973–1974; second edition, Volume I to IX, 1977–1988 (CRC Press, Cleveland). He was also coeditor with A. Laskin for Macrophages and Cellular Immunity (CRC Press, Cleveland), 1972; and Microbial Ecology (CRC Press, Cleveland), 1974.

In addition, Dr. Lechevalier had four patents: (1) neomycin and process of preparation (with S. A. Waksman), July 1957, US Patent no. 2,838,443, and 16 foreign patents on neomycin, (2) candicidin and process of preparation (with S. A. Waksman), 11 July 1961, US Patent no. 2,992,162, (3) process for recovering precious metals (with W. Drobot), September 1981, US Patent no. 4,289,531, and (4) recovery of metals (with W. Drobot), October 1981, US Patent no. 4,293,334.

Hubert has always been grateful for having two outstanding coworkers. One was his wife Midge. The other was Dr. Nancy N. Gerber, a natural products organic chemist, who was interested in pigment and volatile substances produced by actinomycetes. Among other achievements, she isolated and determined the structure of geosmin which is an off-flavor substance which con-taminates the water and fish. Unfortunately, Nancy died of stomach cancer 25 years after joining Hubert’s laboratory.

Hubert has received numerous honors for his life contribu-tion to microbiology. He was elected to Sigma Xi in 1949, associ-ate foreign member of the Société Française de Microbiologie (1972–1987), Honorary Member of the same society in 1987. He was a recipient of Lindback Award for distinguished research

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in 1976, and Charles Thom (1872–1956) Award with his wife for contribution to industrial microbiology in 1982. He was hon-ored with a Doctor of Science degree from Laval University of Canada in 1983. He was elected President of the Rutgers Chapter of Sigma Xi, 1982–1983. He received the Bergey Trust Award for contributions to bacterial taxonomy in 1989. He was also included in the New Jersey Inventors Hall of Fame in 1990. Recently, he was elected an Honorary Member of the Society for Actinomycetes, Japan, 1997.

Hubert served as an Editorial Board member of Applied Microbiology (1959–1976), Annales de Microbiologie of the Pasteur Institute (1974–1989), coeditor of the Handbook of Microbiology (1970–1989), and coeditor of Critical Reviews in Microbiology (1970–1978), CRC Press. He was editor of The Actinomycetes published by the Waksman Institute of Microbiology, 1981–1989. He was a member of the Subcommittee on the Taxonomy of Actinomycetes of the International Committee on Bacteriological Nomenclature, the Subcommittee on Tastes and Odors of the American Water Works Association, and the Joint Task group on the detection of actinomycetes for 15th and 16th edition of Standard Methods for the Examination of Water and Waste Water (American Public Health Association). He was a reviewer for the Waste Water Biology Manual of Practice of Water Pollution Control Federation. He also served as a member of the ASM Archives Committee, 1972–1980, and a member of the Advisory Board of the Center for the History of Microbiology of the ASM. He was also a member of the Subcommittee on the Taxonomy of Nocardia of the International Committee of Systematic Bacteriology, and a member of the Advisory Committee on the Actinomycetes of Bergey’s Manual of Determinative Bacteriology. He was the Chairman of Subcommittee on Actinomycetes of the ASM, 1974–1980; a trustee of the American Type Culture Collection (ATCC), 1973–1979; and served as a Secretary-Treasurer of the ATCC 1978–1979. He was a member of the International Scientific Council for the Organization of the World Congress of Microbiology held in Prague, Czechoslovakia, 1994.

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One of the authors, K. T. Chung has the honor to know him, mostly through his kind advice and editing of many of this author’s writings on the biographies of pioneer microbiologists. He is also an honest, modest man with a good sense of humor. When this author proposed to write his biography, he humbly refused by saying that he had not done much for microbiology. However, he not only has contributed to the welfare of mankind through his discoveries. He has helped to make our lives better. The authors are also specially impressed by the book Three Centuries of Microbiology, which described in detail the pro-gress of microbiology. From this book, we can also see how grateful a person that Hubert is. He always remembers where he came from, and who has helped him.

Hubert has had a successful life. It seems that good fortune always follows him. Nevertheless, we should also learn that his success is the result of smart and efficient work. Although he never admitted that he worked hard, he kept his work effective. He and his wife always devoted some time to hiking, playing golf, bicycling, and skiing in order to keep in healthy. In research, Hubert focused on what he enjoyed. As Pasteur said “Chance favors the prepared mind”. Hubert was always prepared as he knew what he liked to do. He devoted wholeheartedly to pur-sue his interests such as searching for antibiotics and systematics of actinomycetes. He followed the advice of the famous French mathematician, Jacques Hadamard, who said that the key to success was to do what you like. He is not only successful in his profession as described in this article; he also has had a wonder-ful marriage. He and Midge are always together and enjoy a sweet home. Their children can tell a better story of their mar-riage life. Up to this writing, Hubert and Midge are enjoying their retirement at their home in Morristown, Vermont, a place which is not far from Canada where he originally studied, and also in America, to which he contributed his life career. Once retired, Hubert found that he was as busy as ever and he said that he could not understand how he ever had time to work.

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The Lechevaliers’ main occupation is the management of their little 100+ acre forest, which is recognized by the State of Vermont as a Tree Farm. When there is no snow, he cut skiing trails. He enjoys gardening, skiing, painting, and reading. He is a happy man, and really knows how to enjoy life.

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Chapter 53

Thomas D. Brock (1926 to Present): A Successful Modern Microbes Hunter

“The road to Yellowstone was never straight, but it was almost never bumpy, and the view was marvelous all the way”.

—Thomas D. Brock

Source: https://en.wikipedia.org/wiki/Thomas_D._Brock(US Public Domain image)

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Thomas D. Brock is probably the most well-known microbes hunter in the 20th century. He and his associates isolated many microorganisms from different habitats and explored the diversity of microorganisms. He also blossoms in other aspects, including text books writings, publishing, computer, and the history of science. He is a versatile scientist.

Background

Thomas was born on 10 September 1926 in Cleveland, Ohio. His father Thomas Carter Brock came from Toronto, Canada, and was a power engineer, worked in various industries in Cleveland. His mother Helen Sophia Ringwald was from Chillicothe, Ohio. She was a registered nurse. Although Thomas Carter Brock had only eighth grade education, he educated himself to become a power engineer and was able to keep a job during the Depression of 1930s. His mother remained at home during the Depression. Thomas D. was their only child, and had a stable home family in a pleasant neighborhood with lots of friendly playmates. They lived on the top of a hill in a unique cul-de-sac adjacent to an errant farm and a forested park. Tom seemed to have a good childhood although he did not have much opportunity to be exposed to music and art, which he liked. His parents were working-class, and did not have much music and art in their lives.

When Thomas was almost 15, his father got sick and died unexpectedly. Tom and his mother moved back to his mother’s hometown Chillicothe, Ohio, and lived in genteel poverty. Tom had to help the family financially and worked at a variety of clerical jobs in rug, clothing, and grocery stores with a low pay of $0.25 per hour.

Thomas’s father had always recognized his lack of formal education, so always encouraged Tom to get a good education. He taught little Tom how to make electromagnets, radios, and coils from discarded electrical equipment. When Tom was only 10 years old, his father bought him a chemistry set for Christmas

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gift, and helped him set up a simple laboratory in the base-ment. When they moved to Chillicothe, Ohio, Tom met Mr. David Thornburgh who had a great influence on him. Mr. Thornburgh was also interested in chemistry. They would set up a small research laboratory in the loft of a barn behind his house and did a lot of crazy experiments such as explosives and toxic gas. Thornburgh also heard about penicillin and made some fleeting attempts to enrich soil for antibiotics-producing microorganisms. At this stage of life, Tom dreamed of becoming a chemist.

During World War II, Thomas, like many other patriotic youth, joined the military. He was enlisted in an electronic pro-gram in the U.S. Navy, spent 18 months in various Navy Schools in the Chicago areas. He finished his Navy career in Kodiak, Alaska, as a member of the shore patrol. One of his jobs in the town of Kodiak was to clear the bars of sailors at curfew and to make sure the brothels were empty. In the Navy, he spent a lot time in reading and dreamed of becoming a writer.

After getting out of the Navy, he was a military veteran and was eligible for the GI bill. He enrolled at the Ohio State University in the fall of 1946. For one reason or another, he did not pursue his dream as a writer, but went back to science. He was influenced by one of Sinclair Lewis’s book called Arrowsmith, and dreamed of becoming a bacteriologist. However, for unknown reasons, he did not pursue bacteriology; instead, he majored in botany and graduated with honors in 1949.

After a Bachelor’s degree, he was offered a graduate assist-antship in botany at Ohio State University. He then obtained his Masters degree in 1950 with his research on the mushroom (Morchella esculenta) and PhD degree with a dissertation on “Lipid synthesis in Hansenula anomala” in 1952. In the same year, Tom married Louise, who has helped Dr. Brock’s scientific career tremendously. During his student years, Tom was impressed by a well-known field ecologist, Dr. John N. Wolfe (1910–1974), who would take students on field trips to various interesting habitats.

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Beginning of a Profession

Tom was first employed as a temporary research associate at the Ohio Agricultural Experiment Station (Wooster, Ohio) working on soil fungi. Tom did not like the job; so instead, he spent most of his spare time looking for other jobs. Fortunately, he got a position in the Antibiotic Research Department at the Upjohn Company, Kalamazoo, Michigan.

Tom stayed with Upjohn Company for five years. He learned a lot of bacteriology at this time, and did research on the mode of action of antibiotics. He also developed an interest in histori-cal research and knowledge of German. Both of these interests helped his career a lot later on. In 1957, Tom secured a position as an Assistant Professor in the Biology Department of Western Reserve University (WRU) which is now Case WRU. The new job gave him the opportunity to develop his own research pro-gram, a freedom which he did not have previously. The teach-ing load was enormous, and his salary was only half of that of the Upjohn, but Tom felt that teaching was the best way of learning. He had taught general microbiology, nursing microbi-ology, mycology, and medical microbiology. He also obtained two research grants, one from the National Institute of Health (NIH) on the mode of action of antibiotics and one from the National Science Foundation (NSF) on yeast mating. His wife, Louise, worked loyally as a technician and collaborator. They published 13 papers by the time they left the Biology Department at WRU. Tom also spent time in translating German literature, and his book entitled “Milestones in Microbiology” was pub-lished in 1961. This book is a combination of interests in both German and History, which Tom developed while he was in Upjohn. It is this book, which introduced us to Dr. Brock. This book has been very instrumental in our interest in studying the biographies of pioneer microbiologists.

Dr. Brock took whatever opportunities available to improve his knowledge and skill in microbiology. He took a bacterial genetic course at Cold Spring Harbor where he associated with

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many distinguished geneticists and molecular biologists. He also learned much from the faculty and from his own students who took his course or worked in his laboratory while at the WRU. Many students such as Julius Adler, Marshall Nirenberg, Gordon Tompkins, David C. White, and Solomon Bartnicki-Garcia, later became distinguished scientists.

Dr. Brock’s desire to do research was ever growing. He was impressed by the active research activities going on in the department of Microbiology, which was just cross the street from his office and laboratory. He then resigned his assistant professorship in 1959 and worked as a postdoctorate in Dr. Lister O. Krampitz’s (1909–1993) laboratory. Dr. Krampitz was the chairman of the Department of Microbiology and his laboratory was packed with top-flight scientists. Many of these scientists went on to distinguished positions elsewhere. Dr. Brock also proved to be one of these distinguished scientists as we see later. In Dr. Krampitz’s laboratory, Dr. Brock worked on the bio-synthesis of the M protein of group A streptococci. During this period of about a year, Dr. Brock learned much about biochem-istry, immunology, and clinical microbiology. At the same time, Dr. Brock still supervised the research in his own laboratory in the Biology Department for his NIH grant. It was a traumatic year. However, Tom felt that he became a true competent microbiologist after being through this stage.

In the summer of 1958 and 1959, Tom and his wife Louise spent their summer vacation with Louise’s father in Lake Memesagamesing in northern Ontario. Because of this oppor-tunity, Dr. Brock learned about boating and fishing, which introduced him to the aquatic environment and interest in canoeing, kayaking, and the great outdoors. This experience also inspired his later research in marine microbiology, limnol-ogy, and microbial ecology.

Toward the end of his first year as a postdoctorate in 1960, an opportunity arose. Dr. Brock was hired as an Assistant Professor of Microbiology in the Department of Microbiology, Indiana University, Bloomington, Indiana, under the chairmanship of

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Dr. Leland S. McClung (1910–2000) in 1961. This was eight years after his PhD degree; he finally was able to get down to a long-term research plan. This was also a fortunate period, because in the 1960s, the U.S. economy was flourishing, universities were undergoing expansion with special focus on increasing research activity right after the Soviet Union launched Sputnik into space. Training and research grants were not difficult to get because the United States was trying to play “catch up” with the Russian. It was relatively easy to obtain money, students, support, and space at that time in comparison to the present.

In the beginning, Dr. Brock continued to work on the genet-ics and phages of streptococci, amino acid and peptide trans-port in enterococci, and the mode of action of antibiotics, which were supported by previous grants. He was a visiting virologist of American Institute of Biological Sciences during 1961–1962. Slowly, his hibernating interest of microbial ecology began to flourish and brought into blossom. In November 1962, Dr. Brock visited the laboratory of Cornelis B. van Niel (1897–1985) at the Hopkins Marine Station, Pacific Grove, California, which impressed him with the research in the aquatic system. In the Spring of 1963, Dr. Brock made plans to work at the Friday Harbor Laboratories located in Seattle, Washington, through the arrangement of Dr. Brooks Church (1918–) whom Dr. Brock met while visiting Bloomington for a seminar. Dr. Church was a faculty member at the University of Washington, Seattle at that time. Brock worked in the Friday Harbor Laboratory from 1 July to the middle of August in 1963, on the presence of enterococci in marine animals. From this time on, Brock moved forward in the study of microbial ecology and distinguished himself as one of the well-known contemporary microbe hunters in the 20th century.

Dr. Brock studied the streptococci in marine animals for only 2 weeks, and was quickly attracted by the presence of Leucothrix mucor in marine animals. He discovered L. mucor to be a wide-spread marine microorganism. He isolated this organism directly from the marine environment rather than using the traditional

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enrichment culture method. He also demonstrated the unique morphological appearance of the organism — it formed a kind of knot in its physical arrangement. This was a unique and exciting new finding in microbiology. His work on L. mucor appeared on the cover of SCIENCE, and also was featured in the New York Times in 1964. He continued to work on Leucothrix with a number of research papers and generated two PhD students (Dr. Michael Kelly, 1968 and Dr. Judith Bland, 1971). Dr. Brock made his name in the field of microbial ecology, and he became well known internationally.

Since then, Dr. Brock began to focus on what microbial ecology was all about. He consulted with limnologists and macro-ecologists and visualized that the proper approach to microbial ecology was to study microorganisms directly in their natural environment. His fundamental training in plant ecology at Ohio State University also strengthened this concept. Although his major research efforts continued to be in the area of streptococcal genetics, yeast matings, and the mode of action of antibiotics, he continued to develop the idea and con-cept of microbial ecology. Ultimately, he wrote a book entitled Principles of Microbial Ecology, which was published in 1966 by Prentice Hall.

Yellowstone Research and Beyond

In the summer of 1964, on his way back from Friday Harbor to Indiana, he stopped over at the Yellowstone National Park and took a sample of the hot springs to see whether there were Thiothrix. He was astonished to see the enormous development of microorganisms which were present in the runoff channel of the Yellowstone hot springs. As an ecologist, he thought that Yellowstone hot springs were probably a mature habitat for microorganisms and a plan was made to study the microorgan-isms in the Yellowstone hot springs the next year. His research proposal to study the microbial ecosystem of thermal springs was supported by NSF and moved Dr. Brock’s career to a new climax.

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Dr. Brock observed the extensive developments of photo-synthetic organisms in the hot springs of Yellowstone Park; he began to make quantitative measurements of the parameters related to microbial growth, such as chlorophyll, DNA, RNA, and protein contents. He also found Vitreoscilla species living in temperature near boiling temperatures. These microorganisms were isolated from the White Creek area, which they called pool A, Octopus Spring. He did an extensive study on the ther-mophilic microorganisms and published many research papers on them. One of the highlights of findings was the discovery of Thermus aquaticus isolated initially by his undergraduate honor student Hudson Freeze, and later was studied by his laboratory technician Pat Holleman. T. aquaticus turned out to be a very important organism for biotechnology. It produced heat stable enzymes called Taq endonuclease and Taq polymer-ase, which are useful in the polymerase chain reaction (PCR). T. aquaticus attracted the attention of many microbiologists, biochemists, and molecular biologists. By the end of 1990, over 1000 papers on T. aquaticus had been published. Dr. Brock’s contribution was now well-recognized.

From this time on, Dr. Brock found a wide distribution of bacteria in boiling springs (temperature over 90°C). He used the “immersion slide” technique to illustrate the life at high tem-peratures. His findings were invited to be published in Science in 1967 where he illustrated photographically the living creatures in high temperature. His work elicited a lot of interest and led to several fruitful collaborations.

Dr. Brock was now an expert in thermophilic microorgan-isms; he traveled to different areas of the world, including Italy, Iceland, New Zealand, Japan, Central America, and Caribbean where hot springs were located. This was primarily between 1966 and 1972. The original intention was to find boiling springs lower than Yellowstone hot springs. He found bacteria in virtually every boiling springs of neutral and alkaline pH. His work changed the view point about the upper temperature limits for life. He discovered that the upper temperature limit

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for life was somewhat higher than that we normally thought. Dr. Brock’s work led to hypothesize that microbes might be present in somewhat higher temperature ecosystems such as the deep sea thermal vents. It was later discovered that they were indeed flourishing live communities in those environ-ments. The research on Yellowstone hot springs served as a model for the study of other ecosystems. For this subject, Dr. Brock published about 114 research papers on the microbiology related to Yellowstone hot springs and hot temperature ecosys-tem. This work extends the vision of microbiologists and the horizons of microbiology.

As Dr. Brock’s reputation as a distinguished microbiologist was climbing high, he had problems with his first marriage with Louise, which ended in divorce. He then married Katherine Middleton in February of 1971. In the same year, Dr. Brock was invited as an Edwin Broun (1887–1981) Professor of Natural Science, the University of Wisconsin. This was a great honor for him since the University of Wisconsin is a great university although Dr. Brock loved the Indiana University. At the University of Wisconsin, he reached the peak of his scientific career. He con-tinued to do research on the Yellowstone hot springs. He also studied the genera Sulfolobus and Chloroflexus. He also studied limnology and cyanobacterial populations of Lake Mendota. He developed computer models for the determination of natural microbial population in the Lake Mendota, and also in a variety of Lakes throughout Wisconsin. Eventually, the work on Lake Mendota was compiled into a book called A Eutrophic Lake: Lake Mendota, Wisconsin, which was published by Springer-Verlag, New York in 1985.

The Yellowstone research had aroused a tremendous inter-est and excitement among the scientific communities. This was particularly, so, when several of the microorganisms Dr. Brock isolated from the high-temperature environment fitted into the new bacterial category of Archaebacteria (Archea), which was proposed by Dr. Carl Woese (1928–2012) of the University of Illinois. Dr. Brock had cultured and described these

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microorganisms which helped Dr. Woese quickly to extend the Archaebacteria concept. This work also stimulated other labora-tories to work on the microbiology of the deep sea vents. The emerging of Archaebacteria concept and the development of PCR technology had made thermophiles to be a very important group of microorganisms both in microbial evolution and bio-technology. Dr. Brock had made a historical land mark in science.

Book Writing

Dr. Brock is a very talented scientist. Other than research, he was a very successful writer and a good historian. By now, he had written two books. One is Milestone of Microbiology published in 1961 and the other is Principles of Microbial Ecology pub-lished in 1966. These important two books were the only kinds of books available in this area at that time. In 1967, Dr. Brock signed a contract with Prentice-Hall and began to write a text book called Biology of Microorganisms, the first edition was published in 1970. Initially, this book was written for nonmajors, but later major students use this as a text also. This book proved to be very successful. The second, third, and fourth editions were translated into Japanese and Spanish and all did very well throughout the 1970s and 1980s. In 1986, Dr. Brock redesigned the book using new full-color art, and the fifth edition was published in 1988, which was the first full-color microbiology textbook on the mar-ket. Later, other coauthors includes Michael T. Madigan, John M. Martinko, and Jack Parker joined Dr. Brock. The most current edi-tion is the 15th edition, published in 2017, and it is still one of the top selling microbiology textbooks.

History of Microbiology and Others

As mentioned before, Dr. Brock wrote a book called Milestone of Microbiology in 1961. Although he did not claim that was a book related to the history of microbiology, he had an abiding interest in the historical aspects of microbiology. He regards

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historical research as a respectable scholarly activity, and he considers that it can be done without research grants, students, or technicians. With his self-learned German language skill, he wrote a biography of Robert Koch, which was published in 1988 by Springer-Verlag. This book was well received. In the summer of 1959, when Dr. Brock spent several weeks at the Cold Spring Harbor, he made a sketch of the history of genetics. This sketch was finally expanded and completed as a book enti-tled The Emergence of Bacterial Genetics, which was published in 1990 by Cold Spring Harbor Laboratories. Again, this book was well received by the scientific community.

Brock was also interested in local history, particularly the history of the Village of Shorewood Hills, where he had made his home when he first moved to Madison, Wisconsin. With his interest in history and experience in book publishing, he pro-duced on a pro bono basis several publications for Historic Madison, Inc.

Computers

Dr. Brock was also well versed in the computer. He used comput-ers in the analysis of the data when he was involved in lake research. He initially used the University of Wisconsin main-frame computers, later the Apple IIe, and then moved on to CP/M-based systems, and finally to the IBM-PC computer. He was so good with computers that he published numerous articles in microcomputing magazines. He learned many programming languages and wrote extensive programs. He actually became a competent computer scientist. However, he humbly said that the computer is simply a tool for his research and publication.

Publishing

Dr. Brock was good with computers, and he also published so many papers and books, he decided to get into the publishing business himself. He actually set the type, printed, and

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published his book called “Membrane Filters: A User’s Guide and Reference Manual” by himself. He then set up a publishing company for marketing and sales. He not only published his own books, but he also handled production of other publishers. He had published more than 60 titles including the well-known second edition of The Prokaryotes in 1990–1991. For that book, he was involved with the design, copyediting, and production.

Brief Commentary

As can be seen above, Dr. Brock is a multi-talented man and also a man with broad interests which helped him to have a very fulfilling life. He has contributed greatly to the science of microbiology. He has isolated many microorganisms which have a profound impact on the understanding of microbial diversity and biotechnology. He is also good in writing, publishing, and computers. He has published more than 350 research papers and several books. His book The Biology of Microbiology is still the best textbook of microbiology for both majors and nonma-jors. He is also a good teacher, and he has produced more than 34 PhD students and has directed numerous postdoctoral asso-ciates from all over the world. His contribution to microbiology is tremendous.

His life has been full of excitement and color. As he put it “The road to Yellowstone was never straight, but it was never bumpy, and the view was marvelous all the way”. This state-ment describes his marvelous life. At this stage of life, Dr. Brock is still publishing, and still attends scientific meetings, gives lectures, seminars, and writings.

Dr. Brock received many honors and awards in his career. He received the Career Development Award from the NIH in 1962–1968. He was a lecture of Foundation of Microbiology in 1971–1972 and again in 1978–1979. He was a recipient of the Fisher award in 1984 and the Carski Foundation distinguished teaching award of the American Society for Microbiology in 1988. He raised two children, Emily K. and Brian Thomas.

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Chapter 54

Arnold L. Demain (1927 to Present): A Giant of Industrial Microbiology

“We have the unity of biochemistry on one hand, and the diversity of microbial life on the other. We have to understand and appreciate both”.

“Whenever my students work on a topic, they should read everything, and then write a new review on the subject”.

—Arnold L. Demain

Source: Courtesy of Dr. Demain

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Dr. Arnold Lester Demain has done a tremendous amount of work on the application of microbiology, which includes micro-bial toxins, enzyme fermentations, biosynthesis of antibiotics, vitamins, amino acids and nucleotides, microbial nutrition, industrial fermentations, regulatory mechanisms, and genetic engineering of fermentative microorganisms. To call him a giant of industrial microbiology is very appropriate. His life is a witness of the progress of biotechnology; therefore, it is very worthwhile for us to learn about it.

Arnold (“Arny”) was born on 26 April 1927 in Brooklyn, New York. All of his grandparents were immigrants from the Austrian–Hungarian Empire around the turn of the century. The maternal grandparents were from Lembourg (now Lvov in the Ukraine) and the paternal grandparents from Budapest (now Hungary). His father Henry Demain and his mother Gussie Katz both were born in 1904 on Manhattan’s East Side in New York City. This couple’s marriage was an unstable one, both being strong people; they were married and divorced from each other twice. Arny’s father Henry and Grandfather Joseph Demain were both in the pickle business. His mother, Gussie worked initially as a secretary, then a salesperson in a Manhattan department store, and finally as an administrative assistant in a finance company. Arny was essentially raised by his loving mother, his grandmother Fannie Demain, and his aunt Ruth Busch. Although Arny was an only child, he was very close to his 15 first cousins, all of whom lived in New York City. He was raised in Brooklyn and the Bronx in years dominated by the depression. The family moved often and he attended some five elementary schools and three high schools before graduating from Midwood High School in the Flatbush section of Brooklyn in February 1944. During those years, Arny worked very hard to help the family. He had many jobs such as a grocery delivery boy (2 cents per delivery plus an occasional 5 cents tip), and as a stock boy for Lord & Taylor’s Fifth Avenue department store at 40 cents per hour during the school months and $17 per week during the summer. His experiences working with pickles had a great influence on his later career development.

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At the age of 16 (1944), Arny’s mother and father jointly took him by train to Michigan State College (MSC; now University) in East Lansing, Michigan. This was because Henry, his father, knew that the United States’ leading investigator of cucumber fermentations was Professor Frederick W. Fabian (1888–1963) at MSC. Arny was virtually deposited on the steps of Professor Fabian’s food fermentation laboratory at MSC.

After his freshman year, he enlisted in the US Navy for ser-vice in February 1945, several months before his 18th birthday, because the World War II was still going on. He served at the Bainbridge Naval Base in Maryland, the US Navy Hospital in Philadelphia, on the hospital ship USS Consolation and the US Navy Yard in Brooklyn. Although he spent two years in the Navy, he did not participate in wartime action. Arny returned to MSC’s Department of Microbiology and Public Health in 1947 and obtained his BS degree in 1949 and his MS degree in 1950. His Master’s thesis dealt with the microbial spoilage of cucum-bers by softening during fermentation.

During his last year in Michigan, Arny had met incoming freshman Joann (“Jody”) Kaye from Youngstown, Ohio, and fell in love with her. Together, they headed for continued stud-ies in northern California in the fall of 1950.

The reason for Arny’s choosing the University of California, Berkeley, was again influenced by his father’s profession. Henry Demain had established a canning and pickling plant for the Vita Foods Corp. in Chestertown, Maryland, and his brothers Ben and Seymour had opened Demain Foods Co., another pickling operation, in Ayden, North Carolina. Through these associations, Henry and his brothers had met Dr. John Etchells (1909–1981), the acknowledged successor to Professor Fabian as the leading US investigator of cucumber fermentations. “Jack” Etchells had a joint appointment at North Carolina State College (now University) with the US Department of Agriculture as Professor. He had been a student of Prof. Fabian’s ten years before Arny attended MSC. Dr. Etchells knew virtually everyone in the food science area and convinced Arny that the University of California was the place for him to do his PhD. studies. Arny

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was sent by Dr. Etchells to Dr. Emil M. Mrak (1901–1987) of the Food Science Department in Berkeley and did his course work and PhD preliminary examinations there during the years of 1950–1952. His main activity in Berkeley, other than studying, was to transfer and maintain the viability of the cultures of the famous UC yeast collection. During this period of time, Jody attended San Mateo Junior College. Arny and Jody were mar-ried at the St. Moritz Hotel in New York City on 2 August 1952, and began a long happy marriage life, making a home in Davis, California, where the UC Food Science Department had been relocated. Arny then spent two years under the tutelage of the prominent yeast scholar, Dr. Herman J. Phaff (1913–2001), working on yeast polygalacturonase. Arny obtained his PhD in 1954 and his dissertation was on the nature of pectic enzymes, which were responsible for pickle softening. He was the first to employ “affinity chromatography” by using a pectic acid gel column to isolate polygalacturonase from the broth of the pro-ducing yeast, Kluyveromyces fragilis. It not only was a technical adventure, but also helped the interpretation of the mecha-nisms of how this enzyme worked.

Dr. Phaff had a profound influence on Arny’s career. They coauthored seven research papers during this period of time. Dr. Etchells, from North Carolina State University, also affected Arny’s scientific development. Dr. Etchells wrote many articles on pectic enzymes and softening of vegetables, in which Arny was interested. Both Drs. Phaff and Etchells kept abreast of all the literature in their areas of interest. Arny also read the rel-evant literature and wrote new reviews on pectic enzymes. This is one reason that he is so knowledgeable about the progress in many fields. He has written reviews on almost every topic he has ever worked on.

In March of 1954, Arny accepted a position as a research microbiologist at the penicillin factory of the Merck Sharp & Dohme Research Laboratory in Danville, Pennsylvania. This was a big shift for his research, because now he had to focus on penicillin biosynthesis. He educated himself about the penicillin fermentation by reading extensively in the library of Bucknell

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University, especially papers by Professor Marvin J. Johnson (1906–1982) of the University of Wisconsin, and the book Chemical Activities of the Fungi by Professor Jackson W. Foster (1914–1966) of the University of Texas. At Danville, Arny wrote several papers on yeast polygalacturonase and penicillin biosynthesis. At the end of 1955, he moved to the Merck research laboratory in Rahway, New Jersey, where he spent the next 13.5 years. He began his work under the tutelage of Dr. David Hendlin (1920–2006) in the Microbiology Department, which was directed by the well-known Dr. Harold Boyd Woodruff (1917–2017). From both of these men, Arny learned much about nutrition of microorganisms and the discovery and production of antibiotics. During the first 10 years in Rahway, he and his technical assistant Joanne Newkirk made great progress on microbial growth factors, cephalosporin biosynthesis, protein synthesis, and nucleotide fermentation, the results of which were published in a number of papers.

In 1965, he was asked by Merck Vice President Dr. Karl Pfister (1919–) to found a new department which he named “Fermentation Microbiology”. Thus, a new phase of Arny’s life began in which he became a Department Head and had an opportunity to set up a new group of over 30 people dealing with improvement of current Merck fermentations and devel-opment of fermentations for new Merck products. This was an exciting challenge since it gave him the opportunity to hire new people who could help him establish a place for his department in the world of industrial microbiology. Some of those people who later became well known for their research were Louis Kaplan, Edward Inamine, Jerome Birnbaum, Barbara Lago, and Raymond White.

During his employment at Merck, Arny made many scientific contributions, both in basic science and applied science. He innovated methods to enhance the production of secondary metabolites using starved resting cells. He was the first to detect feedback inhibition of penicillin production by the amino acid lysine, and originated the study on the effects of primary

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metabolites on the secondary metabolism of microorganisms. He elucidated the mechanism by which the biosynthesis of cephalo-sporin in Cephalosporium acremonium was stimulated by the presence of methionine, that is, methionine worked as an inducer of enzymes of cephalosporin biosynthetic pathways — a new mechanism which had never been reported before. He also recog-nized that the negative effect of certain nutrients on secondary metabolism was a general effect resembling to that of catabolite repression, and such repression was due to rapid metabolism of carbon, nitrogen, or phosphorus sources during growth.

In 1969, he was invited to be a full professor of industrial microbiology at the Department of Nutrition and Food Science (later renamed the Department of Applied Biological Sciences) of the Massachusetts Institute of Technology (MIT). He set up a fermentation microbiology laboratory and is still carrying out his academic research there almost 3 years after his retirement. At MIT, Arny and his distinguished group of international students and postdoctorates contributed to many areas of research including cell-free-b-lactam formation, which resulted in a breakthrough discovery of a key enzyme in cephalosporin biosynthesis — deacetoxycephalosporin C synthetase (“expand-ase”). The discovery of this enzyme established the role of penicillin as an intermediate in cephalosporin C biosynthesis and disproved the previous hypothesis that these two separate end products of C. acremonium were formed by a branched second-ary metabolic pathway. They were the first to purify isopenicillin N cyclase. They discovered the enzyme isopenicillin N epimerase, an extremely labile enzyme of cephalosporin biosynthesis. They also did ground breaking work on L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACV) synthetase. They showed that the initial step catalyzed by this multifunctional enzyme produced the dipeptide L-a-aminoadipyl-L-cysteine, an enzyme-bound intermediate in all penicillin- and cephalosporin- produc-ing microorganisms.

Arny also postulated that antibiotics and other secondary metabolites are produced in nature, which serves the survival

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of the producing organisms. This theory overthrew the previous concept of Selman A. Waksman (1888–1973), who considered that the production of antibiotics was an artifact of microor-ganism cultured in the laboratory. The antibiotic producing microorganisms also develop means to protect themselves from their own antibiotics, that is, to prevent suicide. This is a very fundamental principle of antibiosis in natural environments.

He also worked on the production of gramicidin S by Bacillus brevis and found that the antibiotic acted as an inhibitor of germination outgrowth of the spore, prolonging this stage in the producing organism. He and an associate showed that the hydrophobicity of Bacillus spores was due to the presence of gramicidin S on the surface of the spores. He observed that the germination outgrowth phase was a heat resistant stage of B. brevis. This finding disproved the common belief that all Bacillus spore lose their heat resistance during the earlier stage of germination initiation.

Arny also carried out studies on microbial nutrition. He was the first to discover that one of the growth stimulating effects of protein digests was due to lipids associated with proteins such as casein, and not always due to their peptides or amino acid components. He was also the first to report that an iron transport factor (siderophore) was an absolute requirement for some bacteria. He demonstrated that Lactobacillus homohiochi and Lactobacillus heterohiochi, contaminants of the wine mak-ing process, not only tolerated over 20% ethanol, but also required ethanol for optimal growth. With a postdoctoral asso-ciate, he showed that betaine’s stimulation of the biosynthesis of vitamin B12 in Pseudomonas denitrificans was due to the induction of δ-aminolevulinic acid synthase. Arny and a col-league discovered that mannan was required for optimum pro-duction of streptomycin by Streptomyces griseus. Mannan induced the synthesis of α-D-mannosidase which converted mannosidostreptomycin to streptomycin. He and his associates also isolated a number of new mycotoxins, which had not been previously discovered.

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In more recent years, Arny and his group have been actively studying the effect of nutrition on the production of red pig-ment by Monascus sp. and of rapamycin by Streptomyces hygroscopicus. His work with students and postdoctorals on the cellulosomal proteins of Clostridium thermocellum established the nucleoptide sequence of the key scaffolding gene, cipA. Arny and his postdoctorals developed important bioconversions, such as the transformation of compactin to pravastatin, and penicillin G to deacetoxycephalosporin G. With a colleague, he discovered that conditions stimulating microgravity interfered with secondary metabolism by bacteria.

Arny has been particularly concerned with environmental factors in growth media that may regulate gene expression and with the development of mutant strains with altered regula-tion for increasing formation of useful products.

At MIT, Arny was significantly aided by two assistants, Nadine Hunt Solomon during the early years and Aiqi Fang in recent years. Over the years, he has been successfully in combining knowledge of basic microbiology and biochemistry and making use of this in understanding basic principles and potential appli-cations. He has published more than 430 research papers, and more are on their way, since he is still actively directing research. He has stated “We have the unity of biochemistry on one hand, and the diversity of microbial life on the other; we have to understand and appreciate both”. His contribution to science is a paradigm of what Louis Pasteur (1822–1895) said “There are not two sciences, there is only one science and its application and these two activities are linked as the fruit is to the tree”.

In addition to his numerous research papers, he is also a coeditor or coauthor of 10 books and has had 19 US patents. He has been in great demand as a guest speaker at international conferences and has been recognized by many countries and scientific organizations. He received the Hotpack Award and the Labatt Award from Canada, Rubro Award from Australia, Award of the Italian Industrial Pharmaceutical Association, Honorary Membership in the Croatian Society of Biotechnology, Honorary

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Membership in the French Microbiological Society, Kitasato Medal and Honorary Membership in the Kitasato Institute and the Society of Actinomycetes of Japan, Fellow of the Institute of Biotechnological Studies (England), Mendel Medal (Czech Republic) and Honorary Membership of the Czechoslovak Microbiological Society, the von Humboldt Foundation Prize (Germany), and the first Hans Knoll Award of the former German Democratic Republic. In the United States, he has also received many awards: American Society for Microbiology (Waksman Award of the New Jersey Branch, Distinguished Service Award, Cetus Biotechnology Award, Alice C. Evans Award, and Fellowship in the American Academy of Microbiology); American Chemical Society (First David Perlman Lectureship Award and Marvin J. Johnson Research Award); and the Society of Industrial Microbiology (SIM; Charles Thom Research Award, Waksman Outstanding Teaching Award, and Charter Fellow).

Arny has served microbiology well, as an officer for scien-tific organizations, as a Visiting Professor (University of British Columbia, National Taiwan University, Osaka University, and Fujian Institute of Microbiology), and as a member of the editorial boards of 33 journals and book series. Those include the Journal of Bacteriology, Applied and Environmental Microbiology, Journal of Antibiotics, Advances in Biochemistry, Antimicrobial Agents and Chemotherapy, Journal of Industrial Microbiology and Biotechnology, Nature Biotechnology, Science, Current Opinion in Microbiology, Critical Reviews in Biotechnology, Applied Biochemistry and Biotechnology, Enzyme and Microbial Technology, and Annual Review of Microbiology. For 15 years, he has been the International Editor for the Americas of the Journal of Applied Microbiology and Biotechnology.

Arny was President of the Society for Industrial Microbiology in 1990. He was also elected as a member of the National Academy of Sciences in 1994, and to the Mexican Academy of Sciences in 1997. He has also received honorary doctorates from

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the University of Leon (Spain), Gent University (Belgium), and the Technion (Israel).

Arny has been interested in teaching and is an outstanding educator. This can be seen from the most cherished honors that he has received. He was named “Best Teacher of the Year” (1971 and 1973) by students of the Nutrition and Food Science Department, and “Teacher of the Year” by the MIT Graduate Students in 1974. He was a major contributor to the annual summer professional course in Fermentation Technology at MIT. He has trained a large number of predoctoral students and postdoctorates from all over the world and has molded them scientifically, mentally, and socially. The name coined for this group of the current and former collaborators is “Arny’s Army” which is an international family of scientists and engineers. Members of this group are in the upper scientific echelons of many of the world’s leading pharmaceutical and chemical com-panies as well as in academic departments of microbiology and biochemical engineering.

Several international meetings have been dedicated to him: The first Symposium on Industrial Microbiology and Biotechnology held in Cambridge, Massachusetts, 1995; the 5th International Conference on the Biotechnology of Microbial Products: Novel Pharmacological and Agrobiological Activities held in Williamsburg, Virginia, in 1997; the 2nd Symposium on Industrial Microbiology and Biotechnology held in Nara, Japan, 1997; the International Symposium: Control of Gene Expression in Antibiotic-Producing Microorganisms held in Madrid, Spain, 1999; and the most recent “Celebration of Arny’s Army and Friends” in Gent, Beligium, July 1999. There are also two papers and a book dedicated to Dr. Demain: Enzymatic Synthesis of Hydrophilic Penicillins (by Luengo, J. M.; in J. Antibiotics 48:1195–1212, 1995), The Cellulosome Concept as an Efficient Microbial Strategy for the Degradation of Insoluble Polysaccharides (by Shoham, Y., Lamed, R., and Bayer, E. A.; in Trends Microbiol. 7:275–280, 1999), and Biotechnology of Antibiotics, 2nd edition (by Strohl, W. R. (editor), Marcel Dekker, New York, 1999).

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Arny and Jody have had a wonderful marriage in which they raised two children. Pamela Robin Demain was born in 1953 in California. She is married to kitchen and bath designer Thomas McCloskey and they have two children, Megan Elizabeth and Andrew James. Pamela now is Executive Director of Global Marketing Research at Merck in Whitehouse Station, New Jersey. Jeffrey Brian Demain was born in 1965 in New Jersey, and now is a trial lawyer and Partner in the law firm of Altshuler, Berzon, Nussbaum, Berzon, and Rubin in San Francisco. Jeffrey is married to clothing manufacturer’s representative Lauren Brener. Arny’s family has consistently supported his activities. Jody frequently accompanies Arny to the annual meetings of the SIM. A proud moment for Arny and Jody was the 1998 SIM meeting in Denver, Colorado, at which Arny and Pamela were both invited speakers.

Arny is a kind gentleman. He always uses his knowledge to help people no matter where they come from. His kindness, knowledge, and wisdom attracted many of the world’s top intellects to join his efforts in research and development. At this moment of writing, at the age of 72, his expertise is still in high demand by many universities, companies, and coun-tries of the world. His has contributed not only to science and technology, but also to humanity as well. We have the honor to know him, indeed a treasure of mankind.

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Chapter 55

Bruce N. Ames (1928 to Present): A Pioneer of Genetic Toxicology and

Molecular Mutagenesis

Source: https://en.wikipedia.org/wiki/Bruce_Ames(US Public Domain image)

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Dr. Bruce N Ames is well known for his famous Ames Salmonella/microsome mutagenicity assay, which is used by more than 3000 laboratories in the world to test chemicals for mutagenic activ-ity in a bacterial system. His method, and also his involvement in the study of identifying mutagens/carcinogens, have influ-enced cancer studies for more than 50 years and will continue to influence research in the future. He is a world leader in molecular genetic toxicology, his research accomplishments having made a great impact on how we think about cancer and other degenerative diseases today.

Bruce Nathan Ames was born on 16 December 1928 in New York City. He has two younger sisters. His father, Dr. M. U. Ames, was a high school chemistry teacher who also had a doctorate in law. His mother, Dorothy Andres Ames, came to the United States as a young child from Poland. His father was a chairman of a high school chemistry department, and later a supervisor of science for all the New York City public schools, and an Assistant Superintendent of New York City schools. They lived in the Washington Heights area of Manhattan in New York City. Every summer they rented a cabin on a lake in the Adirondack Mountains, where they enjoyed hiking, swimming, and other outdoor activities. As a child, Bruce loved the natural world and collected animal specimens. He was also an avid reader. He read many books left lying around the house by his father, and he carried a stack of books home for reading every week from the library.

At the age of 13, he attended the Bronx High School of Science. He was always interested in biology and chemistry. He did some experiments studying the effect of plant hormones on the growth of tomato root tips. [Plant hormone auxins were discovered by Frits H. Went (1903–1990) and Kenneth V. Thimann (1905–1997) at about that time]. In 1946, he enrolled in Cornell University in Ithaca, New York, majoring in chemis-try with a minor in biology. He received his BA degree in 1950, and went directly to the graduate school at the California Institute of Technology (Caltech), Pasadena, California, where

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he worked in Dr. Herschel K. Mitchell’s (1913–2000) laboratory. He was influenced by many distinguished scientists at Caltech. He studied biochemical genetics using mutant strains of the bread mold Neurospora. Using genetic/nutritional mutation techniques to study the biochemical pathway was pioneered by George W. Beadle (1903–1989) and Edward Lawrie Tatum (1909–1975). George Beadle was the chairman of the genetics department from 1946 to 1959 at Caltech. Bruce’s thesis work was a cutting edge subject during those days. He completed his Ph.D. degree in 1953 at age 24. He studied the biosynthesis of the amino acid histidine using different mutant strains of Neurospora. Bruce himself said that, as a student, he was never a straight A student, but he was creative and good at solving problems. We can also see that he has a very good training; he has always been associated with top grade scien-tists. His innovative scientific career had been laid with a good foundation.

After completing his PhD degree at Caltech, Bruce took a postdoctoral position in 1953 and worked at Bernard Horecker’s (1914–2010) laboratory at the National Institute of Health (NIH). From 1954, Bruce became an independent inves-tigator and worked on histidine biosynthesis gene regulation using Salmonella typhimurium. He married Dr. Giovanna Ferro-Luzzi, a postdoctoral student from Italy in 1960. In 1961, he took a sabbatical leave from NIH in Europe. He divided the year between Dr. Francis H. C. Crick’s (1916–2004) laboratory in Cambridge, England, and François Jacob’s (1920–2013) labora-tory at the Pasteur Institute in Paris, France. At that time, Crick was about to receive a Nobel Prize in 1962 for his earlier work on the structure of deoxyribonucleic acid (DNA). Jacob was working on how the messenger ribonucleic acid molecule (mRNA) interacts with DNA to synthesize protein, which was also a frontier research for biology at that time. Jacob, too, received Nobel Prize in 1965. These studies were at the cutting edge of scientific progress. They impacted Dr. Ames and affected his later scientific thought.

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Dr. Ames returned to the United States in 1962 to become chief of microbial genetics at the Laboratory of Molecular Biology at NIH. He focused his research on the regulation of the histidine operon — a specific group of genes involved with the biosynthesis of histidine in Salmonella and the role of transfer ribonucleic acid (tRNA) in this regulation. This work paved the way for his later development of the mutagenicity assay.

One of the special contributions during this period of time was that Dr. Ames co-authored with Dr. Robert G. Martin (1935–) a paper on sucrose gradient centrifugation, a method for determination of the molecular weights of proteins. This technique was very useful for biochemical, molecular biologi-cal, and biotechnological investigations. This technique also contributed to the development of molecular biology because the determination of the molecular weight of proteins is very important for any kind of biochemical research. Another sig-nificant contribution was the study with Martin showing that the histidine biosynthetic genes were turned on and off as a unit and that a single mRNA was produced from the cluster of genes. This work was one of the bases for the operon theory of François Jacob (1920–2013) and Jacques Monod (1910–1976) that led to their Nobel Prize.

Sometime in 1964, Dr. Ames read the list of ingredients on a box of potato chips, and began to wonder whether preserva-tives and other synthetic chemicals could cause genetic damage to human cells. It could be serious if we ingested large quantities of a mutagenic chemical. He began to develop a test for chemi-cal mutagens. Since he mutated bacteria all the time, he used strains of S. typhimurium, which were mutated in a gene for synthesizing histidine (a histidine-requiring mutant). He placed about one billion cells of this bacterial culture into a petri dish with an agar medium without histidine. With such a huge num-ber of bacteria, a few revertant colonies would appear following a short incubation time. This is called spontaneous mutation (mutation back to histidine nonrequiring). Any chemical added to this test system that increases the number of such revertants

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is a “mutagen”. A mutagen increases the rate of mutation. Later, Dr. Ames also added to the mixture a liver homogenate fraction, called the S9 fraction, from rodents. The S9 preparation contains various metabolic enzymes. Some chemicals do not cause mutation themselves, but increase mutation if this liver homogenate is also included in the assay system. This is because the enzymes in the liver homogenate can metabolize the chemi-cal to an active form, which then mutates the bacteria. Chemicals that can cause mutations in the test system without the inclu-sion of this homogenate are called “direct mutagens”. Those chemicals that require this liver homogenate are called “indirect mutagens” that required “metabolic activation”. This simulates how chemicals are metabolized in the liver system.

In 1968, Ames accepted a position as a professor of bio-chemistry at the University of California at Berkeley. He and his students continued to work on chemically induced mutations. Chemicals that cause mutations in bacteria may cause muta-tions in human genes. He first paid attention to those chemical capable of causing human cancers — called carcinogens. His paper published in 1973 in the United States Proceedings of National Academy of Science (PNAS) was titled Carcinogens are mutagens. In a subsequent paper, he reported that 174 of suspected carcinogens, 80% or 90% of them caused mutations. His discovery thrilled the world of genetics and carcinogenesis.

Dr. Ames’s method of assaying the mutagenicity of chemicals is relatively simple, inexpensive and quick, which is in contrast to the time-consuming rodent cancer test assay. His method was quickly adapted by thousands of laboratories in the world. Dr. Ames never patented his method, he also voluntarily pro-vided his tester bacterial strains free of charge. He was also will-ing to teach others how to do the test. Ames soon became world renowned and his method was widely used. His method of detecting mutagens is commonly known as the ”Ames test” by both scientists and the public.

With his knowledge of the histidine operon, Dr. Ames contin-ued to improve the sensitivity of the tester strains. A set of

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different types of histidine requiring tester strains were intro-duced. These strains would not only give answers to whether the test chemicals are mutagenic, they could also help interpret the types of mutations (such as base-pair mutation or frameshift mutation). In 1994, Ames and Dr. Pauline Gee (1953–) developed a new set of six strains that were more sensitive than the old tester strains and diagnosed the 6 possible base pair mutations. The new Ames test not only helps to identify the genotoxic nature of many chemicals, but also helps us to understand the molecular mechanism of mutagenesis. This work contributed tre-mendously to our broad knowledge of genetic toxicology today.

Since the publication of Ames’s work, both the public and government were concerned with the risk of hazardous chemi-cals released to the environment. Ames himself urged close scrutiny of possible environmental cancer hazards. He and his students demonstrated that a flame retardant, tris-BP used in children’s polyester pajamas was a mutagen. As a result of his effort, tris-BP was withdrawn from the market. He also stressed problems associated with the relative risks of various carcino-gens. He noted that the potency of carcinogens could vary well over a million fold and a priority list was necessary to evaluate various carcinogens.

However, as the time went by, and more scientific data piled up, Dr. Ames began to change his view and came to doubt that traces of synthetic chemicals were important as causes of cancer. In the 1980s, California agriculture was threat-ened by the medfly. Many agricultural experts recommended use of insecticide, malathion. But the governor Jerry Brown (1938–) did not agree because he sided with the environmental activists and believed that the wide use of malathion would pose a risk to human health. Ames, however, testified that a significant hazard did not exist from using that pesticide. He suggested that the spraying of malathion would pose no more danger than pouring a can of diet soda on one’s front lawn.

Dr. Ames was not a spokesman for industry and in fact does no consulting or testifying in lawsuits, rather his comments

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were based on new scientific findings and comparative obser-vations. In his paper published in 1983 in Science “Dietary car-cinogens and anticarcinogens”, he found that daily items such as coffee, alcohol and potatoes contain mutagens and carcino-gens with possible hazards much greater than synthetic chemi-cals. He argued that the important thing was that many plants, vegetables, and fruits, we consume also contained anticarcino-gens such as vitamin C, vitamin E, beta- carotene, selenium, and other natural substances. He argued that the higher incidences of cancer in certain human populations might be due to “less than optimum amounts of anticarcinogens and protective fac-tors in the diet”. Dr. Ames views pesticides as a public health advance in making produce less expensive. He strongly advo-cates eating more fruits and vegetables which he believe is the best way to lower risks from cancer and heart disease, other than giving up smoking. Vitamins, antioxidants, and fiber of plant source are important anticarcinogens.

The rodent test for carcinogenicity utilizes maximum tol-erated doses of suspected hazardous synthetic chemicals. Therefore, Ames thought it was necessary to examine the carci-nogenicity of the natural background of chemicals that humans ingest (99.9% of the chemicals) as a control to put synthetic carcinogens in perspective. He and his colleagues, particularly Dr. Lois Gold (1941–2012), had spent much time in developing a “Carcinogen Potency Database” and published a paper enti-tled “Ranking Carcinogenic hazards” in Science, 1987. They created an index called HERP (Human Exposure Dose/Rodent Potency Dose), which compared the average daily dose of chemicals which humans might receive with the dose needed to induce cancer in rodents. The results of HERP show the possible cancer hazard of traces of synthetic chemicals such as pesti-cides, are tiny compared to natural chemicals in the diet. Even the much greater possible hazards from “rodent carcinogens” in natural chemicals should be taken with much skepticism. He and Dr. Gold have shown that half of all chemicals ever tested are “rodent carcinogens”. He and his colleague Lois Gold

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argued that since half of natural chemicals tested were rodent carcinogens, that this finding and considerations of mechanism, suggested that high-dose tests were not relevant for low-dose exposures.

Dr. Ames’s view of the relationship between cancer and pesticides received many critical attacks. The HERP was not intended to be a reliable risk indicator but “only a way of set-ting priorities for concern”, Ames argued. However, Ames and his colleagues concluded that “we lack the knowledge to do low-dose risk assessment”. Dr. Ames’ conclusion on extrapola-tion from high-dose to low-dose effects profoundly affected the scientific investigation of hazardous agents. Skepticism as to low-dose effects of synthetic chemical carcinogens on cancer formation has been spreading among toxicologists.

Despite the attacks and criticism on his view of cancer, Ames insisted on that it is important to keep up with new scientific facts and modify one’s viewpoint, especially in the rapidly changing and difficult area of cancer causes and prevention. He also thinks there are several misconceptions on cancer which need to be clarified. He repeatedly addresses those issues to the general audience which he regards as a part of his duty as a scientist. His basic viewpoints are: (1) concern with hundreds of minor, hypothetical risks is a distraction from major risks such as unbalanced diets and cigarette smoking; (2) many micronutrient deficiencies are radiation mimics and are a major source of DNA damage in the US population; (3) almost half of the test chemi-cals are rodent carcinogens for both synthetic and natural chemicals; (4) high-dose animal testing is unreliable for low-dose extrapolation; (5) human exposure to rodent carcinogens is almost all to natural chemicals; (6) synthetic chemicals pose minuscule risks compared to substances found in nature; (7) correlations does not mean causation, and “cancer clusters” mostly come about by chance alone; and (8) there are undesir-able trade-offs in eliminating pesticides, as pesticides lower the cost of fruits and vegetables, a major protection against cancer.

Like many experts, Ames considers smoking and dietary imbalances the most serious causes of cancer. He also agrees

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with many experts that increasing dietary fruits and vegetables and insuring adequate micronutrients are the best cancer pre-ventive measures, other than decreasing smoking.

His work on mutagenesis and cancer led Dr. Ames to con-sider the nature of human aging. In support of the widely dis-cussed free radical theory of aging, Ames and his students showed that many of the effects of aging may be due to oxida-tive damage caused by free radicals in mitochondria. There is a large amount of oxidative damage just from living. He said “living is like getting irradiated”. We are generating energy when burning fat, carbohydrates, and proteins, which means pulling electrons off them. We add the electrons to oxygen in the mitochondria to generate energy. The four electron addition to one oxygen molecule is safe, but if one electron is added at a time, we make superoxide, hydrogen peroxide, and hydroxy radical. These radicals can cause oxidative dam-age which contributes to degenerative diseases such as cancer, aging, heart disease, cataract, and brain dysfunction. He esti-mated a very high (105 hits/cell/day in the rat) endogenous oxidative DNA damage rate and a fairly high steady level of oxidative DNA damage in individuals. He has found that thymine glycol, thymidine glycol, hydroxymethyluracil, and 8-hydroxydeoxyguanidine in human urine, and suggested that they are probably derived mostly from repair of oxidized DNA in the tissues. These free radicals can be scavenged by various repair mechanisms and enzymes defenses and also by antioxi-dants such as vitamins C and E, selenium, etc. A major defense against cancer and other degenerative diseases is to have ade-quate antioxidants and other micronutrient in the diet. Many of those who get cancer or degenerative disease may be those who are deficient in these important antioxidants and micro-nutrients in their diets, or those whose living style helps enhance oxidative damage such as smoking. The Ames labora-tory’s recent research is focusing on the following: (1) the rela-tionships between nutritional factors, particularly folate, vitamin B12, and B6 deficiencies, and DNA damage and cancer; (2) chronic inflammation as a risk factor for cancer formation.

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The lab is developing analytical methods to measure chronic inflammation in humans, and nutritional intervention to com-bat it; and (3) mitochondrial oxidative decay and aging. He is finding some mitochondrial metabolites reverse mitochondrial decay, which may delay degenerative processes.

In brief, Dr. Ames’s interest is in identifying the important mutagens that will damage human DNA, the defense mecha-nisms to protect us from damage, and studying the conse-quences of DNA damage for cancer formation and aging. His outstanding work not only revealed a great amount of knowl-edge related to molecular genetic toxicology and cancer, but also provided a lead to future investigations, in human health. He has published over 400 scientific papers and is one of the most cited scientists in all fields.

Dr. Ames has often been honored for his achievements. In 1964, he received the Eli Lilly Award of the American Chemical Society. In 1966, he won the Arthur Fleming award. He received the Lewis Rosenthal Award in 1976, the Environmental Mutagen Society Award in 1977, the Cal Tech Distinguished Alumni Award in 1977, the Simon Shubitz Cancer Prize and the Felix Wankel Research Award in 1978, and the John Scott Medal in 1979. In 1983, he was a recipient of the Gairdner Foundation Award from Canada and the Charles S. Mott Prize of the General Motors Cancer Research Foundation. In 1985, he won the Tyler Prize for Environmental Achievement, and in 1989, he was awarded the Roger G. Williams Award in Preventive Nutrition. In 1991, he won the Gold Medal of the American Institute of Chemists. In 1992, he was recipient of the Glenn Foundation Award of the Gerontological Society of America. He won the Lovelace Institute Award for Excellence in Environmental Health Research in 1995 and the Achievement in Excellence Award for the Center for Excellence in Education in 1996, and the Honda Prize of the Honda Foundation (Japan) in 1996. In 1997, he received the Japan Prize. In 1998, he was a recipient of the US National Medal of Science. He is also a recipient of many honors. He is a member of the National Academy of

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Sciences, the American Academy of Arts and Sciences, and is a fellow of the American Association for the advancement of Science. He served as a member of The National Cancer Advisory Board from 1976 to 1982. He has received honorary degrees from Tufts University and the University of Bologna. He is a foreign member of the Royal Swedish Academy of Sciences, and an elected fellow of the Academy of Toxicological Sciences and the American Academy of Microbiology.

Dr. Ames remains at the University of California at Berkeley in the Department of Biochemistry and Molecular Biology. He served as the chairman from 1983 to 1989. Since 1979, he has been the director of the National Institute of Environmental Health Science Center at Berkeley.

Bruce Ames married Giovanna Ferro-Luzzi on 27 August 1960. Dr. Ferro-Luzzi (1936–) is also a professor of biochemistry at Berkeley. They have a daughter, Sofia, and a son, Matteo. Dr. Ferro-Luzzi came from Italy, and they are enjoying a good Mediterranean diet with lot of fruits and vegetables. Dr. Ames practices what he believes in. He has a wonderful marriage and is happily living in Berkeley, California.

One of the authors, K. T. Chung had the opportunity to listen to Dr. Ames’ seminars since 1970 as a graduate student in California. He found Dr. Ames handsome and having a good sense of humor. He is a good and popular speaker for confer-ences or symposiums. One reason for his popularity is that he is always optimistic and his sense of humor, zeal, and enthusiasm, make him not likely to be an enemy of anybody although he received many critical attacks on his work. He is successful in science as he said he is always thinking and challenging assumptions. Although he sounds like a spokesman for indus-try, he never obtains any funding or asks for any personal ben-efit from industry. Science is for science. That is the way it should be and that is the way Dr. Ames practices. He is a true leader.

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Chapter 56

Richard L. Crowell (1930 to Present): Cellular Receptors and Viral Infection

“It is the cellular membrane receptors, not viruses, which determine the type of viral infections”.

—Richard L. Crowell

Richard Lane Crowell dedicated most of his career to perform research on enteroviruses in mammalian cell cultures with emphasis on chronic infections, the viral interference phenomenon, factors that make cells susceptible to viral infections, and the role of coxsackieviruses in heart disease, diabetes, and myositis. He is best known for his studies on cellular receptors, which are important determinants of virus susceptibility of the host cells. He has also been active in many biomedical organizations including being elected President of the Association of Medical School Microbiology Chairs of the United States and Canada (1986) and of the American Society for Microbiology (ASM) (1991). He has helped advance the biomedical education.

Dick, as his family and friends knew him, was born on 27 September 1930 in Springfield, Missouri. His father, Thomas Rolla Crowell, Sr., a Presbyterian minister, and mother Addie Malinda Lane, had two children, Thomas R. Crowell, Jr. (Tom) and Dick. Dick was the younger and following the divorce of his parents, when he was six years of age, both boys lived with their mother. His mother made certain that they would

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eventually go to college by opening savings accounts, so that each quarter ($0.25), or other gifts received on special occasions, would be saved for college. Richard was a bright boy and as he grew up, he was always asking questions to everybody. This inquisitiveness must have been annoying to all, since he remembered being told to be quiet many times. He was good in many subjects in school, usually on the Honor Roll, in addition to being a good athlete. He excelled in high school basketball, track (half mile), and cross country running and played trombone in the marching band and in the orchestra. He was elected to the All-State Orchestra for Western New York. He received nine varsity letters from three different high schools by the time he graduated. The Tonawanda High School basketball team won the Western New York basketball championship his senior year and he was on the starting team, even though only 5 feet, 10 inches tall. He also played the lead roles in both the Junior (Franklin Grove, IL) and Senior High School plays.

At the age of 17, Dick attended the University of Buffalo in Buffalo, New York, eventually majoring in biology. He chose biology, because he received an “A” for a grade, while he received a “D” in chemistry (his major) in his first semester. There just didn’t seem to be enough time to study a hard chemistry course while playing basketball on the Freshman team, whereas, biology seemed more interesting and much more easier to understand. The high point of that year was to beat Niagara University, a well-renowned basketball school in those days. In his sophomore year, he received a tuition scholarship in the Biology Department under Dr. Frederick J. Holl to help prepare laboratory reagents, and so on. In the following two years, he was a teaching assistant for parasitology and also for genetics. It seemed strange to be teaching students who were four years or more older than him, since many veterans from the World War II had returned to school. Upon taking a course in bacteriology under Dr. Wilbert H. Spencer (1899–1991), he found his real interest. He enrolled in a tutorial class in bacteriology, which

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allowed him to begin to do research in the Bacteriology Laboratory at the Millard Fillmore Hospital in Buffalo. Dr. Spencer was the director of the laboratory and Dick would spend many long hours culturing anaerobic bacteria (diphtheriods) and examining them under the microscope.

Sometime during Dick’s third year, his Uncle Walt, with whom he had been living, since his mother died when he was 16, had a heart attack and became a patient at the Millard Fillmore Hospital. This allowed Dick opportunity to visit him more often than usual, since he was studying in the Bacteriology Laboratory. After a number of visits (heart patients were usually kept in the hospital for about a month in those days) he noticed a cute student nurse was taking care of Uncle Walt. Following some kidding by other patients in the ward, Dick phoned Arlene for a date to go put. One thing led to another and eventually Dick and Arlene M. Prell were married. Uncle Walt was recovered and attended the wedding (27 June 1953) in high spirit.

In the meantime, Richard obtained his B.A. degree in Biology with honor (cum laude) in 1952, and was elected to Phi Beta Kappa Society. Another important aspect of college days was the Theta Chi fraternity, Gamma Pi Chapter. Many life-long friends were developed over numerous parties and intramural athletic activities. Special memories were being “Pinned” to Arlene at the fraternity “Sweetheart Dance” and subsequently having a number of “brothers” sing the “Sweetheart of Theta Chi” at their wedding reception.

In 1952, Dick continued on to graduate studies at the University of Minnesota Medical School, Minneapolis, Minnesota, with a major in Bacteriology and Immunology (specialty was virology). During his first year of graduate school, he often corresponded with Miss Arlene Mildred Prell, who was a student nurse at the Millard Fillmore Hospital in Buffalo, New York. Upon completion of the first year of graduate studies, they were married in Buffalo on June 27, 1953. Following a 2-week honeymoon trip to Quebec City, Dick was required to return to

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graduate school and Arlene to her nursing training. A happy marriage strengthened both of their studies. Arlene graduated in September and joined Richard in Minneapolis. Dick obtained his Master of Science degree in June of 1954, and his PhD degree in March of 1958, both from the University of Minnesota under the mentorship of Dr. Jerome T. Syverton (1907–1961), who was Chairman of the Department of Microbiology and Immunology and an expert on the new field of mammalian tissue (cell) cultures and on poliovirology and cancer biology.

Graduate education in microbiology and immunology at Minnesota was rigorous, under Dr. Syverton. For instance, any person with an MD degree was required to complete the MS degree prior to advancement to the PhD degree program. The first laboratory assignment for Dick was to study the possibility of polioviruses surviving over winter in cockroaches. This meant receiving live cockroaches from sewers of Tyler, Texas in metal shipping containers during the winter (1952). Upon opening the first can, the live cockroaches jumped out of the can and disappeared into the woodwork. This was a major concern, so opinions about how to do this properly were sought. Someone suggested placing the container into the CO2 box (dry ice), to anesthetize the cockroaches and then they couldn’t scamper into the room. This was a good advice and led to placing the immobilized roaches under a dissecting microscope to recover pooled specimens of salivary glands, and intestines, respectively. The specimens were assayed for polioviruses in HeLa cells (a continuous line of human cervical epithelial cells), which had just been developed by Dr. Syverton to identify polioviruses in the test tube. To make a long story short, no live poliovirus was ever recovered from any of the cockroaches. Subsequently, others found that no animal, other than humans, provided a host system to account for the survival of polioviruses during winter.

Dick was assigned the new topic of identifying which of the coxsackieviruses would grow in HeLa cells. The coxsackieviruses had only been discovered in 1948. The assays for the 20 different coxsackieviruses (16 in Group A; four in Group B) were

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performed in newborn mice, the only available host to grow these viruses. Thus, hundreds of pregnant mice needed to be watched and cared for, since the coxsackieviruses would only grow in mice less than 24 hours of age. It required one litter of mice for each dilution of virus to be assayed. The results formed the basis of a Master’s degree thesis and were published in the Journal of Immunology (1955) (since there were no virology journals in the United States at the time).

His research required for the PhD degree explored the basis of the muscle cell destruction caused by coxsackieviruses in newborn mice. This led to studies of proteolytic enzyme activation (cathepsins) and to the early release of transaminases at intervals following infections of cells. These studies were performed with infected HeLa cells to obtain a more defined virus-cell interaction, than could be obtained using infected neonatal mice. Unfortunately, these studies were never published, although they were written and rewritten several times prior to Dr. Syverton’s death early in 1961.

All graduate students in the department were required to teach other students, as part of the curriculum for graduate training, so Dick spent many hours in the preparation of teaching laboratories and in the teaching of graduate, medical, and dental students. He also presented the introductory course in microbiology and immunology to undergraduates and the course to dental students several times during his tenure as a graduate student and as a postdoctoral student. In addition to teaching and doing research for his thesis, Dick prepared large pools of antiserums in monkeys against each of the 20 known coxsackievirus immunotypes during the summers, to earn extra money. These antiserums were then assayed for neutralizing antibodies in mice and in cell cultures and then freeze-dried in 1 mL aliquots. These serums served as reagents for distribution to other laboratories for confirmation of the large number of coxsackievirus isolates being discovered in the 1950s.

After Dick received his PhD, he obtained a postdoctoral trainee fellowship from the National Cancer Institute and continued to work in Dr. Syverton’s laboratory. In the meantime,

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he was employed as an instructor in the Department of Microbiology. He furthered his studies by looking for induction of enzyme activities during virus infection in collaboration with Samuel J. Deal, PhD, who had recently completed his training with Professor Herman C. Lichstein, PhD in the same department. Unfortunately, these studies were not published either. However, as a result of interest in chronic viral infections in culture, Dick concurrently began studies of coxsackievirus B3 infection of HeLa cells in the presence of low amounts of antibodies against the virus. He soon discovered that normal appearing cells became chronically infected with the virus. This was a serendipitous observation, since human serum, at 10% concentration, had been used to culture the human HeLa cells (considered to be a necessary requirement for human cell growth), and the pooled human serum contained antibodies to coxsackievirus B3. An extensive series of experiments were conducted to explain the phenomenon of cell survival in the presence of high numbers of infectious virus. The results revealed that virus-antibody complexes bound to the cell surfaces and blockaded the receptors from attaching new infectious virus. In fact, he showed that the persistently infected HeLa cells were resistant to superinfection by each of the five members of the group B coxsackieviruses, but they were not resistant to polioviruses. These results were reported in the Journal of Experimental Medicine, 1961, 113: 419–435, and served to initiate his future career studies of cellular receptors for the coxsackieviruses.

In 1960, Dr. Crowell, and his young family (wife and two children) moved to Ambler, Pennsylvania, when he accepted the position of Assistant Professor in the Department of Microbiology, at Hahnemann Medical College in Philadelphia, under the chairmanship of Dr. Amedeo Bondi (1912–2005). In recollection of his early days in Philadelphia, Dr. Crowell remembers: ”The 20 miles drive down the Schuylkill Expressway for the first time into Philadelphia from Ambler, our new home. Arlene turned to me with tears in her eyes and said ’what have you done to us?’

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The miles of row homes and ugly industry viewed from across the river were not exactly the views we had come to cherish in Minneapolis. With my usual optimism, I replied, ’Philadelphia is not all like that, and you will grow to like it’.”

His laboratory and office were on the third floor of the Old College Building, the one with the stone steps, well-worn from previous faculty and students climbing them for over 70 years. The building had been built in the late 1800s and eventually it was replaced with a new one. He remembered that after five years, he turned one day to Arlene and said, “we can unpack those other boxes now, because I think we are going to like it here”. Little did he realize that the dye had been cast — for he was to become “a lifer” (35 years) at Hahnemann.

Dr. Crowell had a smooth and successful academic career at Hahnemann Medical College (now called Drexel University College of Medicine), he was promoted to Associate Professor in 1964, and professor in 1971. He was the Chairman of the department (later called Department of Microbiology and Immunology) from 1979 to 1995 when he retired.

Dr. Crowell’s research interests were in medical virology. He devoted his career to studies of coxsackieviruses, which can cause severe diseases such as viral meningitis, pleurodynia, heart and skeletal muscle disease, pancreatitis, and hepatitis. He was continuously supported by a grant from the National Institute of Health (NIH) for 32 years. He and his students investigated viral replication, specific viral interference in HeLa cells, and interactions between coxsackievirus B3 and HeLa cells. He also studied the immunological changes in the cell cultures caused by group B coxsackieviruses. One of the most notable contributions of his work was the elucidation of HeLa cell plasma membrane receptor to which the group B coxsackieviruses would attach. Dr. Crowell wondered if different viral receptors on different types of cells could account for the occurrence of completely different types of diseases that were caused by certain closely related viruses. He found that large amounts of a purified virus would bind to all its corresponding receptor

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molecules on the cell surface, and saturating them. These virus-saturated cells were rendered incapable of attaching another virus that competed for the same receptors. Thereby, using virus competition, he showed that a single type of receptor molecule was shared by the three serotypes of polioviruses and that the six group B coxsackieviruses bound to a different receptor from the one which served to bind the polioviruses. He extended these findings to other species of picornaviruses and found that their receptor specificity correlated with the type of disease they caused. Polioviruses caused destruction of neurons in the spinal cord and brain stem to produce poliomyelitis, whereas the group B coxsackieviruses caused a different spectrum of diseases. Ninety serotypes of human rhinoviruses, which are the leading agents to cause the common cold, were found by others to share a common receptor that was distinctly different from those used by the other picornaviruses (the group of small RNA-containing viruses including coxsackieviruses, poliovirus, human rhinovirus, and among others.) These findings helped to prove the theory that cellular receptors are the major determinants of virus tropism. Dr. Crowell also found that the receptors for the group B coxsackieviruses could be digested off the cells by the enzyme chymotrypsin. However, the cell did not die during the process and the receptors were regenerated following protein synthesis. He proved that receptors were located on the outside surface of the plasma membrane, since chymotrypsin-treated cells which were disrupted, showed no evidence of intracellular receptor activity. All these cutting edge findings in molecular medical virology helped explain the mystery of how viruses having specificity for causing human diseases. They also helped lay the foundation for future investigations on the use of viral vectors (adenoviruses) in gene therapy, since he and coworkers found that certain adenoviruses shared the same receptor with the coxsackieviruses of group B for infection of cells.

As a result of his research, he published many papers, reviews, and book chapters. In addition, he edited three books

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entitled: Tumor Virus Infections and Immunity (University Park Press, Baltimore, Maryland, 1976); Virus Attachment and Entry Into Cells (ASM, 1986); and Innovations in Antiviral Development and the Detection of Virus Infections (Plenum Press, New York, NY, in the series of Advances of Experimental Medicine and Biology, 1992).

Dr. Crowell has been very active in medical education. He participated on many committees within the university for which he worked for. He was elected Faculty Representative to the Medical School Council for many years. He served on the Academic Affair Council, the Executive Faculty Committee, Medical School Committee, Educational Coordinating Council, Medical School Admission Council, Research Committee, and Faculty Representative to the Board of Trustee, Medical School By-Laws Committee, Committee on Academic Quality of the Graduate School, Chairman of Promotion Committee, Institutional Life Safety Committee, Medical Students Appeals Committee, and Numerous Search Committees for Chairs and Deans. He had helped build a positive reputation for the Hahnemann University School of Medicine.

Dr. Crowell also was active in academic societies. He was a charter member of the American Society for Virology. He was a member of Phi Beta Kappa Society, Society of Sigma Xi, American Association for the Advancement of Sciences, American Association of Immunologists, Society of General Microbiology (United Kingdom), and Society of Experimental Biology and Medicine. In particular, he was elected President of the Association of Medical School Microbiology Chairmen in 1986, and President of the ASM in 1991. His many contributions to these academic societies served to illustrate his outstanding skill of leadership. Within these organizations, he also served as an associate editor. He was on the editorial board of the Journal of Microbial Pathogenesis.

For over 35 years, Dr. Crowell taught many courses including: General Bacteriology, Medical Bacteriology, Dental Bacteriology, Medical Microbiology, and Virology to medical students. He

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also directed the research programs of many graduate students (17 doctoral students and 11 master students). There were numerous postdoctorates, research faculty, and visiting professors from all over the world, who studied in his laboratory. He was considered to be a very successful teacher as evidenced by the accomplishment of his students. Most of his students and postdoctorates went on to hold important positions in different organizations everywhere in the world.

Dr. Crowell took his wife and four children to Uppsala, Sweden, where he spent a sabbatical year, 1969–1970, performing research at the Wallenberg Institute with Professor Lennart C. Philipson (1929–2011). This was a pivotal year in Dick’s career, since he developed an ability to use radioactive labels in his studies of the coxsackieviruses. The collaboration with Dr. Philipson was a very rewarding one and set the stage for further progress in his research program.

Dr. Crowell was invited as a visiting professor at the University of Buenos Aires, in 1982, and at the University of Cordoba in 1992, both in Argentina. He was also a visiting scientist at the Merck Company Research Laboratories at West Point, Pennsylvania, between 1983 and 1984. In 1985, Dr. Crowell was invited to spend 15 days in China by Professor Gu, President of the Chinese Academy of Medical Sciences, Beijing. Dick delivered numerous lectures on his research and came away with many fond memories and new friends. One of his future doctoral candidates came to study at Hahnemann as a result of that trip.

Dr. Crowell has received many honors. He was a Research Career Development Awardee of the National Institute of Allergy and Infectious Diseases (1962–1972); a recipient of the Christian A. and Mary F. Lindback Foundation Award for Teaching at the Hahnemann Medical College (1967–1968); a Councilor for Eastern Pennsylvania Branch of the ASM (1970–1972), and then the Vice President of the branch in 1972–1973 and President 1973–1975. He served as a Diplomat of the American Academy of Microbiology since 1967; a consultant to

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the National Cancer Institute, Virus-Tissue Resources Branch (1966–1968). He was elected an Associate Member of the Hahnemann Alumni Association, 1973; an Honorary Member of the Premedical Honor Society, Alpha Epsilon Delta (Pennsylvania Zeta Chapter, Villanova University) (1985); an Honorary Member of Argentina Society for Virology in 1982. He also served as a consultant to Smith Kline Corporation (1975–1977), and to Lehn and Fink Products, Inc. (1976–1980); on the Nominating Committee of the American Society for Virology (1987–1988), and an Associate Editor of the Journal of Microbial Pathogenesis since 1986. He was also invited as an Alberta Heritage Foundation Visiting Professor, University of Calgary, Canada from 1987 to 1988; and as a Panel Member of the National Research Council’s Associateship Program from 1988 to 1995. He also served as a member of the Biosafety Committee for the Merck Company since 1989. Of course, the most well-known honors were his elections as President of the Association of Medical School Microbiology Chairmen in 1986 and as President of the ASM, which has more than 40,000 members. After his retirement, Dr. Crowell still served as a consultant to Potomac Institute for Policy Studies, Arlington, Virginia 1996–1998.

Dr. Crowell has been an organizer and cochairman for numerous sessions of the national and international scientific meetings. He has been a speaker for more than 120 times in different national, international, or regional scientific meetings and seminars. For example, a more recent important speech was given at the annual meeting of the Argentine Society for Microbiology in Cordoba, Argentina, in October, 1992, where he was the featured speaker.

Dr. Crowell is an optimistic, responsible, hardworking, and happy man. His pleasant personality makes him welcome in various organizations. His responsible and hardworking nature wins him the trust of his colleagues, students, and friends. His wonderful marriage also helped him achieve success. Arlene always stood beside him and supported his endeavors. They

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raised four children: Steven R, born in 1956; Kathleen M., born in 1958; Barbara L., born in 1962; and Wendy J., born in 1964. They all have successful stories of their own, including the production of eight grandchildren. After his formal retirement from the MCP-Hahnemann University, they are still happily living in the same home in Ambler, Pennsylvania. They enjoyed traveling in their 21 foot conversion campervan, which has taken them to various places in the United States and Canada. They have previously also traveled extensively abroad. Dr. Crowell is still active in consulting, attending scientific meetings, and giving speeches in various occasions. One of the authors, K. T. Chung had the honor to meet with him in a scientific meeting. Even not knowing him well previously, this author could feel the infectious sincerity and responsible demeanor of Dr. Crowell, which undoubtedly has contributed to his successful career.

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Chapter 57

Peter Charles Doherty (1940 to Present): Pioneer of Immunology

Source: http://www.royalsoc.org.au/membership/dist_fellows.htm(US Public Domain image)

The Nobel Prize in Physiology or Medicine in 1996 was awarded to Drs. Peter Charles Doherty and Rolf M. Zinkernagel (1944 to present) for their work on how the human immune system recognizes viral infected cells and then targeted them for destruction. Their discovery has led to a new understanding of

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organ rejection after being transferred, and a better compre-hension of genetic susceptibility to diseases and a new approach for vaccines.

Peter was born on 15 October 1940 in Brisbane, Australia. His father is Eric C. Doherty and mother Linda Byford. He has a young brother Ian. They were greatly influenced by their grandmothers from both their father’s and their mother’s sides. One grandmother was a devout Methodist, and the other was a lapsed Quaker who was born in England. They were from County, Lancashire, and Essex, England, respectively, and migrated to Australia around 1840.

Eric Doherty was a telephone mechanic and an administra-tor involved in the planning of telephone services. Eric had a strong desire to learn but it was not possible because of the financial circumstances. Eric’s father died of pneumonia during the 1919 influenza epidemic, and his wife (Peter’s grand-mother) was left in a poor financial condition. Peter’s mother, Linda Byford, was a piano teacher and spent much of her youth on the tennis court. After marriage, she played Chopin, Debussy, and Beethoven for her children. One of her brothers (Peter’s uncles) died in Singapore when Singapore was captured by the Japanese during World War II, and her other brother contracted malaria during the fighting in New Guinea. Their deaths had a great influence on Peter, who grew to resent wars and infectious diseases.

Young Peter did not acclimate to the outdoor Australian way of life because his skin was fair and was highly susceptible to skin cancer. Instead, he loves to read. He became addicted to reading anything and everything. He inherited his mother’s talent for classical music. He enjoyed going to the beach at least 3 weeks per year with his close relatives and enjoyed bushes and the Pacific coast. He also did fishing in the surf at night. He liked to work as a carpenter at his father’s workshop. He was good at it, and once was successful in manufacturing some very substantial coffee tables. He also enjoyed working on houses as a carpenter. One of his ambitions in his teenage

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years was the construction of a photographic enlarger and darkroom. Unfortunately, his father died in 1961 and their house was sold.

Peter rode a steam train to attend the local public schools and a Methodist Church. In school, he played basketball, and was a sergeant in the army cadet corps. He was good in physics and chemistry, but was more interested in literature and his-tory. He read the works of Aldous Huxley (1894–1962), Jean-Paul Sartre (1905–1980), and Ernest Hemingway (1899–1961). He was somewhat influenced by one of his senior cousins Dr. Ralph Doherty who was to become a leading viral epidemiolo-gist. When Peter was 17, on the first day of school, he attended the University Veterinary School; Peter visited the campus and was impressed. After that, he decided to study veterinary medi-cine. Since he grew up without any money, he intended to get a reasonably good job after graduation. He also thought that a man of action was more realistic than a philosopher.

So, Peter attended the University of Queensland and majored in veterinary medicine. He took many basic science courses. Therefore, he had a good science background. He was intro-duced to the principles of ecology in the first year of zoology. He was fascinated by the course, and almost wanted to switch his major. However, he was also impressed by “infectious diseases” taught by Dr. John Francis, and “immunology” taught by the parasitologist, Dr. John Frederick Adrian Sprent (1915–2010). He also liked the “population genetics” taught by Dr. Glenorchy McBride (1925–1990). He read Sir Frank MacFarlane Burnet (1899–1985) and R. M. Stanley’s books on viruses, and some of Burnet’s writings on immunology and cancer. He was impressed with Dr. Burnet’s teleological Darwinism, the idea that the body is a set of ecosystems and the realization that a good science involves quantitative measurement. In his final year of under-graduate studies, he wrote a thesis on the UV-induced squa-mous eye cell carcinoma of Hereford cattle. He obtained his bachelors of veterinary science in 1962.

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When he graduated, he worked in the Queensland Department of Agriculture and Stock. He spent several months surveying cattle for various venereal diseases and found that Trichomoniasis was present in an area where it was thought to be completely eradicated. Later, he worked at the state veteri-nary laboratory of the Animal Research Institute (ARI), in Yeerongpilly on the epidemiology of bovine leptospirosis. Good luck followed him; he met a very attractive young girl, Penelope Stephens, at the ARI. He fell in love with her and married her in 1965. In 1966, he obtained his Master degree of veterinary science. He published several research papers on leptospirosis.

During this period of time, Peter was sent by Dr. Les Newton, Director of the ARI to Melbourne, where he worked with Dr. Toby St. George in the Commonwealth Scientific and Industrial Research Organization (CSIRO) Laboratory of Dr. E. L. French. He also spent time in the virology group at the Commonwealth Serum Laboratory and visited F. J. Fenner’s Department of Microbiology at the John Curtin School of Medical Research (JCSMR), Canberra. Peter’s intention was to meet Dr. Cedric A. Mims (1924–), who impressed Peter by his thought on viral pathogenesis. Upon returning to Brisbane, Peter realized that his interest in life was to become an experimentalist, not a diagnostic virologist, and he decided to pursue a PhD degree.

Peter initially intended to work in Dr. Mims’ laboratory and pursued the PhD degree under his direction. However, Dr. Mims’ laboratory did not have a vacancy at that time. He was recommended by Dr. J. A. Roberts, who had recently returned from Cornell University; he responded to an adver-tisement from the Department of Experimental Pathology at the Moredun Research Institute, which is affiliated with the University of Edinburgh, Scotland. Peter got that job and sailed for Britain in early 1967 with his wife.

At the Moredun Research Institute, Peter did research and helped run the diagnostic neuropathology program for the Scottish Veterinary Investigation Service. He learned

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neuropathology from the head of the department, Dr. R. M. Barlow, and from Hugh Fraser who was doing research with Alan Dickinson defining the genetics of the scrapie mouse model. Dr. Barlow also taught Peter to write good English.

He was working with the tick borne flavivirus, louping-ill virus, which was considered to cause problem for the vaccine which was developed at the Moredun many years ago. He developed a close collaboration with a young veterinary gradu-ate, Mr. Hugh W. Reid. Mr. Reid did the virological and serologi-cal aspects of the ensuing experiments, whereas Peter focused on the light and ultra-structural pathology related to louping-ill disease. Peter did some inadvertent human experiments. He injected himself in the finger with the louping-ill virus, and Reid helped him to test the presence of the virus and titers of antibody in Peter’s blood.

Peter’s wife Penny also had a research job. She worked with E. C. R. Reeve at the Institute for Animal Genetics until the birth of their first child, James, in 1968. Their second child, Michael, was born in 1970. They seemed to enjoy living in Edinburgh. The Festival and Traverse Theater were their favorites. They also arranged vacations to Europe, including Scandinavia and Stockholm in their Volkswagen van. They also attended the conferences related to virology and neuropathology. Most of all, Peter enjoyed the outdoor activities without worrying about getting sunburn since the climate was just right for him.

Peter was also enrolled as a part-time graduate student at the University of Edinburgh Medical School. The research collaboration with Mr. Reid had resulted in publications of nine research papers. His PhD degree in pathology was granted in 1970. They almost stayed in Edinburgh permanently.

In the December of 1971, Dr. Doherty left the permanent position at the Moredun to accept a postdoctoral fellowship in the Department of Microbiology, JCSMR in Australian National University. He intended to learn basic immunology, and return his research to veterinary science. He also wished to work with the renown scientist, Dr. Cedric Mims. It turned out that

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Dr. Mims decided to take the chair-ship in Microbiology at Guy’s Hospital Medical School. So they overlapped by 6 months or so, they had never formally worked together.

At JCSMR, he first studied the pathogenicity of Semliki Forest virus infection in the mouse, then switched to the lym-phocytic choriomeningitis virus (LCMV) model for immuno-logical analysis. He met Dr. Rolf Martin Zinkernagel (1944–) in 1973, and established a close collaboration with him. During this period, he had a breakthrough discovery on the major histocompatibility complex (MHC) restriction. He also got acquainted with many world renown scientists, including Dr. Bob Gerdes among others.

It was also during this period of time that Dr. Doherty worked out “The single T-cell receptor-altered-self” hypothesis, which was a big breakthrough in the understanding of how self-cells were different from the non self-cells in the T cell-mediated immunity. Dr. Doherty presented his findings at the Second International Immunity Meeting in Brighton, United Kingdom. He was also invited to give talks in about 20 institu-tions in the United States. Among his hosts were Dr. Allan Rosenthal at the NIH and Dr. David Katz at Harvard University. He also gave talks to a number of institutions in England. He even traveled to Stockholm to speak to Drs. Goran (b. 1936) and Erna Moller’s groups at the Wallenberg Laboratory in Lilla Frescati. However, he could not find a permanent position at the JCSMR. Dr. Hilary Koprowski (1916–2013), at the Wistar Institute called on him and offered him an associate profes-sorship at the Wistar Institute. He and his family moved to Philadelphia, Pennsylvania in 1975.

At the Wistar Institute, Dr. Doherty was deeply involved with the Immunology Graduate Group headed by Drs. Darcy Wilson and Norman Klinman at the University of Pennsylvania. He did some research in collaboration with Dr. Walter Gerhard on the virology of influenza, and also collaborated with the late Dr. Wiktor in Dr. Hilary Kaprowski’s research program on immu-nology of rabies. He also participated in the research program

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on sclerosis. Dr. Doherty stayed at the Wistar Institute for seven years (1975–1982), and became an established scientist. He was well known within the eminent immunology group. At the same time, Dr. Doherty’s wife, Penelope, went back to school and developed a new career in the area of drug information. Dr. Doherty was busy engaging research activities, wrote pro-posals, and other scientific endeavors. It seemed that every-thing was going well.

In 1982, things changed. Dr. Doherty had a strong compas-sion toward Australia, and a position opened at the JCSMR, Australia. He decided to accept the appointment as the head of the Department of Experimental Pathology at JCSMR with the intention to build a vital research program. However, things were disappointment for him. Dr. Doherty felt that he spent too much time on nonproductive matters and the situation hopeless. Nevertheless, he felt there were some productive things. He had a good interaction with Dr. Jane Allan and Rhodric Ceredig. In general, from 1982 to 1988, there was a low period in his life and he decided to move back to the scien-tific world.

In 1988, Dr. Doherty moved to Memphis, Tennessee, to work at the St. Jude Children’s Research Hospital (STCRH). This move was engineered by Dr. Alan Granoff of the STCRH, whom he met in 1974, and Dr. Rob Webster who was a close colleague of the JCSMR virologist, Dr. Graeme Laver. Dr. Doherty was offered the position as the Michael F. Tamer Chair of Biomedical Research and Chairman of the department of immunology by Dr. J.V. Simone, then the Director of STCRH. Dr. Doherty was given the resources and environment to devote his research. The research hospital is a superb, open, research environment that is extremely well funded. The problems he had since then were too much sunshine and being too far from the Pacific Ocean.

A lot of good research has been conducted since then, he was also appointed as an Adjunct Professor in the Department of Pathology and Pediatrics, University of Tennessee. Over the

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years, he was mentor for a good number of graduate students and postdoctorals. The most notable one is Dr. Rolf Martin Zinkernagel, who shared with him the Nobel Prize in 1996. Dr. Zinkernagel is now Professor of Experimental Biology at the University of Zurich, Switzerland. Others include Dr. R. B. Effros, J. R. Bennink, N. Greenspan, D. Schwartz, N. Bowern, J. Dixon, Z. Tabi, M. F. Uren, and X. Y. Mo. All of them received Dr. Doherty’s training at Australian National University, Wistar Institute, or at the STCRH, and now have their own successful stories. He has published more than 300 research papers, reviews, and chapters in books up to 2001 when this article is written. He has a very sharp mind and is a very efficient writer. His work on the cell-mediated immunity in viral infections is a milestone contribution to science and human health.

Honors and awards were flooded upon him. He was awarded an honorary Doctorate of Sciences from the University of Queensland, Australia, a year before the Nobel Prize. Since then, he was bestowed the Honorary Doctor of Science from Australian National University (1996); University of Edinburgh, Scotland (1997); Tuft University, Medford, Massachusetts (1997); Rhodes College, Memphis, Tennessee (1998); Kyorin University, Japan (1998); Warsaw Agricultural University, Warsaw, Poland (1998); Latrobe University, Melbourne, Australia (1999); Imperial College, University of London, United Kingdom (2000); Autonomous University of Barcelona, Spain (2000); North Carolina State University, Raleigh-Durham, North Carolina (2000); University of Guelph, Canada (2001); and University of Pennsylvania, Philadelphia, Pennsylvania (2001). From 1998 to 2001, he was elected Professorial Fellow, Eminent Scholars Scheme for the University, Melbourne, Department of Microbiology and Immunology, Australia. He also received many honors and awards. In 1983, he was awarded with a Fellowship from the Australian Academy of Science and Paul Ehrlich Prize, Germany. He received Gairdner International Award for Medical Science, Canada in 1986, was elected Fellow of the Royal Society of London in 1987, and received the Albert Lasker Basic Medical

b2806_Ch-57.indd 557 28-Jul-17 10:12:02 AM


558


Research Award in the United States in 1995. In 1997, he was honored Companion of the Order of Australia. In 1998, he was elected Foreign Associate of the National Academy of Sciences, United States, and was awarded an Honorary Fellowship of the Royal Australian College of Pathologists and Royal Australian College of Physicians.

Dr. Doherty has also tirelessly served as a consultant for many organizations. At present, he is on the Scientific Review Board of the Howard Hughes Medical Institute, the Scientific Board of the Welcome Foundation, and the Scientific Council of the Gairdner International Awards for Biomedical Research.

Dr. Doherty’s research career has been very unconventional. He never had a powerful mentor, so every piece of research came from his own ideas. Ideas interest him. He also loves com-plexity and the unexpected. This may in part stem from his nonconformist upbringing. He has been self-taught and started his independent research when he was 22 years old. He has a sense of being something of an outsider to look for different perceptions in everything from novels, to art, and to experi-mental results. He was influenced by his reading of Arthur Koestler, Edward de Bono, writings of Karl Popper, and Thomas Kuhn, and so on. He would be a very good writer if he was to choose to do so. He has a strong compassion toward his native Australia. He attempted to return to Australia twice. Even now, as he is in the service of science and others, Australia is always on his mind.

One of the authors, K. T. Chung has the honor to visit him in his office, and find him to be a very humble gentleman. He is very independent in his thinking. Intellectually, he marches to the beat of his own drum, and does not like competition. There are too few people working in the area of viral patho-genesis and immunity, there is very little funding, there are many problems, and there is little time. This author would agree with him that everything he said except that he has been extremely well funded for his work.

b2806_Ch-57.indd 558 28-Jul-17 10:12:02 AM


559


Dr. Doherty and his wife Penelope happily live in Memphis, Tennessee. He spends most of his leisure time in skiing, walking, and reading. He is a readaholic. After receiving the Nobel Prize Award, he travels much more extensively than before, but he is still conducting research, directing students, serving as a consultant, and enjoying life.

b2806_Ch-57.indd 559 28-Jul-17 10:12:02 AM


b2530_FM.indd 6 01-Sep-16 11:03:06 AM


561


Name Index

Adolf Hitler (1889–1945), 319Adolf Mayer (1843–1942), 147Adolf von Behring (1854–1917),

115Adrian M. Srb (1917–1997), 390Albert Calmette (1863–1933),

172, 240Albert Einstein (1879–1955), 275Albert Jan Kluyver (1888–1956),

144, 350, 399, 411, 431Albert J. Riker (1894–1982), 343Albert Neisser (1855–1916), 100Albert Schatz (1920–2005), 448Aldo Castellani (1877–1971), 191Alessandro Volta (1745–1827),

32Alexander A. Inostrantzev

(1843–1920), 198Alexander Braun (1805–1877),

71, 85Alexander Fleming (1881–1955),

372, 446, 493Alexander Gordon (1752–1799),

37Alexander K. Butlerov

(1857–1861), 198Alexander Kovalevsky

(1840–1901), 124

Alexandre Yersin (1863–1943), 170, 218

Alfred Fischer (1858–1913), 185Alfred Hershey (1908–1997), 287Alfred H. Sturtevant

(1891–1970), 385Alfred Tennyson (1809–1892),

184Alhazen (965–1039 AD), 2Alice C. Evans (1881–1975), 234Almroth E. Wright (1861–1947),

299Alphonse Dochez (1882–1964),

292Alphonse Laveran (1845–1922),

169Alphonse R. Dochez

(1882–1964), 286Amédée Borrel (1867–1936),

171, 240, 246Amedeo Bondi (1912–2005), 543Anatole France (1844–1924), 351Andre Maurois (1885–1967), 308Andrew Carnegie (1835–1919),

116Andrew J. Moyer (1899–1959),

304Antoine Balard (1802–1876), 52

b2806_Name Index.indd 561 28-Jul-17 10:12:35 AM


562


Antonia Scarpa (1752–1832), 32Antonie van Leeuwenhoek

(1632–1723), 11, 15Antonio Vallisneri (1661–1730),

16Anton Maria Schyrleus de Rheita

(1597–1660), 3Archibald E. Garrod

(1857–1936), 388, 401Archimedes (287–212 BC), 2Arie J. HaageSmit (1900–1977),

386, 400Aristides Agramonte

(1868–1931), 162Arnold Demain (1927–), 433Arthur Balfour (1848–1930), 273Arthur Eastwood (1869–1936),

290Arthur Kornberg (1918–2007),

416Auguste Rodin (1840–1917), 124Augustin Bistrzycki (1862–1936),

272August Strindberg (1849–1912),

351August von Wassermann

(1866–1925), 218

Barbara McClintock (1902–1992), 140, 384

Bartolomeo Gosio (1863–1944), 297

Beketov A. S. Famintzin (1815–1881), 198

Benjamin E. Volcani (1915–1999), 431

Benjamin Jesty (1736–1816), 22Bernard Horecker (1914–2010),

529

Bernard Ogilvie Dodge (1872–1960), 387, 400

Berthold Feiwel (1875–1937), 273

Bjornstijerne Bjornson (1832–1910), 102

Boris Ephrussi (1901–1979), 385Bor Shiun Luh (1916–2001), 433Bruce Ames (1928 to present),

403Bruce Buchnan (1936–), 490Bugie (1921–2001), 448Burt G. Wilder (1841–1925), 225

Calvin Bridges (1889–1938), 385Camille Guerin (1872–1961), 247Camillo Golgi (1843–1926), 136Carl Claus (1835–1899), 127Carl C. Lindegren (1896–1986),

385, 400Carl Djerassi (1923–2015), 490Carl Fraenkel (1861–1915), 115Carl Libermann (1842–1914), 272Carl Lucas Alsberg (1877–1940),

297Carlos Finlay (1833–1915), 161Carl R. Woese (1928–2012), 468Carl Ten Broeck (1885–1966), 233Carl von Nägeli (1817–1891), 86,

199Carl von Rokitansky

(1804–1878), 38Carl Wielhelm Boeck

(1808–1875), 99Carl Woese (1928–2012), 482,

512Carl Zeiss (1816–1888), 112Casimir Davaine (1812–1882),

109


Name Index

563


Cedric A. Mims (1924–), 553Charles Bouchard (1837–1915),

130Charles Cagniard de la Tour

(1777–1859), 53Charles Chamberland

(1851–1908), 169Charles Clifton (1904–1976), 431Charles Darwin (1809–1882), 8,

359Charles Dickens (1812–1870),

184Charles Edouard Chamberland

(1851–1908), 58Charles Eliot (1834–1926), 230Charles F. Wheeler (1842–1910),

184Charles Nicolle (1866–1936), 128Charles Robert Richet

(1850–1935), 231Charles Thom (1872–1956), 303Charles V. Taylor (1895–1946),

410Chester S. Keefer (1897–1972),

304Christian Ehrenberg

(1795–1876), 71Christian Gottfried Ehrenberg

(1795–1876), 85Christian Nees von Esenbeck

(1776–1858), 70Christopher Columbus

(1451–1506), 3Christopher Wren (1630–1723),

11Claudius Ptolemy (100–170 AD),

2Clifford Dobell (1886–1949), 12

Colin Munro MacLeod (1909–1972), 286, 292, 401, 488

Cornelis B. van Niel (1897–1985), 144, 254, 313, 402, 410, 432, 467, 509

Daniel Elmer Salmon (1850–1914), 226

Daniel J. O’Kane (1919–2007), 466David Bruce (1855–1931), 138David Hendlin (1920–2006), 520David Livingstone (1813–1873),

191, 359David Lloyd George

(1863–1945), 274David Nobarro (1874–1958), 192Dean B. Cowie (1913–1977), 491Dennis R. Hoagland

(1884–1949), 412Dmitri I. Mendeleev

(1834–1907), 198

Edmond Joseph Delwiche (1874–1950), 343

Edmond Nocard (1850–1903), 170

Edvard Grieg (1843–1907), 102Edward A. Adelberg

(1920–2009), 412Edward Culpeper (1660–1738), 8Edward Jenner (1749–1823), 39,

58Edward Lawrie Tatum

(1909–1975), 382, 473, 486, 529

Edwin B. Fred (1887–1981), 342, 399



564


Edwin Broun (1887–1981), 512Edwin G. Hastings (1872–1953),

332, 342Edwin Michael Foster

(1917–2013), 335Edwin Ray Lankester

(1847–1929), 123Ehrlich (1854–1915), 225Eilhard Mitscherich (1794–1863),

52Eilhard Mitscherlich

(1794–1863), 71Élie Metchnikoff (1845–1916),

117, 171, 181, 203, 244, 261Emil Christian Hansen (1842–

1909), 148Emil M. Mrak (1901–1987), 431,

519Emil von Behring (1854–1917),

129, 215, 245Emile Duclaux (1840–1904), 168,

187Emile Zola (1840–1902), 351Erich Hoffman (1868–1959), 131,

171Ernest G. Anderson (1891–1973),

385Ernst Abbe (1840–1905), 8, 112Ernst A. Ruska (1906–1988), 8Ernst Boris Chain (1906–1979),

304, 493Ernst Gale (1914–2005), 459Ernst Haeckel (1834–1919), 125Erwin F. Smith (1854–1927), 96Esmond E. Snell (1914–2003),

399Etienne Burnet (1873–1960), 122Eugene Haagen (1898–1972),

365

Felix-Etienne-Pierre Mesnil (1868–1938), 138

Ferdinand Julius Cohn (1828–1898), 111, 199

Ferdinand Magellan (1480–1521), 3

Ferdinand Ritter von Hebra (1816–1880), 38, 43

Florence Nightingale (1820–1910), 39

Franceso Redi (1626–1697), 16Francisco Pizarro (1471 or

1476–1541), 21Francis Crick (1916–2004), 378Francis Drake (1540–1596), 3Francis H. Crick (1916–2004),

282, 388, 529Francis J. Ryan (1916–1963), 403,

487François Jacob (1920–2013), 380,

529Franklin Delano Roosevelt

(1882–1945), 306, 367Franklin D. Keim (1885–1956),

383Frank MacFarlane Burnet

(1899–1985), 488, 552Fred Neufeld (1869–1945), 292Frederick W. Fabian

(1888–1963), 518Friedrich August Johannes

Loeffler (1852–1915), 62, 112, 170

Friedrich Gustav Jakob Henle (1809–1885), 34, 107, 123

Friedrich Wilhelm Scanzoni von Lichtenfels (1821–1891), 43

Friedrich W. Nietzsche (1844–1900), 351


Name Index

565


Frits H. Went (1903–1990), 528Frits Zernike (1888–1966), 8Fritz A. Lipmann (1899–1986),

414Fritz Kogl (1897–1959), 399Fritz Mietzsch (1896–1958), 318Fritz Muller (1821–1897), 124Fritz R. Schaudinn (1871–1906),

171Fritz Schaudinn (1871–1906), 131Fyodor M. Dostoyevsky

(1821–1881), 122

Galileo Galilei (1564–1642), 3, 16, 38

Georg Meissner (1829–1905), 107

Georg Theodor August Gaffky (1850–1918), 112

George Beadle (1903–1989), 486George Bernard Shaw

(1856–1950), 351George F. Sprague (1902–1998),

384George Fresenius (1808–1866),

85George Sternberg (1838–1915),

162George W. Beadle (1903–1989),

398, 473, 529Georges Leclerc count de Buffon

(1707–1788), 17Gerhard Domagk (1895–1964),

303Gerhard Gottschalk (1935–), 415Gerrit van Iterson (1878–1972),

310Gerrit van Iterson, Jr.

(1878–1972), 149, 351

Gijsbertus van Iterson (1878–1970), 144

Giordano da Rivalta (1260–1311), 2

Giovanni Faber (or Johann Faber) (1574–1629), 4

Giovanni Rasori (1766–1837), 32Giuseppe Sanarelli (1864–1940),

161Glenorchy McBride (1925–1990),

552Gregor Johann Mendel

(1822–1884), 38, 375

Halvor O. Halvorson (1897–1975), 467

Hans Lippershey (1570–1619), 3Hans Molisch (1856–1937), 310Hans Zinsser (1878–1940), 234,

361Harland G. Wood (1907–1991),

399Harold Boyd Woodruff

(1917–2017), 520Harold J. Conn (1886–1975), 209Harriett Ephrussi-Taylor

(1918–1968), 286Harry Emerson Fosdick

(1878–1969), 283Harry G. Jopson (1911–2012),

465Harry Sinclair Lewis (1885–1951),

325Harry Truman (1884–1972), 277Harry W. Graybill (1875–1938),

232Hector Cameron (1843–1928), 78Heinrich Anton de Bary

(1831–1888), 35, 148, 198



566


Heinrich Göppert (1800–1884), 70

Hendrick van Swinden (1746–1823), 2

Henrik Ibsen (1828–1906), 102, 351

Henrique da Rocha-Lima (1879–1956), 269

Henry Cline (1750–1827), 28Henry Longfellow (1807–1882),

184Herman J. Phaff (1913–2001),

519Hernando Cortes (1485–1547),

21Herschel K. Mitchell

(1913–2000), 529Hilary Koprowski (1916–2013),

555Hippolyte Taines (1828–1893),

370Horace A. Barker (1907–2000),

432, 467Howard Ricketts (1871–1910),

263Howard Walter Florey

(1898–1968), 304, 447, 493Hsien-Wen Li (1902–1976), 384Hugo Marie de Vries

(1848–1935), 147Hugo von Mohl (1805–1872), 71,

86

Ignaz Semmelweis (1818–1865), 57, 76

Ira L. Baldwin (1895–1999), 210Irwin C. Gunsalus (1912–2008),

467Isidore Straus (1845–1898), 170

Ivan Petrovich Pavlov (1849–1936), 203

Jack N. P. Davies (1915–1998), 192

Jack Schultz (1904–1971), 385Jackson W. Foster (1914–1966),

448Jacob Lipman (1874–1939), 445Jacobus Metius (1571–1631), 2Jacques de Kadt (1897–1988),

351Jacques Monod (1910–1976),

379Jakob Kolletschka (1803–1847),

41James Carroll (1854–1907), 162James Cook (1728–1779), 24James D. Watson (1928–), 282,

378, 388James Hutton (1726–1797), 8James Simpson (1811–1870), 45,

78James Symes (1799–1870), 77Jean-Baptiste Biot (1774–1862),

52Jean- Baptiste Dumas

(1800–1884), 51Jerome T. Syverton (1907–1961),

541Jesse W. Lazear (1866–1900), 162J. H. van ’t Hoff Jr. (1852–1911),

146Joan Bennett (1942–), 152Johan Lukas Schoneleion

(1793–1864), 34Johann Klein (1788–1856), 39Johannes Muller (1801–1858),

71, 85


Name Index

567


John B. Haldane (1892–1964), 388John Desmond Bernal

(1901–1971), 12John Etchells (1909–1981), 518John Frederick Adrian Sprent

(1915–2010), 552John N. Wolfe (1910–1974), 506John Needham (1713–1781), 17John P. Altgeld (1847–1902), 94John Shaw Billings (1838–1913),

159John W. Powell (1834–1902), 92Jonathan Burrill (1839–1916), 185Josef Skoda (1805–1881), 38Joseph A. Sewall (1830–1917), 91Joseph Banks (1743–1820), 24Joseph C. Hutchison

(1822–1887), 157Joseph Dutton (1874–1905), 192Joseph Klarer (1898–1953), 318Joseph Lawrence (1836–1909),

81Joseph Lister (1827–1912), 39,

57, 113, 140, 173, 190Joseph Meister (1876–1940), 119Joshua Lederberg (1925–2008),

382, 398, 472Josiah Clark Nott (1804–1873),

161Jules Bordet (1870–1961), 128,

222Jules Joubert (1834–1910), 58,

169Julius Friedrich Cohnheim

(1839–1884), 111

Karl Ewald Hasse (1810–1902), 107

Karl F. Kesseler (1815–1881), 198

Karl J. Belar (1895–1931), 385Karl Kunth (1788–1850), 71Karl Landsteiner (1868–1943),

222Karl Pfister (1919–), 520Karl Rudolf Leuckart

(1822–1898), 123Karl von Siebold (1804–1885),

123Karl Wilhelm von Nageli

(1817–1891), 71Kenneth V. Thimann

(1904–1997), 386, 528Kitasato Shibasaburo

(1853–1931), 115, 129, 176, 239, 245

Kiyoshi Shiga (1871–1957), 218

Lajos Markusovszky (1815–1893), 40

Laura Bassi (1711–1778), 16Laurence Graber (1887–1977),

343Lazzaro Spallanzani

(1729–1799), 32L.E. Den Dooren de Jong

(1890–1980), 147Leland S. McClung (1910–2000),

509Leonardo da Vinci (1452–1519),

38Leo Tolstoy (1828–1910), 131LeRoy Abrams (1874–1956), 409Lien-Teh Wu (1879–1960), 219Linus Pauling (1901–1994), 386Lister O. Krampitz (1909–1993),

508Lloyd G. Stevenson (1918–1988),

175



568


Lois Gold (1941–2012), 533Lore A. Rogers (1875–1975), 334Louis Ferdinand Thuillier

(1856–1883), 61, 119, 170Louis Herbert Manceaux

(1865–1934), 264Louis Joblot (1645–1723), 17Louis Kahlenberg (1870–1941),

334Louis Martin (1864–1946), 171Louis Pasteur (1822–1895), 19,

21, 35, 70, 107, 122, 140, 144, 168, 185, 190, 196, 238, 244, 261, 371, 376, 428, 494, 523

Lourens G. M. Baas-Becking (1895–1963), 352

Ludvig Hektoen (1863–1951), 266

Maclyn McCarty (1911–2005), 286, 292, 401, 488

Marcus Rhoades (1903–1991), 384Martha Chase (1927–2003), 287Martin Buber (1878–1965), 273Martin D. Kamen (1913–2002),

413Martinus W. Beijerinck

(1851–1931), 62, 74, 196, 350Marvin Johnson (1906–1982),

343Marvin P. Bryant (1925–2000),

468Mary Wortley Montagu

(1689–1762), 25Matthias Schleiden

(1804–1881), 71Maurice Wilkins (1916–2004),

378, 388

Max Knoll (1897–1969), 8Max Schultze (1825–1874), 71Max Theiter (1899–1972), 164Max Tishler (1906–1989), 446Maynard Alexander Joslyn

(1904–1984), 412, 431Meyer J. (Mike) Wolin, 481Michael Doudoroff (1911–1975),

412, 432Michael Heidelberger

(1888–1991), 286Mikhail S. Voronin (1838–1903),

198Miles J. Berkeley (1803–1889),

34, 86Minoru Yoneyama (1924–), 435Morris Solotorovsky

(1913–1992), 494Moshe Shifrine (1928–), 433

Nancy N. Gerber (1929–1985), 493

Napoleon (1769–1821), 29, 50Nehemiah Grew (1641–1712), 6Nero (37–68 AD), 42Nicolas Louis Sohngen

(1878–1934), 411Nicolaus Copernicus

(1473–1543), 3, 38Nikolai A. Menshutkin

(1842–1907), 198Nikolai Gamaleia (1859–1949),

127Nils T. E. Fries (1912–1994), 399Norman Heatley (1911–2004),

447Norton D. Zinder (1928–2012),

489


Name Index

569


Oliver Wendell Holmes (1809–1894), 37

Oscar N. Allen (1905–1976), 343Oswald T. Avery (1877–1955),

292, 401, 488Otto Kandler (1920–), 469

Patrick Manson (1844–1922), 138, 191, 243

Paul Clarke, 343Paul de Kruif (1890–1971), 12, 15Paul Ehrlich (1854–1915), 111,

131, 178, 301, 323Paul Frosch (1860–1928), 62Pauline Gee (1953–), 532Perry Wilson (1902–1981), 343Phillip Gerhardt (1921–2008),

343Pierre Allorge (1891–1944), 494Pierre Borel (1620–1671), 3Pierre Chassaignac (1804–1879),

81Pierre Dangeard (1895–1970),

495Pierre Emile Duclaux

(1840–1909), 138Pierre-Marie-Alexis Millardet

(1838–1902), 89Pierre Paul Emile Roux (1853–

1933), 58, 128, 138, 180, 238, 244, 261

Ralph Wolfe (1921–), 415, 480Ray Lankester (1847–1929), 192Rebecca Craighill Lancefield

(1895–1981), 286Reese H. Vaughn (1908–1988),

412

Regnier de Graaf (1641–1673), 5René Jules Dubos (1901–1982),

286, 493Rene Pomerleau (1904–1993), 495Rhazes (865–925), 21Richard Friedrich Johannes

Pfeiffer (1858–1945), 115Robert Boyle (1627–1691), 11Robert E. Hungate (1906–2004),

355, 410, 477Robert G. Martin (1935–), 530Robert Hooke (1635–1703), 6Robert Johnson (1845–1910), 82Robert Koch (1843–1910), 19, 21,

35, 37, 57, 70, 122, 140, 144, 176, 190, 196, 215, 225, 238, 239, 428

Robert Lyman Starkey (1899–1991), 431, 446, 496

Robert P. Hanson (1918–1987), 422

Roger Bacon (1219–1292), 2Roger Y. Stanier (1916–1982),

354, 412Rolf Martin Zinkernagel (1944–),

555Rollins A. Emerson (1873–1947),

384Ronald Ross (1857–1932), 138,

325Rudolf Virchow (1821–1901), 45,

115, 127Rufus Cole (1872–1966), 285

Sachi Sri Kantha (1953–), 221Salvador E. Luria (1912–1991),

467Salvino d’Armati (1258–1317), 2



570


Sam Ruben (1913–1943), 413Samuel Molyneux (1689–1728), 6Sara Josephine Baker

(1873–1945), 37Selman A. Waksman

(1888–1973), 198, 371, 445, 493, 522

Sergei N. Winogradsky (1856–1953), 74, 89, 145

Sewall G. Wright (1889–1988), 388

Simeon B. Wolbach (1880–1954), 269

Simon Flexner (1863–1946), 116, 232

Simon H. Gage (1851–1944), 225Solomon Spiegleman

(1914–1983), 467Stanislaus von Prowazek

(1875–1915), 263Sterling Emerson (1900–1988),

385

Themistocles Zammit (1864–1935), 190

Theobald Smith (1859–1934), 116, 185, 428

Theodore Schwann (1810–1882), 53

Théodore Turquet de Mayerne (1573–1655), 4

Theodosius Dobzhansky (1900–1975), 385

Theophil Mitchell Prudden (1849–1924), 115

Thérèse Tréfouël (1892–1978), 319

Thomas Hunt Morgan (1866–1945), 385

Toju Hata (1908–2004), 454

Ulysses S. Grant (1822–1885), 81

Voltaire (1694–1778), 16

Wallace E. Herrell (1909–1992), 304

Walter Reed (1851–1902), 362Walther Hesse (1846–1911), 113Wayne Umbreit (1913–2007),

445Wesley G. Hutchinson, 465Wilbert H. Spencer (1899–1991),

539Wilbur G. Downs (1913–1991),

367Wilbur Sawyer (1879–1951), 363Wilhelm Dönitz (1838–1912),

115Wilhelm Krause (1833–1910),

107William Boog Leishman

(1865–1926), 139William Gilson Farlow

(1844–1919), 89William H. Peterson

(1880–1960), 343, 399William Henry Welch

(1850–1934), 115, 116, 158, 233

William McDonald Scott (1884–1941), 290

William Morton (1819–1868), 78William Stovall (1887–1971), 343


Name Index

571


William V. Cruess (1886–1968), 431

William Z. Hassid (1899–1974), 414

Winston Churchill (1874–1965), 274

Yukichi Fukuzawa (1835–1901), 217

Zacharias Janssen (1580–1638), 3



b2530_FM.indd 6 01-Sep-16 11:03:06 AM


573


Subject Index

17D strain, 365

acetone, 272acetyl-CoA, 415actinomycetes, 493actinomycin, 446Aedes aegypti, 161affinity chromatography, 519agar-agar, 113agricultural microbiology, 339Ames test, 531amoeboid cells, 125anaerobic metabolic pathways,

409anaerobic roll tube technique,

479animalcules, 6ankylostomiasis, 248Anopheles, 138anthrax, 57, 108, 169antibiotics, 297antitoxin, 171, 177, 216, 230antivenom serum, 246Arny’s Army, 525aseptic surgery, 79attenuated virus, 245attenuation, 58

autotrophic bacteria, 202auxotroph, 474

babesiosis, 229Bacillus anthracis, 169Bacillus Calmette Guerin (BCG),

243 bacterial conjugation, 486badge of honor, 40Bergey’s Manual, 334big four in bacteriology, 144brucellosis, 189bubonic plague, 218, 238, 246butanol, 272

calcinaccio, 33calcino, 33candicidin, 493capsule, 286carbolic acid, 80cell, 11cellular receptors, 538central dogma, 379cercaria, 420chiasmata, 377chloroform, 78cholera, 170

b2806_Subject Index.indd 573 28-Jul-17 10:12:50 AM


574


chromosomes, 374coenzyme M (CoM), 468cowpox, 22coxsackieviruses, 544cross immunity, 249cross over, 377crown gall, 186cytogenetics, 384

Delft School of microbiology, 350

diphtheria, 175, 215diphtheria toxins, 170double-helix, 282Drosophila, 385Dutch School and the rise of

general microbiology, 310

enrichment culture techniques, 213

enteroviruses, 538epidemic typhus, 263epidemiology, 42erysipelas, 135ether, 78excellent teacher, 313exotoxins, 245

F+, 486fermentation, 53fermentation microbiology, 520filterable virus, 61fire-blight, 91food microbiology, 329foot-and-mouth disease (FMD),

423

gangrene, 135genetic transmission, 282

giant of industrial microbiology, 517

green bacteria, 352

handwashing, 44hanging drop slide, 109hansenarium, 100HeLa cells, 541HERP (Human Exposure Dose/

Rodent Potency Dose), 533Hfr, 486histidine biosynthesis gene

regulation, 529histidine operon, 530history of microbiology, 493hog cholera, 226human body louse, 260human-pox inoculation, 22

industrial microbiologist, 272infectious microbiology, 420inoculation, 26interspecies hydrogen transfer,

481

jumping genes, 379

Kitasato Institute, 220Koch’s postulates, 99

lactobacilli, 130lambda phage, 473leishmaniasis, 263leprosy, 98Listerine®, 82lysozyme, 302

major histocompatibility complex (MHC), 555


Subject Index

575


Malaria, 134mal del segno, 33Malta fever, 189Merck & Co., 446mesoderm, 125methanogenesis, 353, 464Mexican typhus, 266microbe hunter, 12, 198, 505microbial ecology, 510Micrographia, 11micrographs, 7molds, 297murine typhus, 263mutagen, 531mutant, 474Mycobacterium leprae, 98mycology, 85

neomycin, 493Neurospora, 387, 400nitrogen fixation, 144nitrogen-fixing bacteria, 204notatum, 303

one gene-one enzyme theory, 397optical isomers, 52

P factor, 486Pasteur Institute, 168pasteurization, 235penicillin, 297Penicillium, 303phagocyte, 127photomicrographs, 112photosynthesis, 350phytopathology, 85, 93plant galls, 147polymerase chain reaction (PCR),

511

polysaccharide capsule, 291Private 606, 301Prontosil, 316, 318Prontosil Rubrum, 318protoplasm theory of life, 72puerperal fever, 40pure culture, 18purple bacteria, 351

rabies, 170radioactive carbon, 413replica plating, 473R-form, 286Rhizobium, 342rickettsial diseases, 266Rocky Mountain spotted fever,

266root nodule, 148, 343rumen microbiologists, 477

S9 fraction, 531Salmonella typhimurium, 529sensationalism, 324septicemia, 135serum therapy, 175

formed, 286siderophore, 522simple microscopes, 4sleeping sickness, 138, 189single T-cell receptor-altered-self

hypothesis, 555smallpox, 21Smith Hall, 183spontaneous generation theory,

16St. Mary’s Hospital School, 299strangling angel, 176sulfanilamide, 319sulfate reducer, 144



576


sulfur drug, 316swamp soil, 371symbiosis, 88Synthetic Zionism, 274syphilis, 130, 171

Taq endonuclease, 511tartaric acid, 52teleutospores, 87tetanus, 215Texas cattle fever, 225The Microbe Hunters, 323thermophilic microorganisms,

511thermo-resistant spores, 73tick, 228, 297toxoplasmosis, 264transformation, 290transposition, 377trypanosome, 191trypanosomiasis, 189tsetse fly, 191tuberculin, 115tuberculosis (TB), 243

Type II (pneumococcus), 291Type III (pneumococcus), 291typhus, 260tyrothricin, 371

unity in biochemistry, 312urediospore, 87

V+ hormone, 400vaccinia, 22variola, 21Vegetative Force, 18Vesicular Stomatitis, 423

yeast-cactus-Drosophila ecosystem, 437

yeasts, 430yellow fever, 155, 362yellow fever vaccine, 359Yellowstone National Park, 510Yersinia pestis, 240

zoonosis, 32, 420


Pioneers In Microbiology: The Human Side Of Science

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