A RESOURCE UNIT ON ANIMAL REGENERATION

A THESIS

SUBMITTED TO THE FACULTY OF THE SCHOOL OF EDUCATION

ATLANTA UNIVERSITY, IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR THE DEGREE

OF MASTER OF ARTS

BY

BOBBIE L. JOHNSON

SCHOOL OF EDUCATION

ATLANTA UNIVERSITY

ATLANTA, GEORGIA

AUGUST 1962

3 k 9^

DEDICATION

This thesis is affectionately dedicated to my

wife, Mrs. Patricia Grace Johnson, for her under¬

standing and encouragement, and to my children -

Michael, Michele and Kassondra,

B.L.J#

ii

ACKNOWLEDGMENTS

The author wishes to thank all persons who have made

the success of this venture possible* Special words of

appreciation, however, must go to Drs, E. K, Weaver and

L* Boyd of the School of Education; Drs* G, E. Riley and

M, L. Reddick of the Biology Department, Atlanta

University; Dr, K, A, Huggins, Director of the National

Science Foundation Academic Tear Institute; Drs* H, C.

McBay and Roy Hunter, Jr, of Morehouse College, and Dr,

Joe Hall, Superintendent of the Dade County (Florida)

Public Schools,

B,L,J,

iii

TABLE OF CONTENTS

Page

DEDICATION Ü

ACKNOWLEDGMENTS iii

Chapter

I. INTRODUCTION ...... 1

Rationale • 1 Evolution of the Problem • •••...••.•• 3 Contribution to Educational Knowledge. . . . . . 3 Statement of the Problem U Purpose of the Study . U Limitation of the Study. U Definition of Terms U Materials ..... .•••• 5 Operational Steps 6 Method of Research «•••• •••• 8 Survey of Related Literature. ••••••••• 8

H. THE RESOURCE UNIT 13

Outline . . . * 13 Introductory Comments. •••.••••••••. 13 Orientation for the Teacher* Animal Regeneration 18

Introduction ....... 18 Universality of Regeneration 20 Source of the Cells of the Regenerate. . . . . 23 Extrinsic Factors in Regeneration, 2k Stimulus for Regeneration .......... 2$ Organization of the Regenerate ... 26 Release of Block to Regeneration 28 Regenerative Differences in Closely Related

Forms 29 Summary ................... 29 References ....... 31

Specific Treatises and Books with Chapters on Regeneration. ••••••••••••• 31

Articles 32 Protozoa ..... .•••• 32 Coelenterates (Hydra and Tubularia). , . . 32

iv

V

TABLE OF CONTENTS (CONTINUED)

Chapter Page

Flatwonns (Planaria) • •••••••••. 33 Annelids (Earthworms)* •••••••••• 33 Crustaceans (Crayfish) * 34 Vertebrates ••••* . . . . • 34

Statement on the Objectives. .......... 37 Introduction 37 Principles of Biology Associated with

Regeneration Phenomenon. *••.•••••* 37 Suggested Activities for Securing the Objectives 39

Introduction . • 39 Questions on Regeneration 40 Experiments for Answering Question 1 42 Experiments for Answering Question 2 45 Experiments for Answering Question 3 . . • * • 46 Experiments for Answering Question 4 • • * . • 47 Experiments for Answering Question 5 « • • • • 49 Experiments for Answering Question 6 , * * . * 50 Experiments for Answering Question 7 • • • • • 51 Experiments for Answering Question 8 £2 Experiments for Answering Question 9 • • • • « 53 Experiments for Answering Question 10. .... 54 Experiments for Answering Question 11. . * . . 55

Evaluation of the Experiences from the Activities of the Unit 56 Ability to Accurately Observe and Record Data. 56 Operation Skills Developed •••••..••• 56 Ability to Propose new Experiments for Testing

Validity of Other Principles Not Included In This Unit 56 Examination 56

Appendix for the Unit. . 56 Animals Required •«••• 56 Methods for Culturing and Caring for Animals

in the laboratory 57 Equipment and Supplies Needed. 58 Non-Specific Items •••.•••. 58

III. SUMMARY AND CONCLUSIONS 59

Recapitulation of Experimental . 59 Summary of Literature. * 60 Resume of Findings 6l Conclusions. 63 Recommendations 63

BIBLIOGRAPHY 64

CHAPTER I

INTRODUCTION

Rationale.—The phenomenon of regeneration (the restoration of lost

parts) has attracted the attention of man for many years. Much of this

concern resulted from the "closeness" of the phenomenon. That is to say,

man could not escape the recognition of regeneration because all around

him, and even within himself, he could see its manifestations* such as

the healing of wounds and the replacement of finger nails and hair. Too,

he was most certainly led to wonder why some animals could replace a lost

limb and he could not. How amazed were the oyster fishermen when they

found they could not destroy the oyster's natural enemy, the starfish, by

tearing off its limb. Each of these limbs, when broken in a certain

manner, would form an entirely new starfish.

After two centuries of work and thought, men of science, as well as

the laity, are still puzzled by the regenerative powers of some animals.

There is, however, one clear point: the ability to replace lost parts has

decreased with advancing evolution of more complex animals. In other

words, animals in the "lower phyla" (large categories for classifying

living organisms) show more power of regeneration than those in the "higher

phyla."

It is also true that, with increasing age, the ability to repair and

to replace is progressively lost, for repair is a measure of the growth-

energies in an individual. These energies are greatest during the early

1

2

stages of the life span. As age accumulates growth-energies diminish.^"

How are lost nails replaced? What causes a wound to heal such that

it is often difficult to point out the original site of the injury? How

can a fragment of an animal (like a planarian) give rise to a complete

and well organized animal? How is it that man cannot restore a lost

finger or leg when some other animals can? These questions are obvious

to man because he sees the sources of the questions. Yet there are num¬

erous instances of the replacement of lost parts going on daily within his

body about which he is unaware. The lining of the stomach is being re¬

placed almost constantly, and worn-out blood cells are being restored, as

well as replaced.

Beyond the "selfish” aspects of the phenomenon of regeneration is its

tremendous biological importance. What is the source of the cells which

will form the regenerated part? Why is it that animals so closely related

as frogs and salamanders show such different regenerative abilities? Why

does regeneration occur more readily only in the young of some species,

and in both young and old of other species? What triggers the regenerative

process itself — is it the marshaling of the necessary cells and synthetic

processes with the ability to duplicate the organization already observed

prior to injury?

It would appear, therefore, that a unit of study on this topic will

be most rewarding to a pupil in gaining some insight into a problem so

basic and so close to himself. The teacher will be in a position to, in a

single unit, touch upon several key biological principles - cell death,

-

L. J. Milne and M. J. Milne, The Biotic World and Man (2d. ed,;

Englewood Cliffs, New Jersey: Prentice-Hall, Inc., 19^8),p. 272.

3

cell renewal, tissue organization. Furthermore, it will be possible to

observe the effects of environmental factors (temperatures, etc.) on the

regenerative process.

Evolution of the Problem.—This problem grew out of a series of ex¬

periments on animal regeneration conducted by the writer in a course in

Experimental Biology at Atlanta University. The impact of the phenomenon

of regeneration itself, as well as the numerous biological principles in¬

herent in the phenomenon, led the writer to feel that such studies would

provide needed impetus to high school biology courses. The pupil would

undoubtedly be fascinated by direct observations on regenerating systems

to the extent of wanting to raise many important questions about the

process. Several short-term experiments could then be set up to provide

answers to many of the questions. The writer believes that as teacher of

such a group, he could then lead them to a greater understanding of key

issues in biology as well as to an appreciation of the tasks involved in

adequately interpreting the results of the experiments.

Contribution to Educational Knowledge.—The problem to be explored

by the writer will contribute greatly to the pupils' knowledge about some

of the mysterious phenomena happening around themj phenomena which they

can see and marvel at. Too, the pupils will be made more cognizant of

many of the unseen events going on constantly within their bodies. Such

events which they take for granted pose baffling problems for biologists

and would offer significant clues to problems of life itself if they could

be solved. Hence, a practical contribution can be made: the pupils come

to appreciate the tediousness and difficulty involved in the interpretation

of scientific data as well as the setting up of suitable experiments to

U

answer important questions»

Statement of the Problem»—The problem here is to identify certain

phenomena associated with animal regeneration and then to utilize these

as the bases for preparation of a resource unit on regeneration to be used

in teaching high school biology. Such a resource unit will include data

on the kinds of animals best suited for short-term experiments on regenera¬

tion and on specific experiments designed to answer key questions.

Purpose of the Study»—The ultimate objective of this study is pre¬

paring a prospectus for teaching certain selected principles in a course in

high school biology. The prospectus will be one that is "alive" and

challenges the imagination and thinking of the student, while fascinating

him. Furthermore, the student will become directly involved in what he is

doing and, in all probability, will exhibit more intellectual curiosity

about what is happening, especially since he will be able to design means

of directing the course of his experiments. The teacher can, therefore,

take advantage of this kind of pupil exuberance to get over many more basic

principles of biology.

Limitation of the Study,—The material presented in this study will

touch directly upon the gross aspects of the regenerative process. In

order to gain a deeper insight into the minute details of the phenomenon,

inferences will have to be drawn from the gross observations. The study is

further limited in that the entire animal kingdom cannot be dealt with,

hence only certain phyla and/or classes will be studied.

Definition of Terms.—Some of the significant descriptive terms to be

used in the study are defined below:

"Resource Unit" - A systematic and comprehensive survey, analysis, and

5

organization of the possible resources (e,g., problems, issues, activities,

bibliographies, and the like) which a teacher might utilize in planning,

developing, and evaluating a learning unit.^

'•Regeneration" - The repair by growth and differentiation of damage

2 suffered by an organism past the phase of primordial development,

"Mitosis" - A form of cell division characterized by ,., exact

chromosome duplication,

"Metabolism" - A group of life-sustaining processes including prin¬

cipally nutrition, production of energy (respiration), and synthesis of

more protoplasm,

3 "Growth" - Increase in cell size or in cell number,

"Vertebrates" - Animals possessing a cranium and a spinal column of

vetebrae which form the chief skeletal axis of the body. It

"Invetebrates" - Animals without a spinal column of vetebrae.

Materials,—The materials to be used in the experiments proposed in

this study are the following:

(l) Invetebrates

(a) Stentor - a protozoan

1 Harold Alberty, "How to Make A Resource Unit" (unpublished bulletin,

College of Education, The Ohio State University Press, 19WJ), p. 5« 2 P. Weiss, Principles of Development (New York: Henry Holt Company,

1939), p. U58. 3 P. B, Weisz, The Science of Biology (New York: McGraw-Hill Book

Company, 1959), p. 583. k

C, P, Hickman, Integrated Principles of Zoology (St, Louis, Missouri: C, V, Mosby Company, 1955), p. 3$lu

6

(b) Hydra - a freshwater coelenterate

(c) Tubularia - a marine coelenterate

(d) Planaria - a free-living fiatworm

(e) Earthworm. - an annelid worm

(f) Leech - an annelid worm

(g) Crayfish - a crustacean (artfchropod)

(h) Starfish - an echinoderm

(2) Vertebrates

(a) Goldfish - a bony fish

(b) Frogs (tadpoles and adults) - tailless amphibians

(c) Salamanders (adults) - tailed amphibians

(d) Mouse - a mammal

Operational Steps.—-The operational steps in this study will be as

follows:

1. Several questions will be posed to test certain key hypotheses

about animal regeneration.

2. Suitable animals, representing "lower" and "higher" groups, will

be selected. The suitability of the animals will be based on

their known ability to show rather rapid regenerative powers.

(a) These animals will then be categorized as follows:

(1) Ability of a fragment to give rise to a whole animal

(2) Ability of an animal to restore that part only

3. Experiments will be carried out to establish the rate of re¬

generation under controlled conditions of temperature, growth

medium constituents.

U. In order to show the significance of these factors in growing

systems, experiments will next be conducted wherein temperature

7

and changes in growth medium will be varied. The information

obtained here will be correlated with the "controls" of number

three.

5>. Each of the above experiments will be prefaced with particular

questions to be answered by the results of the experiments.

6. A Resource Unit will be prepared centered around the following

biological principles :

Regeneration is almost universal among living thingsj from

the simple to the more complex animals, the abilities to

regenerate lost parts and to reproduce asexually, fall off,

gradually and independently, as the body becomes more

specialized.

Growth and repair are fundamental activities for all protoplasm.

Growth and development in organisms is essentially a cellular

phenomenon, a direct result of mitotic cell division.

All cells arise through the division of previous cells (or protoplasm), back to the primitive ancestral cell (or pro¬

toplasm). Cell division is the essential mechanism of

reproduction, of heredity, and to a large extent, of organic

evolution.

Cells are organized into tissues, tissues into organs and organs

into systems, the better to carry on the functions of complex

organisms.

The protoplasm of a cell carries on continuously all the general processes of any living bodyj the processes concerned

in the growth and repair or upbuilding of protoplasm (anabolism)

and the processes concerned with the breaking down of protoplasm

and elimination of wastes from the cell (catabolism). The sum of all these chemical and physical processes is metabolism.

Prom the lower to the higher forms of life, there is an in¬

creasing complexity of structure, and this is accompanied by

a progressive increase in division of labor. In all organisms,

the higher the organization the greater the degree of differ¬ entiation and division of labor and of the dependency of one

part upon another.

Cell division is the essential mechanism of reproduction of

heredity, and to a large extent, of organic evolution.

8

The environment acts upon living things, and living things act upon their environment. Since the environment of living things changes continually, these creatures are continually engaged in a struggle with their environment.

The range of temperature for life activities is very narrow as compared with the range of possible temperatures. There is a minimum temperature below which, and a maximum temperature above which, no life processes are carried on. The temperature range for life processes is from many degrees below 0° C, to nearly the boiling point of water.

Adult organisms that differ greatly from one another but which show fundamental similarities in embryological development, have originated from similar ancestors.

Animals resemble each other more and more closely, the farther back we pursue them in embryological development.

Method of Research,--The principle-question-experiment method will be

employed in this study. Such a procedure will permit the student to

realize at once the principles involved. Then specific questions may be

proposed about the principle. The experiment will provide experiences that

may be discussed in the light of the principle, with appropriate conclusions

to be drawn from the results.

Survey of Related Literature,—The success or failure in the teaching

of any subject rests, to a large degree, in the ability of the teacher to

make each lesson vitally interesting and worthwhile to the pupil. Suf¬

ficient experimental evidence indicates that if there is abundant present-

day life activity in the classroom, the child is more likely to develop

a lively interest in the subject field. Success in the promotion of this

type of lesson depends largely in the development of certain fundamental

1 M, J, McKibben, "An Analysis of Principles and Activities of

Importance for General Biology Courses in High Schools,” Science Education, XXXIX (April, 1955), pp. 187-196,

9

techniques in the learning process.

One of the main objectives of science instruction is to develop in

students the habits of thought and action inherent in the so-called

methods and attitudes of science. There is a need in science courses for

setting classroom situations which will be conducive to the development

of the ability to communicate and to participate effectively in group dis-

2 eussions. One of the best ways to secure these objectives is through the

medium of the laboratory exercise. The value of problem-solving through

laboratory work in the school does not lie in the factual knowledge that

may result from it but in the attitudes and habits of reflective thinking

it encourages and in the understanding it gives of how the knowledge of

science gained by the student from description was attained in the first

3 place. Appropriate criticisms of research can be made more intelligible

when one is made aware of underlying principles.

Every school is, in essence and purpose, and experimental school. All

of us are trying to improve our teaching. Our effectiveness in this process

of improving our instruction depends upon our philosophy, and upon our

skills in implementing our philosophy through provision of opportunities

for students to learn...In the curriculum there is definite need for the

development of procedures and techniques for directing effectively the

_

M. P. Simmons, "A Model Lesson in General Science," Science Education, XXIII (March, 1939), p. 133.

2 J. M. Mason and W. G. Warrington, "An Experiment in Using Current

Scientific Articles in Classroom Teaching," Science Education, XXXVIII (October, 19&), p. 299.

3 Science in General Education. Report of the Committee on the Function

of Science in General Education (New York: D. Appleton Centüry Co., 1938), p. 317.

10

activities of the learner. The resource unit is the procedure for directing

the activities of the learners.^ The unit to be described in this study

will provide a directive for students and teachers in high school biology

courses with the hope that such a directive will represent a somewhat

radical departure from existing methods in such courses.

Most of the experiments presented in present-day science manuals and

carried out in science laboratory periods, preclude the possibility of

much reflective thinking on the part of the student. Too often the

student's job is to follow direction in an effort to achieve accuracy in

results he already knows. Too often the subject of the experiment has

little or no real interest and motivation for the student, who, as a con¬

sequence, blindly follows the directions without question or understanding.

Under these circumstances it can hardly be expected that the student will

2 develop resourcefulness and facility in employing the scientific nethod.

The study of regeneration phenomenon promises to open many fruitful

avenues of approach toward the solution of such basic problems of develop¬

ment as growth, differentiation, morphogenesis, and secondary adjustments

3 between parts and the rest of the organism. The word regeneration is used

to refer to the processes by which an animal restores, or tends to restore,

any regions which may be removed. At one extreme an adult mammal which

has suffered the loss of a small part, such as a finger, or a larger part,

_

Edward K. Weaver, "How to Make a Resource Unit" (unpublished com¬ pilation, The State Teachers College, Montgomery, Alabama, Summer Session, 1946), p. 2.

2 R. W. Burnett, "Vitalizing the Laboratory to Encourage Reflective

Thinking," Science Education, XXIII (March, 1939), p. 138. 3 Weiss, op. cit., p. 478.

11

such as a finger, or a larger part, such as a limb, can do more in the way

of regeneration than merely repair the wound and close the cut surface.

At the other extreme a very small part of the normal body of a coelen-

terate, a flat worm, or a starfish, can restore the whole large region

which is missing and become a complete individual. There are all grades

1 in between these two extremes.

On the whole, one gains the impression that regenerative capacity

tends to vary inversely as the scale of organization. Generally speaking,

the percentage of good regenerators is lower among the higher forms than

among the lower, more simply organized forms. But when it comes to par¬

ticulars, there is not a single group of animals whose regenerative

capacity could be safely predicted from its position on the evolutionary

scale alone. There are lowly forms with practically no regenerative

capacity, for example, the ctenophores and rotifers; while, on the other

hand, some higher forms, such as the crustaceans and amphibians, re¬

generate amazingly well. Moreover, closely related forms, such as the

earthworms and the leeches, or the tailed and tailless amphibians, may

often represent diametrical extremes with regard to regenerative capacity:

the earthworm and urodele amphibians are excellent regenerators, while

2 the leeches and anurans are among the poorest,

A basic problem in biology is that concerning the stimulus for re¬

activating developmental processes after development has been, as it were,

halted. In other words, is an adult organism a fixed structure, incapable

1 C, H. Waddington, Principles of Qribryology (London: George Allen

and Unwin, 1957), p. 302, 2 Weiss, op, cit,, p, U59.

of change, or does it still show some of the labile organization so

1 characteristic of the egg? The act of regeneration clearly indicates that

an adult organism is capable of change. The process of regeneration gives

rise to some new problems in development. One of them concerns the origin

of the cells which give rise to the regenerating structure. We might

visualize either that the new structure comes from the old cells by

migration or cell division or that there may be reserve cells, embryonic

in nature, which are undifferentiated and which give rise to the new

structure,^

1

L. B, Barth, Embryology (New York: Henry Holt and Company, 1953),

P. 327.

CHAPTER II

THE RESOURCE UNIT

This chapter presents the resource unit on "Regeneration" for High

School Biology Teachers, The basic factual materials for this unit were

primarily derived from a series of "experimental" studies carried on in

the Biology Department of the Atlanta University Schools of Arts and

Sciences during the summer sessions of 19f>7 and I960, All other materials

presented in the resource unit were gathered through copious reading and

reference work in the Trevor Arnett Library, Atlanta University, during

the first and second semesters of the 1961-1962 academic year, A brief

outline of the contents of the unit is provided below, together with

certain introductory and other explanatory comments.

Since all of us are trying to improve our teaching, our effective¬

ness in this process will depend upon our philosophy and upon our skills

in implementing our philosophy through the provision of opportunities for

students to learn. The resource unit is the procedure for directing the

activities of the learners. The unit to be described will provide a

directive for teachers in high school biology courses, with the hope that

such a directive will represent a somewhat radical departure from existing

methods in such courses.

Outline

I, Introductory Comments

II# Orientation for the Teachers Animal Regeneration

13

lli

A. Introduction

B. Universality of Regeneration

C* Source of the Cells of the Regenerate

D. Extrinsic Factors in Regeneration

E. Stimulus for Regeneration

F. Organization of the Regenerate

G. Release of Block to Regeneration

H. Regenerative Differences in Closely Related Forms

I. Summary

J. References

1. Specific Treatises and Books with Chapters on Regeneration

2. Articles

a. Protozoa

b. Coelenterates

c. Flatworms

d. Annelids

e. Crustaceans

f. Vertebrates

III, Statements on the Objectives

A, Introduction

B, Principles of Biology Associated with Regeneration Phenomena

TV, Suggested Activities for Securing the Objectives

A, Introduction

B, Questions on Regeneration

C, Experiments for Answering the Questions

D, Evaluation of the Experiences from the Activities of the Unit

15

1. Ability to Accurately Observe and Record Data

2. Operation Skills Developed

3. Ability to Propose New Experiments for Testing Validity

of Other Principles Not Included in This Unit

U. Examination

V* Appendix for the Unit

A. Animals Required

B. Methods for Culturing and Caring for Animals in the

Laboratory

C* Equipment and Supplies Needed

D. Non-Specific Items

Introductory Comments

Any scientific study or activity, involving phenomena which not only

“strike one's fancy" but which are of tremendous scientific importance as

well, is likely to be most rewarding to the participant. A student in a

biology course who can, by active and well-planned experimentation, come

to grips with a phenomenon so close to him as regeneration of damaged or

lost body parts, will acquire immeasurable knowledge about some key

biological principles. This is eo because phenomena associated with re¬

generation itself touch upon and, to be sure, are part of several funda¬

mental principles of biology in general. For example, it is well known

that adult frogs and salamanders (both are amphibians) show different re¬

generative abilities. When a salamander's limb is amputated, a new one

forms. This does not occur normally when a frog's leg is amputated. Hoi*,

ever, both frog and salamander larvae show remarkable capacities for re¬

generation. Such a simple observation on such closely related animals as

16

frogs and salamanders clearly illustrates the principle that animals

resemble each other more and more closely the farther back we pursue them

in embryological development,

A salamander's limb may be de-boned (that is, all bones are removed,

by surgical procedures) and amputated. The amputated surface will heal

and, in time, a new limb regenerates, complete with bony partsJJ Where did

the new cells come from for the "boned” new limb which regenerated from

a "de-boned" stump? First of all, one of the basic principles of biology

is that all cells arise through the division of previous cells. Since this

is true, the above observation on limb regeneration suggests that cells

which were originally one thing can become something else; for it is

clear, that some of the remaining cells in the de-boned stump becane , as

it were, triggered into becoming bone cells. This fact leads up to the

principle that growth and development and repair in organisms is essentially

a cellular phenomenon, a direct result of mitotic cell division.

Finally, a tiny fragment of an animal like a planarian can give rise

to a whole organism; however, if a dog's tail is cut off at any stage in

its life, no new tail forms, A planarian is a flatworm, an irorertebrate

animal; a dog is a mammal, a vertebrate. Such an occurrence fits another

of the generalizations in biology: While regeneration is almost universal

among living things, from the simple to the more complex animals, re¬

generative abilities fall off as the body becomes more specialized.

It would appear, therefore, that a resource unit on animal regeneration

will provide many experiences that can be quite beneficial and rewarding

to a pupil. The usefulness of such an approach to teaching biological

principles gains support from the following: There appears to be a growing

17

concern over the country about the development of scientific attitudes and

the abilities of critical thinking or problem solving ...Some of the

needed studies recommended...in the area of problem solving ares a. Tech¬

niques of setting up problems to be solved in a unit of workj b. Learning

experiences designed to help pupils identify problems; c. Processes in¬

volved in proposing and testing hypotheses; d. Learning experiences to

help pupils interpret evidence; e. Learning experiences designed to

1 help pupils draw conclusions from data.

From such a unit proposed in this thesis, the student can gain in¬

sight into a problem so basic to biology and yet so close to himself. Too,

he will obtain satisfaction from verifying accepted principles for himself,

as a result of carrying out the proposed experiments. In this way the

student visualizes the facts instead of just memorizing than. The teacher

will be in a position to take advantage of such a learning situation and

stress several key biological principles from a single ’'living" unit of

study.

Prior to the use of the suggested activities in this unit, it would

be well for the teacher to make a survey of the pupils' awareness of such

happenings in nature, including those within their own bodies. That is

to say, he (the teacher) might askî Have you ever heard of an animal

losing a part of its body and having it restored? What kinds of animals

seem to possess this ability? Have you ever wondered why it is that a

human being does not restore an amputated limb? Are there any parts of

your body which are being repaired or restored? Do you look upon this

-

E. S. Oboura, L. H. Darnell, G. Davis, and E. K. Weaver, "Fifth Annual

Review of Research in Science Teaching," Science Education, XLI (December,

19S7), p. UOU.

18

restoration activity as being a simple or complex phenomenon? What are

some of the problems involved in such activity? How would you go about

trying to determine if all kinds of animals, simple and complex ones, have

the ability to restore lost or repair damaged parts?

Following this brief survey, the teacher can go into details about

certain aspects of regeneration (as selected from the Orientation to the

Teacher); after which the class can be divided into small groups to carry

out selected experiments designed to stress basic principles of biology.

The students should be allowed to draw conclusions from their observations,

discuss them with their other classmates, and all of these activities are

to be coordinated by the teacher.

Orientation for the Teacher: Animal Regeneration

Introduction

One of the significant qualities of many animals, during their embry-

ogeny , is that of plasticity. That is to say, certain parts of the embryo

are not irreversibly fixed, but can be altered to such an extent that these

parts can become something entirely different from their expected fate. In

time, however, as the embryo ages, there is a progressive decrease in the

“lability” and an increase in the "stability” of embryonic parts. In

other words, the older the developing organism becomes, the more fixed

(or determined) will be its many parts.

Once the organism has reached an advanced stage in its life history

(adulthood, for example), can those "fixed” areas repair or restore them¬

selves if damaged or lost? Will there always be a reservoir of the "labile"

embryonic cells on hand to re-form any losses suffered by the adult? Gan

the "stable" cells of the adult be stimulated to become "labile" cells?

How "fixed" is an adult animal? Is it so rigidly fixed or stable that it

19

cannot exhibit any degree of plasticity or lability?

The evidence is clear that the parts of adult animals are not rigidly

determined. They can repair and restore damaged or lost parts in numerous

instances. This fact is so obvious from our general knowledge about our

own body as to be taken for granted. We cut ourselves and the wound heals.

We lose a nail and a new one develops. We break a leg bone and it repairs

itself. We are told that billions of our red blood cells become worn out

daily, and yet we do not suffer (under normal conditions) any ill effect

since these cells are replaced.

On the other hand we are equally aware that when we cut off the tail

of our pet dog or cat, no new tail forms. Should one of our legs or arms

be amputated, there is no restoration of the lost part. Yet when the

fisherman cuts his bait (an earthworm) into two parts, he finds that after

a time there are two earthworms. The chief enemy of the oyster industry,

the starfish, if severed into five parts, may after a while form an entirely

whole organism from each of the five fragments. Just as noticeable is

the ability of the little boy's pet salamander to restore a missing limb,

or his tadpole to replace part of a severed tail.

These and many other rather obvious occurrences are rightfully puzzling

to any man. Yet, to the student of life phenomena these incidences of

regeneration present new problems of animal development. Some of these

problems are as follows:

1. Is there any direct correlation between the ability of an

organism to regenerate and the position it occupies on the evo¬

lutionary scale of animal organization? If so, are there both

quantitative and qualitative manifestations in this capacity

20

to replace or repair lost or damaged parts?

2. What is the source of the cellular materials involved in re¬

storing lost cellular areas? Do they rise anew or are they

produced by thetransformation of cells already present?

3, Can the capacity for regeneration be controlled by extrinsic

forces, or is it an expression of intrinsic factors only?

U* What initiates a re-awakening of the ability of a seemingly

well determined and stable organism to exhibit, once again,

capacities usually reserved for the embryo?

Does the regenerated part acquire the same, in all details,

organization associated with the normal structure?

6, How valid is the statement that the "ability of organisms to

regenerate is not lost but that certain conditions of healing

may create a block to cellular activities in the area of the

severed structure?" If such a block is removed, may regeneration

occur in those forms which normally do not regenerate?

7* May closely related organisms show strikingly different capa¬

cities for regeneration?

The information to follow attempts to relate the evidence from experimental

morphology which seeks to provide clearer insight into these problems if

not answers.

Universality of Regeneration

Regeneration appears to be a universal phenomenon within the

animal kingdom. Although present in nearly all animal groups, the

ability to regenerate missing portions of the body differs in scope and

its course within specific groups, Trembley (around 171*0) first decribed this

act (regeneration) in the fresh water coelenterate, Hydra, One of these

21

organisms may be cut in two or more parts and each will reconstitute itself

into a new and complete individual, although somewhat diminished in size*

Numerous other invertebrates form (including other coelenterates) are known

in which major portions of the body can be repeatedly restored after loss.

Among such organisms are representative protozoa, flatworms, nemerteans,

annelids, arthropods, echinoderms, and tunicates. In most of these a

fragment measuring only a small fraction of the original body can become a

complete individual.

Experiments with Stentor, a protozoan, have shown that regeneration

can occur in fragments containing portions of the nucleus. Even here, how¬

ever, there must be a minimal volume relationship between the nucleus and

the cytoplasm in order to get regeneration. In coelenterates like Hydra,

small sections of the body, comprising as little as 1/200 part of the

original individual, can regenerate a complete whole, Flatworms such as

Planaria show remarkable regenerative abilities. If one of these animals

is transversely cut into halve», within a period of a few days visible

signs of regeneration are already manifest. This "bud’1 grows steadily and

soon replaces the lost part of the body. That is to say, a new fore-part

is formed on the caudal half, and a new hind-part on the anterior half,

Whether the cut is made in the middle, or in the front or hind part of the

worm, the result is always the same*

Segmented worms (annelids) like the earthworms show a somewhat similar

type of regenerative capacity. It has been found in such animals that the

regeneration-bud which develops at a front edge will always differentiate

into a fixed number of segments, which agree with those found in the rostral

end of the body, the Hheadw of a normal worm. The number of segments in

the regenerate is entirely independent of the place of the cut. In arth¬

ropods regeneration is confined to the renewal of lost appendages. For

22

example, in most crustaceans the limbs may regenerate at any stage of de¬

velopment including the adult; however, in insects, limb restoration occurs

only in the larval stages, and the regenerated limb usually does not reach

the size of a normal limb. Among the echinoderms, the starfishes can

regenerate arms and parts of the central disc. The arms appear to be lost

rather frequently in the natural environment of these animals, as individuals

regenerating one or more arms are found quite often.

The regenerative capacity of vertebrates is much less extensive than

that observed in invertebrates. Even in the most favorable cases, such

ability appears to be confined to the replacement of organs, never entire

bodies! Among fishes the restoration of fins and scales has been studied

widely. In amphibians regenerative power is high during the larval stages

of nearly all forms; and in tailed amphibians, it persists even in the

adult stage. These animals can adequately replace parts removed from eyes,

jaws, limbs, gills, tail, and several of the internal organs. While an

adult tailed amphibian like a salamander can replace a lost limb, an adult

tailless form like a frog cannot.

Reptiles (lizards, e.g.) can restore lost tail parts. Even birds and

mammals are not without some regenerative abilities. Lost feathers may be

restored in birds; in mammals, the phenomenon of wound healing is quite

obvious, and so is the restoration of a lost nail, deer antlers, bone

repair, and others.

From the information provided above it is quite clear that in some

forms regenerative capacity is enormous, while in others it is almost

neglible. On the whole, one gains the impression that regenerative ability

tends to vary inversely as the scale of organization. That is to say, in

23

general terms, the percentage of good regenerators is lower among the

higher forms than among the lower, more simply organized, forms. Never¬

theless, when it comes to particular instances, there is not a single group

of animals whose regenerative ability could be safely predicted from its

position on the evolutionary scale alone. There are lowly forms with

practically no restorative capacity, for example, ctenophores and rotifers;

while, on the other hand, some incomparably higher forms, like crustaceans

and amphibians, regenerate amazingly well.

Source of the Cells of the Regenerate

When a portion of an organism is removed, there are three sources from

which the cells which build up the regenerated part may be derived:

1. The tissues of the stump may grow out and form the new organ

or part.

2. The body may contain a reserve of undifferentiated or embryonic

cells which accumulate at the wound and then differentiate into

the tissues of the regenrated part.

3. Some of the already differentiated tissues may lose their

differentiation and assume a more plastic condition from which

they are able to re-differentiate into the specialized tissues

of the regenerate.

Observations from experimental morphology have provided evidence that

in animals such as coelenterates, flatworms and some annelid worms, the

body contains a supply of reserve cells (called neoblasts) which play a

part in regeneration. The stimulus of the wound activities these undif¬

ferentiated cells. In vertebrated animals, however, there is still no full

agreement as to the origin of the cells of the regenerate. There appears

2k

rather overwhelming evidence that there is a dedifferentiation of the

tissues in the near neighborhood of the wound, and that this is by far

the most important source of the regenerating cells.

Extrinsic Factors in Regeneration

Extrinsic forces may play significant roles in regeneration. The rate

of regeneration is dependent on temperatures, as most biological processes

are. Increase of temperature, up to a certain point, accelerates re¬

generation. For example, in Planaria regeneration is scarcely possible at

a temperature of 3°G. Of six individuals kept at this temperature only

one regenerated a head, and this was defective} the eyes and brain were

not fully differentiated after six months. Regeneration was most rapid

at 29.7°C.J at this temperature new heads developed in U.6 days. A tem¬

perature of 31.5>°C. was too high, and the heads regenerated after 8,5

days. A temperature of 32°C. proved to be lethal for the animals.

Food, on the other hand, does not affect regeneration very much. Even

a fasting animal will regenerate at the expense of its own internal re¬

sources. In such diverse cases as rats regenerating parts of the liver,

salamanders regenerating limbs or hydras and planarians regenerating parts

of their body, depriving the animals of food does not prevent regeneration

and may even accelerate it to a certain extent. If planarians are deprived

of food for a long time, they can live by metabolizing constituents of

their own bocfcr. The animal, of course, diminishes in size as a consequence.

In this state a planarian can still regenerate. Although the over-all size

increases, the missing parts are gradually rebuilt so that a complete, even

if small, worm is eventually developed. Although restriction of feeding

25

seems to be favorable for regeneration, if anything, extreme degrees of

emaciation by starving prevent regeneration except in organisms such as the

planarian which is able to utilize its own body as a source of energy with¬

out deleterious results.

Stimulus for Regeneration

In order for regeneration to take place, certain conditions must be

met. First of all there must be a stimulus to provoke the restorative

activities. There must be cellular material with an adequate supply of

"embryonic" potencies to produce all the new tissues necessary. There must

be organizing factors to make the regenerate appear exactly like the normal

structure or organisms.

According to definition, regeneration is the replacement of lost

parts. One could expect, therefore, that the loss of some part of the body

would be the adequate stimulus to set in motion the mechanism which re¬

stores the part, and thus restores the normal structure of the animal.

This is by no means always the case. If a deep incision is made on the

side of a salamander^ limb, or on the side of the body of an earthworm or

a planarian, a regeneration bud (blastema) may be formed on the out surface.

The bud then proceeds to grow and develop into a mew part, as in ordinary

regeneration. In the case of a limb the new part thus developed will be

the distal part of the limb, from the wound level outward. The development

of the regenerating part proceeds just as if the entire distal part of the

limb were cut off.

In the case of a planarian a lateral incision may cause the develop¬

ment from the wound surface of either a new head or a new tail, or both.

If both a head and a tail are regenerated, the head forms from that part

26

of the wound surface facing posteriorly. This results, of course, in the

regenerated head lying more anterior than the regenerating tail. A some¬

what similar reaction is produced by lateral incisions in the earthworm,

with the restriction that lateral incisions near the head end of the worm

give rise to additional heads, incisions in the middle part of the animal

cause the development of both heads and tails, while incisions in the pos¬

terior part of the animal^ body cause the formation of tails only. Another

peculiarity, in the case of the earthworms, is that the incision must be deep

enough to sever the ventral nerve chain if any regeneration at all is to

take place.

Careful study of each of the above mentioned cases will show that the

original parts of the animal (heads, tails, limbs) had not been removed,

so that the regenerated parts are additional and/or superfluous to the

animal. The experiments allow us to draw the conclusion that not the

absence of an organ but the presence of a wound is the stimulus for re-

generation. Thus it would seem that the capacity for regeneration is not

a novel and secondarily acquired adeptness of the organism at meeting later

accidents by adequate repairs, but is simply a residue of the original

capacities for growth, organization, and differentiation through which the

individual was first formed. Hence, the extent of regenerative capacity

is limited by the extent to which formative capacities survive the on¬

togenetic phase.

Organization of the Regenerate

Unless the regenerate is structured exactly like the normal structure,

one may question its occurrence. In a hydroid, similar to Hydra (Having a

base for attachment, a long body, and a set of tentacles which take in

27

food), if we cut it transversely into two parts and follow the development

of each of these two parts, we find that one end will form the tentacles

and the other end will simply form a base. It is the upper end which forms

tentacles; the lower forms a base.

If an animal of simple structure, e.g., a planarian, is cut into two

parts transversely, a bud-like out-growth, within a few days, will develop

on the cut surface in each half. (This is the so-called regeneration-bud.)

It grows steadily, and replaces the lost part of the body, i.e. a new fore¬

part is formed on the caudal half, and a new hind-part on the anterior

half. The position of the cut is of no, or at most of secondary, im¬

portance. Whether it is made in the middle, or in the front or hind part

of the worm, the result is always the same. Even if the worm is divided

into several parts by a series of transverse cuts, each of them will re¬

generate a head at its front edge, and a tail at its hind edge.

In earthworms, such as Eisenia foetida. a number of regions with dif¬

ferent powers of regeneration can be distinguished. In the foremost six

segments of the body, a head is regenerated at a front edge, but no re¬

generation takes place at a hind edge. In the succeeding zone of about

eleven segments, heads are formed by both front and hind edges. Next

comes a region, extending to segment fifty-four, in which only tails are

regenerated at both edges. Finally, in the zone hehind segment fifty-

four, a tail is formed by a hind edge, but no regeneration occurs at a

front edge. This earthworm (Eisenia), even in young stages, has 100 seg¬

ments. When the worm is amputated at segment fifty, new segments re¬

generate posteriorly until the total is 100j amputated at eighty, twenty

new segments regenerate posteriorly.

It has been found that in many annelid worms the regeneration-bud

28

which develops at a front edge will always differentiate into a fixed

number of segments, which agree with those formed in the rostral end of the

body, the ’'head" of a normal worm. The number of segments in the re¬

generate is entirely independent of the place of the cut. This shows that

here regeneration does not lead to restitution of the missing part of the

body, but produces only a new apical end by an autonomous differentiation.

The same applies to planarians. Here, again, regeneration at a front edge,

irrespective of its position, produces only a head, whereas that at a hind

edge leads to complete restituttion of the missing parts.

Release 6f Block to Regeneration

Normally the adult frog limb after amputation will heal, but no re¬

generation blastema will form. If, however, one stimulates the amputated

surface with salt solutions, the limb forms a blastema. Later the

blastema begins to differentiate into the missing limb. Hence, it appears

that the usual stimulus of simply cutting through the limb is not suf¬

ficient. Probably in the amputated stump of a frog limb restorative

processes are blocked in some way. As already observed, such a block is

not associated with an amputated salamander linibj it normally regenerates

after amputation.

Failure of a frog limb to regenerate appears to be related to a

failure of the tissues near the cut surface to undergo necessary changes.

The skin merely heals over the wound, and no further action occurs. In

amputated salamander limbs, if the skin is pulled over the cut surface,

no regeneration occurs. That is to say, in an animal in which regeneration

normally occurs, if the wound is covered with skin immediately after

amputation, regeneration is blocked. Such a phenomenon might explain the

29

absence of regeneration in adult frog limbs because it is a normal occur¬

rence for the skin (both outer and inner layers) to close over the wound

quite rapidly during the healing process. When salt treatment is applied,

the inner layer of the skin fails to cover the wound, and under such

treatment regeneration occurs in the frog limb. Hence, it seems that when

both inner and outer layers of the skin close over the wound, no regeneration

takes place; when only the outer layer covers the cut surface, regeneration

ensues. The ability of organisms to restore lost parts does not seem to

be lost as such, but conditions of healing arise which present a block to

necessary changes in the cells around the cut surface. If such a block

can be alleviated by chemical (or other) treatment (increasing nerve.’

supply), restorative processes take place.

Regenerative Differences in Closely Related Forms

All of these studies on regeneration fail to tell us why the fishes

regenerate much less than amphibians, which, during the course of evolution,

emerged from fish ancestors. Too, no answer is forthcoming to the striking

differences in regenerative capacities between amphibians with tails

(salamanders) and those without (frogs), or the truly segmented worm

(annelids) like earthworms and leeches. By and large, however, when one

looks at the gross picture of regeneration, it would appear that "the

whole is less than the sum of its parts"; for in certain organisms even a

tiny fragment can give rise to a whole and completely organized organism.

The truth is, however, that we have not come to understand fully "the

wholeness of the whole."

Summary

It is apparent, therefore, that the phenomenon of regeneration is

30

■universal among animals. From protozoa to mammals, some kind of res¬

torative processes occur. The quantity of repair or restoration does seem

to correlate with the histological complexity of the organism and its

position on the evolutionary scale of organization. That is to say,

generally speaking, the best regenerators are the simplest organized

organisms. On the other hand, the evidence is quite clear that the quan¬

tity and quality of regenerative processes may be controlled by extrinsic

factors.

References

Specific Treatises and Books with Chapters on Regeneration

Adams, A. E. 1959 Studies in Experimental Zoology (Regeneration,, Experi¬ mental Qnbryology, Endocrinology). Edwards Brothers, Inc,, Ann Arbor,

pp. 7-19*

Balinsky, B. I, i960 An Introduction to Bribryology. W. B. Saunders Company, Philadelphia, pp, 472-508.

Barth, L. G. 1953 Embryology, rev, ed, Henry Holt and Company, New

York, pp. 327-338*

Barth, L, G, Regeneration: Invertebrates, In: Analysis of Development

(B, H. Willier, P, Weiss, and V, Hamburger, eds.), W, B. Saunders

Company, pp, 664-672.

Berrill, N, J, 1961 Growth, Development, and Pattern, W, H, Freeman and

Company, Inc,, San Francisco, pp. 244-320, 358-1*02.

von Bertalanffy, L, 1962. Modern Theories of Development: An Introduction

to Theoretical Biology" Harper and Brothers, New York, pp. 168-172."

Bliss, De. I960 Autonomy and regeneration. In: The Physiology of

Crustacea, Vol. I (T. H. Waterman, ed,), Academic Press, Inc, New York,

Bodenstein, D, 1953 Regeneration. In: Insect Physiology (K. D, Boeder,

ed.), John Wiley and Sons, Inc., New York,

Goss, R. J. 1961 Regeneration of vertebrate appendages. In: Advances in

Morphogenesis, Vol. 1 (M. Abercrombie and J. Brachet, eds.), Academic

Press, Inc,, New York, pp, 103-149,

Needham, A, E, 1952 Regeneration and Wound-Healing. John Wiley and Sons,

Inc., New York.

Needham, J. 1942 Biochemistry and Morphogenesis. Macmillan Company,

New York, pp. 430-456,

Nicholas, J. S. 1955 Regeneration: Vertebrates. In: Analysis of De¬

velopment (B. H. Willier, P. Weiss, and V. Hamburger, eds.), W. B.

Saunders Company, Philadelphia, pp. 674-693*

Raven, C. P. 1959 An Outline of Developmental Physiology. Pergamon Press,

Inc., New York, pp. 166-189*

Thornton, C. S. (ed.) 1959 Regeneration in Vertebrates. University of

Chicago Press, Chicago.

Vorontsova, M. A., and L. D. Liosner. I960 Asexual Propagation and Re¬

generation. Pergamon Press, Inc., New York,

31

32

Waddington, C. H. 1957 Principles of Embryology, George Allen and Unwin, Ltd., London, pp. 302—32U.

Weiss » P. 1939 Principles of Development. Henry Holt and Company, New York, pp. H5«-U78.

Articles

Protozoa

Balumuth, W. 19h0 Regeneration in protozoa: a problem in morphogenesis. Quart. Rev. Biol., 15: 290-337*

Lillie, F. R. 1897 On the smallest parts of Stentor capable of re¬ generation, a contribution on the limits of divisibility of living matter. Jour. Morph., 12s 239-2U9.

Coelenterates (Hydra and Tubularia)

Barth, L. G. 19I4.O The process of regeneration in hydroids. Biol. Rev., 15: U05-U20.

Barth, L. B. 19UU The determination of the regenerating hydranth in Tubularia. Physiol. Zool., 17: 355-366.

Berrill, N. J. 19U8 Temperature and size in the reorganization of Tubu¬ laria. Jour. Exper. Zool., 107: U55-U6U.

Berrill, N. J. 1957 The indestructable hydra* Scien. Amer*, 197: 118-125*

Brien, P. i960 The fresh-water hydra. Amer. Sci., lj.8: U61-U75*

Burnett, A. L. 1959 Histophysiology of growth in hydra. Jour. Exper. Zool., II4.O: 281-3U2.

Chalkey, H. W. 19U5 Quantitative relation between the number of organized centers and tissue volume in regeneration masses of minced body sections of Hydra, Jour. Nat. Can, Inst., 6: 191-195*

Curtis, W. C. 19^0* The histologic basis of regeneration and reassociation in lower invertebrates. Amer. Nat,, 7U* U87-500.

Goldfarb, A. J, 1909 The influence of the nervous system in regeneration. Jour. Exper. Zool., 7: 6U3-722.

Ham, R. G. 1958 Chemically-induced loss of regenerative capacity in hydra. Fed. Proc., 17: 236.

Ham, R. G., and R. E. Eakin 1956 Effects of lithium ion on regeneration of Hydra in a chemically defined environment. Jour. Exper. Zool., 133: 559-572.

33

Ham, R. G., and R. E. Eakin 1958 Time sequence of certain physiological

events during regenerative in hydra. Jour. Bxper. Zool., 139s

33-51.

Lillie, F. R. 1900 The source of material of new parts and limits of

size. Amer. Hat., 3U* 173-177.

Moore, J. A. 1939 The role of temperature in hydranth formation in

Tubularia. Biol. Bull., 76: 10U-107.

Rafferty, K. A. 1935 The fates of segments from Tubularia primordia. Biol. Bull.. 108: 196-205.

Roudàbush, R. L. 1933 Phenomenon of regeneration in everted hydra. Biol. Bull., 6U: 253-258,

Steinberg, M. S, 1955 Cell movement, rate of regeneration, and the axial

gradient in Tubularia. Biol. Bull., 108: 219-23U.

Tardent, P. 1959 Principles governing the process of regeneration in

hydroids. In: Developing Cell Systems and Their Control (D. Rudnick, ed., 18th Growth Symposium), Ronald Press Co., New York, pp. 21—UU.

Tweedell, K. S. 1958 Inhibitors of regeneration in Tubularia. Biol. Bull., llU: 255-269.

Flat-worms (Planarla)

Bardeen, C. R. 1901 On the physiology of Planaria maculata. with especial

reference to the phenomena of regeneration. Amer. Jour. Physiol.,

5: 1-55.

Bmsted, H. V. 1955 Planarian regeneration. Biol. Rev., 30: 65-125.

Curtis, W. C., and L. M. Schulze 193U Studies on regeneration. I.

Contrasting powers of regeneration in Planaria and Procotyla. Jour.

Porphol., 55s U77-512.

Rulon, 0. 19U0 The environmental control of regeneration in Euplanaria.

Amer. Nat., 7Us 501-512.

Santos, F. V. 1931 Studies on transplantation in planarians. Physiol.

Zool., hi 111-161;.

Annelids (Earthworms)

Berrill, N. J. 1952 Regeneration and budding in worms. Biol. Rev., 27s

U01-U38.

3U

Gates, G. E. 19l*8 On segment formation in normal and regenerative growth of earthworms. Growth, 12: l6j^»l80.

Hyman, L. H. 19ljl Aspects of regeneration in Annelids. Biol. Symposia. 2: 21*1-256.

Liebmann, E. 191*3 New light on regeneration of Eisenia foetida. Jour. Morphol., 73: 583-610.

Moment, G. B. 191*6 A study of growth limitation in earthworms. Jour. Exper. Zool., 103: 1*87-506.

Moment, G. B. 1950 A contribution to the anatomy of growth in earthworms. Jour. Morphol., 86: 59-72.

Moment, G, B. 1953 The relation of body level, temperature, and nutrition to regenerative growth. Physiol. Zool., 2o: 108-117.

Crustaceans (Crayfish)

Emmel, V. E. 1910 Differentiation of tissue in the regenerating crust¬ acean limb. Amer, Jour. Anat., 19: 109-156.

Needham, A. E. 1953 The central nervous system and regeneration in Crustacea. Jour. Exper. Biol., 30: 155.-159.

Vertebrates

Birnie, J. H. 1931* Regeneration of the taü-fins of Fundulus embryos, Biol. Bull., 66: 316-325.

Brynes, E. F. 1901* On the skeleton of regenerated anterior limbs in the frog. Biol. Bull., 7: 166-169.

Collins, H, H. 1932 Regeneration of hindlimbs in the vemilion-spotted newt, Triturus viridescens. Anat, Rec., 5U, Suppl., 58 (Abs.).

Durbin, M. L. 1909 An analysis of the rate of regeneration throughout the regenerative process;. Jour. Exper. Zool., 7: 397-1*20.

Dent, J. N., and E. L, Hunt 191*8 The relationship of temperature to the rate of regeneration of epidermis in Triturus viridescens. Anat. Rec., 100: 651*.

Ellis, M. M. 1908 Some notes on the factors controlling the rate of re¬ generation in tadpoles of Rana clamitans-Daudin. Biol. Bull., ll*: 281-283.

35

Ellis, M, M. 1909 The relation of the amount of tail regenerated to the amount removed in tadpoles of Rana clamitans. Jour, Exper, Zool.,

7ï 1*21-1*55.

Eycleshymer, A, C, 1907-08 The closing of wounds in the larval Necturus.

Amer, Jour, Anat., 7* 317-325,

Goss, R. J,, and M. W, Stagg 1957 The regeneration of fins and fin rays in Fundulus heteroclitus. Jour, Exper, Zool., 136: 1*87-508.

Gidge, N, M,, and S. M. Rose 19kh The role of larval skin in promoting limb regeneration in adult Anura, Jour, Exper. Zool., 97? 71-93,

Morgan, T, H., and S. E, Davis 1902 The internal factors in the re¬

generation of the tail of the tadpole. Arch, f, Entw-mech., 15: 3lU-

318.

Needham, J, 1936 Biochemistry and causal morphology in amphibian re¬ generation, Science Progress, 31» Ul—5U,

Nabrit, S, M, 1938 Regeneration in the tail fins of embryo fishes

(Opsanus and Fundulus), Jour, Exper. Zool., 79* 299-308.

Polezhayev, L. W. 191*6 The loss and restoration of regenerative capacity

in the limbs of tailless Anphibia. Biol, Rev., 21s H4I—llt7,

Reed, M. A. 1903 The regeneration of a whole foot from the cut end of a

leg containing only the tibia. Arch, f, Bitw-mech., 17: 150-15U.

Rose, S. M. 191*2 A method !>r inducing lirrib regeneration in adult Anura,

Proc, Soc, Exper. Biol, and Med., 1*9* 1*08-1*10.

Rose, S. M. 19l*l* Methods of initiating limb regeneration in adult Anura. Jour, Exper, Zool., 95* 11*9-170,

Rose, S. M, 191*5 The effect of NaCl in stimulating regeneration of limbs of frogs. Jour, Morphol,, 77* 119-11*0.

Schotte, 0. E., and E. G, Butler 191*1* Phases in regeneration of urodele limb and their dependence upon the nervous system. Jour, Exper.

Zool., 97s 95-121.

Schotte, 0. E., and M. Harland 19l*3 Amputation level and regeneration in

limbs of late Rana clamitans tadpoles. Jour, Morphol,, 73* 329-363,

Thornton, G. S. 191*2 Studies on the origin of the regeneration blastema

in Triturus viridescens. Jour. Exper. Zool., 89: 375-391.

Zelney, G, 1909 The effect of successive removal upon the rate of re¬

generation. Jour, Exper, Zool., 7* 1*77-512.

36

Zelney, G. 1909 The relation between degree of injury and rate of re¬ generation - additional observations and general discussion. Jour.

Exper. Zool., 7î 913-961.

Zelney, C. 1909 Some experiments on the effect of age upon the rate of

regeneration. Jour. Exper. Zool,, 7* 563-593»

Zelney, C. 1916 A comparison of the rates of regeneration from old and

from new tissue. Proc. Nat. Acad. Sci.f 2î I4.8I4.-U86.

Zelney, C. 1917 The effect of degree of injury, level of cut and time

within the regenerative cycle upon the rate of regeneration. Proc.

Nat. Acad. Sci., 3: 211-217.

37

Statements on the Objectives

Introduction

Perhaps more than any other science, biology is a staggeringly large

collection of facts. This poses a great problem in an elementary course

of biology because the mere accumulation of these facts is a large task

and leaves little time to see what they mean and how they fit together.

It is not unheard of for students to fail utterly to see the point...j

they may never see it, and the unhinged facts may soon wash away from their

minds.^ One way out of such chaos of detail is to generalize, where

possible. Such principles (or generalizations) based on several facts

can therefore be stressed, rather than mere memorization of details with¬

out any understanding of the underlying idea.

The principles used in this unit were originally selected from

2 McKibben1s analysis and subsequently scrutinized by a group of biologists

in the Atlanta University Center. All agreed to the importance of these

principles and the significance of studies on regeneration as being directly

related to an understanding of these principles.

Principles of Biology Associated with Regeneration Phenomena

A. Principles: Regeneration is almost universal among living things. Prom the simple to the more complex animals, the abilities

to regenerate lost parts and to reproduce asexually, fall

off, gradually and independently, as the body becomes

more specialized.

Growth and repair are fundamental activities for all pro¬

toplasm. Prom the lower to the higher forms of life,

Ï “

J. T. Bonner, The Idea of Biology (New York: Harper and Brothers, 1962), p. ix.

^M. J. McKibben, op. cit.

38

there is an increasing complexity of structure, and this

is accompanied by a progressive increase in division of

labor. In all organisms, the higher the organization

the greater the degree of differentiation and division

of labor and of the dependency of one part upon another.

Associated

Phenomena: Apparent direct correlation between the ability of an organism to regenerate and the position it occupies on

the evolutionary scale of animal organization. Quan¬ titative and Qualitative gradations in regenerative

abilities between lowest and highest animal phyla.

B. Principles: Growth and development in organisms is essentially a

cellular phenomenon, a direct result of mitotic cell

division. Cells are organized into tissues, tissues into

organs, and organs into systems, the better to carry on

the functions of complex organisms.

All cells arise through the division of previous cells.

Cell division is the essential mechanism of reproduction,

of heredity, and to a large extent, of organic evolution.

Associated Phenomena: Source of the cellular materials involved in restoring

lost cellular areas: Do they arise anew or are they

produced by the transformation of cell types already

present? Precise organization of the regenerated part.

C. Principles: The environment acts upon living things, and living

things act upon their environment. Since the environment of living things changes continually, these creatures

are continually engaged in a struggle with their environ¬

ment.

The range of temperature for life activities is very narrow as compared with the range of possible temperature.

There is a minimum temperature below which, and a maximum

temperature above which, no life processes: are carried on. The temperature range for life processes is.from

many degrees below 0°C. to nearly the boiling point of

water.

Associated

Phenomena: The apparent control of regenerative rates by extrinsic

forces. The re-initiating of regeneration, by external

means, in animals that lose, after some time, their ability to regenerate certain parts of their body.

The re-awakening, by externally applied factors, of the ability of a seemingly well-determined and stable organism

39

to exhibit once again, capacities usually reserved for

the embryo; that is, abilities of adult animals to restore lost and repair damaged parts.

D. Principles: Adult organisms that differ greatly from one another but which show fundamental similarities in embryological development, have originated from similar ancestors.

Animals resemble each other more and more closely the

farther back we pursue them in embryological development.

Associated

Phenomena: The strikingly different abilities for regeneration of lost limbs in such closely related animals (amphibians)

as adult frogs and salamanders.

The strikingly similar abilities for regeneration of lost parts in the tadpoles (young stages) of frogs and

salamanders.

E. Principles: The protoplasm of a cell carries on continuously all the general processes of any living body; the processes

concerned in the growth and repair or upbuilding of

protoplasm (anabolism) and the processes concerned with

the breaking down of the protoplasm and elimination of

wastes from the cell (catabolism). The sum of all these

chemical and physical processes is metabolism.

Associated

Phenomena: Apparent correlation between age of the organism and the capacity for regeneration, or the rate of regeneration.

Suggested Activities for Securing the Objectives

Introduction

The objectives previously stated are to be carried out by the

Question-Experiment method. The significance of experimentation in high

school biology courses has gained significant support from the American

Institute of Biological Sciences. Glass has recently commented on this

problem:

The biological sciences are currently advancing at so rapid

Uo

a rate as to double the amount of significant knowledge in every ten to fifteen years. While this fact makes it imperative to revise our courses and methods of teaching at more and more frequent intervals, it also makes it increasingly impossible to •cover' in any course all that-is significant and that a general citizen might profitably know.

How can one truly understand the nature of science as in¬ vestigation and inquiry without some active participation in the experimental attack upon a new and unsolved problem? No matter how much you learn about the facts of science, you will never quite understand what makes science the force it is in human history, or the scientists the sorts of people they are, until you have shared with them such an experience. The laboratory and the fields are the scientists' workshops. Much reading and discussion are necessary in scientific work, but it is in the laboratory and field that hypotheses are tested. Properly to realize this aim, the student's experience must involve real, not make-believe, scientific investigation.

Questions on Regeneration

Question 1. Do "lower" (invertebrates) and "higher" (vertebrates) animal groups show the ability to regenerate?

Experiments on Invertebrates: Stentor - a protozoan Hydra - a coelenterate Planaria - a flatworm Earthworm - a segmented worm (annelid) Crayfish - an arthropod (crustacean) Starfish - an echinoderm

Experiments on Vertebrates: Goldfish - a bony fish Frog tadpole - an amphibian Mouse - a mammal

Question 2. Is there any correlation between the quantity (or extent) of regeneration and the simplicity or complexity of the organism?

Experiments on: Planaria - a flatworm Lumbricus - an earthworm (annelid) Frog tadpole - an amphibian

Question 3. When an animal is cut into two equal parts, does each re¬ generate attain the size of the original organism?

"^B. Glass, "Perspectives: A New High School Biology Program," American Scientist. XLIX (December, 1961), p. !?2J>.

p. 529.

la

Experiments on: Planaria - a flatworm Luribricus - an earthworm (annelid)

Question li. When part of an animal is removed, will the regenerated portion reach the size of the part originally removed?

Experiments on: Planaria - a flatworm Crayfish - an arthropod (crustacean) Lumbricus - an earthworm (annelid)

v Frog tadpole - an amphibian

Question Is the ability of an organism to regenerate organized along an axial gradient (antero-porterior, posterior-anterior polarity)?

Experiments on: Planaria - a flatworm Tubularia - a coelenterate Earthworm - an annelid

Question 6. Can a ‘'fragment" give rise to a whole and thoroughly organized organism?

Experiments on: Planaria - a flatworm Tubularia (or Hydra) - a coelenterate Lumbricus - an earthworm (annelid)

Question 7. Does the type of cut made on the animal have any effect on the morphology of the regenerated part?

Experiments on: Planaria (a flatworm) longitudinal cut oblique cut transverse cut partial

longitudinal oblique transverse

Question 8. Is the "material" for regeneration dispersed or localized within the animal?

Experiments on: Planaria a flatworm Tubularia (or Hydra) - a coelenterate

Question 9. Are extrinsic forces of significance in regeneration?

Experiments on the Effects of Food - Planaria Experiments on the Effect of Temper attire - planaria Experiments on the Effects of Crowding - Planaria

Question 10. Are intrinsic forces of significance in regeneration?

k2

Experiments on the Effects of Age - Frog Tadpole Experiments on Previous History of Regeneration - Flanaria

Experiments on Effects of Nerve Supply- Lumbricus

Question 11. May closely related organisms (evolutionary, i. e.) show different capacities for regeneration?

Experiments on Annelids: Earthworm (Lumbricus) and Leech Experiments on Amohibians: Adult Salamander and Adult Frog

Experiments for Answering Question 1

Stentor (a protozoan): Place s single protozian in a small amount of

water or culture medium on a depression slide on the stage of a bin¬

ocular microscope. With an £*ibryo knife or fine needle sharpened

to a thin blade, cut the specimen transversely into two parts (an

anterior and a posterior one) with a fairly quick motion. Add more

water or culture medium and place the slide in a moist chamber for

daily observation. More definite observations may be made if the cut

pieces are separated by using another depression slide (previously

marked) noting whether the piece is an anterior or a posterior part.

(See Figure 1).

Hydra (a coelenterate): Take three (3) Hydra and place each in a separate

container of culture fluid. Obtain a small flask of additional culture

and six more glass containers (small dishes). Label the dishes con¬

taining each Hydra A, B, C, Label the other dishes as follows: A-

base, A-hydranthj B-base, B-hydranth; C-base, C-hydranth. Pour into

each of the dishes some culture medium. Cut each hydra through a

region slightly below the hydranth (containing the tentacles), and

slightly above the base, thus leaving the stem portion. Place the base

and hydranth pieces in their proper dishes and leave the stem portion

~har-

h3

in the original dishes. Set the dishes aside and make daily ob¬

servations, Sketch any changes you may note at the cut surfaces of

all of the pieces. Does each piece become a whole Hydra? How long

does it take for complete regeneration to occur in each piece? (See

Figure 2).

Flanaria (a flatworm) : Using a camel hair brush, place a single specimen

in a small dish on the stage of the binocular microscope. Add a small

amount of pond water, enough to cover the body surface. With a sharp

scalpel, cut the specimen transversely into equal halves (i.e,, an

anterior and a posterior part). Add more water. Label and cover the

dishes (placing each piece in a separate dish and noting whether it

is anterior or posterior). Record the progress of the cut pieces,

(See Figure 3),

Lunibricus (earthworm; an annelid): Place a group of earthworms in a

finger bowl filled with damp paper towel strips to remove the grit

and dirt from the body surface of the animal, (The animals may be

cleaned in groups,) Allow the animals to clean for thirty to sixty

hours. Remove the animals from the cleaning bowl one at a time and

place in a petri dish on the stage of a binocular microscope. Add

a small amount of spring water, enough to cover the body surface.

Two drops of chloretone (2 per cent solution) should be placed in the

the medium to anaesthetize the animal. With a sharp razor blade, cut

the specimen transversely into equal parts (anterior and a posterior

part). Place each piece in an appropriately labeled bowl that contains

dirt from the natural environment of the worm and cover the bowl with

• 2- ~ f-jyJr-<3

33531 PïiC

ï~i f 3 PUnar 'ta

kh

damp, punctured paper towels. The toweling and dirt must be kept

moistened. Make daily observations on the pieces, (See Figure U).

Gambarus (crayfish: an arthropod crustacean): Take three (3) crayfish

and place them in separate jars containing a small amoung of water.

Label the jars A, B, C, Remove the antenna of the animal in jar A

by cutting through the point (X) shown in Figure 5, Sever a walking

leg (Point 0 of same Figure) of the animals in jars B and C, Set

jars aside and record the changes which take place in the animals

whose antennae and walking legs (portions) have been removed,

Asterias (starfish; an echinoderm): Sever two (2) starfishes in the

manner described in Figure 6, Place the pieces in appropriately

labeled containers and set aside for subsequent inspection. Record

all changes and make sketches of any external signs of restoration.

Goldfish (a bony fish): Take three (3) goldfish and place each of them in

a separate container of water. Remove and discard the t ail fin of

each animal (see Figure 7), Set the containers aside and record all

subsequent changes.

Frog tadpole (an amphibian): Place a single tadpole in a petri dish con¬

taining no pond water. With a sharp razor blade cut off and discard

about 2ram from the posterior most end of the tail. Place each tadpole

in a separate battery jar containing pond water and record all sub¬

sequent developments, (See Figure 8.)

Mouse (a mammal): Place a mouse into a glass jar. Close the jar opening

with a top to which a wad of cotton has been attached. Prior to

/Ai/oiwa

f-ïf 7- QroUfiïU

fc f- ~ TjdjOo/a.

closing, the cotton should be dipped in ether. In this way an--

aeâthètizethe mouse but do not kill it by allowing too long an ex¬

posure to the ether. When the mouse has been overcome by the ether

fumes, remove it from the jar and shave a region of the dorsal side

of the body. With a sterile, well sharpened scalpel, inflict a

skin-deep wound within the shaven area. Return the animal to a

separate container. Record superficial changes in the wounded area.

Experiments for Answering Question 2

Flanaria (a flatworm): Using a camel hair brush, place a single specimen

in a small stender dish on the stage of a binocular microscope.

Add a small amount of pond water, enough to cover the body surface.

With a sharp razor blade cut the tip of the posterior end (approxi¬

mately one-fourth of the total body size). Add more culture medium

(pond water). Label and cover the dishes. In all cases, place the

posterior piece in one dish of water and the anterior three-fourths

in another. Set aside and record all observations,

Lumbricus (earthwormj an annelid): Place a group of earthwoms into a

finger bowl filled with damp paper towel strips to remove the grit

and dirt from the body surface of the animals. Allow the animals to

clean for thirty to sixty hours. Remove the animals from the cleaning

bowl one at a time and place in a petri dish on the stage of a

binocular microscope. Add a small amount of pond water, enough to

cover the body surface. Two drops of chloretone (2 per cent

solution) should be placed in the medium to anaesthetize the animal.

With a sharp razor blade, cut the tip of the posterior end (approximately

U6

one-fourth of the total body length). Add more culture medium

(pond water). Each piece should be placed in appropriately labeled

finger bowls containing dirt from the natural environment and the

bowls kept covered with damp, punctured paper towels, Make periodic

observations on the progress of each cut as it attempts to restore

itself.

Frog Tadpole (an amphibian): Place single tadpole in a petri dish con¬

taining no pond water. With a sharp razor blade cut through the tip

of the posterior end (appriximately one-fourth of the total body

length). Discard the severed tip; place the amputated specimen into

a battery jar containing pond water, and record all subsequent de¬

velopment.

Experiments for Answering Question 3

Planaria (a flatworm): Using a camel hair brush, place a single specimen

in a small dish on the stage of a binocular microscope. Add a small

amount of pond water, enough to cover the body surface. With a

sharp razor blade, cut the animal transversely into equal parts (thus

an anterior half and a posterior half). Add more spring water.

Place each half in a separate container, appropriately labeled. Prior

to cutting, measure the length of the planarian. Record all sub¬

sequent transformations of the cut pieces,

Lumbricus (earthworms an annelid): Place a group of earthworms into a

finger bowl filled with damp paper towel strips to remove the grit

and dirt from the body surface of the animals. Remove a single

specimen from the cleaning bowl and place in a petri dish on the

1*7

stage of a binocular microscope. Add a small amount of spring water,

enough to cover the body surface. Two drops of a 2 per cent

chloretone solution should be placed in the medium to anaesthetize

the animal. With a sharp razor blade, cut the animal transversely

into two equal parts. Each half is then placed into a bowl, ap¬

propriately labeled, that contains dirt from the natural environ¬

ment and covered with danqp, punctured paper towles. Keep an accurate

record of the periodic observations made on the progress of each cut

half.

Experiments for Answering Question It

Planarian (a flatworm): Using a camel hair brush, place a single specimen

into a stender dish on the stage of a binocular microscope. Add a

small amount of pond water, enough to cover the body surface. With

a sharp razor blade, cut one side of the anterior half of the

organism, as shown below (a). Another animal may be cut, as in (b),

while another as in (c). Place all pieces in appropriately labeled

dishes containing some pond water, ^ake daily observations and

record all changes.

Lubricus (earthwormj an annelid): Place a group of earthworms into a

finger bowl filled with damp paper towel strips to remove the grit

and dirt from the body surface of the animals. Remove the animals

from the cleaning bowl one at a time and place in a petri dish. Add

a small amount of pond water, enough to cover the body surface. Place

two drops of 2 per cent chloretone solution into the medium to an¬

aesthetize the animal. With a sharp razor blade, cut off five

segments from the anterior end of an animal marked A, and ten segments

from the posterior end of an animal marked B. Prior to cutting, make

a record of the number of segments anterior to the clitellum and the

number posterior. In this way, should regeneration occur you will

know if the correct number of segments has been restored. Place all

pieces in correctly labeled bowls containing dirt from the natural

environment and covered with damp, punctured paper towels. Record

all periodic inspections.

Cambarus (crayfishj an arthropod crustacean): Place a crayfish into a

finger bowl containing pond water. With a sharp razor blade, cut

off and discard all the right or left walking legs at various levels.

Place the amputated specimens into battery jars (or jointly into an

aquarium), with proper labeling on each jar. Observe the animals

every two or three days and record all observations.

Frog Tadpole (an amphibian): Place a single tadpole into a petri dish

containing no pond water. With a sharp razor blade, cut off 3-5mm

of the posterior part of the tail. Put the amputated tadpole into

battery jars containing pond water, set jars aside, and make daily

observations.

Experiments for Answering Question $

Tubularia or Hydra (coelenterate): Cut a single specimen into several

parts (as indicated below). Give each cut region a label and place

each niece into a separate container of sea water (for Tubularia)

or de-ionized water plus Calcium (for Hydra), talcing care to label

each container appropriately. Set the containers asi.de and make

hourly observations. Does each piece form a complete organism,

having a stem, base and a hydranth?

Planaria (a flatworm): Cut a single planarian into several parts (as

diagrammed below). Give each region a label and place all pieces

into separate containers of pond water. Each container should be ap¬

propriately labeled. Set the vessels aside and make periodic ob¬

servations on the progress of each piece. Do extreme tail pieces

form a head? Do extreme head, pieces form a tail? Are the middle

regions more capable of forming both head and tail?

Lumbricus (earthworm; an annelid); Anaesthetize an earthworm with 2 per

cent chloretone and cut into several pieces according to the diagram

50

below. Place each piece into an appropriately labeled dish containing

some dirt from the natural environment of the worm. Cover the dishes

with damp, punctured paper towels. Record all subsequent observations.

Are the posterior-most segments capable of forming heads? Are the

anterior-most segments capable of forming tails? As one progresses

from the anterior to the posterior segments, in which regions will

both head and tail- formation occur most frequently?

Hydra or Tubularia (coelenterate): Cut out a fragment of the stem of one

of these hydroids, as indicated below. Discard the anterior part

containing the hydranth and the posterior part bearing the base.

Place the fragment in a separate dish of culture fluid (sea water or

de-ionized water with calcium added) and make frequent observations.

Sara.

Planaria (a flatworm): Cut out a fragment of the worm, as indicated below.

Discard the other portions. Place the fragment in a dish of pond

water, set aside and make daily observations. ^

51

Lumbricus (earthworm; an annelid): Cut out a piece of the worm from the

posterior-most end of the clitellum to twenty-five segments back.

Discard all other pieces. Return the fragment to a dish of moistened

dirt (kept moist with damp paper towels). Record all subsequent

observations.

Sa-r-c,

Experiments for Answering Question 7

Planaria (a flatworm): At least seven (7) planarias are needed in this

experiment. ’The worms should be labeled 1-7. The manner of

cutting for each worm is described in the diagram beloxj:

52

Worm Number 1 - Arrow A - B

Worm Number 2 - Arrow C - D

Worm Number 3 - Arrow E - F

Worm Number k - Arrow A - 0 (and or B - 0)

Worn Number 5 - Arrow C - 0 (and or D - 0)

Worm Number 6 - Arrow E - 0 or F - 0

Worm Number 7 - Zig-Zag line indicated by Arrow X - XX

Keep both pieces (where necessary) in the same container of pond

water. Make daily observations and record all changes.

Experiments for Answering Question 8

ELanaria (a flatworra): Gut a single planarian into as many parts as are

shown in the following diagram. Label each piece and place it in an

appropriately labeled dish containing pond water. Set aside and

make daily observations. What is the fate of each piece? Does each

piece become a whole planarian?

53

Experiments for Answering Question 9

Effects of Food: Use Planaria (a flatworm) - Starve five planarias for

varying periods of time ( two days, five days, seven days, etc.).

Gut the starved animals transversely into an anterior and a posterior

half. Place each cut piece into a separate container of pond water

and label each appropriately. Make daily observations. Compare the

time required for total generation to occur with the animals re¬

generating according to Experiments for Questions three on planaria.

Take three planarias that have been allowed to grow in a medium

containing an excess amount of food (such that the animals are never

without food) for a period of four to seven days. Cut the overly

fed animals into equal halves with a transverse cut. Place each

piece into a container of pond water, with each container being

appropriately labeled. Make daily observations. Compare the time

required for total regeneration to occur with that in Experiments

for Questions three and nine on planaria.

Effects of Temperature: Use Planaria (a flatworm) - Place pieces of

transversely cut planarias into appropriately labeled containers of

pond water and allow subsequent development to occur at the following

temperatures:

3 “ 5°C. 10 - 15°C.

2h - 26°C. 27 - 28°C. 29 - 30°C. 32 - 3U°C.

Make daily observations and compare the time required for total

regeneration to occur with the time required for the planarias in

Question three to regenerate,

Effects of Crowding (Population Density): Use Planaria (a flatworm) -

Repeat the Experiments on Planaria in Question five. Instead of

placing each piece into a separete container, place all pieces into

a single container of the same size, Hake daily observations and

compare time required for total regeneration to occur with the value

obtained for the regenerating planaria in Question five.

Experiments for Answering Question 10

Effect of Age: Use Frog Tadpole (an amphibian) - Repeat the Experiment

on the Frog Tadpole in Question four, using tadpoles of different

ages (based on total body lengthj the older ones being longer and

larger). Place each tadpole in a separate container, taking care

to label each vessel appropriately. Make daily observations and

compare the time required for tail regeneration to occur with values

obtained from Question four.

Effect of Previous History of Regeneration: Use Planaria (a flatworm) -

Repeat Experiments on planaria in Question three several times,

always using one of the regenerated organisms in subsequent cuttings.

Make a careful study on the time required for total regeneration to

occur in a First Regenerate, Second Regenerate, Third Regenerate,

Fourth Regenerate, and a Fifth Regenerate, Do Fifth Generation Re¬

generates become restituted at the same rate as First Generation

Regenerates?

Effect of Nerve Supply: Use Lumbricus (earthworm; an annelid) - With

55

a sharp razor blade, make a partial transverse cut in an earthworm

as indicated by Arrow A (Figure U). As seen in the Figure, this cut

will not reach the ventral nerve cord. In another earthworm, make

a partial transverse cut as indicated by Arrow B in Figure [>, This

cut will penetrate the ventral nerve cord. Place each animal in an

appropriately labeled container of moistened dirt from the environ¬

ment of the animal, hake daily observations and record changes

occurring in both.

Experiments for Answering Question 11

Annelids (an earthworm and a Leech): Make transverse sections (as indi¬

cated by the arrows in the diagram belox*) through an earthworm and a

leech. Place each piece in an appropriately labeled container. Set

aside and make daily observations. Record all changes. Do the earth¬

worm and leech fragments regenerate, giving complete worms?

Amphibians (a salamander and a frog): Cut off the forelimb of an adult

salamander (as shown in Figure A below) and an adult frog (as shown

in Figure B), Place each amputated animal in a separate jar, set

aside and make periodic observations. Record all changes. Do both of

these animals show similar abilities to restore portions of amputated

limbs?

56

Evaluation of the Experiences from the Activities of the Unit

Ability to Accurately Observe and Record Data»—Make a constant check

on each student to determine the sharpness of his ability to make ob¬

servations. Watch for growth in the student’s ability to make careful

observations* ones which do not often "meet the eye” at first glance.

Have each student turn in periodically a written record of his observations

and the analysis of his data. Stress throughout the activity the necessity

for scientific accuracy in recording data.

Operation Skills Developed.—Always be cognizant of the skills of

each student in making proper operations* The student is to be made aware

of the importance of handling live materials and the care one must take in

carrying out his dissecting.

Ability to Propose few Experiments for Testing Validity of Other

Principles not Included in This Unit.—Have the student propose new ex¬

periments to test the validity of other biological principles not covered

in the unit.

Examination.—Prepare an examination to test how well the student has

grasped, not only the broader picture of the experiments and their results,

but details as well.

Appendix for the Unit

Animals Required

The following animals represent suitable experimental material for

1 studies on regeneration:

Ï " Organisms 1-8 are invertebrates, while 9-12 are vertebrates.

Si

1* Stentor - a ciliated protozoan

2. Hydra - a fresh-water coelenterate

3. Tubularia - a marine coelenterate

lu Planaria - a free-living flatworm

5* Earthworm - a free-living annelid (segmented) worm

6, Leech - an ecto-parasitic annelid (segmented) worm

7. Crayfish - a crustacean (arthropod)

8. Starfish - an echinoderm

9, Goldfish - a bony fish

10. Salamander - a tailed amphibian

11. Frog (adult and tadpole) - a tailless amphibian (adult, that is)

12. Mouse • a mammal (rodent)

Methods for Culturing and Caring for Animals in the Laboratory

The accompanying leaflets, published by the General Biological Supply

House,^ will serve as appropriate guides for laboratory care of the animals

required for the experiments suggested in this Unit, There is a leaflet

for every animal listed above, except Tubularia and Starfish and Goldfish.

Tubularia and Starfish live in a marine environment} hence, the leaflet

entitled "Notes on Marine Aquaria" should be used for these organisms* The

leaflet entitled "Starting and Maintaining a Fresh-Water Aquarium" will

suffice for the Goldfish* General operation techniques are cited in the

leaflet on "Laboratory Dissection." In addition, since some of the

animals thrive on algae, there is a special leaflet on "Growing Fresh-

Water Algae in the Laboratory."

Ï “

General Biological Supply House, 8200 South Hoyne Avenue, Chicago 20,

Illinois.

î>8

Equipment and Supplies Needed

All of the equipment and supplies called for in executing the experi¬

ments may be obtained from the General Biological Supply House* 8200 South

Hoyne Avenue, Chicago 20, Illinois. The leaflet "Basic Laboratory Equip¬

ment for High School Biology Course" contains the pertinent information

for ordering these and other supplies. Another leaflet, “Preserving

Zoological Specimens: Narcotization, Fixation and Preservation", provides

adequate information about narcotizing (or anaesthetizing) the animals

prior to operation. Furthermore, should anyone desire to preserve a

specimen used in his experiments, appropriate suggestions are provided in

the textual materials included in this unit.

Non-Specific Items

In order to provide further assistance to the teacher who may desire

to do additional work not cited in the Unit, several other leaflets are

included: "Special Projects for Biology Students," "Demonstration and

Display Materials," "Embryology in the High School Biology Course," "A

Selected List of Books for the Biology Library."

TURTOX SERVICE LEAFLET No. 4

THE CARE OF PROTOZOAN CULTURES IN THE LABORATORY

Care of Cultures Protozoa are almost infinite in form,

in habit and in their distribution, and a wide range of kinds can be found in any hay infusion or culture of decaying pond weeds. Simply because such “Mixed Cul¬ tures” contain a great many forms which are not easily recognized is no argument for ignoring them in the school labora¬ tory. Their variety and the changes in protozoan population which occur from day to day, the cycles through which a mixed culture passes and the great num¬ ber of different forms present, are all extremely interesting to one who wishes to know something of the science which we call Biology.

Field collecting in any pond or stream will produce an abundance of protozoa from which pure cultures of some forms may be obtained. This takes time, how¬ ever, for after inoculation a pure culture usually requires several weeks to develop to a point at which it is best for labora¬ tory study; and, furthermore, a “pure” culture seldom remains pure for any long period of time.

Since laboratory work is given at the beginning of the fall term, and it takes time to rear protozoan cultures, the teacher finds himself faced with the prob¬ lem of supplying his students with living material at the very beginning of the school year. It is usually advisable, there¬ fore, to purchase pure cultures from Tur- tox and keep these going as long as pos¬ sible after they have served their purpose in the course.

Turtox cultures are shipped in tightly capped two-ounce jars. As soon as they are received at the school the screw- caps should be removed and the bottles kept at a temperate of not above 70° F and out of direct sunlight. The cultures can be left in the shipping jars until they are used in the laboratory. Anieba, Paramecium, Euglena, Stentor and others will keep in good condition for days without any attention, but mixed protozoan cultures should be studied immediately since the larger forms eat the smaller and the variety is quickly reduced.

Material which has been purchased should be examined immediately upon unpacking to be certain the protozoans have not died in transit. Shipments leave

our laboratory in excellent condition but Sometimes encounter delays or are ex¬ posed to temperature extremes which kill the specimens. Cultures received in poor condition should be reported at once and at the same time a delivery date should be given for the replacement. If no delivery date is indicated the re¬ placement will be made promptly.

After the cultures have been received and opened allow them to remain un¬ disturbed for an hour or more. In search¬ ing for the protozoan in the culture jar the characteristics of the species should be kept in mind and much fruitless searching with pipette can be eliminated. Ameba settle to the bottom or on the sides of tlie container. Paramecium, Euglena, Didinium, Blepharisma, Euplotes and Colpidium are found swimming freely throughout the entire culture. They can usually be concentrated by wrapping the bottle with dark paper leaving only the surface of the water exposed to the light. Stentor and Vorticella attach themselves to food, sides and bottom of the bottle.

To check the protozoan culture or study the forms in class place a drop of the culture water on a clean microscope slide and cover with a clean coverglass. Ex¬ amine under the low power objective and be sure the light is well stopped down. Cultures of protozoa used in class work usually contain more specimens than needed and the remaining material can he used in propagating cultures in the laboratory. The same procedure can be used in starting pure cultures by isolat¬ ing species found in a mixed protozoa collection.

In culturing protozoa the important factors to consider are temperature, light, kind and quantity of food, type of culture jar, and freedom from contamination. These factors vary widely of course and will be treated separately for each specific form.

Ameba If the teacher wishes to culture Amebae

from a local source, some decaying water plants (water lily leaves are particularly good) and pond water should be ob¬ tained. A little of the plant material and about 50 cc of pond water may be placed in a finger bowl dish and observed daily. To save time it is usually best to start

TURTOX Service Department Copyright, 1959. by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

several cultures, using pond weeds and water from several localities. If Ameba are present, they should appear in quan¬ tities in about two weeks, being most plentiful on the bottom of the culture dish. In looking for them, pipette a few drops of the bottom sediment onto a slide and examine under the microscope.

Ameba appears to do best with a mod¬ erate temperature, diffused light and very little food. At 50° F. they are inactive and sluggish, at 75° F. they develop rap¬ idly and at a temperature of 90° or higher there is little chance of their sur¬ vival.

Direct sunlight is harmful to Ameba and an over abundance of light encour¬ ages the development of the other more vigorous protozoa, such as Euglena and Paramecium, which result in rapid con¬ tamination of the culture. As a general rule, windows located on the side of the room opposite that side on which the cultures are placed will provide suf¬ ficient light for Ameba.

Ordinary stacking finger bowls make excellent culture dishes. Several dishes may be stacked one upon the other and the top dish may be covered with a piece of glass. Wheat kernels and pieces of timothy hay about 2 inches in length, which have been boiled for five minutes, supply the source of food.

Each bowl is filled with 75cc. distilled water to which 1 grain of wheat (cut in two) and one 2 inch piece of hay (cut into )4" lengths). Using a clean pipette, inoculate immediately with amoeba from a pure culture. After 2 weeks discard one half of the fluid and add enough distilled water to bring the volume to lOOcc. Add the same amount of food as at the first if the culture is “clean”— with not too many paramecium and not too much bacterial growth. Reduce the amount of the second feeding if the culture shows these signs of over-rich¬ ness. An even simpler medium consists of just three grains of boiled wheat per culture dish treated in the same way as indicated above.

These cultures are examined every 2 weeks to observe the food supply and the abundance of the Amebae. The water mold which invariably develops on the wheat and hay is not detrimental to the culture. After six weeks the Ameba will have divided and increased to make a heavy culture and may now be used for class study. If the cultures have been inoculated with one of the larger strains, the Ameba can be seen plainly with the naked eye as tiny milky-white specks on the bottom and side of the dish.

Chaos chaos Chaos chaos, the largest ameba known to science, was first seen by Roesel von Rosenhof in Germany in 1755. It has been

found in nature only five times since then; in 1900 by H. V. Wilson in North Carolina; in 1902 by E. Penard in Swit¬ zerland; in 1916 by W. A. Kepner in Virginia; and by A. A. Schaeffer in Ten¬ nessee and by A. A. Schaeffer in 1936 in a marsh in New Jersey. Since rediscover¬ ing this ameba in 1936, Dr. Schaeffer has succeeded in culturing it and, during the 1937-38 school year, his pure cultures were made available to hundreds of teach¬ ers—General Biological Supply House acting as Dr. Schaeffer’s exclusive dis¬ tributor.

Chaos chaos is of a very large size, fre¬ quently attaining a length of from 4 mm to 9 mm. when in locomotion. It is easily seen with the unaided eye. However, this large size causes the amebas to break up in shipment and there seems to be no way of preventing this fragmentation which occasionally occurs. The result is not a dead culture, but a living culture which contains small instead of large amebas.

Caring for the Culture. Unscrew the culture jar immediately and allow it to stand undisturbed for an hour in a cool place (60° to 70° F.), not in strong light. Then examine the culture, looking for the amebas under a dissecting microscope or under the lowest power of the compound microscope. If large specimens are pres¬ ent, they will be seen without difficulty. If the amebas appear to be of small size, allow the culture to remain in the un¬ capped jar for three or four days; in most instances they will increase in size greatly during the period.

Sub-culturing. Chaos chaos is a heavy feeder and for rapid growth it must have abundant food such as Paramecium or other ciliates. New cultures of Chaos chaos can be started by the method com¬ monly used for culturing Paramecium. When the new culture is rich in Parame¬ cium inoculate with the Chaos chaos. In three to six weeks the culture will show good growth.

Study Suggestions. Examine this ameba in a shallow culture dish, with the naked eye and with a low power (5X) magnifier. Then observe under the low power of the compound microscope, and study a por¬ tion of one specimen under high power. Note the many nuclei. Using a microm¬ eter eyepiece (or any other convenient method) measure the length of a speci¬ men “at rest” and the length of one in locomotion. Study its method of engulfing (eating) a Paramecium.

Paramecium The culturing of Paramecium is not

so difficult as culturing Ameba. They require little attention and develop very rapidly. Ordinary room temperature is

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

satisfactory for good continuous growth of the culture, but a temperature of 80° to 85° will bring the culture to its maxi¬ mum growth in a shorter time. The best culture dishes for Paramecium are finger bowls, both the 4% and 7% inch diam¬ eter, because of the greater surface area and less depth of water. However, any type of glass container will do for this protozoan. Timothy, rice and wheat boiled about five minutes are good food for the culture. The culture will grow in direct and indirect light; however, the latter is best because direct sunlight en¬ courages other protozoa and results in contamination.

The dishes for culturing should be thoroughly cleaned. If the 4y2 inch di¬ ameter finger bowls are used fill the bowl two-thirds full with distilled water and add 4 kernels of boiled wheat or rice and 12 to 15 pieces of cooked timothy 15 to 20 mm. long. If larger finger bowls, or other containers are used, increase the amount of food in proportion to the amount of water.

The media are ready for immediate inoculation with paramecium from a concentrated culture found locally or purchased from a reliable source. The older method of allowing media to age prior to inoculation is no longer recom¬ mended since there is often an observ¬ able tendency for the food organism (bacteria) to outgrow and crowd out the cultured organism. The film of bac¬ teria which sometimes forms on the surface of the medium should be broken up whenever it is encountered. Its con¬ tinuance is detrimental to paramecium.

Stack finger bowls to any desired height, cover the top one with a glass plate and place where they will receive plenty of indirect light.

Three or four days after inoculation the Paramecium will be seen concen¬ trated near the surface of the water and around the food. In ten days or two weeks the concentration will be at its maximum. When the Paramecium begin to die down a little boiled timothy, wheat or rice should be added to main¬ tain the culture. Such cultures can be kept in pure condition from four to six weeks.

Pure-line Cultures of Paramecium for General Study and Demonstration of

Conjugation Practically all species of Paramecium

grow well in hay infusion. Because of the ease with which this culture medium can be made, full directions will be given. All glassware that is to be used must be scrubbed clean and sterilization should be done by autoclaving. If an autoclave is not available, glassware after it is scrubbed may be placed in 10% nitric

acid for a short time, rinsed well with distilled water and then allowed to dry. Obtain clean dry timothy hay and cut the spikes as well as the stems and leaves into segments approximately one inch in length. Place 1 to iy2 grams of the cut hay into 250 cc. Erlenmeyer flask, add a knife-tip of precipitated CaCOa, fill to the neck with distilled water, then cover the mouth of the flask with an inverted snugly fitting beaker. Flasks so prepared are heated and the contents boiled for 15 minutes. The flasks or cooked infusion are now allowed to stand (ripen) for ap¬ proximately 24 hours, then the medium is ready for use. This medium is an ex¬ cellent one for the culturing of Parame¬ cium caudatum, P. multimicronucleatum and P. trichium.

Although Paramecium bursaria and P. aurelia will grow in dilute hay infusion better results, especially in demonstrat¬ ing the mating reaction, can be obtained by using the lettuce infusion as described by Drs. Jennings and Sonneborn. This may be prepared by obtaining a clean head of lettuce and placing the individual leaves in an oven. When the leaves be¬ come brown and brittle they are crushed to a powder with a mortar and pestle. One and one-half grams of the desiccated lettuce powder is added to one liter of distilled water and boiled for five min¬ utes then filtered into 250 cc. flasks while hot, stoppered with cotton and if pos¬ sible, autoclaved. While the flasks are still warm (but not hot) small square pieces of “parafilm” are used to cover and seal the plugged mouths of the flasks. This is the stock fluid and flasks so prepared may be stored for weeks in the refrigera¬ tor. Equal parts of distilled water and the stock fluid are mixed and when the mixture reaches room temperature (about 21° C.) the fluid is ready for use.

Flasks of pure-line mass cultures of Paramecium may be kept on shelves at room temperature for months and still contain many individuals. To maintain the pure-line culture it is only necessary to inoculate new flasks of infusion. If larger numbers of the ciliates are needed, use large battery jars and, of course, proportionately larger amounts of hay and distilled water. Demonstrating the Mating Reaction and

Conjugation Two pure-line cultures of Paramecium

bursaria are used for this demonstration. These two cultures are mixed together, preferably at or about noon on a sunny day. Immediately a few are seen to stick together, then the clump becomes larger and larger until clumps of many indi¬ viduals are formed. These large clumps then become smaller until, after about six hours, only small masses of individu¬ als will be observed. Finally individual pairs of conjugants are present and re-

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

main joined for from 24 to 48 hours. It is well to allow two days for this

demonstration, for the individual con¬ jugating pairs will sometimes be best on the second day. The demonstrations should be made in watch glasses or other shallow dishes so that the entire process can be studied under a low power micro¬ scope. IMPORTANT ! Use this material as soon as possible ! Results cannot be guaranteed unless the conjugation cul¬ tures are used within 24 hours after delivery.

Euglena Euglena is the easiest protozoan to cul¬

ture because it requires plenty of food, light and little attention. Battery jars or other tall jars are excellent for cultur¬ ing and a window sill is an ideal place to keep cultures since direct sunlight is beneficial. The temperature can range from normal to 90° with little damage to culture. Boiled wheat, rice or timothy can be used for food.

A clean 6x8 inch battery jar should be filled with distilled water and a large handful of timothy or 100 kernels of wheat or rice added. Place culture on window sill and after a week add a few drops of concentrated Euglena from a pure culture which can be collected very easily in stagnant pools. In two weeks the entire culture will begin to have a greenish appearance and the surface of the water will be covered with a scum containing large numbers of Euglena.

The culture will last for four weeks without any attention. If more food is added every two weeks the culture will last three or four months.

Stentor The culture of Stentor can be carried

on in any type of disli such as finger bowls or battery jars. Because Stentor thrive in rich protozoan cultures contain¬ ing such forms as Euplotes, Colpidium and Chilomonas, it is best to prepare media with the idea of mixed protozoa cultures in mind. This can be done fol¬ lowing tlie same procedure as that given for Paramecium. When the cultûre is ready for inoculation, instead of using Paramecium, use mixed protozoa. When the culture is well established add the Stentor, keep the culture in diffused light and in a temperature of 65“ to 75°. It will be necessary from time to time to sub-culture to new media.

The Culture of Other Protozoa Sometimes Studied

Before concluding the discussions on culturing protozoa, we believe a few ad¬ ditional remarks about the culturing of less familiar forms might prove worth¬ while.

Colpidium, Euplotes and Halteria are commonly found in pond collections and can be grown in pure culture as easily as the other types already discussed. The

culture medium is prepared similar to that suggested for culturing Paramecium. These forms may be easily isolated from the pond collection and used to inoculate the prepared culture medium. They de¬ velop rapidly and will have increaseu considerably a week after inoculation when they will be found in large numbers in the surface scum and upper half of the culture. Sub-cultures should be made every three weeks.

Didinium is an interesting protozoan because of its carnivorous habits. It lives mainly on Paramecium. Consequently paramecium are necessary in the culture of Didinium. These paramecium cultures are developed as previously suggested and when the paramecium are abundant the Didinium can be added. The culture will last as long as there is a sufficient food supply. About every ten days more paramecium should be added or new paramecium cultures should be inocu¬ lated.

The culture of the slow moving peach- colored protozoan, Blepharisma, can best be carried on in shallow culture dishes in media similar to that used for Ameba. A little more boiled wheat is necessary because Blepharisma grows better in rich cultures. Inoculation can take place two weeks after culture has been started. Blepharisma develop quickly in cultures and require little attention from four to six weeks. They are usually found swimming slowly about near the bottom of the dish. Arcella is common in most old pond- water cultures and will often be found on the bottom of the dishes containing old hay infusions from which the para- moecia have died out. Arcella is slow moving and easily studied, but the light' should be carefully regulated when ex¬ amining them with the compound micro¬ scope.

“Both wheat and hay infusions are good, but a mixture is best. To 100 cc. of pond water, add 2 grains of wheat and y2 gram of hay. Inoculate with Chilomonas if available, although Arcella will grow by feeding merely on the de¬ composing infusion. After two or three days, add Arcella which may be found on the bottom of many old cultures or in the bottom ooze of any shallow pond. Isolate them from the ooze and other Protozoa with a fine pipette and place them in the culture in a shallow dish.”

—John P. Turner, University of Min¬ nesota. Quoted from “Culture Methods for Invertebrate Animals” by permission of Comstock Publishing Company, Inc.

Culture Concentrates. Turtox concentrated media for the cul¬

ture of protozoans, algae and such forms as Volvox and Chlamydomonas, is now available. Write for price list and for the Special Turtox Bulletin No. 61V170.

CM)

TURTOX SERVICE LEAFLET No. 39

THE FRESH WATER HYDRAS

The hydras are of interest to almost every teacher of Biology for several rea¬ sons. Hydras are studied in practically every beginning Biology and Zoology course and are also of unusual interest because they are the only common fresh¬ water representatives of the group of Coelenterata to which the marine jelly¬ fishes and corals belong. Many teachers, however, show their students only pre¬ served specimens in alcohol or hydras mounted on slides, overlooking the fas¬ cinating studies which are possible if a few living hydras are available in the laboratory.

Form and Appearance. Hydras are not

large but after one has become familiar with their appear¬ ance and knows where to look for them they can be seen and studied fairly well even without the use of a lens. A hydra has been described as resembling a “short piece of string with one end frayed out,” and this description is a good one if the student is made to realize that a very small bit of string is meant. The frayed ends are the tentacles which radiate out from the mouth region of the animal. When fully expanded a hydra may be an inch or more in length and when contracted the entire animal looks like a tiny drop of flesh-colored jelly. There are several species of Hydra, all of which are similar in general ap¬ pearance, although they may vary greatly as to color. The commoner (and larger) species are grayish, yellowish or brownish in color, although one hydra (Hydra viridissima) is a vivid green. When fully expanded the grayish and brownish kinds are nearly transparent. Food sometimes causes peculiar colorations and on one occasion we collected a quantity of bril¬ liant red hydras, so colored because of a

microscope red crustacean upon which they had been feeding.

As pictures of hydras and discussions of their structures are given in nearly every Zoology text, these will not be in¬ cluded here.

Occurrence. Hydras are found in nearly all perma¬

nent bodies of fresh water, but are much more abundant in some places than in

other localities where the conditions are seemingly just as favorable for their growth. In general they are likely to be found in lakes, slow- flowing rivers and reservoirs, rather than in springs and swiftly-flowing streams.

Where to Collect. It will be rather

difficult for the be¬ ginner to locate Hy¬ dra in the field as the specimens con¬ tract when the wa¬ ter weeds to which they are attached are removed from the

water. Look for this form in permanent pools or small lakes or even flowing streams or rivers. In the Chicago area one species is found upon the rocks along the shores of Lake Michigan where the waves continually pound the shore. When collecting look for Hydra on the sub¬ merged water weeds. Place a few of the weeds in a quart jar of clean pond water and examine by holding it up to the light. The specimens will usually re¬ main contracted for some time and will appear as tiny lumps of jelly with the shortened tentacles projecting outward from the free end of this lump. Do not give up the quest quickly, but make a thorough search. At certain seasons of the year the Hydra may be very abund¬ ant, while at other seasons only an occasional specimen will be seen.

If specimens are located, collect a quantity of the water plants to which

First Things to Do Upon Receipt of Hydra

(1) Before opening jar, shake it gently to detach Hydra which may be attached to the glass or cap above normal surface of water.

(2) Open jar and allow it to stand quietly for a few minutes.

(3) If Hydra are to be used soon, leave them in the jar, placing it in a cool location.

(4) Do not change the water in the jar unless it appears cloudy. If new water must be added, use only clear aquarium or pond water.

(5) To keep Hydra permanently, release them in an aquarium which contains growing plants.

TURTOX

Service Department Copyright, 1960, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue

Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

Thousands of living Hydra in one of the Turtox laboratory tanks.

they are attached and take them to the laboratory in a pail of water. Place these weeds in pans of water and the Hydra will gradually expand so that they can be seen, and they may even come to the sur¬ face and hang suspended from the under side of the surface film. If a large glass aquarium tank is available, a quantity of weeds may be placed in this aquarium and the tank filled with water. After the specimens have expanded, they may be seen easily by looking through the aquar¬ ium from the side. To obtain individual specimens, clip off small portions of wa¬ ter plants to which Hydra are attached and place in another container of water.

How to Care for Hydra in the Laboratory.

If hydra are wanted only for the time the class will be studying them, the living specimens may be kept in a small-sized battrey jar or in a finger bowl full of pond or aquarium water. However, if the hydra are to be kept for a considerable period a fairly large balanced aquarium containing growing water plants (but no fish !) will be needed. Release the hydra in it and they soon attach themselves to the water plants and the glass sides of the tank. In handling living hydra two important points must be kept in mind; first, water from most city water supplies will prove fatal, and they should there¬ fore be provided with natural pond water or water taken from a balanced aqua¬ rium; and second, rapid temperature changes are usually dangerous. Hydra must be kept cool, for they can stand high (70°F) water temperatures only when the water temperature has in¬ creased very gradually.

To grow Hydra successfully, two things are necessary—a good supply of patience and plenty of Dahpnia. One of

the most difficult problems connected with the rearing of Hydra is combating the periods of depression (see next page) which affect them. When hydra become depressed, the tentacles become shortened and the body contracted. They are un¬ able to eat and will soon die. A change of water will sometimes bring them out of this state in a few days, providing the water is taken from a culture in which Hydra is flourishing. We have also found that water taken from an aquarium in which plants are growing is very bene¬ ficial. This condition is most apt to appear soon after the specimens are brought into the laboratory, and it may take considerable care to bring them through the period.

Hydra seems to do well in water which contains a slight amount of boiled aqua¬ tic plants. A very slight amount of fer¬ mentation seems to be beneficial if the water remains perfectly clear. Water thus prepared should stand for several days before the Hydra are added. How¬ ever, we have been equally successful in rearing them in a small balanced aqua¬ rium of one-gallon capacity in which were growing one plant of Vallisneria, one of Sagittaria and one of Elodea.

Hydra is a hearty eater and, if the culture is to do well, it must be fed plenty of Daphnia. This means that an active culture of Daphnia is necessary when the Hydra are to be kept over winter.

Feeding.

As has already been pointed out, Dahp¬ nia are an excellent food for hydra, but any other small crustacean such as Cyclops is acceptable. The small white- worm Enchytraeus albidus is also a good food. It is impossible to keep living hydra in the laboratory for any great

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

length of time unless a regular supply of living food can be maintained for them.

To demonstrate the feeding habits of Hydra to a group of student, pipette a few drops of water into a Syracuse watch glass and place a Hydra in this. Then add one living Daphnia or an en- chytreid and the Hydra will soon en¬ tangle the organism in its tentacles, draw it into its oral cavity and consume it.

Reproduction. Hydra reproduces by means of buds

and by sex cells, but usually the two types of reproduction occur at different times. When well fed, they will develop buds very rapidly and in the course of a few days these buds become detached from the parents and begin their inde¬ pendent existence as distinct individuals.

Usually the sex organs (spermary and ovary) are found on different animals, but occasionally both will occur on the same individual. Fertilization and the early development occur while the egg is still attached to the parent. Hydra also produce winter eggs which are fertilized in the autumn and lie dormant until spring.

Depression. Anyone who attempts to keep living

Hydra for a considerable period will sooner or later find that in spite of plentiful food and other favorable con¬ ditions they will begin to die off. This is usually due to “depression,” the causes of which are not clearly understood. De¬ pression is that slow contraction often found in hydras, where the body stalk shortens and the tentacles contract until

they become nothing but stumps, this continuing until the hydra becomes a small round ball and dies. During this period they refuse to eat and it was once thought that starvation was the cause of depression. Dr. I,. H. Hyman, of The American Museum of Natural History suggests four possible reasons for de¬ pression: (1) The temperature of the water rising to more than 20°C. (2) insufficient oxygen in the water. (3) Ex¬ cess fermentation present. (4) Changing of the hydra to water differing from that in which they were collected.

In our own laboratory we have noticed depression as a result of all of these conditions, but it also occurs at times when no reason is apparent.

Notes on Culturing Daphnia. Since Daphnia are used so commonly

in feeding Hydra as well as aquarium fishes it is well to keep a culture on hand. A few suggestions are given to help those interested.

If Daphnia are collected locally use same water for culturing them as that in which they are found. Pond or aquarium conditioned water should be used if the specimens are not collected locally.

So far as we have been able to deter¬ mine by experimentation, there has been no “sure-fire” method discovered of keep¬ ing a culture of Daphnia permanently in the laboratory. Many methods have been tried and some of them with a fair degree of success, but no perfect culture method which will work under all conditions has yet been found. The more successful methods use rather large containers— usually wooden tubs or small barrels

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

having a capacity of from twenty-five to fifty gallons. It is also well to keep the cultures in subdued light and in a place where the temperature is fairly constant. The food varies greatly. Sheep manure, bone meal, living algae, lettuce leaves and boiled water weeds have been used successfully for varying periods.

Many other small fresh-water crusta¬ ceans in addition to Daphnia are excel¬ lent for feeding Hydra. Cyclops, Oypris, small Gammarus and other similar forms are all good. One or several of these are usually found in abundance in waters

where Hydra are plentiful. One of the easiest foods to rear in the

laboratory for Hydra is the larva of the Brine Shrimp, Artemia salina. See Tur- tox Service Leaflet No. 27.

References: Morgan, “Filed Book of Ponds and

Streams.”

Ward £ Whipple, “Fresh-water Biol¬ ogy.”

See other useful references in Turtox Service Leaflet No. 14.

Materials for Study of Hydra LIVING

3V21 Hydra oligactis (Pelmatohy- dra). These large Hydra are col¬ lected in the Northern lakes and are the largest species we have seen. They are ideal for laboratory study and will be found much superior to the smaller white Hydra. Available at all seasons. Bottle of 50. .$5.00 Large. Per dozen 2.50 Large. Bottle of 25 3.50

9V11 Daphnia. Living Culture with instructions 3.50

9V1105 Brine Shrimp Eggs. Vial of about 50,000 viable eggs, vial of salt crystals for making the brine solution and cultural directions. Price 1.75

PRESERVED

3X111 Hydra. Hydra oligactis, large size, preserved with tentacles well extended. Dozen 1.00

Hundred 8.00

3X112 Hydra. Large selected speci¬ mens showing budding. Each 50

Dozen 2.50

3X113 Hydra. Large, selected speci¬ mens showing male sex organs. Each 55

Dozen 5.50

3X114 Hydra. Large mature speci¬ mens showing female sex organs. Each 65

Dozen 6.50

3X1141 Hydra Reproduction Set. In¬ cludes one of each (1) normal specimens, (2) budding specimens. (3) male and (4) female, in separate vials 1.90

MICROSCOPE SLIDES

Z3.111 Hydra, extended specimen for general body structure, w.m.. $0.85

Z3.112 Hydra, adult with bud, w.m. 1.25

Z3.113 Hydra, male, showing gonads, w.m 1.50

Z3.114 Hydra, female, showing go¬ nads, w.m 2.50

Z3.121 Hydra, x.s. showing detailed structure of ectoderm and endo-

derm 90

Z3.122 Hydra, x.s. through male gonad 1.00

Z3.125 Hydra, x.s. through female gonad - 1.10

Z3.131 Hydra, l.s., general structure. 90

Z3.132 Hydra, l.s. through adult and bud 1.25

Z3.137 Hydra smear preparations with nematocysts discharged and bud 1.20

CHARTS

Refer to your Turtox Biology Cata¬ log for illustrations and descriptions of Charts, Key Cards and Quiz Sheets dealing with the structure and devel¬ opment of Hydra.

All prices are f.o.b. our laboratories and are subject to change without notice

(3n-4)

TURTOX SERVICE LEAFLET No. 16

THE CULTURE OF PLANARIA AND ITS USE IN REGENERATION EXPERIMENTS

Living planaria are easily collected and kept in the laboratory and their remark¬ able powers of regeneration make them of unusual value in laboratory work. By following the simple steps outlined here, little difficulty will be experienced in keep¬ ing live specimens and demonstrating the phenomenon of regeneration.

1. Obtain living specimens by collect¬ ing in the field or by purchasing. Turtox can supply these in any quantity at all times of year. The smaller species, Planaria maculata, are easily collected in most localities. Look for them on the under sides of stones in pools or streams. The larger species, Planaria agilis or Planaria dorotocephala, are found in cold springs or spring-fed pools or streams, and can be captured by laying pieces of raw beef at the margins of the water. When the worms attach themselves to the meat they may be shaken into the collect¬ ing jar.

2. When the worms have been brought to the laboratory, place them in an enam¬ eled dishpan about half filled with clear water. (Pond water is better, but aerated tap water may be used.) Cut a piece of tin to serve as a cover to shut out the light.

3. Feed the worms every four or five days by placing in the pan small pieces of fresh calf or beef liver. It is inter¬ esting to note how quickly the worms can locafe the food. After two or three hours the worms will become gorged and leave the meat. At this time the food must be removed and the water changed, because Planaria cannot live in water which contains any decaying organic ma¬ terial. Agitate the water so that the animals will drop to the bottom of the pan, clean the organic material from the sides of the container and decant all the water. Pour on fresh water and, if not clear, repeat the process. Be sure every bit of meat is removed.

General Laboratory Study To observe the general appearance and

behavior place one or two live worms in a watch glass containing a small quantity of water and examine with the hand lens. To study the detailed structure place a few grains of chloretone in the water. When the worm has become motionless, place it, dorsal side down, on a slide and

l. Eye, S. brain, S. Auricle, k. Ventral nerve cord, 5. Intestine—anterior trunk, 6. Pharyn¬ geal chamber, 7. Pharynx, 8. Mouth, 9. In¬

testine—posterior trunk.

cover with a cover glass. Gently flatten the animal, but do not exert too much pressure. Examine this mount under the low power of the microscope.

Regeneration Experiments The flatworm shows remarkable powers

of regeneration. If possible, give each student a few specimens, so that he can make several experiments. If material is not abundant, let several students work together. Take several Planaria and cut

TURTOX Service Department Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed In U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Diagrams illustrating various cuts which may be made to demonstrate regeneration in Planaria.

them into pieces, as indicated by the ac¬ companying diagrams.

Planaria can be cut by allowing them to extend fully on a glass plate and then cutting with a sharp razor blade or Gil¬ lette knife.

Experiment 1. Cut forward from the caudal end of the body, to a point just ahead of the mouth. Place specimen in a dish of water. Prevent the cut halves from growing together by recutting if the wound closes. Two tails should de¬ velop as a result of this experiment.

Experiment 2. Remove head at “cut 1,” then make “cut 2” from the anterior end of the body posteriorly to a point just back of the mouth. The two halves should develop two heads. Prevent halves from fusing for a few hours as described in experiment 1.

Experiment 3. Cut a 25-millimeter specimen into eight pieces. Place each piece into a separate watch glass of water and label so that it will be possible to tell from which section of the body each piece came. The results of this experiment vary with the species used.

Experiment 4. Make two diagonal cuts as indicated in the above diagram. The center oblique section should develop a head antero-laterally and a tail should

Living Planaria

form postero-laterally. Experiment 5. Remove head at “cut

1” and then cut a triangular piece out of the anterior third as illustrated in the diagram. A normal head should develop at “cut 1” and a very small head at the side.

NOTE: Various abnormalities will oc¬ cur in these experiments. All of the re¬ sults should be recorded by means of diagrammatic sketches.

Living Material

5V11 Planaria (Dugesia). Large size. This is the fresh-water planarian commonly used for experimental purposes. Dozen $2.25 Fifty 8.00 Hundred 15.00

Preserved Material

5X15 Planaria (Dugesia). Large fresh-water planarian. 15 mm. or more in length. The large size and comparatively small amount of pigment in the body make these speci¬ mens ideal for laboratory work. Flattened. Each 35 Dozen 2.00 Hundred 14.00

Microscope Slides

Z5.ll Planaria (Dugesia), w.m. Animal is excellently fixed in an expanded condi¬ tion. The digestive system is completely in¬ jected with carmine, in contrast with the lightly hematoxylin-stained background of the body $1.10

Z5.12 Planaria, x.s. through anterior, middle (pharynx) and posterior regions 1.35

Z5.13 Planaria. l.s. Each slide contains at least one section through the pharynx. 1.50

Z5.15 Planaria, w.m. of injected specimen and uninjected specimen stained for cellular structure. Both on one slide 1.60

All prices are F.O.B. our laboratories; subject to change without notice.

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TURTOX SERVICE LEAFLET No. 41

COLLECTION AND CULTURE OF EARTHWORMS AND OTHER ANNELIDS

The large earthworm, Lumbricus terrestris, commonly used for dissection in schools, is a somewhat delicate animal, not easily maintained under artificial conditions in large concentrations. How¬ ever, if large containers and the correct soil mixture (see below) are provided, and if at least one cubic foot of soil is provided for each 40 earthworms, good results can be secured. If the earth¬ worms are very large, it is often better to limit the population to 25 worms per cubic foot.

Smaller earthworms (there are numer¬ ous species) are hardier and can be maintained in greater concentrations. The manure worm, Eisenia foetida, is very hardy and easy to maintain in laboratory cultures. This worm is a good size for use in feeding toads, frogs, snakes, lizards and aquarium fishes; and it produces abundant cocoons from which very small worms emerge.

Collection

Earthworms (Lumbricus terrestris) may be collected in quantity on rainy nights during the spring and fall. In summer the rains are likely to be short and hard followed by quick drying, giving the worms little opportunity to emerge. The best collecting season is in the spring. During the winter the worms are confined below the frozen earth, but with the spring rains and the thawing of this frozen crust they swarm to the surface. In the Chicago region this swarming usually occurs during the months of April and May, at which time spells of rainy weather of two to three or more days duration are common, with the precipitation heaviest during the night. It is during the time of these cold spring rains that the earthworm collector equipped with rubber boots, raincoat, bucket and flashlight, repairs to the lawns and shrubbery. On hands and knees he moves cautiously, and with straining eyes endeavors to distinguish the worms from the twigs and leaves with which the ground is covered. As visual adjustment comes, the worms may be seen lying with the anterior half

of the body out of the hole. By a quick dart of the hand the worm is seized just where its body emerges from the hole and with a steady pull the posterior end is drawn out and the worm dropped into the bucket. Should the pull be too vigorous the collector feels a sudden giving way and finds himself holding a half worm, the other half being securely anchored in the hole. It is the collector’s hope that the rain will continue regard¬ less of its chilling effect and despite the fact that the ground over which he must crawl is becoming muddier and muddier, for should the rain cease, the worms soon retreat into their holes. Earthworms must be stalked with some care, for they are sensitive to the jar of the earth caused by the approaching collector and will jerk back into their holes like a flash at the faintest vibration of broken stick or incautiously placed hand, knee or pail. Should the rain pour down in a steady, chilling sheet, the collector picks worms with both hands, and, with con¬ tinuous rain, many of the worms leave their holes entirely. Copulating pairs are now numerous and two worms can be scooped up with a single motion. Should the rain continue all or most of the night and into the next morning, the streets and roads may become covered with worms that have crawled or been washed onto the hard surface and are unable to escape by burrowing.

Culture

In many parts of the United States earthworms of one or more species are plentiful and small quantities can be collected during the spring, summer or autumn by digging for them in gardens, barnyards or moist woodlands. If large numbers of worms are required, outdoor beds can be established, or large indoor containers can be maintained.

A very satisfactory and productive outdoor “earthworm farm” can be made as follows:

1. Select a shady place where the soil is well drained and excavate an area about 6 by 6 feet (or larger if desired) and about three feet deep. If the under-

TURTQr^BUCTS

TURTOX Service Department

Copyright, 1960, by

GENERAL BIOLOGICAL SUPPLY HOUSE (Incorporated)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SION OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

lying soil is heavy clay, line the ex¬ cavated area with six or eight inches of coarse sand and gravel.

2. Fill the reminder of the bed with a mixture of equal parts of light loam, well rotted manure and leaf mold. The leaf mold should contain a good propor¬ tion of half-rotted leaves.

3. After this has settled, wet it thoroughly and place the worms on the surface of the bed.

If the surrounding soil is heavy clay, sand or rather barren and dry, the worms will remain pretty well within the bed, breeding and increasing over a period of years. However, if the surrounding soil is a rich, light loam, the worms are likely to spread out and migrate slowly to surrounding areas. This can be pre¬ vented, at considerable cost, by lining the excavated area with a wooden framework covered with fine-meshed copper or bronze screening.

It is well to introduce small quantities of well-rotted manure and rotted leaves into the bed once or twice a year, and to keep the surface moist and covered at all times with a layer of leaf mold several inches deep. In the northern states, a covering of a foot or so of straw, weighted down with poles or boards, will retard frost to some extent and make the living worms available during the winter months.

Containers for maintaining earthworms indoors are more convenient than an outdoor bed if large quantities of worms are not required. The essentials of indoor earthworm culture are (1) a large con¬ tainer, 4 to 6 cubic feet minimum; (2) a temperature lower than 60° F; and (3) proper control of moisture. Earth¬ worms cannot be crowded and not more than 40 medium or large specimens should occupy one cubic foot. A con¬ venient and practical container is a wooden box measuring 4x4x2 feet; or an oak barrel or large keg can be used. For best results the temperature must be kept between 40° F. and 55° F. The container should be filled with a mixture of very light loam (never use clay), partly rotted leaves, leaf mold and well rotted cow or horse manure. This mixture should be kept slightly moist (but never wet or “soggy”) at all times. To retard evaporation and help maintain an even temperature, the container can be covered with a plate of glass or a piece of well varnished (to prevent warping) plywood or wallboard. If the soil contains plenty of dead leaves, no special feeding will be necessary.

In a container of this type, one can keep considerable quantities of adult worms, and some egg capsules or co¬

coons can usually be found at all seasons.

Enchytrae Worms

Every fish fancier knows “Enchytrae” as one of the best foods for his fish. Equally important is it for food for fish in laboratory aquaria and for the small salamanders such as red-spotted newts. The scientific name of this “domesticated” annelid is Enchytraeus albidus.

The cultural methods for this form are quite similar to those of earthworms. Secure a tight wooden box and fill it up to within two inches of the top with rich humus, sifted to remove coarse particles. Place the original culture in a hole in the center. Cover it over with humus and then cover the box with a wooden or glass cover to prevent it from becoming dry. Place the box in a cool basement, the temperature should not become higher than from 60° to 65 °F. Add water sparingly—just enough to keep the humus damp.

Feed Enchytrae bread soaked in milk by working small holes in the humus, filling them with the food and covering over with an inch of humus. Alternate foods are cooked oatmeal, mashed potatoes, mashed potatoes mixed with rich chicken broth, and the like. Feed worms sparingly about twice a week, but never until previous food has been consumed. If food sours, remove at once.

For freeing the worms of dirt when ready to feed them to laboratory animals, place a quantity of them, dirt and all, in a glass jar or paper cup and fill it one-quarter full of water. Within 30 minutes, the worms will have crawled up the sides, where they may be secured free of all dirt.

Aquatic Earthworms

Tubifex and other aquatic earthworms are found in the mud at the bottom and along the shores of most bodies of fresh water. They can be seen waving aimlessly above the mud and when disturbed retreat into their slime and mud tube. Some species live in decaying vegetation and others are found in considerable numbers in floating masses of algae.

Aquatic earthworms are collected with the mud or decaying debris in which they live and then washed free of the mud by placing the collected material in cheese cloth or fine-screened sieves.

It is easy to keep a culture of these worms growing in an aquarium. A layer of mud about an inch deep, containing Tubifex or other aquatic earthworms is placed on the bottom of the tank and covered with a half inch layer of sand.

(41-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

A pair of copulating earthworms (Photographed late on a rainy night)

The tank is prepared as a balanced aquarium with plants and animals. After the water has cleared the Tubifex will be seen, their bodies extending above the sandy layer. Jarring the aquarium will cause them to retreat momentarily. It is wise to keep fish out of the tank in which Tubifex and other aquatic earthworms are kept.

Aquatic earthworms are good fish food. To obtain them in quantity free of mud they should either be washed clean as stated above or the mud containing the Tubifex and other species should be placed in shallow pans over a warm radiator or low flame. The warmth drives the worms to the surface.

Leeches

Leeches are to be found in almost every standing pond, ditch, lake, and sluggish stream. Many of them are beautifully colored and add much attractiveness to the ordinary balanced fresh water aquar¬ ium. Their care is a simple matter and yet their behavior is of great interest. Although a number of leeches are to be found in local ponds, the one most com¬ monly studied in the laboratory, both preserved and living, is the imported medicinal leech, Ilirndo mediciualis. Of particular interest is the giant leech, Haemopis grandis, the largest American species, which is now much used in school laboratories.

Hirudo medicinalis is still used in medi¬ cine to a small extent. This practice was so prevalent in Europe many years ago that “leech” became practically synony¬ mous with “physician.” Care in the Laboratory. Each year, Tur- tox imports large numbers of the medici¬ nal leeches from France. They usually reach us packed in damp earth. In fact, they can live in this condition for rela¬ tively long periods of time. For actual study, it is much better to keep them in balanced fresh water aquaria. Care should be taken that the aquarium is well supplied with the large oxygenating plants such as Elodea, and Vallisneria. Place the aquarium so that it receives very little direct sunlight as leeches pre¬ fer darkness. Provide stones, leaves and other opaque objects under which they may hide to escape the light.

The usual animal population of the aquarium, such as small fish (bullheads, sunfish, and the like), tadpoles, small crayfish, salamanders, snails, and insect larvae, should be kept in the aquarium. The excess food given the fish promotes growth of the small forms, which in turn furnish some of the food of the leeches.

Most leeches will partake of blood at some time during their life. Some are parasitic upon turtles, frogs, salaman¬ ders, and fish. Although it isn’t essential that they have blood to live for only a month or two, it is interesting to know just what kinds of blood they prefer. The turtle is frequently used to supply

(41-3'

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

the blood for live leeches (the leeches are placed upon the soft skin of the tur¬ tle). Rabbits may also be used for this purpose by shaving the hair from a small area of the ventral skin. Leeches will sometimes feed upon fresh liver if placed In a fingerbowl along with it. These and other methods determined by experimen¬ tation will be helpful when leeches must be kept for long periods of time. Leeches have lived in our aquaria, practically Without attention, for as long as a year. Teachers will experience no difficulty in

maintaining them for experimental pur¬ poses if but slight attention is given.

Marine Annelids Students living near the seacoast can

collect living marine worms such as Nereis, Amphitrite, and Arenicola. Some of these will live for several weeks in a salt-water aquarium, and are far more interesting than preserved specimens. Refer to Turtox Service Leaflet No. 20, Notes on Marine Aquaria.

MATERIALS FOR THE STUDY OF ANNELIDS

8V11 Lumbricus. Earthworms. Large mature specimens, each with clitel- lum. Usually available at all sea¬ sons, but must be ordered two weeks in advance of delivery date. Dozen $3.00 Hundred 14.00

8V112 Earthworms. Small specimens, suitable as food for snakes, sala¬ manders, turtles and the larger aquarium fishes. Order two weeks in advance. Lot of fifty with direc¬ tions for establishing a permanent culture 4.00 Per hundred 7.00

8V115 Earthworm Cocoons. The co¬ coons or capsules contain eggs or minute earthworms in various stages of development. Easily kept in moist filter paper for study; culture and instruction sheet sent with each shipment. Available from November to May. Per dozen 1.25 Per hundred 6.00

8V12 Enchytrae. A small, thread-like, white worm about 25 to 30 mm. in length, especially useful as a food for small aquarium animals. Instruc¬ tions for the care of the living Enchytrae are sent with each ship¬ ment. Large portion, sufficient to start a permanent “culture”. . .$3.50

8V14 Tubifex. Small aquatic earth¬ worms found in pond mud. Large portion with instructions for start¬ ing permanent culture 4.00

8X264 Lumbricus. Preserved Earth¬ worms. Large size, 9" to 12" for dis¬ section and laboratory study. Dozen 2.10 Hundred 16.00

8X266 Lumbricus. Preserved Earth¬ worms. Mature specimens, size 7" to 9". Dozen 1.70 Hundred 12.00

The special 64-page catalog, Turtox Dependable Biological Supplies lists all of the material needed in beginning courses in Biology, Botany and Zoology. This catalog is especially helpful in planning requisitions for purchases under Title III of the National Defense Education Act.

A comprehensive selection of Turtox Charts and Turtox Key Cards of Earthworm, Leech and other Annelids is available. Write for the free Turtox Three-Way Checklist of charts and biological drawings.

All prices are f.o.b. our laboratories and are subject to change without notice.

(41-4)

TURTOX SERVICE LEAFLET No. 27

BRINE SHRIMP AND OTHER CRUSTACEANS

This leaflet discusses very briefly the care of the brine shrimp, Artemia salina, and some of the fresh-water crustaceans commonly studied in school laboratories. For information on barnacles and ma¬ rine crabs, refer to Turtox Service Leaf¬ let No. 20, “Notes on Marine Aquaria.”

Brine Shrimp The brine shrimp, Artemia salina, is a

phyllopod crustacean of very general distribution. It is extremely interesting as a marine crustacean which can be easily reared and studied in the school laboratory, and it has great practical value as a constant source of living food for Hydra and small aquarium fishes.

The resting eggs of the brine shrimp float in the water and require drying before they will hatch. This feature makes them available at all seasons, for the dried eggs remain viable (alive) for several years if kept in a dry and fairly cool place.

Hatching the Eggs. The eggs hatch very quickly (within 24 to 48 hours) after being placed in a brine solution which is kept at a temperature of 70° to 75° F. Natural or artificial sea-water may be used if available. However, the brine solution used as a hatching medium may be almost any concentration from about 0.1% to 6%. When sea-water is not available make up a brine solution by adding two teaspoonfuls of common table salt to one quart of water. After hatching, the larval stages (nauplii) may be used as a food (see below) or may be transferred to other containers for fur¬ ther development. If thousands are al¬ lowed to remain in one container they will die after a few days for lack of food and sufficient oxygen.

Rearing the Shrimp. If you wish to rear Artemia to maturity and establish a permanent culture, place only a few of the larvae in a quart or larger con¬ tainer of brine solution and supply them with food. Yeast is a satisfactory food, as are also various of the one-celled floating algae. The latter will usually appear in a culture of natural sea water rather soon and will eventually appear in any brine solution in which the brine shrimp eggs (together with the natural

debris attached to them) are placed. For quicker results scrape some of the green slime from the glass side of a fresh-water aquarium and place a little of this in the brine shrimp culture. (Many of these unicellular fresh-water algae will grow in a weak brine solution as readily as they grow in fresh water.)

Use as Food. For feeding Hydra, the larval brine shrimp may be concentrated by straining the culture solution through a piece of fine-meshed cloth. They are then placed in the fresh-water tank con¬ taining the Hydra, where they will live for several hours, usually long enough to provide the Hydra with ample food. Be¬ cause of the very low cost of the eggs and the great rapidity with which they hatch, new cultures can be started every few days if a constant food supply is wanted.

The earliest larval stages are also very useful as food for small aquarium fishes; or the brine shrimp may be allowed to develop and grow larger if they are de¬ sired as a living food for larger fishes.

Laboratory Study. Be sure to examine the eggs (both dry and hatching) and the nauplius larvae under the micro¬ scope. It is a most interesting study and it permits you to show your students the continuous development of living examples of a marine crustacean.

Glass Shrimp This fresh-water shrimp, Palaemonetes

exilipes, or “glass shrimp”, as it is often called because of its remarkably trans¬ parent body, is an interesting crustacean for laboratory study, and a valuable scavenger for the balanced aquarium. It lives well in either a small or a large balanced aquarium, but prefers a tank containing plenty of vegetation. It lives harmoniously with most small fishes, but must not be placed in an aquarium con¬ taining fishes large enough to eat it. Palaemonetes will sometimes breed if a considerable number of individuals are kept in a large aquarium, and the fe¬ males carrying eggs are particularly in¬ teresting for study. When hatched, the young shrimps are very small and will form food for even the smaller fishes un¬ less they are immediately transferred to

TURTOX Service Department Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE ( IX CORPORATED )

8200 South Hoyne Avenue Chicago 20, Illinois

THE SION OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Larva of the Brine Shrimp.

a tank where this danger does not exist. The glass shrimp is largely a scaven¬

ger in its feeding habits and is therefore useful in keeping the aquarium clean. It will eat particles of fish food and may occasionally be fed tiny pieces of oyster, raw fish or raw meat.

Fairy Shrimp During the late winter or early spring,

it is not uncommon to find small pools containing thousands of fairy shrimps. All disappear after a few weeks and the pools in which they lived, being of a temporary type, may dry up completely during the summer months. Not so many years ago, the fairy shrimp was pointed to as a splendid proof of the theory of “spontaneous generation”; it appeared in unbelievable numbers in pools formed by the melting snow, it disappeared as suddenly as it had come and then in a few weeks the pool in which it had lived became a dry and dusty bit of ground.

The explanation, of course, as every teacher now knows, is that this creature produces resting eggs which sink to the bottom and live in a dormant stage for long periods. Indeed, some of these eggs appear to require drying before they can develop, and all fairy shrimp eggs can go through extended periods (several years) of drought and freezing without harm. When conditions are again suitable (when the spring thaws flood the pools), they hatch and thousands of fairy

shrimps again swim about. Eubranchipus when adult is about one

inch in length and reddish or bronze in general coloration. The females are larger and usually much more plentiful than the males. All species of fairy shrimps swim on their backs, propelling themselves rather gracefully by means of their eleven pairs of waving gill-feet. They feed upon diatoms, protozoans and other microscopic forms.

We have never been able to discover any dependable way of maintaining per¬ manent laboratory cultures of Eubran¬ chipus, although dormant eggs brought into the laboratory and placed in water will usually hatch at any season. Speci¬ mens collected in the spring will usually live for several weeks in jars or aquar¬ ium tanks. The temperature of the water should be kept as low as possible, as high temperatures hasten their devel¬ opment and shorten their life span.

Daphnia Daphnia (or “water-fleas”) of various

species can be collected in small quanti¬ ties ordinarily by swishing a fine-meshed net among the aquatic plants growing in shallow water. At some seasons, often during the autumn, Daphnia and other small crustaceans occur in great abun¬ dance in the shallow waters of stagnant ponds and ditches. Since Daphnia are used so commonly in feeding hydras and small aquarium fishes, it is well to keep

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Female Cyclops with egg sacs.

cultures of them in the school laboratory. If Daphnia are collected locally, cul¬

ture them in the water in which they are found. If the specimens are not obtained locally, use pond water or wa¬ ter from an established and well bal¬ anced aquarium.

It is only fair to admit that we know of no “sure-fire” method of maintaining permanent cultures of Daphnia. Many methods have been tried and some of them with a good degree of success, but no perfect culture method which will work under all conditions has yet been discovered. The more successful methods use rather large containers — usually wooden tubs or barrels having a capac¬ ity of from twenty-five to one hundred gallons. Four or five cultures should be maintained, for then there is a good chance that at least one culture will con¬ tain numerous Daphnia at any given time.

Professor Harold Heath of Stanford University recommends the following method: “Use one ounce (by weight) of dried sheep manure to one gallon of water. A wooden trough holding twenty- five gallons of water is used as a con¬ tainer. After the sheep manure and water have been in the trough for two or three days, the Daphnia are added. A few lettuce leaves are placed in the culture from time to time and are re¬ placed by fresh leaves as they become thoroughly decayed.”

Other foods frequently used include bone meal, decayed masses of algae and

decayed aquatic plants. Keep the cul¬ ture in subdued light and in a place where the temperature is fairly con¬ stant at'65° to 70° F.

Other Fresh-Water Crustaceans

Cultures of mixed crustaceans may include many small forms which occur in ponds and slow-flowing streams. The cultures which we furnish during the autumn and winter months usually con¬ tain Daphnia, Cyclops, Cypris, Latona, Gammarrus and others. Most of these will live well in cultures or in small bal¬ anced aquaria.

A small aquarium of from one to five gallons capacity is suitable for mixed crustaceans. Such an aquarium should contain sand and growing plants and some bottom debris (dead leaves, mud, etc.) from a pond. It can contain a few snails and small clams, but must not include fish which would feed upon the crustaceans.

Gammarus will live well and repro¬ duce abundantly in a small tank con¬ taining a few dead leaves and other decaying vegetable matter. These smaller crustaceans need little attention and will continue in fair numbers in any small tank containing pond debris and some algal growth.

Crayfish

Crayfish are of very general distribut- tion and adult specimens can usually be obtained in lakes, ponds, and streams

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Living Daphnia photographed in a culture.

without difficulty. Being to some extent nocturnal, they hide during the day and will be found by lifting up flat stones or other objects under which they retreat for protection. In places where cray¬ fish are plentiful it is usually possible to secure small specimens by swishing a net around among the aquatic plants or by dipping up netfuls of bottom debris. Crayfish which are to be kept in aquaria in the laboratory should be collected from ponds or quiet rivers, as those taken from fast-flowing streams are less likely to live in captivity.

In the late autumn or early spring look for the females carrying eggs at¬ tached to their swimmerets. If one is found, take her to the laboratory, place her in an aquarium and watch the eggs develop into young crayfish. Often fe¬ males carrying young will be found in the early spring, although within a few days the fully developed young will leave their mother’s swimmerets to shift for themselves.

The crayfish is one of the most inter¬ esting forms to keep in the laboratory and two or three should be included in a large aquarium. Students will learn more of the habits of this animal by ob¬ serving a few living specimens than they will by reading chapters of textbook material. An adult may be placed in a gallon battery jar containing clear wa¬ ter and the students can observe the movements of the mouth parts, antennae and appendages. Feed the specimen a small piece of raw fish or beef and ob¬ serve its method of taking food.

Crayfish often uproot or otherwise dis¬ turb the plants in an aquarium unless they are provided with stones under which they may retreat. Therefore, it is a good plan to have a loose pile of good-sized stones in one corner of the aquarium, and also to select small cray¬ fish as aquarium inhabitants. Two or three specimens measuring about two inches or less in length are enough for a six gallon tank.

Refer to your Turtox Biology Catalog for current listings and prices of living crustaceans. Brine shrimp eggs are available as follows.

9V1105 Brine Shrimp Eggs. Vial of about 50,000 viable eggs of the brine shrimp, Artemia salina, a vial of salt crystals for making the brine solution and complete directions for culturing. These eggs are available at all seasons and will hatch within two days after being placed in the brine solution. Excellent for the living study of the development of a marine crustacean and as a food for Hydra and both fresh-water and marine aquarium fishes. Complete culture set as described.. .$1.75

All prices are f.o.b. our laboratories and are subject to change without notice.

(27-4)

TURTOX SERVICE LEAFLET No. 7

THE CARE OF FROGS AND OTHER AMPHIBIANS

Frogs Grassfrogs (Rana pipieus and others). One of the most frequent inquiries reach¬ ing tile Turtox Service Department is the one which asks, “How can I keep living grassfrogs in my laboratory?” The answer is not as simple as one might suspect, for different methods are used, depending on the number of frogs to be kept, Uie length of time they are to be maintained, and the time of year the stock is purchased or collected.

The small laboratory may wish to keep only four to six specimens for a relatively long lime. A large woodland terrarium would be almost ideal, if there were a sunken dish in it in which water is kept. Or, the semi-aquatic terrarium can be used with success. In either case, you will have provided the approximate environment afforded in nature. These are meadow frogs, frequenting almost dry fields in the summer in quest of in¬ sect food, and returning to the ponds for hibernation and subsequent egg-lay¬ ing the following spring. Feed them living insects such as mealworms, cock¬ roaches, small grasshoppers if available, Hies, caterpillars, and the like. They ean sometimes be trained to accept beef liver, lean beef, etc., moved before them on the end of a string or broom straw. See that eacli individual receives some food.

Some of the laboratories of our large universities wish to keep great numbers of frogs available for several weeks. Their care is often a real problem. We recommend large wooden tanks (cypress is perhaps most economical in the long run) with a gradual slope of pebbles, about %" diameter, in one end. The tank is to be equipped with an inlet so that water is constantly trickling through it, and an outlet which drains all but about %" of water in the bottom. Not much water is needed, especially when the frogs are kept during the summer. That which flows through the tank tends to keep it clean and free from wastes. However, flush it out thoroughly every four or five days. It is best to locate such a tank in the base¬ ment, where it will remain cool at all

times. Keep it dark and do not disturb the frogs often. Inspect at least every third day (oftener if there has been a disease) and remove any frogs that have died or are in a weakened condition. If disease should become rampant, sort over all specimens and segregate those not in the best condition. Flush out the tank thoroughly, remove specimens to a temporary storage receptacle and ster¬ ilize the tank with a strong salt solution, a formalin solution or lime. Return the frogs only after a thorough washing out of the disinfectant. Always disinfect tanks thoroughly after each lot of frogs is used and also just before another batcli is to be introduced.

In such concentrations, it is seldom advisable to feed the frogs, as it would be an unending task. If kept cool, un¬ disturbed, and dark, there will be but little activity and, hence, but little necessity for food. During the winter months the frogs can stand much more water and the tank described above may be altered so as to keep a constant level of water about one to two inches deep.

Another method for maintaining liv¬ ing frogs consists of a tank, (or any other suitable container) provided with a layer of damp (not wet) sphagnum moss. (The dry baled moss used by florists is suitable.) The slight amount of iodine in the moss appears to prevent red-leg and other infections. The sphagnum moss tank should contain only the damp moss—no water.

Care of Frogs upon Arrival. Just as soon as you have received your frogs, whether there be three or three hundred, open the package carefully and then inspect each one to be sure it has not suffered from its long journey. It is well to have a pail of fresh water handy and to wash the animals off before plac¬ ing them in your tanks. Any frog which appears to be diseased should be segregated.

Bullfrogs and other aquatic frogs. Bullfrogs may be collected in swamps and lakes and they occur more or less throughout all the eastern half of the United States. However, most bull-

TURTOXmOIUCTS

TURTOX Service Department

Copyright, 1960, by GENERAL BIOLOGICAL SUPPLY HOUSE

(IXCORPOEATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

frogs sold by dealers and supply houses come from the Gulf States, as these southern frogs are larger and bullfrogs are much more plentiful in the South. It is not uncommon for them to be rather sluggish when they arrive at northern destinations during the winter and spring months. They will become lively as soon as they are placed in water and allowed to warm up gradually, say, to about 76“ F.

Upon arrival, the bullfrogs should be washed in clear water to free their bodies of any wastes which might have ac¬ cumulated during shipment. Place them then in a large aquarium; probably one equipped with running water would be best. It should be screened over to prevent the frogs from leaping out and dying when no one is in the laboratory. These animals can float on the water for long periods of time but should be pro¬ vided with rocks or other supports upon which they can rest. If large quantities of bullfrogs are to be kept, the wooden tanks recommended in the section deal¬ ing with grassfrogs are very desirable.

Feed bullfrogs upon small crayfish, minnows, earthworms, young grassfrogs and the like. Green frogs may be fed earthworms, mealworms and flies. It is possible to train either of these frogs to take food to which it is unaccustomed in nature—lean beef, beef liver and even canned shrimp if it is dangled before the animal so that it appears In have motion.

Green frogs (Rana clamitans and other aquatic species) require about the same care as bullfrogs. Being much smaller, however, a few individuals will live nice¬ ly in a swamp or semi-aquatic terrarium.

Tree Frogs

Tree frogs, or tree “toads” (ITyla) as they are commonly called, are plentiful in many sections of the United States, but because of their secretive habits and protective coloration they are seldom seen by the casual observer. Tree frogs live in moist situations, spending much of their time in trees and hushes and in the case of most species, resorting to ponds only during the breeding season. They live remarkably well in a school terrarium and are interesting amphibi¬ ans for laboratory observation.

Tree frogs must be maintained in a humid situation where sufficient moisture is available at all times; the woodland type of terrarium is well suited to them. (Note: See Turtox Service Leaflet No. 10, “The School Terrarium.”) Screened ter¬ raria are not satisfactory; the best type is a terrarium housed in a standard rectangular aquarium tank with a glass

cover. In a terrarium of this kind the humidity is easily controlled and the tree frogs are protected from drafts and sudden temperature changes. The ter¬ rarium should contain loam and leaf mold and may be planted with mosses, ferns, liverworts and other woodland plants.

Tree frogs require living food and will readily accept almost any small in¬ sects. They will occasionally take worms and will sometimes accept tiny pieces of raw meat presented on the end of a toothpick. During the winter months living Drosophila (fruit flies) and cockroaches (both of which are easily reared in laboratory cultures) are quite satisfactory as food. Small meal¬ worms may also be used.

Frog Eggs

The eggs of the grassfrog, Rana pipiens, are found in ponds and ditches in early spring. Eggs of the spring peeper, Ilyla crucifer, appear at the same time, but toad eggs are not found until the first part of May, and bullfrog eggs are usually not found until June.

It is interesting to follow the devel¬ opment of the frog from the egg, through the tadpole stage, to the adult frog. Any makeshift aquarium can be used for this study and since many schools have a fresh-water aquarium we suggest placing a few eggs in it for ob¬ servation.

Frog eggs are found in shallow water. When they are collected they should be brought to the laboratory in pails of the pond water in which they are found.

In the laboratory the eggs may be placed in shallow pans, or a small cluster can be added to the fresh-water aquarium. It is best not to place an entire clump of frog eggs in the tank lest there be more tadpoles in the aquarium than the water can support. Approximately twenty-five to fifty eggs are enough for a demonstra¬ tion. Small lots of eggs can be kept in finger bowls, provided that the water is changed frequently. Large masses of eggs can be kept in dishpans which are artificially aerated until the eggs have hatched, but then they must be spread around in other containers or better still the majority of tadpoles should be re¬ turned to the pond where they have a much better chance for survival.

The rate of development of the frog egg is determined almost entirely by the temperature of the water. A temper¬ ature range of 50“ to 60“ F. is satisfac¬ tory, but a water temperature of 70“ to 75“ will cause more rapid develop¬ ment especially where the -food supply is adequate. Overheating of the water will cause the eggs to die.

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

At a temperature of 70° to 75° the young tadpoles will attain swimming size in about two days. Watch the egg mass closely and should it turn white, it is an indication that the eggs are unfer¬ tilized or dead. The whole mass will pol- 'ute the water and should be discarded. Healthy eggs soon turn dark all over as they develop.

Toads The common toads do well in certain

terraria. Size is an important matter in keeping them; the small to medium- sized animals usually being sought as they adapt themselves better to the small area afforded in a terrarium. Too, a toad will burrow into the soil and, the larger he is, the more trouble one will encounter in keeping plants growing on the soil surface of the terrarium.

Terraria are planted in the usual way with some soil in the bottom covered over with mosses, lichens, and the like. Ferns, the dwarf varieties, are planted in one end. A group of rocks may be arranged in the opposite end and, if there are some cracks, in which the toad may hide, perhaps he will take to them rather than resort to burrowing. Do not disturb the animal for quite some time after establishing it in its new home. Provide a shallow drinking pan.

Feed them living specimens at first— cockroaches, caterpillars, mealworms, Hies if available, ants and young spiders. Later, you may have the toad take sev¬ eral angle worms or even lean beef held in front of it at the end of a toothpick or broom straw. There is no danger of over-feeding, although it is not neces¬ sary to feed every day. Two or three times per week is generally sufficient.

In small terraria, it is not advisable to keep salamanders with toads. The poison from the toad skin is quite detrimental to other small amphibia.

Toads must be kept warm if they are to lie active. When room temperatures fall below 70° F., toads usually burrow under and may not be seen for long periods. An electric light bulb suspended above the terrarium frequently has a Deneficial effect upon them. They may not come out into the bright light, but it warms them up so that, as soon as the light is removed, they will usually hop out of their retreat and feed.

Salamanders There are many kinds of salamanders

which may be kept in the laboratory and we give here brief directions on the care of those which are more generally main¬ tained. Other salamanders may be

prevalent in your locality and since all have much in common it will be a simple matter to vary our suggestions to care for the species in which you may be interested.

Newts. The common red-spotted newt receives our first attention, because, without a doubt it is the one most gen¬ erally seen in the laboratory. There are two distinct phases in the life of this animal; the red eft or land phase and the olive green, red spotted aquatic phase. Tlie red eft is an ideal woodland terrarium animal; its brilliant color con¬ trasting so vividly with that of the moss- covered terrarium floor. This phase does nut require the aquatic habitat and should never be placed in an aquarium. It feeds upon very small insects, young spiders, small ants and similar living food. White worms (Enchytraeus) are relished. Bits of lean beef and calf liver can be fed with forceps. The animals being very small, require but infinites¬ imal amounts of food.

The aquatic phase of the newt should be kept in an aquarium. It is well if the tank is heavily planted so that the leaves form mats in some places on the surface of the water, as the newts like to crawl out upon them at will. Feed this animal by removing it from the tank to a lingerbowl of tepid water into which has been dispersed small particles of lean beef or liver. Leave it here until it has eaten as much us it likes, then wash it off and return it to the aquarium. About once a week is satisfactory for feeding unless the animal has a voracious appetite, in which case you may feed as often as it appears to lie hungry. Once the animal lias become accustomed to a regular feeding schedule and to this type of food, one may be successful in feeding it in the aquarium. The only danger is that food particles introduced might not be eaten and then decay, thus fouling the aquarium. This point cannot be over-emphasized in feeding any aquarium inhabitants—excess food is very apt to spoil the entire set-up.

The Large Adult Salamanders. The tiger salamander (Ambystoma tiyrinum), the spotted (A mby.stoma maculatum) and the marbled (A. opacum) are com¬ monly in stock in the proper season in the Turtox Laboratories. They make good terrarium inhabitants, thriving in either the woodland or semi-aquatic habitais. They frequently burrow beneath I he moss covering to hide but are less likely to do so if some crevices between rocks or small “logs” are provided. This type of environment is also suitable to the giant newt of the Pacific Coast, Trilurus tornsus. They feed upon meal-

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

The Tiger Salamander, Ambystoma tigrinum.

worms and other small insects but one can train them to accept, quite willingly, large amounts of hamburger. They have to be fed with forceps at first but even¬ tually will feed out of a small dish. Feed at regular times and less trouble will be encountered. Vary the diet with liver and experiment with various other raw meats.

Triturus pyrrhogaster. This form (the red-bellied salamander) lives well in the ordinary balanced aquarium and may be cared for in the same manner as described above for the aquatic phase of the red-spotted newt.

Plethodons. These small salamanders make good terrarium animals, preferring the moist but not wet woodland environ¬ ment. They are delicate and therefore

must be given special attention. Their care approximates that indicated above for the red eft.

Necturus, Cryptobranchus and Amphi- uma. These large amphibia are not generally suitable for balanced aquaria (unless they be of small size and the aquarium of large capacity) and are most successfully maintained in the run¬ ning-water aquarium. They may be kept for long times in such tanks, even in large wooden tanks where the water depth is maintained at a constant level of about four inches (a continuous flow being provided). They will eat earth¬ worms, minnows, small crayfish, large water bugs and calf liver. Do not allow the water to become warm during the summer months.

Additional Turtox Service Leaflets offering information on the care of Amphibians are:

No. 10 The School Terrarium

No. 23 Feeding Aquarium and Terrarium Animals

13V25 Triturus viridescens. The Red-spotted Newt, aquatic phase. Two for $1.35 Dozen 7.50

13VS3 Tree Frogs. Will live well in a moist woodland terrarium. Three for 3.50 Dozen 10.50

Write for current prices of grassfrogs, bullfrogs, Ambystoma, Necturus and other amphibians.

All prices are f.o.b. our laboratories and are subject to change without notice.

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TURTOX SERVICE LEAFLET No. 40

THE CARE OF RATS, MICE AND GUINEA PIGS Rats:

The albino rat has long been used in anatomical, physiological and psycho¬ logical experimental work, and because so many data are available it is con¬ sidered a standard laboratory animal.

A pair of rats may be kept in a small cage, measuring ten inches in height, ten inches in diameter, and constructed of galvanized hardware cloth of three- eighths inch mesh for top and sides. The bottom should be made of one-half inch mesh to allow the feces to drop through. The top of the cage should be removable or hinged, to facilitate removing the animals for observation and for clean¬ ing the cage. A shallow pan should be placed underneath the cage to catch the urine and fecal matter. This pan must be cleaned daily to avoid any undesir¬ able odors in the laboratory.

If more than one pair of animals are to be kept, a larger size cage is required. It should be constructed of the same material, but it should be nineteen inches long, twelve inches wide and nine inches high. The cage also should be equipped with a pan, and the top hinged, or a long door made on one side. Larger cages than the above size may be used, but they are difficult to handle in the laboratory.

The best location for the cage is out of all drafts and direct sunlight. Rats should receive their sunlight only in the form of diffused radiation. The air must be kept fresh and a temperature of 68° to 72° is adequate. Sudden changes in temperature are harmful and should be avoided as much as possible. Some ar¬ rangements must be made for keeping the animals warm in the school room at night and on weekends during the winter months.

Each rat cage should be equipped with a food cup, water fountain, and, if feed¬ ing or breeding records are to be kept, a data card holder. The food cup can be a small metal or glass cup if the food is a dry powder type. However, if the biscuit type of food is used, a small shallow wire-mesh basket can be attached to one side of the cage.

Water can be supplied in either a small glass receptacle or a glass water fountain. Such a fountain can be con¬ structed in a few minutes by using either a test tube or a small round bottle, a

one-hole rubber stopper and a short piece of glass tubing. One end of the tubing is sealed in a flame. Continue to apply heat to it uniformly while blowing through the other end so as to produce a bulbous enlargement in the heated portion. This bulb will burst, leaving a small opening which is flamed. Heat the tube about one inch away from the enlargement and bend it slightly. In¬ sert the glass tube into the one-hole rub¬ ber stopper which is then inserted into either the bottle or test tube. The filled fountain is inverted and attached to the outside of the cage. Be careful that the glass tip protrudes slightly through the open mesh into the interior. It should be at convenient height for the animals.

Food and water are of primary im¬ portance in the growth and well-being of all laboratory animals. The stomach of the rat is so small that it cannot take in enough water to last for a long time. Therefore, an abundant water sup¬ ply should be available.

Because the rat is an omnivorous creature, its food requirements are pro¬ vided easily. Table scraps are a very satisfactory food. A well balanced diet containing all the elements necessary for normal growth can be prepared as follows:

Whole wheat flour 21 parts Yellow corn meal 21 parts Whole oat flour 20 parts Whole milk powder 20 parts Linseed Oil meal 10 parts Casein 3 parts Yeast 3 parts Calcium carbonate 1 part Sodium chloride 1 part

This food is a dry powder and must be thoroughly mixed. Any reserve supply of food should be stored in a cool place. It is possible to supplement the diet with carrots, lettuce, and other leafy vegetables.

The addition of vitamin E (wheat germ, either dry or the oil) to the diet of female rats used for breeding pur¬ poses is very beneficial in producing healthy litters of young.

Laboratory rats frequently handled, become quite gentle, especially if they are cage-bred animals. Should it be necessary to observe the animal, grasp it firmly, but not quickly, by the middle

TURTOX Service Department

Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (IN'CO HP ORATED)

8200 South Hoyne Avenue

Chicago 20, Illinois

THE SION OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Young White Rats

of the tail and allow the animal to rest its feet upon the palm of the other hand. Another method of holding is to place the index finger under the neck, just in front of the fore limbs and the other fingers about the belly between the fore and hind limbs—the thumb around the back of the neck. Always refrain from jerky or rapid movements when handling animals, otherwise they may become frightened.

Use only healthy young adult rats for breeding purposes. The female matures sexually at about eighty days, but it is best to begin breeding when she is one hundred days old. The litters will be larger and the young will be of better size. The oestrous cycle of the rat is ap¬ proximately four days, subject to varia¬ tion up to seven or eight days. The gesta¬ tion period is twenty-one to twenty-two days, also subject to some variation.

Females will usually mate immediately after casting a litter, if the male is pres¬ ent. It is best to remove the male until the young are weaned before the females are bred again.

Provide the pregnant female rat with an abundant lot of shredded newspaper with which to build her nest. After the young are born handle the mother rat very gently. Any major disturbance may result in her eating the young.

The young are quite immature at birth ; their eyes are completely closed and re¬ main so for fifteen days. At the time the eyes open, one can detect size differ¬ ences and any runts should be discarded. When twenty-eight to thirty-five days old

the young may be weaned. They will have eaten some food prior to this time so that weaning is not too severe. The same stock diet as that given the mother will be relished by the youngsters. The young rats may be left together until they are eight weeks old, when the sexes should be separated to prevent breeding at this young age.

Anyone working with young animals will need to know how to distinguish between the sexes at an early age. The most reliable criterion is the distance between the anus and the genital papilla; it is greater in the male than in the fe¬ male of equal age. In mature specimens sex differences are easily recognized.

The two sexes start out at much the same weight at birth. There is a discern¬ ible difference at forty days and after that the male gradually increases in weight over the female.

Rats normally live to be about three years old. Their most reproductive pe¬ riod is from three months to the end of the first year. Commercially it is unwise to keep and breed the worn out stock. Such animals should be elminated from the breeding colony when they have passed their prime.

Mice:

The albino mouse also has been used extensively in anatomical and physiolo¬ gical experimental work. A great deal of research on cancer has been done with white mice. Besides the white there are other colored varieties such as chocolate

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

220A431

and black which can be used in cross breeding experiments, with the white mouse, to demonstrate inheritance of color.

Small wire cages such as recommended for rats can be used for mice. If wooden boxes are used, one-half inch of dry saw¬ dust should be placed om the bottom. All cages should be provided with dry hay or shredded paper. The cages should be k*pt dry at all times. Keep the ani¬ mals at average room temperature and out of all drafts.

Mice thrive on all sorts of dry grains, corn, oats and wheat, also apples, lettuce and other green vegetables. They will eat meat, but that should be avoided because they may eat their young.

The following mixture may be fed to both rats and mice:

20 parts cracked oats 10 parts buckwheat

5 parts cracked corn 5 parts whole wheat

2 '/2 parts sunflower seed 1 '/2 parts millet

1 part peanuts Water should be available at all times;

the glass water fountain is best since there is less chance of spilling.

Specimens should be sixty days old before they are bred. The sexes may be distinguished by the distance between the anus and the genital papilla; it Is greater in the male than in the female. The gestation period is twenty to twenty- two days. The males should be removed from the cage when the young are born. The young should be weaned when twenty-one to twenty-eight days old.

Guinea-Pigs: Guinea-pigs are rodents native to

South America, but they have enjoyed a world-wide distribution due to their great desirability as laboratory specimens. They are docile animals, prolific and easi¬ ly cared for.

220A528

Guinea-pigs make excellent laboratory subjects for experiments in heredity and for testing the potency of vaccines, se¬ rums and germ cultures. They are espe¬ cially recommended in elementary stu¬ dies on reproduction because their care is so simple and they may be handled by the students with little fear of being bitten or scratched.

Cages similar to those used for rats, but larger, are required. A cage twenty- four by sixteen by twelve inches with sides and top of three-eighths inch wire mesh, and bottom of one-half inch wire mesh is satisfactory. A door should be constructed either on top or on one side in order to remove the animal easily. The eages should be placed in a shallow metal pan to catch tne waste materials This pan should be cleaned frequently.

Keep the animals at average room tem¬ perature about 72° F and out of all drafts. Colds are usually due to sudden temperature and humidity changes as well as unclean quarters.

Guinea-pigs are herbivorous in feeding habits and will eat a wide variety or grains, nuts and greens. A satisfactory diet is one composed of a mixt«re of grains, such as corn, oats and wheat with succulent vegetables such as carrots, apples, lettuce, sweet potatoes, fresh grass and alfalfa hay. A mixture consist¬ ing of equal parts of oats, wheat and barley, and a sufficient quantity of soy bean or linseed meal, to form ten per¬ cent of the total, is a satisfactory food. When vegetables and fruits are plenti¬ ful the animals do not require water. However, it is best to provide each cage with a non-spillable water dish.

Allow the adults to become fully ma¬ ture before breeding so that better lit¬ ters will be produced. They should not be bred before they are nine months old. The male may be allowed to remain in the cage with the pregnant female if there is ample room, but if other cages are available it is better to separate them. The gestation period is sixty-five to seventy days. The young are well

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

developed at birth and can shift for themselves at an early age. They begin to eat solid food when quite young, and tender leafy vegetables should be on hand a few days after birth. They should be weaned when four weeks old.

Guinea-pigs are ordinarily healthy and

hardy creatures. The animals sometimes have external parasites which can be detected by the guinea-pigs’ scratching. The animals should be dusted with pryethrum powder and the cages thor¬ oughly cleaned and sterilized.

Please note: Postal regulations prohibit the shipment of live animals such as rats and mice by mail. Therefore, these animals can be delivered only via railway express. New express regulations set a minimum rate of $4.50 for any live animal shipment, however small. This rate is not excessive if six or more animals are ordered, but the cost of shipping one animal will be at least $4.50.

1SV72 Mice, White. Mature animals of pure strain but not pedigreed. Suitable for breeding or feeding experiments. Pair (male and female) $4.75

1SV74 Rats, White, Healthy, mature animals of pure strain but not pedi¬ greed. Suitable for average breeding or feeding experiments. Dozen.. .23.00 Pair 4.50

ISVTJfZ Adult Hooded Rat. A select strain of pure line “hooded” rats. De¬ sirable in introductory heredity ex¬ periments—although they are not pedi¬ greed. Easily reared and serve wher¬ ever rats are indicated in biology ex¬ periment. Each (specify sex) 3.75 Per pair (male and female) 6.50

220A431 Turtox Rat Cage. Cage is round, 9 inches high by 9 inches in diameter. Made of heavy galvanized mesh with removable bottom pan. Each 10.95 Per unit of two cages 21.00

220A528 Turtox Economy Cage for mice and hamsters. Easy to clean. Size 8%" x 6" with a 4)4" opening. Each 2.25

220Alp Animal Cage Breeding. A cage suitable for breeding rats or guinea pigs or for group feeding experiments. Size 18 x 12 inches by 9 inches high. Made of % inch mesh galvanized iron with bottom of inch mesh, resting in detachable galvanized iron pan. Top is hinged. Card holder for 3 x 5 data card.

220A525 220A525 Turtox Activity Cage. A rec¬

tangular, all-metal cage with a large revolving cylinder. This is a well-con¬ structed, roomy cage of our own man¬ ufacture. The wire mesh will retain mice or hamsters and the cage is large enough to accommodate white rats and small squirrels. Size 16 in¬ ches long, 10'4 inches high and 9 in¬ ches wide. Complete with removable metal try and sanitary water fountain. Each $19.95

55V80 Rat Grozc'iny Ration. A high grade ration for feeding growing rats or mice. Contains all the necessary proteins, carbohydrates, fats, minerals and vitamins. We recommend this as a diet for growing animals. Mixed and ready for use. Per lb 1.00 Per 5-lb. package 4.25

55V32 Turtox Guinea Pig and Rabbit Food. A prepared food containing the essential nutrients for growth and re¬ production of guinea pigs. By using this food, greens need be fed the ani¬ mals only once or twice a week. Lab¬ oratory tested. Directions for feeding supplied with each shipment. 1-pound package 60 5-pound bag 2!50 25-pound bag 9.50

All prices are f.o.b. our laboratories and are subject to change without notice.

(40-4)

TURTOX SERVICE LEAFLET No. 20

NOTES ON MARINE AQUARIA

The biology student living on the sea- coast has a great advantage over his fellow in an inland laboratory, for there can be no comparison made between the interest-arousing qualities of a star¬ fish dripping with and smelling of formalin and those of a living starfish crawling over the submerged rocks in a clear tide-pool. There is no reason, however, why students living far from the seacoast should not have the opportunity of seeing living examples of some of the smaller marine animals. Salt-water aquaria are now used in hundreds of inland schools and the living marine animals can be shipped successfully at any time during the colder months of the year.

Collecting and Shipping

Teachers who are fortunate enough to live near the seashore are usually able to collect an interesting variety of living marine animals at any season of the year, and, if the school laboratory is located nearby, the transportation of the speci¬ mens presents no problems. Wooden or enameled pails are best for use in marine collecting, and, of course, glass jars of various sizes are suitable for small forms. (Do not use metal containers.) Collect small-sized specimens and do not crowd too many into a small amount of sea¬ water. It is far better to return to the laboratory with one small starfish alive than with a pail-full of dead or dying specimens.

The Aquarium Tank

The best container for a small marine aquarium is a rectangular all-glass tank, although the standard metal-framed tanks with slate bottoms and glass sides may be used provided they are so constructed that no water comes into direct contact with any metal parts. The best size for a beginner is a tank of from six- to ten- gallon capacity. After one has had some experience with a small aquarium, the larger sizes may be used.

The tank or tanks to be used should be

thoroughly cleaned and made ready be¬ fore the shipment of sea-water and living specimens arrives. The tanks should be located in a place where a fairly low temperature can be maintained and where they will receive little or no direct sun¬ light.

It is a good idea to divide the shipment of sea-water and living specimens be¬ tween two tanks. This allows one to de¬ termine which animals will live together in harmony and, should any forms be injured, they may be kept in a separate aquarium until they recover.

Sand If you use sand other than that secured

from an ocean beach, be sure that it is a pure silica sand and wash it thoroughly and repeatedly to remove any mud or organic matter before it is placed in your aquarium.

Sea-water Natural salt-water from the ocean is

supplied with our aquarium sets and we do not advise the use of synthetic sea¬ water unless large quantities are re¬ quired. However, some teachers will find it economical to prepare synthetic sea¬ water and, in such instances, the following formula may be used:

Distilled (or rain) water 10 gal. Sodium chloride, C. P 45V2 oz. Potassium chloride 1)4 oz. Calcium chloride 2 oz. Magnesium chloride (dry) 8% oz. Magnesium sulphate 11% oz. Bicarbonate of soda 1/5 oz.

After thoroughly mixing the above, add:

Potassium nitrate 1/5 oz. Sodium phosphate 10 grains Iron chloride 5 grains Natural sea-water 1 gal.

The reason for adding the natural sea¬ water is not entirely clear; but it is un¬ questionably necessary. Some vital ele¬ ments are apparently lacking in the manufactured sea-water and the addition of the natural water remedies this. It

TURTOX Service Department

Copyright, 1959, by GENERAL BIOLOGICAL SUPPLY HOUSE

(Incorporated)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed In U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

has been suggested that natural sea-water contains a substance corresponding roughly to the vitamins in foods.

After the synthetic sea-water has been made up, it should be placed in tightly corked glass carboys and kept in a dark place until wanted for use.

Maintaining the Right Concentration When the tank is filled with sea-water,

the level of the water should be marked on the outside of the glass by drawing a line to coincide with the surface of the water. As evaporation takes place, pure distilled water should be added to bring the water up to the original level. In large tanks, the water should be tested from time to time to see if distilled water is needed to replace the loss by evapora¬ tion. Natural sea-water should show a reading of 1.025 when tested with a hydrometer. Any needed adjustment should be made weekly or oftener.

Temperature of the Water The proper temperature control is of

utmost importance in the success of a marine aquarium. In general, the water temperature should be maintained at 55° to 60° F and a temperature ten degrees lower than that is often better.

Sudden changes of temperature are usually fatal to aquatic animals. If your shipment of living marine animals ar¬ rives during the winter when the weather is cold, the temperature of the sea water in their container will probably be very little above 32° F. This should be raised very gradually and, in no case, should the animals be transferred suddenly to much warmer water. A good plan is to allow a period of at least 24 hours in which to allow the temperature of the water to rise gradually.

Many suggestions have been offered in regard to the matter of keeping the school marine aquarium water cool; but the one considered most feasible is that in which the aquarium is placed on the window sill where it will not receive direct sun¬ light. The window is then opened until about two or three inches of the glass side of the aquarium are exposed to the out-of-door conditions. The open space on either side of the tank is then blocked off with wooden panels and strips of felt so that the room temperature will not be affected. The advantage of such a system as this is that it is possible to maintain a lower degree of temperature in the aquarium than could otherwise be at¬ tained.

Tropical forms must, of course, be provided for according to the conditions found in the water where they are collected.

Aeration of the Water

The first and most important rule to follow in planning salt-water aquaria is to remember that most marine animals require more oxygen than do most fresh¬ water forms.

The main reason for this is that the marine forms used in aquaria are usually collected in tide-pools along surf-swept beaches where the oxygen content of the water is unusually high. Therefore, fewer inhabitants should be placed in each tank than would be the case if one were deal¬ ing witli fresh-water animals. The tend¬ ency is always to overcrowd an aquarium and, although this may cause eventual failure of a fresh-water tank, it is quickly and completely fatal in a salt-water aquarium.

Large public exhibition aquaria are usually planned so that there is a large reservoir of salt-water furnishing enough water for a continuous, though rather slow, flow through the exhibition tanks. Compressed air vents are usually placed so that the water is aerated thoroughly while in the main storage reservoir. How¬ ever, the size and cost of such a system as this renders it impossible for the average school laboratory.

Several small and inexpensive aerating pumps are now available and one of these will be of great help in aerating the water in your aquarium, especially dur¬ ing the first week or so until the animals become “acclimated” to their new sur¬ roundings. (Turtox will furnish informa¬ tion and prices on suitable aerators for any size tank.)

A marine aquarium may, of course, be “balanced” just like a fresh-water aqua¬ rium if the oxygen given off by the plants is sufficient to supply the needs of the animals. The green alga, Ulva, commonly called sea-lettuce, is helpful as an oxygen producer, as is also Cladophora. Marine diatoms are particularly desirable and we now include them in the sand supplied with our larger sets. In general, however, the “balancing” of a marine aquarium comes about gradually and, at the start, it is highly desirable to aerate the water by artificial means.

Light

Salt-water aquaria require much less light than do fresh-water aquaria and they should receive very little direct sunlight. Except when it is desirable to watch the inhabitants, the aquarium should be shielded by cardboard on three sides to keep out very strong light.

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

A small marine aquarium containing starfish, sea urchins, sea anemones, Ulva and Fucus.

The Animals With the aquarium tank made ready

and in its permanent location, the pre¬ liminary preparations are completed and the living marine animals may be or¬ dered. Under the ideal conditions exist¬ ing in very large aquaria, almost any marine forms will live; but the marine- aquarium enthusiast who is experiment¬ ing with small tanks should attempt to secure the more hardy animals which will live for a while, at least, under somewhat adverse conditions. Among the best small-aquarium inhabitants are small marine snails and barnacles. Starfish, sea urchins, sea cucumbers and sea ane¬ mones will usually live for a few weeks and under carefully controlled condi¬ tions may be kept in aquaria for much longer periods. Small crabs will often thrive where more exacting forms die, and Obelia will often live and reproduce new colonies in very small aquaria.

Dr. Lyell J. Thomas, who has done some very interesting work with marine aquaria at the Zoological Laboratory of The University of Illinois, lists the fol¬ lowing animals which have been found to do well in small marine aquaria under laboratory conditions:

“Protozoa—a long list have been identi¬ fied and offer the inland protozoologist a new and fertile field for study; Porifera

—silicon sponges ; Coelenterata—Obelja, Clava, Sea anemones, Corals ; Platyhel- minthes—Small Turbellarians found on Ulva; Nemathelminthes—free living; Ro- tifera; Bryozoa; Annelida—Serpulids or tube worms, Nereis, Climenella; Mol- lusca—Snails, Limpets, the nudibranch Aeolis, Clams (both Mytilus and burrow¬ ing forms), Pecten, Chitons; Arthropoda —Barnacles and many small Crustacea; Chordata—Ascidians; all coming from pool or low tide areas.”

Feeding the Animals To feed the animals, it is sometimes

best to remove them to a separate dish filled with salt water of the same tem¬ perature as that of the aquarium. Wood¬ en forceps are best for handling the ani¬ mals. Twice a week is often enough to feed the various forms. Small pieces of macerated oysters, clams or fish make a fine food and they should be dropped near the mouth of the animal by means of forceps. The juice of oysters and clams also makes a fine food and can be dropped by means of a pipette into the mouths of such animals as the Metridi- um, Thyone and Cucumaria. Fresh water clams may be used as well as salt water clams and even small pieces of fresh water fish will be readily devoured. As soon as the animals have been fed, they may be returned to the aquarium and

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

the glass plate put back on top of the tank.

If you wish to feed the animals with¬ out removing them from the main tank (and this is advisable, of course, in many cases), put in very small amounts of food and remove promptly any that is not eaten, or otherwise it will decompose and quickly foul the aquarium.

Temporary Marine Aquaria Even though it may require consider¬

able effort to maintain salt-water aquaria for long periods of time in a small lab¬ oratory, the teacher will find that a few attempts along this line are well worth while. The tanks containing sea-water can be maintained throughout the school year, and from time to time, assortments of a few living marine forms can be ordered. Even though some animals are short-lived, the students will have had an opportunity to study living starfish, sea urchins, anemones and other marine forms which they might otherwise never have seen. The result of even a few hours spent in this way will be a real interest that preserved specimens could never have awakened.

Summary The really essential points to keep in

mind are few; but they are very impor¬ tant: (1) Use small specimens and few

of them; (2) Keep the water temperature low; (3) Aerate the water constantly and (4) Feed sparingly. The Turtox Service Department will gladly answer your questions and offer help in the es¬ tablishing and caring for marine aquaria of any size. Write to us if there are any points on which you wish additional in¬ formation.

Literature “Culture Methods for Invertebrate

Animals.” Published by Comstock Pub¬ lishing Company.

“The Aquarium Book,” by E. G. Boulinger. Published by D. Appleton & Co.

“Goldfishes and Tropical Fishes,” by W. T. Innés, published by Innés and Sons. Philadelphia.

“Fishes in the Home,” by Mellen, pub¬ lished by New York Zoological Society, New York City.

“Guide to the New York Aquarium,” published by New York Zoological So¬ ciety, New York City.

“Living Marine Animals for our In land Laboratories.” A report by Lyel) J. Thomas, appearing in the January, 1925, issue of Transactions of American Microscopical Society.

“Care of Small Salt-water Aquaria,” by I. M. Mellen. Published by New York Zoological Society, New York Aquarium, New York City.

For information on shipments of living marine specimens write to:

Miami Tropical Aquarium, 982 South¬ west 3rd St., Miami 36, Florida

Supply Department, Marine Biological Laboratory, Woods Hole, Massachusetts.

Coral Reef Exhibits, P.O. Box 23, Coco¬ nut Grove 33, Florida

Greater Miami Fisheries, 3475 N.W. 187th St., Opa-Locka, Florida

Global Aquarium, 65 Mt. Vernon St., Ridgefield Park, N. J.

205A20 Turtox Special Aquarium. Size 18" long, 10%" wide and 9%" high. Capacity, 6 gallons. Metal frame with sides, ends and bottom of heavy glass $15.95

205A 201 Turtox Special Aquarium. Same as Number 205A20, but larger, of 10 gallon capacity 19.95

205AU0 Aquarium Forceps. Wood, 18%" long. Each 2.25

205A578 Temperature Control Unit. A combination unit, consisting of 75 watt heater, thermostat and indicator light. For 110-120 volts, A.C. only 9.50

205A6 Aquaditioner Air Pump. A good low-price aerator for tanks up to 50 gallons. Operates on 110 to 120 volts, A.C. or D.C. $6.50

205A601 White Mist Aerator. An ex¬ cellent aerator for use on 110 volts, A.C. Complete with 5 feet of rubber tubing and one air breaker 21.00

205A 6 6 Aquarium Cement. Pound can 95

All prices are f.o.b. our laboratories and are subject to change without notice.

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TURTOX SERVICE LEAFLET No. 5

STARTING AND MAINTAINING A FRESH-WATER AQUARIUM

Photograph of the No. JtSVll Turtox Aquarium Outfit, as listed on page four of this leaflet.

Starting and maintaining a fresh-water aquarium in the school laboratory is easily accomplished by anyone who is willing to devote a little thought and care to the work. The pleasure and practical teaching advantages to be derived from one or more aquaria containing interesting plant and animal specimens is well worth the time and effort expended. Before starting your aquarium, however, make sure that you have the correct type of tank, the right quantities of aquatic plants and animals. Keep in mind the basic requirements of the specimens to be included in such a demonstration, because a fresh-water aquarium is a biological association of plant and animal life.

The following brief outline states the procedure to follow in establishing a fresh¬ water aquarium. If these directions are followed closely, no particular difficulty should be encountered.

TURTOTMIUCTS

TURTOX Service Department

Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed In U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Equipment. The only absolutely essen¬ tial piece of equipment is a suitable aquarium tank; a rectangular tank with metal frame, glass ends and sides, of about 6 to 9 gallon capacity is recom¬ mended. Small, round aquaria are not satisfactory (visibility is poor through curved glass), and the round (ish globes are useless. In addition to a suitable tank, the following items will prove their usefulness: dip-net for trans¬ ferring fish, one pair of long aquarium forceps, one pair of plant snips and a syphon tube.

HOW TO START THE FRESH-WATER AQUARIUM

1. Clean the tank thoroughly, remov ing all dust, grease, etc., from the glass. If the tank has previously been used as an aquarium wash very thoroughly with soap and ammonia water. Rinse with clear water three or four times.

2. Clean thoroughly, in running water, sufficient aquarium sand or gravel to cover the bottom of the tant to a depth of one to two inches. Con tinue washing until all debris and sol¬ uble matter has been removed. Place the sand in the aquarium.

3. Now add the water. Use clear pond water if possible; if this cannot be obtained, use tap water which has stood in open containers for a day or so. Pour the water into the aquarium (using a sheet of paper as shown in illustration) until it is 8 or 8 inches deep. After it has been planted add more water to fill the aquarium. (Im¬ portant: Chlorinated city water is not safe to use unless it is allowed to stand in open containers for 48 hours.)

4. The aquarium should be placed where it will be exposed to strong dif¬ fused light. Direct sunlight for an hour or so each day is usually not harmful, but neither is it necessary. North or East exposures are usually best.

5. The aquarium is now ready for planting. Plants are used for a variety of functions in an aquarium. They pro¬ vide a natural habitat and protection for small fishes. Plants utilize some of the excreta thrown off by the fish and other animals while they, in turn, are used as food by some species of fish and snails. They also promote the growth of microorganisms and other small animals.1

It is well to use plants rather spar¬ ingly at first, remembering that they will soon grow and spread. If too many are crowded in at the beginning some

(S-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

may die and decay, thereby fouling the water. All plants will not thrive in an aquarium and it is best to secure tank- grown (not wild) aquatic plants of kinds that are known to grow well in aquaria. Vallisneria, Sagittaria, Elodea (Anacharis), Myriophyllum and Ca- bomba are all good. Many other plants can be grown successfully in an aquari¬ um, but the ones mentioned give variety and are dependable.

6. Plant the aquarium as follows: Take several of the rooted plants (such as Vallisneria and Sagittaria), spread the roots out on the sand and cover them up to the crowns, pressing them down to secure anchorage. Add several stalks of the non-rooted plants (Elodea, Myriophyllum and Cabomba), weighting the lower ends down with small stones or with pieces of lead. Arrange the upper parts of the plants so that they float freely in the water, and remove all dead or broken leaves. Now fill the aquarium with water to within one inch of the top.

If the conditions are right, growth should be noticeable in about two weeks after planting. If the water does not become clear in a day or so it is usually an indication that it contains some dead and decaying plants or other organic matter.

7. Allow the aquarium to stand for a few days until the water has cleared, when it will be ready for its animal population. (Snails, fishes, etc., can be placed in the aquarium immediately after planting if necessary, but it is best to wait until the water is clear.)

8. In stocking an aquarium several things must be borne in mind. First, use only animals which get along to¬ gether; predaceous forms must be kept by themselves. Second, do not use ani¬ mals (or plants) which naturally live only in running water, as they will not live in the close confinement of an aquarium. Third, do not overcrowd. Fourth, do not use animals which will stir up the sand on the bottom and keep the water cloudy.

9. Animals. Fish. It is important not to over-crowd an aquarium with fish. A good rule to follow is one inch of fish to one gallon of water. The average six-gallon tank will support six one-inch fish or three two-inch fish. This rule cannot be followed in all cases. The number of fish used must be governed by the number of other animals to be included in the

5. Obtain dome rooting planta from Turtox

tlwoeria oagitUria Ludtfigia

6. Also dome non-rooting planta

Anacharis Cabomba Myriophyllum

'y, Place tank in permanent location. Partly fill \dith clear Water, set plants in grav'd

Ô Complete filling of tank with water and cover wtthglass plate. 'Add no animals until water is clear and the plants are growing.

GDIERAL BIOLOGICAL SUPPLY HOUSE-7 6HÜ.AaT Ç^lilAcE-CHIC Ago.

(5-3)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

aquarium and the surface area of the water. It is usually advisable to use several small fish rather than one large one. Small native fishes (minnows, dace, bullheads and sunfish) are far more in¬ teresting than the sluggish goldfish. If fish are not obtainable, snails, newts and aquatic insects will add much life and interest to the aquarium.

Snails. Six to a dozen snails may be kept in a 6-gallon aquarium. Snails are valuable as scavengers and will help keep the algae from the glass. Various species of pond snails (egg layers), and red snails, may be used. If possible, one or two viviparous (liv<" bearing) snails should be included.

Clams. One small clam, an inch to two inches long, may be used. This is an interesting form to study, for the inhalant and exhalant siphons show perfectly. The clam should be watched closely to see that it is living, as it will disintegrate quickly and foul the aquarium if it should die.

Newts. Several small newts can be kept in the aquarium. They are active, inter¬ esting and relish small bits of raw meat as food. Reference: 1 Atz, J. W. "The Functions of Plants in Aqua¬

ria", the Aquarium Journal, Vol. 21, No. 2 and 3, February and March, 1960, pp. 40-43; 66-60.

10. Watch the aquarium carefully for the first few weeks. Remove at once any dead animals and clip off all dead parts of plants. After the plants have become established it may be necessary to remove some of them to prevent over-crowding.

FEEDING THE AQUARIUM ANIMALS

This subject is discussed in Turtox Service Leaflet No. 23 and will be men¬ tioned only briefly here. The most im¬ portant rule to be followed in caring for aquarium animals is—Feed Sparingly. Because higher animals must be fed often it is but natural that one should view with alarm the prolonged fasts of the cold-blooded animals. However, these forms can go for long intervals without food and remain perfectly healthy. Most of the small native fishes will do well on a balanced food, such as the Turtox Natural Fish Food. They also relish living crustaceans, such as Daplmia or Cyclops, and finely chopped live earth¬ worms. Newts will eat tiny pieces of raw meat, raw fish or small pieces of living earthworms. The snails and clams neeii not be fed, as they can shift for them¬ selves in an aquarium containing plenty of living plants. They also feed upon the excess fish food.

4SV10 Turtox Aquarium Set. A thor¬ oughly dependable collection of plants and animals for the balanced fresh-water aquarium. Includes suf¬ ficient material to stock a standard 6 to 9-gallon aquarium tank.

Animals: Snails of two kinds Two aquatic Newts One small clam (Or other aquatic animal.)

Plants: Sagittaria Ludwigia Vallisneria Cabomba Myriophyllum Water Poppy (Or other plants may be substituted.)

Set as described $4.75

45V11 Turtox Aquarium Outfit. In¬ cludes the special new Turtox Aqua¬ rium of 6-gallon capacity made of polished bulb-edged glass with slate bottom, one-piece metal frame and removable glass top (see page 1), 1 bag Turtox washed aquarium gravel, 1 jar Turtox Natural Fish Food, and 1 aquarium dip net. Complete outfit with instruction leaflet .. 18.50

45 VI2 Complete Aquarium Outfit with Plants and Animals. Consists of Nos. 45V10 and 45V11 de¬ scribed above. Both for 23.00

205A41 Aquarium Forceps, Wooden forceps, 18% inches long. Each $1.10

205A54 Aquarium Dip Net, Each 75

205A47 Aquarium Siphon. Flow of water starts automatically. Made of glass with rubber connections. Length, 12 inches. Each 1.40

Write for current information on tanks, aerating pumps, air breakers, heaters and thermostats.

FOODS AND REMEDIES

55V21 Turtox Natural Fish Food. A balanced food. Per 100 cc jar .75

55V24 Shredded Shrimp. Per 100 CC.

jar 50

55V41 Fungus Cure for Fishes. Per ounce of concentrated crystals. .50

55V42 Salt Bath for Fishes. A com¬ bination of three mineral salts. Per ounce 50

55V44 Medicated Aquarium Hi-Ball. For neutralizing acidity in the water. Each 25

All prices are f.o.b our laboratories and are subject to change without notice. (5-4)

TURTOX SERVICE LEAFLET No. 42

LABORATORY DISSECTIONS

To the scientist, the word “dissection” has a meaning far different from the idea that it conveys to the beginning student in Biology. The word literally means “to cut apart,” with the word ‘'cut” given as a synonym. However, when one learns that synonyms for “cut” include such an array as “carve, chop, cleave, gash, hack, hew, sever, shear, slice, sunder and whittle” as well as “dissect,” it is obvious that the instructor does not have all of these in mind when he calls for a careful dissection of some laboratory form. The word anatomize expresses more clearly what the scientist has in mind when he speaks of a dissection, for this implies the separation of organs and tissues in such a manner as to give a true understanding of the various parts of the specimen.

Prerequisites of a Good Dissection 1. Good Materials. The first pre¬

requisite of a good dissection is good material. Specimens that are imperfectly preserved, with organs and tissues in various stages of disintegration, obviously are unfit for dissecting purposes. It is also desirable that the specimens be properly straightened for convenience in pinning out or fastening in the position in which they will be dissected. For ex¬ ample, an earthworm that is not straightened and relaxed before preserv¬ ing, or a frog that is badly distorted, makes dissection just that much more difficult.

2. Proper Instruments. By proper instruments it is not meant that they must be expensive. It does mean that one should have the necessary instruments for certain purposes. For ordinary dis¬ sections called for in elementary Zoology courses one pair of fairly fine-pointed scissors, one scalpel, one forceps, two needles and one probe are sufficient. Where iiigher vertebrates are dissected various types of the above instruments are desirable, with the possible addition of bone forceps and other special pur¬ pose instruments. The purpose of the in¬ strument and the use to which it is to be put is most important, although the quality should be good. For student use dependable instruments may be procured at a reasonable price.

3. The Right Frame of Mind. Every teacher of Zoology with a class of stu¬ dents taking the subject for the first time finds certain members of either sex who have a natural or feigned aversion for handling laboratory specimens. Such students usually receive little sympathy from the average teacher, for he knows that interest in the specimen and in the subject soon submerges any aversion on the part of the student. The student himself should proceed with his dissec¬ tion with the exploring spirit—here is something new of which he has a limited knowledge and he should be determined to learn by himself the structure, mech¬ anism and functions of the specimen be¬ fore him.

Fig. 1. Diagram of proper method of pinning an earthworm for dissection.

Dissecting the Specimen In practically all cases the specimen

should be fastened to the wax-bottomed dissecting tray by means of pins. The pins should be inserted obliquely as shown in Figure 1. There are three reasons for this—First, when inserted obliquely the pins are braced against any tension set up in the specimen and will not easily be pulled out. Second, the

TURTOX Service Department Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (Incorporated)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SION OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

specimen does not have a tendency to "creep” up the pins and change position. Third, the pins are out of the way of the hands and instruments, thus permitting free access for working on the specimen.

Opening the Specimen. After the speci¬ men is securely pinned the first step usually is to open the body cavity. At this time great care should be taken. Always note carefully the laboratory di¬ rections for opening the specimen and follow them explicitly. If scissors are used, carefully insert the point so that it does not penetrate deeply. Then hold¬ ing the ‘‘finger ring” of the scissors down low, thus keeping the point up against the inside of the body wall, care¬ fully extend the cut to the point indicated in the directions. The thumb and middle finger should be used in the scissor rings, with the index finger serving to steady the instrument in cutting. Certain small specimens, as the earthworm, should be pinned out progressively as the cut is made.

Further Dissection. Once the body wall has been cut through and the speci¬ men properly pinned, the greater part of Ihe dissection should be carried on with non-cutting instruments, thus avoiding the destruction of important structures by cutting. Once parts are severed, especially blood vessels and nerves, their correct relationships are likely to be lost. Instead of cutting, use the closed points of the forceps for pressing one way against one of the parts to be separated and the probe on the other part for ef¬ fecting the separation. In this way the connective tissues that bind the parts to¬ gether can be loosened and the structures clearly exposed. In separating muscles, blood vessels and nerves pull the probe

with a slight spreading motion in the di¬ rection taken by the parts being dis¬ sected. The needles may be used at times in the same way, depending on the delicateness of the structures involved. Use the scalpel or scissors only when ab¬ solutely necessary. Avoid pulling, pinch ing and picking at the specimen in an aimless manner. Make every move with a definite end in view. As O. W. Holmes has so aptly said: “Let the eye go before the hand, and the mind before the eye.”

Orderliness and Cleanliness Conducive factors in good laboratory

work ai-e orderliness and cleanliness. Determine the most efficient arrangement of all materials and instruments used and keep them in good working order. Cleanliness of instruments and hands and the disposal of dissected parts, or speci¬ mens no longer needed, help to keep one in the proper frame of mind for good work. Slovenliness in the care of ma¬ terials and instruments is only too likely to be a reflection of the student’s state of mind.

It is not amiss at this point to remind the student that the essential feature of laboratory work is to learn to observe correctly, to remember what he observes and with his assimilation of facts to com¬ pare the various specimens that he studies in the laboratory. For these processes there is no time when he can study and retain facts with greater facility than while he is actually making the dissection. The student who follows his laboratory directions intelligently and who faithfully executes those direc¬ tions, studying and understanding as he goes, is the one who will get the most out of his laboratory work.

TURTOX ELEMENTARY DISSECTING SET. 308AS01 Elementary Dissecting Set. Our most popular dissecting set,

many thousands of winch are sold each year. The instruments and case are of dependable quality, and this set has proved very satisfactory for beginning zoology and biology classes, in both high schools and colleges. The following instruments are in¬ cluded: One pair scissors, one pair forceps, two teasing needles, one all-steel scalpel, one 6-inch English and Metric ruler and one high quality waterproof case. Price each $3.95

310A5310 Dissecting Pan. Made of non-rusting galvanized sheet metal with J4 inch wax liner for pin¬ ning. Complete with rings to which specimens can be fastened. Size, 7 by 11 x 1J4 inches. Each 2.75

Dozen 31.25 310A56 Dissecting Pins.

Extra heavy, 1)4 inches long Per % lb. box (approximately 600) 1.85

For a complete listing of dissecting instruments, refer to your Turtox Catalog. Drawings of the anatomy and skeletons of all of the laboratory animals are now available. Ask for the Three-Way Checklist of Turtox charts and biological drawings. All prices are f.o.b. our laboratories and are subject to change without notice.

(42-2)

TURTOX SERVICE LEAFLET No. 6

GROWING FRESH WATER ALGAE IN THE LABORATORY

Fresh-water algae are of very gen¬ eral distribution and are found in nearly every type of damp and aquatic habitat. Ditches, ponds, rivers, lakes and marshes of any locality abound in a great variety of forms, both of the unicellular and multicellular types, Some algae grow on damp earth or rocks and some kinds make up the greenish covering which appears on the bark of trees. Most of the more conspicuous forms are inde¬ pendent, either free floating or attached in tufts or mats to the substratum, but there are also epiphytic and endophytic species. Many of the smaller forms are attached to the other water plants or are found in the loose sediment and debris of the substratum.

Many kinds of algae are easily cul¬ tured in the laboratory. Some kinds will grow well if brought indoors and placed in containers of pond water or in a balanced aquarium; other field-collected algae are more difficult to culture and can be maintained over long periods only by the use of nutrient solutions, and the pure-culture technique. For most kinds of algae, large finger bowls or battery jars are good culture containers; some robust forms such as Spirogyra and Cladophora are best grown in aquarium tanks.

When possible, use the water in which the algae were growing, since sudden changes in kinds of water are injurious. Where additional water is needed use distilled water, or let the tap water run for several minutes before filling the jar, since water standing in pipes, or other metal containers for that matter, is harmful to most algae. Add new water gradually, to compensate evaporation.

Do not put too much material into a jar. Actively growing material will in crease and gradually accommodate itself to conditions. The excess in an over¬ abundant supply will be choked off and the consequent decay will cause fermen¬ tation and general fouling of the culture, ultimately killing the whole. Likewise,

luxuriant forms like Cladophora may soon overstock themselves and the ex¬ cess material must be removed from time to time.

Cultures may be started at any time of the year. In winter bring in some mud over which the desired form was growing the previous season. Sticks or stones may also be used. Place it in a jar and add tap water, distilled water, or rain water as the case may require. Cultures of some forms, such as Chara and Nitella, have been obtained from mud collected several years before. Cul¬ tures started in this manner usually yield a variety of material.

If you have healthy culture, do not throw it out if the algae should disappear Seasons of dormancy occur in nature— look in the bottom of the jar for spores. Let the water evaporate, cover the jar for protection, and set it aside. After a period of a month or two add more water and you will likely have your cul¬ tures again. Lengths of dormancy pe¬ riods vary. In Volvox it is quite long; in Oscillatoria it is only a couple of months; in Cladophora there may be no dormancy. Warm weather makes it dif¬ ficult to keep some cultures of algae in good condition. Refrigeration, after col¬ lection, will make it possible to study the material for a week or more instead of just a day or two.

The method used in collecting algae is different from that used for aquatic zo¬ ological specimens. Care must be taken to avoid metal containers, specially on long, warm trips. Ample water must be used if the containers are to be sealed. However, forms like Spirogyra, Mou- geotia, Zygnema, Cladophora, Hormiscia and Vaucheria may be rolled in wet newspapers or magazines and carried thus.

Blue-Green Algae

Oscillatoria is readily found in stag¬ nant water, watering-troughs, damp

UCTS

TURTOX Service Department

Copyright, 1958, by GENERAL BIOLOGICAL SUPPLY HOUSE

(INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

earth, flower pots, and in many other habitats. It is recognized by its dark blue-green, or blackish color. Oscillatoria is one of the easiest of plants to keep in the laboratory. Place a little of the material in a container partly filled with water; cover with a lid to avoid unneces¬ sary contamination from dust. By add¬ ing water from time to time to compen¬ sate for evaporation, the cultures should keep indefinitely.

Nostoc is frequently found in lakes and also occurs on damp earth. It is easy to maintain in laboratory cultures, and will keep in good condition for a month or more if kept under refrigeration at a temperature of about 40° F.

Rivularia is found attached to the leaves and stems of various aquatic plants. It often occurs in laboratory aquaria.

Oloeocapsa is found in gelatinous masses, sometimes floating in ponds, but more often as a coating on wet rocks. It is easily maintained in laboratory cultures. Green Algae

Volvocales. Most of the members of this group and some of the related fami¬ lies are best grown in pure culture, using nutrient solutions. The Turtox “Universal” Concentrate has been de¬ veloped for culturing many of these forms. Its use (described below) en¬ ables anyone to grow and maintain per¬ manent pure-line cultures, including such difficult forms as Volvox and Eu- dorina. (The Turtox strain of Volvox aureas has been cultured continuously on this medium from the spring of 1949 until now, 1958.)

Turtox 4-Unit “Universal” Concen¬ trate 61V170 Set “A”. Makes one liter of medium for growing Volvox, Eudo- rina, Pandorina, Chlamydomonas, Spiro- gyra, Zygnema, etc.: The medium is made from the concentrate by mixing all of the Unit I with 925cc. of distilled water. This is followed by the addition of Unit II and, after further mixing, by Units III and IV. The culture fluid is then ready for immediate use.

Turtox 4-Unit “Universal” Concen¬ trate 61V170 Set “B”. Makes five liters of medium. Prepare in the same way as Set “A”, but add the units to 4625cc. of distilled water. Cultures may be put up in battery jars (with glass plate covers to prevent the en¬ trance of gross contaminants) or in small, stacking finger bowls—allowing about % inch of air space in each bowl, and covering the uppermost dish with a glass plate. All glassware previously used for culturing of algae should first be washed and then steamed 30 min¬ utes to avoid contamination. New glass¬ ware may be made ready for use by washing it with detergent and hot water, followed by a rinse in weak hydrochloric acid solution and a final rinse in tap

water. Droppers (for transfers) may be steamed and kept in a wide-mouth screw-cap jar.

Store cultures in a room where there is a plentiful supply of daylight, at tem¬ peratures not in excess of 80° F. (Op¬ timum 70°-75°F.) If, because of the season or for other reasons it Becomes necessary to use artificial light, we recommend the Sylvania 150W, 120V Spot Bulb (Turtox No. 375A97) at a distance of about eight feet from the culture shelf.

For those who wish to compound their own media, the following have been suggested in the literature, and we recommend reading the references cited at the end of this leaflet.

Knop’s solution, for forms that prefer an acid medium:

Magnesium sulphate, MgS04.7H20 0.25 g.

Potassium phosphate, KH2P04 0.25 g.

Potassium chloride, KC1 0.12 g. Calcium nitrate,

Ca(N02)2.4H20 1.00 g. To prepare, measure out one liter of

distilled water and dispense about 250cc. into each of 4 flasks. Dissolve the chem¬ icals separately in these portions of wa¬ ter and combine into one volume—add¬ ing the calcium nitrate last. There should be no evidence of precipitation if the individual solutions are well mixed as they are combined. To complete the me¬ dium, add one drop of freshly prepared 1% ferric chloride solution. Experimen¬ tation will best determine the most suit¬ able concentration for any particular alga—which may vary from full strength to a 1:10 dilution. The pH of this solu¬ tion will be about 6.4.

Knop’s solution (modified), pH 7.6. Magnesium sulphate,

MgS04.7H20 0.1 g. Potassium phosphate,

K2HP04 0.2 g. Potassium nitrate, KN03 1.0 g. Calcium nitrate,

Ca(N02)2.4H20 0.1 g. Prepare as outlined above and add

one drop of 1% ferric chloride solution. When combined with agar, the fol¬

lowing formula is well suited for the propagation of Chlorella, Gloeocapsa, Pleurococcus, Desmids, Diatoms, etc.

Ammonium nitrate, NH4NO, 0.5 g.

Potassium phosphate, KH2P04 0.2 g.

Magnesium sulphate, MgS04.7H20 0.2 g.

Calcium chloride, CaCl2.2H20 0.1 g.

Agar 12.0 g. Distilled water 1000.0 cc. 1% ferric chloride solution 0.1 cc.

(Note: the agar may be omitted if a liquid medium is desired.)

(6-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Soil-Water Medium (a modification of the Pringsheim method) for cultur¬ ing the Yolvocales and many of the filamentous forms: To prepare, take a gallon battery jar, add 0.5 gram of pre¬ cipitated calcium carbonate, enough humus or garden soil to make a layer from y2 to % inches deep, and suf¬ ficient distilled water to bring the level within an inch of the top. The culture jar, covered witli a glass plate, to¬ gether with its contents, is steamed (at normal atmospheric pressure) in a pressure cooker or Arnold Sterilizer for an hour on each of two consecutive days before inoculation. (Develop steam slowly to avoid breakage of the battery jar, and time the operation from the first appearance of steam.) The most ef¬ fective illumination for this type of culture is provided by two 40 Watt fluorescent tubes located at a six-inch distance for the first week and at an increased distance of 18 inches subse¬ quently for periods of 16 hours daily—in addition to normal diffuse daylight. An electric timer (Turtox No. 205A719) can be placed in the line to switch the current on and off for the daily illumi¬ nation period.

Spirogyra. Spirogyra is found in the field in the quiet waters of ponds, ditches and lagoons, where it often forms large green mats covering the surface of the water. Occasionally it occurs in running water, while still less commonly it is found attached to rocks and piles.

Spirogyra is not easily cultured for prolonged periods, but it will sometimes grow well in a balanced aquarium, and some species live and increase nicely in nu¬ trient solutions. Solutions made of dis¬ tilled water containing minute quantities of dissolved commercial fertilizers (such as “Vigoro”) often work well. Spirogyra cultures should receive plenty of day¬ light and at least some direct sunlight. The cultures should be started with dis¬ tilled or natural pond water, as city water treated with chlorine or other chemicals is very destructive to Spiro¬ gyra.

The related algae, Mougeotia and Zygnema, can be cultured by the same methods that are used for Spirogyra.

Hydrodictyon is found suspended in ponds and lakes. When brought into the laboratory it should be placed in an aquarium which receives an abundance of direct sunlight; then it should grow well. It will also grow in some of the nutrient solutions.

Vaucheria. One kind of Vaucheria can usually be found in greenhouses, where it forms a “green felt” on flower pots and damp benches. Such material is usually in the vegetative condition. An¬ other species, Vaucheria geminata, is often found in ponds and ditches. This species will grow fairly well in labora¬ tory cultures.

The formation of zoospores may be induced by cultivating the material in the dark, using a 2 per cent cane-sugar solution. The formation of oogonia and antheridia may be induced by cultivat¬ ing in bright sunlight, using a 2 to 4 per cent cane-sugar solution. Sex organs will not be formed in partial light or in darkness.

Cladophora. This alga is found grow¬ ing attached to sticks and stones in quiet or running water. For cultures, select the forms found in quiet water, and place in one or two-gallon aquaria. In larger aquaria Cladophora is likely to grow too luxuriantly ; even with the smaller containers, care must be taken to re¬ move the excess material from time to time.

Cladophora is one of the “easy” algae to culture. It can be maintained with so little trouble that pure cultures in nutrient solutions are not usually worth bothering with.

Oedogonium. Most species of Oedo- gonium are found in the quiet waters of ponds and ditches. Forms with dwarf males are exceedingly small, and appear as a fuzzy covering on submerged twigs, cat-tails, and various other plants. Larger filamentous species often form floating mats, which resemble Spirogyra, but they are not so slippery.

Chamberlain reports that in the case of Oedogonium diplandrum, “Klebs found that a change from a lower to a higher temperature would induce the production of zoospores. A culture which has been kept in a cold room with a tem¬ perature varying from 6 degrees to 0 degree Centigrade, when brought into a warmer room with a temperature vary¬ ing from 12 degrees to 16 degrees Centi¬ grade, produced an abundance of zo¬ ospores within two days. Light does not seem to have any influence on the forma¬ tion of zoospores in this species, but light is necessary for the formation of antheridia and oogonia.”

Although Oedogonium sometimes grows well in an ordinary aquarium, if it is desired to maintain cultures for the entire year, nutrient growing solutions are usually desirable.

Pleurococcus. Good material for study can be secured by collecting pieces ot tree bark on which this alga is growing. Such material may be stored dry ; if placed in a moist chamber for 24 hours, the Pleurococcus will begin active growth and is then in good condition to study. It is also possible to grow Pleurococcus on nutrient agar slants.

Chara and Nitella. Both of these algae are fairly easy to grow in large con¬ tainers in the laboratory. Prepare a good-sized battery jar or an aquarium

(6-3)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

tank with a couple of inches of sand and pond mud in the bottom and fill with natural pond water. Plant a small amount of freshly-collected material in this and place the container where it will get plenty of light and some direct sunlight.

Mud taken from the bottom of ponds in which Chara and Nitella are known to grow (even when the ponds have com¬ pletely dried up) will often produce ex¬ cellent new growths.

Diatoms are usually found in large quantities around springs and in pools and ponds, clinging in great numbers to filamentous algae, or forming gelatin¬ ous masses on various submerged plants. The surface mud of a pond, ditch or lagoon will always yield some forms. Fresh-water diatoms appear in greatest abundance in the spring, are compara¬ tively scarce in summer, but reappear again in the autumn.

Diatoms show their characteristic movements best when transferred from cooler to warmer water. This phase of motility is therefore well illustrated shortly after being brought into thé warm laboratory.

Diatoms are often cultured in aquaria with other algae. Cocconeis, an ellipti- cally shaped diatom, is frequently found covering Cladophora; Vaucheria is often covered with smaller forms. References:

Chamberlain, C. J. Methods in Plant Histol¬ ogy. University of Chi¬ cago Press.

Johansen, D. A. Plant Microtechnique. McGraw-Hill 1940.

Loosanoff& Eagle Use of Complete Fertiliz¬ ers in Cultivation of Mi¬ cro-organisms. Science, 95; 487, 1942.

Nieuwland, J. A. Hints on Collecting and Growing Algae for Class Work. Midland Naturalist, 1:85, 1909.

Pringsheim, E. G. Pure Cultures of Algae Cambridge 1946.

Smith, Gilbert M. The Fresh-water Algae of the United States. McGraw-Hill 1933.

Ward & Whipple Fresh-water Biology. John Wiley & Sons, Inc.

Concentrated Media for the Culture of Algae and Ciliates

This new and easy-to-use concentrate makes it practical to grow such difficult forms as Volvox, Pandorina and Spirogyra. 61V170 Turtox “Universal” Concentrated Algae Medium. Especially formulated

to grow Volvox, Eudorina, Pandorina, Chlamydomonas, Spirogyra, Zyg- nema, etc.

Set A; Consists of four units and makes one liter of growing medium in distilled water. Set of four units (distilled water is not included) $3.50

Set B: Consists of four units and makes five liters of growing medium in distilled water. Set of four units (distilled water is not included) 8.50

All prices are f.o.b our laboratories and are subject to change without notice.

Algae Cultures Price, per culture, sufficient for 12 students $3.50 Price, per culture, sufficient for 25 students 4.00 Price, per culture, sufficient for 50 students 5.00 Price, per culture, sufficient for 100 students 8.50

Blue-Green Algae

*Gloeocapsa *Oscillatoria (P.C.)

*Nostoc (P.C.) *Plant Plankton (mostly blue-greens)

Green Algae mChlorella (P.C.)

*Chlamydomonas (P.C.) *Pandorina (P.C.) *Volvox (P.C.) *Cladophora (P.C.)

*Oedogonium Hydrodicfyon (P.C.)

*Pleurococcus (on bark) Tetraspora

*Spirogyra (P.C.)

Zygnema *Desmids *Scenedestnus (P.C.) * Vaucheria *Diatoms (P.C.) *Eudorina (P.C.)

(*)—Available at all times (P.C.)—Pure cultures

61V55 Living Fresh-Water Algae. An assort¬ ment of three commonly studied species, each alga correctly named and in a labeled jar. We reserve the right to select the species for this assortment, but guarantee to include forms commonly studied and repre¬ sentatives of both the Green and Blue-Green Algae. Three named cultures, each sufficient for 10 to 25 students $4.75

(6-4)

TURTOX SERVICE LEAFLET NO. 59

Quantity

1 1 1

1

1 1 1

1 1

1 1 1 1 set 1 1 1 1

1

1

1

1 1

1 1 1

1 1

1

1

1

1 1

1

1

1

1

1

1

BASIC LABORATORY EQUIPMENT FOR HIGH SCHOOL BIOLOGY COURSE We offer the following list of equipment suggestions in response to many requests

from biology teachers. Those articles marked with an asterisk (*) represent materials which would fulfill the minimum needs of the elementary or high school biology depart¬ ment operating on a limited budget. The complete compilation of equipment is sug¬ gested for groups o,f twenty-four students during a one year course of general biology. It is assumed that standard equipment—such as desks, lights, water and gas outlets—• are available.. Prices are accurate at the time of publication but may be changed with¬ out notice. Charges for transportation (f.o.b. our Chicago laboratories) and special containers are not included. For preserved and living specimens refer to our catalog or write for list of suggestions. IMPORTANT: Write for current price quotations before ordering the materials listed on this leaflet.

Microscope Slides Catalog

No. Material Lot

Price Quantity Catalog

No. Material Lot

Price

BC1.10 * Type bacteria 81.50 1 E17.17 Cat ovary $1.95 BC1.85 * Spirillum volutans .. 1.95 1 E17.12 Rat testis 1.65 BC4.2 Typhoid flagella 1 H2.735 * Bone, human,

stain 2.50 ground thin 1.95 BC4.71 * Bacillus subtilis 1 H3.25 * Muscle, human,

spore stain 1.90 striated, smooth, & B1.221 * Volvox 1.65 heart, l.s 2.25 B1.252 Desmids .90 1 H2.851 * Human blood B1.256 Spirogyra in con- smear .85

jugation 1.00 1 H2.824 * Frog blood smear .. .80 B1.421 * Diatoms .80 1 H10.54 Ear internal B2.322 * Rhizopus. nigri- (Organ of Corti) .. 2.00

cans, zygospores .. .95 1 H10.62 Rabbit eye, l.s. ... 2.00 B2.511 Yeast budding .75 1 P5.271 * Taenia, pisiformis, B2.52 * Pénicillium .85 tapeworm 3.00 B2.531 * Aspergillus .85 1 P6.43 Necator american- BWR Wheat rust (3 slides) 3.00 us, male & female, B3.13 * Lichen .85 w.m 3.50 B5.816 Fern protnallium .. 1.75 1 P9.6813 Stigmata (spiracles) B5.765 * Fern sporangia .85 of house fly larva .85 B6.343 * Male pine cone 1 Z1.21 * Euglena .85

with pollen .80 1 Z1.311 Paramecium .85 B7.410 * Stems monocot & 1 Z2.21 Sponge, commer-

dicot, x.s 1.35 cial, skeleton .85 B7.509 * Leaves, monocot & 1 Z2.31 Grantia, x.s .90

dicot, x.s 1.25 1 Z2.62 Spongilla, gem- B7.3816 Tilia stem, x., rad.. mules, w.m 1.00

& tang.s 1.50 1 Z3.131 * Hydra, l.s .90 B7.492 Corn stem, x.s .85 1 Z4.17 Asterias. w.m 1.25 B7.210 * Roots, monocot & 1 Z5.ll * Planaria, w.m 1.20

dicot 1.25 1 Z8.41 Earthworm, x.s. ... .95 B7.165 Root hairs 1.60 1 Z9.516 House fly, pro- B8.294 Pollen tubes 1.65 boscis 1.00 E4.51 * Asterias embryolo- 1 Z9.638 House fly. cornea .95

gy—all stages' 3.00 1 Z9.641 * Insect tracheal E13.78 * Whiteflsh mitosis .. 2.25 system .95 E14.39 Frog tadpole,

serial x.s. 3.25 Museum Preparations

10M282 * Harmful weeds. 1 9D854 Insect wing types herbarium sheets Bio-gram 6.25 (set of 10) 14.00 1 25D151 Slime molds Bio-

8M145 Insectivorous plant gram 9.00 set (set of 4) 3.75 1 25D2556 Mushroom Bio-

15D102 * Systematic demo- gram 5.50 section collection, 1 25D2721 Puccinia (wheat animal 25.50 rust) Bio-gram .... 6.00

2D14 Grantia Bio-gram .. 5.50 1 25D32 Lichen Bio-gram ... 5.50 3D1342 Gonionemus Bio- 1 25D3512 Polytrichum moss

gram 5.50 Bio-gram 6.00 3D422 Pleurobrachia 1 25D3412 Marchantia Bio-

Bio-gram 6.00 gram 7 25 5D22 Liver fluke 1 25D4102 Fern Bio-gram 5.50 Bio-gram 6.00 1 25D60 5D344 Tapeworm (Taenia) Bio-gram 6.00 Bio-gram 6.75 1 8F1 8D221 * Earthworm Bio- (complete teaching

gram 12.00 unit) 3 00 9D1262 * Crayfish Bio- 1 9D677 Honey bee life

gram 5.50 history (Riker 9D5211 Lubber grasshopper mount) 9.00 Bio-gram 6.25

TURT0M.Q|UCTS

TURTOX Service Department

Copyright, 1958, by GENERAL BIOLOGICAL SUPPLY HOUSE

( IN CORPORA TED )

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

Skeletons Quan¬

tity Cat. No. Material Price

Quan¬ tity

Cat. No. Material Price

1 14S10102 Human skeleton & 1 13S61 Turtle $39.50 steel cabinet $319.00 1 13S71 Chicken 46.00

1 13S452 Grass frog , 16.00 1 13S81 Cat 46.00 1 13S352 Perch 22.00

Models 1 TM-1 * Model of typical 1 TM-15 * Model of hydra 35.70

cell 21.00 1 TM-412 Anatomical model 1 set TM-10 Ten models of of human eye .... 55.00

mitosis , 95.00 1 TM-418 Anatomical model i TM-11 * Model of parame- of human ear .... 44.00

cium , 28.75 1 TM-462 Anatomical model i TM-115 * Model of amoeba ... 30.00 of human trunk

Models of frog em- with head, sexless 275.00 1 set TM-149 bryology , 97.00

Charts—Qu iz Sheets

Catalog No. Catalog No. Key Quiz C.R. Key Quiz C.R. Card Pad Chart Card Pad Chart

1.01 1.01 CR 1 Typical cell 16.113 16.113 CR29.2 Chick development 1.021 1.021 CR 2 Types of animal 17.3 17.3 CR30 Cat dissection

cells 18.1 181 CR50.0 Bacteria types 1.031 1.031 CR 3 Animal mitosis 18.2 18.2 CR51 Spirogyra 1.04 1.04 CR 3.1 Spermatogenesis & 18.3 18.3 CR52 Ulothrix

Oogenesis 18.4 18.4 CR53 Oedogonium 1.06 1.06 CR 3.3 Animal kingdom 19.1 19.1 CR58.01 Slime molds 1.2 1.2 CR 4 Ameba 19.4 19.4 CR58 Rhizopus, bread 1.4 1.4 CR 5 Paramecium mold 1.42 1.42 CR 5.2 Euglena 21.3 21.3 CR64 Fern life history 2.3 2.3 CR 6 Grantia 24.2 24.2 CR71 Flower, generalized 3.3 3.3 CR 7 Hydra, l.s., x.s. 22.5 22.5 CR67.5 Lily life history 4.1 4.1 CR 9 Starfish 24.3 24.3 CR72 Privet leaf 5.2 5.2 CR10 Planaria 24.25 24.25 CR71.5 Seed dispersal 5.5 5.5 CR 8.4 Tapeworm, 23.1 23,1 CR68 Monocots & dicots

Moniezia compared 8.3 8.3 CR11 Earthworm 24.61 24.61 CR73.5 Dicot stem anatomy 9.2 9.2 CR12 Crayfish dissection 24.62 24.62 CR73.6 Monocot stem 9.8 9.8 CR18 Grasshopper anatomy

dissection 28.1 28.1 CR90 Six monohybrid 9.85 9.85 CR20.5 Termite life history matings 10.2 10.2 CR21 Clam dissection 28.2 28.2 CR91 Di-hybrid matings 12.3 12.3 CR21.4 Amphioxus dissec- 28.3 28.3 CR92 Independent in-

tion heritance 13.02 13.02 CR21.53 Arterial arch circu- 29.3 29.3 CR100 Sex-linked in-

lation in vertebrates heritance 13.06 13.06 CR21.55 Lamprey anatomy 13.1 13.1 CR21.6 Shark dissection 13.6 13.6 CR21.50 Vertebrate brains 14.6 14.6 CR23 Frog dissection 48 pads Quiz Sheets (25 per pad) : . $26.00 14.9 14.9 CR26 Frog arterial system

. 7.68 14.12 14.12 CR27.2 Frog, male and fe- 48 Key Cards: male urogenital sys¬ tem

48 Classroom Charts 17 x 22": . 45.60

14.14 14.14 CR27 Frog development 1 390D613 *Easel Chart Stand . 5.00 16.4 16.4 CR29 Pigeon dissection

Catalog Lot Quantity No. Material Price

24 385D185 Turtox sexless human manikins $5.00 24 385D13 Turtox earthworm animalkins 5.00

Kodachrome Lantern Slides

Size 2" x 2" Kodachrome Lantern Slides, each $1.00

60L111 Amoeba proteus 60L312 Paramecium caudatum, fission 60L313 Paramecium caudatum, con¬

jugating 16L017 Grantia growing on seaweed 16L026 Physalia pelagica 16L033 Astrangia danae 61L211 Hydra, entire with bud 61L221 Obelia, entire colony 16L0732 Asterias forbesii, feeding 16L087 Lytechinus variegatus, sea

urchin 16L25 Nereis virens, sand worm 62L361 Clonorchis sinensis, liver fluke 62L14 Planaria, x.s. through phar¬

ynx 62L621 Trichinella spiralis, in

muscle 64L15 Cyclops, with egg sacs 16L413 Callinectes sapidus, blue

crab 18L832 Cecropia life cycle 64L83 Insect antennae, types

14L365 Bacteria nodules on roots 35L41 Algae (Biochrome chart) 52L21 Yeast, sporulating 7L24 Arcyria, slime mold

53L122 Physcia, detail of cup portion 7L77 Cladonia pixidata, with cups 7L381 Coprinus atramentarius,

cluster 8L11 Marchantia, female heads

with young sporophytes 8L416 Mnium, large patch with

sporophytes 8L87 Equisetum arvense, horsetail 8L55 Polypodium hesperium 9L36 Pinus ponderosa, male cones 9L37 Pinus ponderosa, female

cones

58L21 Lily anther, x.s. 38L321 Lily, megaspore, mother cell 10L651 Lilium flower, dissected 57L235 Onion root tip, mitosis 57L383 Tilia, three-year stem, x.s. 57L431 Corn stem, x.s. 57L561 Privet leaf, x.s.

(59-2)

Catalog Quantity No. Material

Apparatus

Lot Catalog Price Quantity No. Material

Lot Price

6 105A10N 2 105A20N

24

6 1800

250 24

4 2

200

24 1

1 1 1 1 4

2 4 1 1 1

1

24

1

24

105A46

110A15

110A115

110A30 110A33

110A325 110A51 120A10 120A20 120A36

130A193 205A20

205A465 205A551 210A1501B 210A17 220A111

220A4311

220A604 250A171

250A40

24 308A511 1 lb. 310A56

308A181 308A24 308A36 308A38 308A541

308A60

310A5310

310A51

310A535

1 gal. 310A547

4 oz. 310A5475 100 310A57 24 310A625

4 oz. 310A545

1 308A81A 1 320A82

* Insect nets .nylon .. Water dip nets.

nylon * Water faucet plank¬

ton gatherer * Carbon tetrachloride

insect killing jars.. Cyanide lepidoptera jars

* Spreading boards .. * Insect pins (200 ea.

size) * Spreading pins * Insect boxes

V.asculums * Plant presses * Plant mounting

sheets Gitsknives

* Aquarium, 6 gallon

* Dip tube * Aquarium net * Footed terrarium ..

Hydroponic outfit .. Entomological breeding chambers.

Rat cage assem¬ blies for nutri¬

tional experiments.. Dietetic scale

* Seed germinating boxes

Fern spore germ¬ inating outfit

* Dissecting sets * Dissecting pins .... * Sharpening stones ..

Large forceps Bone shears Bone saw Instructor’s dis¬ secting set

Steel cabinet for small instruments ..

* Dissecting pans, waxed Dissecting pan, large

* Aluminum dissect¬ ing & utility pans

Formalin Fume- lock

$25.50

9.90

2.25

27.50

7.50 9.60

17.50 2.00

72.00 60.00 13.90

9.50 22.80

15.95 1.25

.85 8.25 9.45

11.60

23.95 8.95

7.00

2.25 68.40 3.70 3.20

12.00 10.00 10.50

10.75

17.00

62.50

4.75

16.50

1.95

1 1 1 1

24 2

4 12

1

1

1

1

1

1

1

1 12

24 1

2 24

1

1

12

1

1 1

12 20 ft.

20 ft.

2 lb.

320A802 320A892 320A96 320A105 325A101 325A4055F

325A4222

330A36 325A72

330A20

330A386

330A67

330A54D

330A545B

335A12

335A342

335A381A 375A895

335A57

335A621 335A7

335A79 350A30 325A91

350A75

350A511

375A11

375A411 375A81 375A31 375A64B

375A64C

375A655

ÿ Section razor " Razor hone

Interval timer Slide making kit ...

s Tripod magnifiers .. Wide-field tube microscopes

s Microscopes with case

* Turtox Scope-aid .. 18 Universal Clamp-on

Lamps Tri-Simplex Micro-Projector

Projector for 2x2 slides, with case ..

Filing cabinet for 2x2 slides

Metal tripod screen, glass bead surface.

Tripod screen carrying case ....

Incubator oven com¬ bination

: Steam pressure sterilizer

Double boiler Test tube racks, wood Culture tube baskets

Inoculating loops .. Blood typing kit (80 tests)

Haemoglobin scales Measuring slides

! Frog holder (for circulation demon¬ strations Respiration appa-

1 Photosynthesis light screens

! Harvard trip balance

Bunsen burner Tripod support Test tube brushes ..

: Rubber tubing, 3/16”

: Rubber tubing, 1/4”

: Rubber stoppers assorted

Deodorant 2.25 100 375A711 Corks, assorted .... Waterproof tags . 1.75 1 375A80 * Ring stand Plastic specimen 1 375A885 Aspirator bags . 9.90 2 375A892 Beaker tongs Kerodex Barrier 24 380A20 Drawing paper pads Cream (K71 for formalin) 1.65 24 380A30

(20 sheets each) .. Drawing pencils, 2H

Utility cart . 32.95 24 380A30 Drawing pencils, 4H Hand microtome . . 49.50 1 pkg. 420A11C * Filter paper, 25cm.

.$ 6.95 3.25

15.00 18.50 49.80

114.00

552.00 31.80

51.00

150.00

119.50

4.25

38.95

6.90

60.95

25.00 6.00

27.00

9.30 12.10

9.50 2.50 5.00

1.75

13.95

13.50

25.00 1.35 4.00 2.75

2.20

2.80

3.70 2.95 7.25 9.75 3.50

12.00 4.00 4.00 1.50

Glassware and Plastic

12 315A109E * Pyrex beakers, 4 dz. 320A50 * Dropping pipettes .. 2.00 250cc 6.60 1 315A9310 Bell jar 9.75

1 350A72 * Osmosis apparatus .. 6.50 1 gr. 320A10 * Microscope slides .. 3.15 4 315A330 Graduates, assorted 24 320A17 Hanging drop slides 3.40

10cc., 50cc., 250cc., 320A201F * Coverglasses, 22mm. & lOOOcc 22.60 sq. No. 2 4.50

2 315A229C&E Pyrex Erlenmeyer 1 315A25C Filtering flask with flasks, 250cc., & side tube, 250cc 1.85 lOOOcc 2.05 1 376A24 Clinical thermom-

2 315A921 Desiccators 6.20 eter 1.75 1 315A17D Bunsen’s funnel .... 2.75 4 376A23 * Laboratory ther-

24 315A40D * Petri dishes 17.50 mometers 9.60 12 320A64A Culture bowls. 48 315A55A * Specimen jars,

(fingerbowls) 13.75 3/4 oz 4.00 24 320A63 Stacking watch 24 315A55B * Specimen jars,

glasses 8.50 3-1/8 oz 3.20 1 315A558 * Storage jar, 3 gal. .. 3.95 48 315A55C * Specimen jars, 8 oz. 8.00 1 315A5275A * Storage pail. 48 315A55D * Specimen jars.

Polyethylene 3.00 16 oz 10.00 1 gr. 315A82C * Test tubes 6x%’’ .. 11.92 24 315A5D * Display jars, 8 oz. 6.00 4 lb. 315A96C * Glass tubing, 24 315A5E * Display jars, 16 oz. 7.00

7-8mm. O.D 7.00 24 315A5F * Display jars, 30 oz. 11.00 4 lb. 315A96D * Glass tubing. 24 315A585F * Clear shell vials

10-12mm. O.D 6.00 with polethylene 6 315A55F Culturing jars, stoppers 2.20

1 gal 5.40 12 320A24 Glass marking pen- 24 oz. 315A821 * Plugging cotton, cils, red 2.50

bacteriological .... 3.10 (59-3)

Chemicals

Quantity Catalog

No. Material Lot

Price Quantity Catalog

No. Material

1 lb. Acetic acid, glacial 4 oz. Wright’s blood C.P $2.90 stain

1 lb. Acid carbolic, 4 oz. * Methyl cellulose uu U.S.P.. cryst reduce movement

1 lb. Acid hydrochloric, of protozoa), 10% C.P 2.55 strength

1 gal. * Alcohol iso-Propyl, 2 lbs. Paraffin 53 M.P anhydrous 2.25 5 lbs. * Paradichloro-

5 lb. Calcium carbonate, benzene U.S.P 4.40 23 gr. * Bacto Nutrient Agar

1 lb. Ether. U.S.P 1.80 (Bl) to make 1 lb. * Formaldehyde. 40% .40 1 liter 1 lb. * Glycerine. U.S.P. .. 1.35 2 vi. 420A21 * Litmus paper, red .. 4 oz. Iodine, U.S.P., 2 vi. 420A21 * Litmus paper,

cryst 2.15 blue 4 oz. Potassium iodide, 2 vi. 420A21 * Litmus paper. U.S.P., cryst 1.50 neutral 1 pint Xylol, pure .55 1 vi. 420A301 P.T.C. Tasle-tesl 4 oz. Benedict's solution papers qualitative .90 1 vi. 420A31 * Potassium iodide—

1 lb. Bouin's fluid 2.75 starch paper Eosin solution. 1 380A78A Turtox Plastic aqueous 1% 1.00 Embedding kit

N. D. E. A. Most of the biological materials, equipment and teaching aids authorized for purchase

under Title III of the National Defense Education Act are described in the Turtox Catalogs.

The special 64-page catalog of Turtox Dependable Biological Supplies will be es¬ pecially helpful. This catalog lists the teaching aids most generally used in beginning courses in Biology, Botany and Zoology.

BIBLIOGRAPHY Bulletin 1952, No. 9. “The Teaching of General Biology in the Public High Schools of the United States.” Bulletin Mise. No. 17. “Science Facilities for Secondary Schools.” (For information concerning the above and other free or low cost publications, address Office of Education, the United States Department of Health, Education and Welfare, Washington 25, D.C.)

AMERICAN ASSOCIATION OF SCHOOL ADMINISTRATORS. American School Buildings, Twenty- Seventh Yearbook, Washington, D.C., The American Association of School Administrators, 1949.

BEUSCHLEIN, MURIEL, and SANDERS, JAMES M. Storage of Classroom Visual Materials, School Science and Mathematics, 50:402-404, May 1950.

BURSCH, CHARLES. Providing Appropriate Housing for Schools. Chapter VII of the 45fh Year¬ book, Part I of the National Society for the Study of Education. Chicago, The University of Chicago Press, 1946.

BUTTERFIELD, FRANCES WESTGATE. Teaching Nature in New York's Science Centers. Amer¬ ican School Board Journal. 113:23, October 1946.

BYERLEY, J. ROY. Planning and Equipping the Science Laboratory. American School Board Journal, 98: 59-62, January 1939.

CAMPSEN, HERMAN M., Jr. Purchase, Construction, and Use of Science Laboratory Apparatus. The American School and University, Seventieth Edition, 1945, p. 382-386.

COLEMAN, H. S. ed. Laboratory Design. New York, Reinhold Publishing Corp, 1951. 393 p. Educational Exhibits, How To Prepare and Use Them. Washington, U.S. Government Printing

Office, 1948. U.S. Department of Agriculture, Miscellaneous Publication No. 634, January 1948. ENGELHARDT, N. L. Changes in Secondary School Buildings. School Executive, 68:25, Aug. 1949. ENGLEHARDT, N. L., Sr., ENGLEHARDT, N. L., Jr., and LEGGETT, STANTON. Planning Secon¬

dary School Buildings. New York, Reinhold Publishing Corp., 330 42nd Street, 1949. GREENE, CLARENCE W. Master Lists and Storage of Equipment Used in High School Courses

in Science. American School Board Journal, Part III, Biology, 113:47-48, November, and 48-49, December 1946.

HEISS, ELWOOD d., OBOURN, ELLSWORTH S., and HOFFMAN, CHARLES W. Modern Science Teaching, Chapter 11, The Science Classroom and Laboratory. New York, The Macmillan Company, 1950.

JOHNSON, PHILLIP G. Science Facilities for Secondary Schools. Washington, U.S. Government Printing Office, 1956. (U.S. Department of Health, Education, and Welfare; Office of Educa¬ tion, Bulletin Mise. No. 17.)

McLEOD, JOHN W. Storage Cabinet Assemblies as Dividing Partitions. American School and University, 21st Annual Edition, p. 223, 1949-50.

MARTIN, W. EDGAR. The Teaching of General Biology in the Public High Schools of the United States,U. S. Government Printing Office, 1952. (Federal Security Agency, Office of Education, Bulletin 1952, No. 9.)

MEISTER, MORRIS. Science Rooms for Secondary Schools. The Science Teacher, 15:75-76, 87, April 1948.

Lot Price

$2.00

1.

1.

3.

2.00 .30

.30

.30

1.00

.25

6.50

(59-4)

88

8

TURTOX SERVICE LEAFLET No. 2

PRESERVING ZOOLOGICAL SPECIMENS NARCOTIZATION, FIXATION AND PRESERVATION

A list of laboratory specimens is published in this leaflet with individual references to one or more narcotizing agents, killing or fixing agents and preservatives. Two tables are supplied: Table No. 1 outlines the recommended narcotization procedures and Table No. 2 furnishes the formulas for general fixatives and preservatives—including those specifically referred to in the list. Where no specific mention is made of a killing agent, the indicated fixative will serve the purpose. It will be noted in most instances that fixative and preservative are identical. Whenever more than one good fixation and preservation method was encountered for a particular specimen, we have offered a choice of several.

Numbers Refer to

Table No. 1

Narcoti-

Numbers Refer to Table No. 2

Killing & Fix-

3, 11

Preser- zation ation * vation *

Protozoa 1, 18, 20 22 From 1 to 2 Epistylis 10 19, 20 10 Opercularia 10 19, 20 10 Spirostomum 11 22 From 1 to 2 Stentor 12 22 From 1 to 2 Vortioella 1, 17 19, 20 10 Sponges, fresh water 2 2 Hydra 2, 14 3 (hot) From 1 to 2 Obelia 16 11 11 Anemones 2, 14 3, 11 11 Campanularia 16 11 11 Gonothyrea 16 11 11 Syncoryne 14 11 11 Tubularia 14 11 11 Actinozoa 14 11 11 Pleurobrachia 14 11 11 Starfish 2, 14, 15 11 11 Sea Cucumber 2, 14, 15 11 11 Planaria 3, 13, 15, 19, 21A 12 10 Flukes 3, 13, 15, 19, 21A 10 10 Microstomum 3, 13, 15, 19, 21A 10 10

Ascaris 98°C. H O (1 sec.) 10 10, 2 Nemerteans 3 8 8 Rotifers 1, 8, 9, 11, 19 19, 20, 11 10 Bryozoa 3, 8, 15, 16 10 10 Pectinatella 3 hot (kill) 2 Plumatella 3 hot (kill) 2 Oligochaetes (f. w.) 4, 5 11, 25 10, 25 Lumbricus 7 6, 10 6, 10 Leeches 3, 5, 14 11, 25 10, 25 Daphnia 5, 17, 21A 11 11 Crayfish 2, 11 (kill) 2, 11 Ticks & Mites 2 2 Centipedes & Millipedes 15 15 Insects 2, 15 2, 15 Slugs Boiled, Cool H,0 2, 11 (inject) 2, 11 Snails (acquatic) • 14 11 11 Clams 11 (peg) 11 Dolichoglossus 14 11, 6 11 Salpa 14 11, 6 11 Cynthia 14 11, 6 11 Lamprey 11 (inject) 11 Fish 13 11 11 Grass frogs 13A, 21 2 (drown), 10 inj. 10 Frog Larvae & Eggs 4, 13A 10 10 Salamanders 13A, 21 10 10 Salamander Larvae 4, 13A 10 10 Reptiles 6 (Inject) 11, 2 (drown) 11 Rabbit 5, 6, (21 B-10cc.) Gas or drown 7, 11 Guinea Pig 5, 6, (21B-2cc.) Gas or drown 7, 11 Rat 5, 6, (21B-2cc.) Gas or drown 7, 11 Mouse 5, 6, (21 B-O.lcc.) Gas or drown 7, 11 Large Mammals Gas or drown 11 Vertebrate Embryos 3 2 *Inject body cavities of all larger specimens.

(The fixing agent is, in most cases, also the killing agent.)

TURTirMlUCTS

TURTOX Service Department

Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Table No. 1. Narcotization Procedures for Invertebrates and Vertebrates Listed on

page one.

(To slow down movement for study purposes or to produce relaxation in a contractile specimen as a prelude to killing and fixing. In most cases, the killing agent is also the fixing agent.)

Note: Choices of methods are indicated where more than one number is given. It is often advisable to modify methods in order to produce desired results, and we recommend experimentation to find procedures that fit the particular factors and conditions in your laboratory.

1 Butyn (Butacaine Sulfate). Add 0.1% aqueous sol. (freshly made) drop by drop until desired effect is obtained.

2 Carbon Dioxide. Add ordinary charged water to fluid containing specimens.

3 Chloral Hydrate. Add crystals (or small amounts of 2% sol.) to fluid containing specimens.

4 Chloretone. Use a 1% sol.

5 Chloroform. For aquatic specimens add small quantities to the surface of water in the form of a spray and cover container. Or treat specimens with vapor under a bell jar. For non-aquatic animals use screw-cap jars with impregnated cotton attached to under¬ side of cap.

6 Ether. Use like No. 5, but add small amounts directly to habitat water.

7 Ethyl Alcohol (70%). Transfer worms to a dish of water ,and add the alcohol drop by drop over a period of IV2 hours until the quantity of the added alcohol is 1/10 that of the water. Test by pinching tail.

8 Hanley’s Solution: Water 90 cc., Ethyl Cellosolve 10 ce., Eucaine Hydrochloride 0.3 gm. To every 10 cc. of culture fluid add 1 drop of H S. Repeat at 10 min. intervals until desired effects are obtained.

9 Hydrochloride of Cocaine (1% sol.) 2 parts, Methyl Alcohol (70%) % part, Distilled Water 4 parts. Butyn or Hydroxylamine Hydrochloride may be substituted in strengths V2 as cone.

10 Hydrogen Peroxide (3%). Add to habitat water.

11 Hydroxylamine Hydrochloride. Use 1% sol.

12 Hydroxylamine—Magnesium Sulphate. Make 2 solutions: 1) 5% Magnesium Sulphate, 2) 0.25% Hydroxylamine Hydrochloride. Adjust pH of sol. No. 2 to 6.4. Then add enough dry MgS04 to sol. No. 2 to make 3%. Concentrate organisms in small volume of fluid. Add V2 as much sol. No. 1. When speci¬ mens again exhibit motility, add same amount of No. 1 sol. After 5 min. remove most of fluid and add % this amount of sol. No. 2.

13 M.S. 222 Sandoz (Tricane Methanesulfonate). Use 0.1 gram to 1 gram per gal. of habitat water—acting from 1 to 5 min. A 0.1% sol. in sea water may be sprayed on freshly caught marine spec.

13A M.S. 222 Sandoz. Use 1 gm. per gal. of habitat water. Use 2 to 15 min. for immature forms, several hours to several days for adult forms.

14 Magnesium Sulphate. Add saturated sol. drop by drop.

15 Menthol Cryst. Place a few crystals on surface of water containing specimens. Apply cover to conserve fumes.

16 Menthol and Chloral Hydrate (Galligher). Grind to a paste one teaspoon of menthol crystals with a medium-sized crystal of chloral hydrate and a little water. Place specimens in a minimum of habitat water and add enough of the mixture to form a thin layer.

17 Methyl Alcohol. Add 10% sol. drop by drop.

18 Methyl Cellulose. An inert restrictive medium used only to restrain movements.

19 Strychnine Sulphate 2% sol.

20 Tobacco Smoke. Invert hanging drop preparation over smoke-filled test tubes. Observe under low magnification.

21 Urethane. Pour 5% sol. on specimen. Wash in water when sufficiently narcotized. (Use 0.25% sol. on larval forms.)

21A Urethane 1% sol.

21B Urethane. Use 10% subcutaneously in amounts indicated.

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

TABLE No. 2

FIXATIVES AND PRESERVATIVES (Qeneral)

(Numbers Apply to List of Reagents on page 4.)

1 2 3 4 5 6 7 8 9

5cT cc.

10 II 12 Mise.

Numbers Remarks

1. Alcohol, 50% #13 50 cc.

2. Alcohol, 70% 30 cc.

#13 70 cc. Preservative

3. Bouln's Fluid 20 cc.

5 cc.

#I7A 75 cc. Gen. Fixative Histology Embryology

4. Carnoy's 5 cc.

#13 15 cc. Rapid Action Fixative

5. Carnoy's Fluid No. 2

15 cc.

5 cc.

#13 30 cc. Ascaris Ova Insects Avoid Over-Fix.

6. Duboscq - 10 cc.

5 cc.

Ts cc.

#I7B 60 cc. Alcoholic Bouin's

7. Embalming Fluid

2.5 cc.

86 cc.

1.5 cc.

T~ cc.

10 cc.

Universal Fixative

Improved by Small % qlyc.

8. FAA 25 cc.

#13 25 cc.

9. Fleming's Sol.

60 cc.

4 cc.

#16 16 cc. Cytology ( Deteriorates)

10. Formalin 5% Sol.

95 cc.

5 cc.

General Fix. & Preserv.

11. Formalin 10% Sol. —

90 cc.

10 cc.

General Fix. & Preserv.

12. Gilson's Fluid

20 gms.

880 cc.

4 cc.

#13 100 cc. (60%)

#15 15 cc.

General Fix.

13. Greening Sol.

~2 0~ gms.

—

0.2 gms.

0.2 gms.

20 cc.

Too cc.

40 gms.

#14 20 gms. Preserves Green Color in Plants

14. Hollande's 2.5 ams.

10 cc.

1.5 cc.

|#I7 4 gms. For Flagellates

!5. Insect Preserv.

280 cc.

60 cc.

20 cc.

50 cc.

#13 175 cc. Improved by small % of Glycerin

16. Kahle's Sol.

30" cc.

6 cc.

1 cc.

#13 15 cc. Insects Embryology

17. Kleinenberg’s 200 cc.

#I7A 98 cc. #20 2 cc.

Embryology

18. Muller's Fluid —

—

100 cc.

—

#18 2.5 gms. #19 1 gm.

Nervous Syst. (Keep in dark)

19. Osmic Acid 9 cc.

—

#16 1 cc. Protozoan Fixative

20. Olney's Nitric Acid Fumes

#15 (See foot¬

note)

To replace Osmic Acid Fixation

21. Perenyi's Sol.

4A 30 cc.

#13 30 cc. (90%)

#I5A 40 cc.

Protozoa

22. Schaudinn's Fluid

5A 40 cc.

225

1 cc.

#13 10 cc. Protozoa Add No. II

just before use. Use @ 70OC.

23. Tellyesnîcky's Fluid

100 cc.

200 cc.

T~ cc.

~25~

#18 3 gms. Bryophyta Amphîb & Fish Embryos

24. Worcester's Fluid

14 gms.

Plant Tissues Protozoa

25. Zenker's Fluid

2.5~ gms.

-

5 gms.

100 cc.

5 #18 2.5 gms. #19 1 gm.

General Fixative

26. Zirkle's Fluid

2 gms.

400 cc.

#18 2.5 gms. Mitochondria (Fix 24 hrs.)

(2-3)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Preparing frogs in one of the Turtox Laboratories

List of Reagents Used in Table No. 2 1 Ammonium Bichromate, cryst. 12 Glycerine 2 Carbolic Acid, cryst. 13 Isopropyl Alcohol 3 Chloroform 14 Lactic Acid 4 Chromic Acid, 1% aqueous 15 Nitric Acid, cone.* 4A Chromic Acid, 0.5% aqu. 15A Nitric Acid, 10% 5 Corrosive Sublimate, cryst. 16 Osmic Acid, 2% aqu. (DANGEROUS) 5A Corrosive Sublimate, sat. aqu. 17 Picric Acid, cryst. 6 Cupric Acetate, cryst. 17A Picric Acid, sat aqu. 7 Cupric Chloride, cryst. 17B Picric Acid, 1% in 95% ale. 8 Cupric Sulphate, cryst. 18 Potassium Bichromate 9 Distilled Water 19 Sodium Sulphate, cryst.

10 Formalin, full str. 40% 20 Sulphuric Acid, cone. 11 Glacial Acetic

*01ney’s Method: To y2 in. of Nitric Acid in small ground stoppered bottle, add a (Turtox News, piece of copper scrap. Invert hanging drop over fumes for 20

Jan. 1953, p. 29) sec. Rinse spec.

References:

Bolles Lee’s The Microtomist’s Vade Mecum. Blakiston, 1950.

Peter Gray The Microtomist’s Formulary and Guide. Blakiston, 1954.

(2-4)

TURTOX SERVICE LEAFLET No. 19

SPECIAL PROJECTS FOR BIOLOGY STUDENTS

So many projects are available for high-school students who are interested in Biology that this leaflet can do little more than list a few of the numerous possibilities. Most of the suggestions are of a general enough nature to prove suit¬ able for schools in all sections of the country, and many can be carried on by students in the smaller high schools where laboratory facilities are rather meager. The resourceful teacher will be able to add greatly to this list and will be able to stimulate student interest in many special projects that are possible because of certain favorable local conditions.

One point should be strongly empha¬ sized. It is highly desirable that every student taking the biology course should be held responsible for some special work to be done “on his own” with the mini¬ mum of help from the teacher. The in¬ terested students will ask for such work; it should be the first concern of the teacher to arouse the interest of the less willing students and to get them to select a special project of some kind. A good plan is to prepare a list of fifty or more projects (even though the class enroll¬ ment is much less than that) and allow each student to make his own selection. In this way almost every student will find something to suit his tastes and in¬ terests, and the completion of the various projects during the term or semester will sustain class interest and create a great amount of wholesome competition.

General Projects

(1) There might be some question as to the classification of a Biology Club as a project for students, yet it seems to us that this classification is quite logical be¬ cause, after all, it takes work to carry on a successful club and by far, the greatest amount should be done by the students themselves. By all means, if your school does not have an active club now, plan to organize one within the near future. Turtox will gladly send you a copy of our Service Leaflet No. 57 on “The Organiza¬ tion and Activities of a Biology Club.” We give this project first place because it is of prime importance and will be a

great factor in forwarding interest in other projects which follow.

(2) One of the most interesting proj¬ ects for a biology class, and one in which any number of students may participate, is the making of a biological survey of the region in which the school is located. Field work of this kind, when properly directed, has the tremendous advantage of making students realize that “biology is at their doorstep” ; they cease consider¬ ing biology as a dry textbook subject when they awaken to the fact that count¬ less new and interesting happenings in the plant and animal world are taking place within a mile or so of their school.

In making a neighborhood biological survey one student or a small group of students should specialize on each divi¬ sion such as (a) Birds, (b) reptiles, (c) insects, (d) flowering plants, etc. Rec¬ ords should be kept of the species iden¬ tified and when possible specimens should be brought in to the school laboratory. The permanent record should credit each student as collector or observer of the species reported by him.

(3) The average biology class usually has several members who have their minds set on becoming doctors. For these students a general survey of local sani¬ tation and health conditions makes a good project. In connection with this the sewage disposal system, water works, etc., may come under observation, as well as studies of various other measures taken for the protection of health.

In making such a survey, valuable help can often be obtained from local officials. The record books of the city hall can also contribute to the work by showing what ordinances have been passed for the pro¬ tection of health. These will include, not only such items as regulation of sewage disposal, but also measures to guard against accidents.

Field Projects

(1) In connection with the local bio¬ logical survey mentioned above it should be possible to make good systematic col¬ lections of such forms as insects, fishes, amphibians, reptiles, ferns, fungi, flow-

TURTOX Service Department

Copyight, 1960, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

ers, etc., for the school laboratory. Each specimen should bear a label showing col¬ lection number, locality, date, collector and remarks. Refer to Turtox Service Leaflets 2 and 3.

(2) Identifying of trees in winter and the making of a collection of twigs in their winter condition.

(3) Identifying of trees in summer and the making of a collection of pressed and mounted leaves.

(4) Bird study offers many possi¬ bilities for the entire class as well as for individual students. In the spring a “mi¬ gration calendar” should be kept, such a record showing (a) name of bird, (b) date first seen, (c) locality, (d) date of greatest abundance, (e) date last seen (if the bird merely passes through as a spring migrant), (f) remarks. Other worthwhile phases of bird study include the studying of nests in winter, making and putting up of bird houses, maintain¬ ing of feeding stations during the winter months.

Write for leaflets, etc., to the National Audubon Society, 1000 Fifth Avenue, New York City.

(5) Herbarium specimens are valu¬ able teaching aids, and students should be encouraged to bring in specimens for the permanent school collection. In addi¬ tion to pressed and mounted specimens of flowering plants, ferns, etc., pressed tree leaves and dried specimens of the woody fungi should be included in any well arranged herbarium. Refer to Tur¬ tox Service Leaflet No. 24, “Preparing and Caring for a Herbarium Collection.”

(6) Students who live on farms or who have backyard gardens should be encouraged to try a little simple work in plant breeding. Crossing different varie¬ ties of corn, beans or peas is not difficult and often produces interesting results. Bulletins on this subject may be secured from the United States Department of Agriculture at Washington, D. C.

(7) Collecting cocoons of various moths and the egg cases of spiders for study in the laboratory. Best time is early autumn.

(8) In the early spring there are few things of more interest than collecting frog eggs. Bring them to the laboratory and place them in an aquarium where the rapid development into young tadpoles can be seen by all members of the class. Frogs (in the Chicago region) usually lay their eggs about April first. Toads lay about June first.

Living Material Projects

(1) Rearing insects in the school labo¬ ratory is possible in many instances and such experiments enable the class to see all stages of the life cycles living under approximately natural conditions. Good subjects for such projects are the meal¬ worm (a beetle), drosophila or fruit fly (a true fly), silkworm moth (a typical moth), and the cockroach (a roach). Refer to any of the better texts on Entomology and to the following Turtox Service leaflets: No. IS, “Rearing the Silkworm Moth.” No. 1-5, “The Culture of Drosophila Flies and Their Use in Dem¬ onstrating Mendel’s Law of Heredity.”

(2) Maintaining and studying the in¬ habitants of a balanced fresh-water aquarium. In this connection remember that a perfectly balanced aquarium (without fish or other large animal in¬ habitants) can be maintained in a con¬ tainer as small as a quart mason jar. In a quart jar Cyclops, small snails and countless other minute examples of pond life will thrive for long periods of time. Refer to Turtox Service Leaflet No. 5, “Starting and Maintaining a Balanced Fresh-water Aquarium,” and to “Guide to the Study of Fresh-water Biology,” by Needham.

(3) Rearing of protozoa. Students will be interested in starting cultures of Par— amecia, Euglena, etc., and in studying the protozoa found in stagnant pond water. Refer to Turtox Service Leaflet No. 4> “The Care of Protozoan Cultures in the Laboratory,” and to “Living Speci¬ mens in the School Laboratory” (Price $1.00).

(4) Rearing of Daphnia to use for feeding the fish in the laboratory aquaria. Directions are given in Turtox Service Leaflet No. 23, “Feeding Aquarium and Terrarium Animals.”

(5) Growing and studying molds. Place some moist bread under an in¬ verted finger bowl. When does the mold first appear? What is it? How did it originate? Drawings and notes should be made each day. Microscopic examina¬ tion will be necessary. Refer to Turtox Service Leaflet No. 32, “The Culture and Microscopy of Molds.”

(6) Bacteria. Fill four petri dishes (or saucers) with culture media of nutrient agar (ordinary gelatine will do fairly well) and proceed as follown: Cover one dish with a clean sheet of stiff paper and place it aside immedi¬ ately. Touch the tips of the fingers to the surface of the gelatine in the second

(19-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

dish. Cough into the third dish. Scatter a little street dust onto the gelatine in the fourth dish. Label each dish, according to the above procedure, and when bac¬ terial growth appears note where it is most abundant. Why? What conclusions should be drawn? A microscopic exami¬ nation will not be necessary.

(7) Establishing and caring for terra¬ ria typical of different ecological groups. Refer to Turtox Service Leaflet No. 10, “The School Terrarium."

(8) Seed germination offers many in¬ teresting experiments. Among the prob¬ lems wortli considering are (a) time for germination of different seeds, (b) tem¬ perature effect on germination, (c) mois¬ ture effect on germination, (d) percent¬ age of fertility, (e) contents of “grass seed” mixtures, etc.

Projects for the Laboratory

(1) Regeneration experiments with living planaria (flatworms). Refer to Turtox Service Leaflet No. 16, “The Cul¬ ture of Planaria and Its Use in Regen¬ eration Experiments.”

(2) Making skeletons of such animals as frog, turtle, cat, etc. See Turtox Serv¬ ice Leaflet No. 9, “How to Make Skele¬ tons.”

(3) Making permanent microscope slides. See Turtox Service Leaflet No. 8, “Making Microscope Slides of Simple Objects.”

(4) Mushroom spore prints. In the autumn, or at any season when fungi are fairly abundant, collections of spore prints may be made as follows: Cut off the cap of the mushroom and lay it, right side up, on a piece of clean white paper. Cover it with an inverted dish and leave for 24 hours. The spores will settle to the paper in the form of a “print” char¬ acteristic of the shape of the mushroom. The spores are of many colors; mush¬ rooms having white or very light colored spores had best be laid on black paper. I'he prints may be made permanent by painting them with a thin white shellac.

(5) Embedding specimens in plastic for permanent museum demonstrations. Refer to Turtox Service Leaflet No. 33, “Embedding Specimens in Transparent Plastic.”

(6) Taxidermy is often of interest to boys, and while the killing of birds is unlawful and should be discouraged, there is no harm in collecting small mam¬ mals for mounting or making into mu¬ seum skins. Refer to the two following booklets which can be purchased from the American Museum of Natural His¬ tory in New York City. “The Capture and Preservation of Small Mammals for

Study” and “The Preparation of Birds for Study.”

(7) Students who are interested in biology and who have even average abil¬ ity in drawing can make charts which will be useful teaching aids for the teacher. Refer to Turtox Service Leaflet No. 26, “Making Biology Charts.”

(8) The making of lantern slides of biological subjects is most interesting and worthwhile. In many schools students are used for preparing a great number of lantern slides. Under the guidance of the teacher, students will experience no great difficulty in following directions given in Service Leaflet No. 45, “Lantern Slides Any Teacher Can Make.”

(9) Ants are remarkably interesting creatures and are easily collected almost everywhere. Study and keep a record of the activities of a colony of ants in an observation nest. Refer to Turtox Service Leaflet No. 35, “Studying Ants in Ob¬ servation Nests.”

(10) ) Experiments with plant hor¬ mones, colchicine and gibberellic acid on seeds, seedlings and growing plants. Re¬ fer to Turtox Service Leaflets Numbers 47, 54 and 60.

(11) Phptomicrography and other phases of photography offer many in¬ teresting projects to the camera-minded student. Refer to Turtox Service Leaflet No. 56 “Simplified Photomicrography.” Write for list of special bulletins and booklets to Eastman Kodak Company, Rochester, N. Y.

(12) Modeling. Enlarged and scale models of many plant and animal struc¬ tures can be made of Permoplast or other modeling compounds. Models can be carved from blocks of wax or of plaster.

(13) Incubate chicken eggs and study of developing embryos. Refer to Turtox Service Leaflet No. 17, “Incubation, Fix¬ ation and Mounting of Chick Embryos.”

(14) Make micro-replicas of snow¬ flakes and of leaf surfaces, surface struc¬ tures of skin, hairs, etc. Refer to Turtox Service Leaflet No. 31.

Note. Many additional student projects are suggested in the sixty Turtox Service Leaflets.

Seasonal listings of projects appear below : (1) September

This is usually a busy month for the teacher, but an ideal time nevertheless to interest the students in a series of projects to be started now and carried on throughout the school year. The resourceful teacher will suggest many possibilities and permit each student, as far as possible, to select the project that most appeals to his individual taste.

(a) Insect Collections. Let one student specialize on beetles, another on butterflies, etc., to arouse competitive interest. Collections start¬ ed now can, of course, be continued during the spring.

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

MONTHLY PROJECTS (b) Bird Study, with lists of the autumn mi¬

grants. (c) Hebarium Collections. Flowers are plentiful

and some important families of flowering plants not found blooming in the spring are now abundantly represented, notably the asters, goldenrods, and thistles.

(d) Start bulbs and other plants for indoor study in the laboratory during the winter months.

(e) Collect clumps of mosses, liverworts, small ferns, etc., for planting in the school ter¬ rarium.

(f) Leaf skeletons. May be made at any time, but the fresh leaves should be collected and pressed now.

(g) Wild seeds and fruits. Collect as many kinds as possible and arrange them in groups, based upon mode of dispersal.

(2) October Field work should be continued during October

if climatic weather conditions permit. There will be ample time for indoor activities when the colder weather sets in.

(a) Deserted Birds’ Nests may now be brought, in. Note the widely different types, the vari¬ ous methods of construction and the great variety of building materials used.

(b) Collect snails, aquatic insects and small na¬ tive fishes to place in the school aquarium.

(c) Look for the silken egg cases of spiders and the large cocoons of the Cecropia, Prométhia, and Polyphemus moths. If brought into a warm room now, the moths will emerge from their cocoons in January and February.

(d) Start an indoor nursery of trees and shrubs, planting seeds that can be found locally.

(e) Mammals. What native wild animals are still found in your region? Which of these (and of those formerly found there) hiber¬ nate, and are active throughout the winter months? , .

(f) Collect and study the seeds of noxious weeds. Why have these plants been able to spread so widely and maintain their abundance in spite of all efforts to exterminate them?

(g) Collect several lots of algae and establish cultures in battery jars. Examine these with a microscope each week. What changes take place?

(h) Make a series of Protozoan cultures, using hay, rice, wheat, boiled lettuce and meat. What forms appear first in each? What follows?

(3) November (a) Grow cultures of at least two common molds. (b) Look up sources of free literature for the

biology library. Write to Superintendent of Documents, Washington, D. C., for lists of scientific publications.

(c) Collect and identify leaves (needles) from the coniferous trees found in your locality.

(d) Establish a “culture” of mealworms (a Beetle) and study its various life history

(e) Make a sky-chart of the November heavens and learn to locate the large constellations.

(f) Secure some natural (not chemically treated) cider vinegar and start a culture of vinegar eels.

(4) December (a) Collect and mount for study, examples of

the winter buds (twigs) of the common trees of your locality.

(b) Make a series of simple biology charts by the projection-tracing method, using either paper or chart-making cloth.

(c) Make microscope slide mounts of stems, buds, etc., by the free hand sectioning method.

(d) Students interested in taxidermy can trap small mammals (field mice, wood mice, shrews, moles, etc.) and make up the skins and skulls for the school study collection.

(e) Take a census of the resident winter birds. (f) Dip up part of a large ant colony and

establish it in an observation ant nest. (5) January

(a) Look for insect galls on oaks, roses, golden- rod stems, poplar, etc. Section these with a sharp knife and study their structure.

(b) Bring into the laboratory a piece of solidly frozen surface soil from field or woodlot. Place it in a clear-glass battery jar, cover the top with a glass plate and place upon a warm, sunny windowsill. What changes take place? How soon? What plants develop and grow? What insects or other animal life appear?

(c) Supplement the school collection with needed lantern slides made with etched glass or cellophane.

(d) Grow some fern prothallia, germinating fern spores on porous earthenware.

(e) Perform a series of simple osmosis experi¬ ments, using (1) parchment paper, (2) animal bladder, (3) the inner membrane

from an egg, and (4) some other suitable substance.

(f) Model in clay certain simple subjects of a biological nature.

(g) Cast in plaster such things as a clam, a small snake, etc.

(6) February Even in the more northern states, regular

field trips should be undertaken by the latter part of February or the first part of March.

(a) Run a series of seed germination experi¬ ments, using some unusual and hard to grow seeds in addition to the standard textbook forms.

(b) Run a series of growth experiments with bulbs and tubers, using the onion, potato, sweet potato, and dahlia.

(c) Start a date record of the first appearance of the various spring flowers. Do not overlook the trees, some of which—like the silver maple and willow—shed pollen very early in the season.

(d) Collect and bring into the laboratory, eggs of Ambystoma and other salamanders. Place them in a large jar of pond water and record the progressive stages of development.

(e) Incubate a dozen eggs (hen) in a small electric or gas-heated incubator. Open one egg every other day and preserve the series of embryos.

(f) Make a series of experiments with the com¬ mon bacteria. (If standard material is not available, an ordinary double boiler, porcelain saucers and gelatin may be used.)

(7) March (a) Collect freshly-laid frog eggs from a nearby

pond and place them in a balanced aquarium. (b) Collect samples of pollen from at least a

dozen varieties of flowers and examine each of these microscopically. Make drawings of each kind.

(c) Secure small samples of various types of local soils (clay, sand, loam, etc). Test for acidity (litmus), for solubility, and examine microscopically.

(d) Put up bird nesting boxes in suitable lo¬ calities near the school.

(e) Make a special collection of the flowers of the more common shrubs and trees.

(f) Let your students make a natural history survey of the locality near your school. A considerable amount of friendly rivalry and interest can be maintained if one group lists the trees, another the herbaceous plants, another the insects, etc.

(8) April An ideal month for field work. Regular trips

afield should be taken, with the entire class attending,

(a) Collect and identify specimens of the local amphibians (frogs, toads, and salamanders). All of these can be kept alive and studied in aquaria or terraria.

(b) Install an observation hive of honey bees outside of one of the school windows.

(c) Start now (or earlier) the making of an authentic herbarium collection of the flower¬ ing plants of your region.

(d) Make a series of leaf-prints of trees, using blueprints or regular photographic paper.

(e) Collect earthworms and study their noc¬ turnal habits.

(f) Hatch some silkworm eggs and raise the larvae, feeding them young leaves of the mulberry or osage orange.

(g) Use Elodea (or some other suitable water plant) to demonstrate that green plants pro¬ duce and give off oxygen in considerable quantities.

(9) May (a) Collect living toad eggs and study their

development in a balanced aquarium. In what ways do they differ from frog eggs?

(b) Make spore-prints of gilled mushrooms. (c) Write to the United States Biological Survey,

Washington, D. C., for information about the studies made through the banding of our native birds.

(d) Make photographs of flowers, ferns, etc. for use as lantern slides. Local pictures are always of more interest than those secured in other ways.

(e) Make a survey of the common household pests of your community. Secure information about the eradication of them and pass this along to any interested families.

(f) Label the trees of your general locality so that the general public will take an interest in them. On the label, give some informa¬ tion as to the common name, scientific name, commercial value, whether native or introduced, etc.

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TURTOX SERVICE LEAFLET No. 12

DEMONSTRATION AND DISPLAY MATERIALS

Most teachers have neither the time nor tile special skill necessary for the creation of large display collections. But nearly every teacher occasionally wishes to save some particularly interesting specimen and the purpose of this leaflet is to suggest tlie simpler ways of prepar¬ ing and mounting specimens for display or demonstration purposes.

There are, of course, countless ways of preparing display collections and of mounting plant and animal specimens for demonstration in the school labora¬ tory. Some of these methods (taxidermy, for example) require careful study and long practice; such techniques will not be discussed here, for the interested teacher can refer to many available books deal¬ ing with museum methods. Other meth¬ ods deal with definite types of specimens or specialized techniques (making insect collections, mounting herbarium speci¬ mens, preparing skeletons, and embed¬ ding specimens in transparent plastic) ; and these methods will not be discussed here, for they have already been covered in oilier Turtox Service leaflets

The ordinary specimens which the aver¬ age teacher may wish to preserve and keep for display and demonstration use, fall into two general groups:

(1) those specimens which can be dried and kept in a dry state, and

(2) those specimens which must be preserved in liquid and stored per¬ manently in a liquid preservative.

I. Dry Specimens Under this heading might be men¬

tioned such items as seeds, wood and bark samples, woody fungi, fossils, shells, skulls and other bones, moth cocoons, and such marine animals as the hard sponges, sea-fans, starfishes and sea ur¬ chins; there are hundreds of others. ( However, at this point, it must be stated that no specimen should be saved unless it can serve a definite purpose and have teaching value. The miscellaneous and uncataloged lots of plain “junk” which sometimes occupy valuable space and continue to be referred to as “the school

museum” have no place in modern edu¬ cation.)

Most dry specimens require little pre¬ liminary preparation except thorough drying. Needless to say, partially dried specimens which still contain considera¬ ble moisture should never be mounted or stored in air-tight containers. First, dry them thoroughly and if speed is desir¬ able, use a low-temperature oven or sev¬ eral infra-red heat lamps. Some marine specimens, such as starfish, are likely to retain an unpleasant odor even after drying. This can be prevented if such specimens are first soaked for twenty- four hours in 70 percent alcohol or in 10 percent formalin, then rinsed in water and dried. Soaking starfish and sea urchins in saturated borax solution be¬ fore drying will protect sucK specimens against destruction by museum beetles and other insects.

Small crayfish, crabs and similar crustaceans may be preserved by soak¬ ing them in a saturated solution of borax and water. Make openings in all the joint membranes and then place the specimens in the borax solution for twen¬ ty-four hours. Then rinse in fresh water and dry in the position in which the specimens are to remain permanently. The wet specimens can be arranged on sheets of cork or balsa wood and pinned out for drying. After becoming thor¬ oughly dry, such specimens are odorless and will keep for many years. The origi¬ nal natural colors will not be preserved, but the dry specimens may be varnished and tinted with oil paints.

Most dry specimens are best left in their natural condition. However, cut and polished wood samples are improved by a coat of clear varnish. Some shells can be preserved and their colors height¬ ened by the application of a very thin coat of white varnish or colorless lac¬ quer. Bones which have been thoroughly degreased and bleached can also be treated with colorless lacquer, prefer¬ ably sprayed on. Du Pont’s clear lac¬ quer, thinned to water consistency, is good for this purpose.

TURTSMOIUCTS

TURTOX Service Department

Copyright, 1947 by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

The storing and mounting of dry dem¬ onstration specimens is largely a matter of personal taste. Most large specimens do not require mounting but if desired they can be fastened to plaques made of plywood or heavy cardboard. Smaller specimens are best if protected under glass. Rilcer mounts are suitable, as are also the various sizes of partitioned dis¬ play cases with glass tops. Such cases accommodate specimens having a thick¬ ness up to about three inches. Small specimens can be mounted with suitable labels on upright glass plates in jars as explained below under Liquid Specimens.

II. Liquid Specimens

Included in this group are such ani¬ mal specimens as sea anemones, spiders, soft-bodied insects, frogs, salamanders, snakes, fleshy plants and countless plant and animal forms which are best dis¬ played in a liquid preservative. Good laboratory dissections, or parts thereof, are often worth preserving and mounting in demonstration jars.

If fresh (living) specimens are to be prepared for display, they should be carefully preserved. After being killed, the specimen should be arranged and pinned out in the desired position in a deep wax-lined tray and then covered with preservative. In addition, animals such as frogs, turtles, etc. should have the preservative injected into the body cavity and into the larger muscles. After being thoroughly preserved (this usually requires several days in the preserva¬ tive), the specimen may be removed and mounted in a glass jar of suitable size and shape.

Almost any type of clear glass jar may be used. The straight-sided cylindri¬ cal or rectangular museum jars are de¬ sirable, but these jars have been very scarce since the war and are not obtain¬ able as this leaflet is being written. Screw-capped jars of clear glass with straight sides are available in a variety of shapes and sizes, and such jars are recommended for school use. They cost comparatively little and may be sealed easily and quickly. (The museum jars usually come with flat glass covers which must be cemented onto the top of the jar.)

After selecting a jar of the right shape and size, prepare a glass plate to support the specimen you are mounting. This rectangular piece of glass should fit as tightly as possible inside the jar, so that it will remain in an upright posi¬ tion. It may be cut from (a) transparent window glass, (b) opaque white glass or (c) opaque black glass; the choice de¬ pends upon the type of specimen being

mounted. Tapeworms, for instance, are most effectively displayed on black glass.

To attach the specimen to the glass plate it is usually most satisfactory to use fairly heavy cotton or linen thread— white or black. Using a needle, the thread may be passed through the speci¬ men and tied securely at the back of the plate. Specimens mounted in this manner usually remain firmly attached to the display plate. Printed or written paper labels may be attached to the plate by means of Murrayite or any other water- and alcohol-proof cement. After speci¬ mens and labels have been attached to the plates and after the plate has been placed in the display jar, the jar should be filled with liquid preservative. Forma¬ lin solution of eight percent strength and seventy percent alcohol are the two preservatives most generally used; the formalin solution is cheapest and also evaporates less readily than alcohol. In either case, the solution should be filtered carefully (to improve clarity) before be¬ ing placed in the display jar.

The jars may now be sealed, either with a screwed-on metal cap or a ce- mented-on glass cover.

This glass plate and display jar meth¬ od is also frequently used for dry speci¬ mens. The procedure outlined above may be followed except that the speci¬ mens can usually be glued or cemented to the glass plate and no preservative is required. Insects and other specimens mounted in this way are protected from tlie ravages of museum beetles and other pests if the jar is kept tightly sealed.

The Care of Demonstration Specimens

It is well to inspect the display or dem¬ onstration collection two or three times each year. A little care and attention will often prevent deterioration and pro¬ long the life of valuable teaching ma¬ terial.

Mounted birds, bird skins, mammal skins, herbarium collections and particu¬ larly insect collections are very likely to be attacked by moths, museum (Der¬ mes tid) beetles and other destructive pests. Such damage can be prevented in large measure by keeping such speci¬ mens in air-tight containers, or if this is not possible, in fairly tight display cases. The latter also serve to protect specimens from dust and careless handling.

The cabinets or other containers in which such collections are stored should hold small trays or other open boxes filled with a good fumigant or insecti¬ cide. One of the best is paradichloroben- zene, known by the trade name of “Di- chloricide.” This actually kills insects and their eggs. Moth balls and naphthalene

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Dry corals displayed in a deep, glass-covered display case. Cases of this type, with or without partitions, are available in many sizes and in depths up to about three inches. They

are suitable for displaying many kinds of dry specimens.

Mounting specimens on white plates in museum jars.

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GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Various ways of mounting demonstration specimens. Display cases, riker mounts, glass boxes, screw-cap jars and sealed museum jars are shown in this group.

flakes may discourage insects, but will not kill them. The Dichloricide evapo¬ rates when exposed to the air; therefore, the supply must be renewed every few months.

If the collection becomes badly infest¬ ed, heroic measures are usually indicated. The infested specimens can be placed in tightly closed containers with a quantity of (a) Dichloricide or (b) carbon bisul¬ phide and left there for several days. This treatment will kill all museum pests. (Caution, the fumes of carbon bisul¬ phide are highly inflammable and explo¬ sive. It must be used with great care). The Dichloricide is slower, but much safer to use.

Museum and display jars containing liquid should be examined once or twice

each year for possible leaks and evapora¬ tion. Even carefully sealed jars some¬ times develop leaks. Jars containing al¬ cohol should be watched with special care.

Bones and other skeletal preparations often become dirty and discolored when exposed to dust and handled frequently. Such preparations can be cleaned by washing (preferably merely rinsing) very carefully in soapy water. If after such washing, they remain discolored, they may be bleached in hydrogen peroxide solution. Bones which are very greasy can be degreased and whitened by treat¬ ing them with carbon tetrachloride. Place the hones in carbon tetrachloride for sev¬ eral hours, then rinse in hot water and dry. (Note: Use carbon tetrachloride only in a well ventilated room.)

110A513 Parudiclilorobenzeue. The fumes are harmless to humans, but will kill moths, museum beetles and most other insects. The best protection for insect collections. Per pound $0.70 Five pounds 3.00

110A555 Display Case. Double frame. Glass top. A low priced entomological case designed primarily for the display of pinned insects but equally valuable for other exhibits such as dried plants, hereditary preparations, botanical life histories and other material which may be pinned to the bottom of a case. The case has a false bottom of tackboard composition for pinning insects. It is covered inside with high grade glossy white paper. The outside is covered with durable black paper.

Size No. Size, inches Each Dozen A 8x12x2 * $3.50 $38.50 B 12x16x2* 3.75 41.25

130A381 Compartment Display Case. Glass top. A durable display case with compartments for separating the vari¬ ous items of a collection placed in it. The partitions are permanently fas¬ tened to the bottom of the case and extend to the top, to prevent the con¬ tents of one compartment shifting to another, when the case is moved.

Size Size, Number of No. inches Compartments Each A 8x12x2* 24 $3.00 B 8x12x2* 12 2.90 C 12x16x2* 30 3.80 D 12x16x2* 12 3.75

Refer also to Turtox Service Leaflet No. 33, “Embedding Specimens in Transparent Plastic.”

All prices are f.o.b. our laboratories and are subject to change without notice.

(12-4)

TURTOX SERVICE LEAFLET No. 43

EMBRYOLOGY IN THE HIGH SCHOOL BIOLOGY COURSE

Written embryology holds little in¬ interest for the beginner in Biology, but practical embryology along with the theoretical will usually arouse keen in¬ terest. For this reason it is well to bring into a course in Biology as much work as is possible on the study of the develop¬ ment of living forms, and of prepared slides. This article is written in order to give the Biology teacher some ideas which will be helpful in teaching the phase of Biology dealing with develop¬ ment.

Field trips should be planned and so conducted that the student will get some general ideas as to adult life, differences in sex, environmental conditions affect¬ ing the laying, fertilization and growth of the egg. Comparison of various forms in their development is interesting and essential to an understanding of embry¬ ology.

Starfish Starfish development lends itself read¬

ily to class room study since the earlier stages of cleavage are simple and give the student the basic facts of division. Inland schools do not have the advantage of using living material, but slides show¬ ing the various steps in development may be used as a substitute. The fol¬ lowing stages should be studied and attention called to various structures:

The unfertilized egg; protective mem¬ branes surrounding the egg, cell wall, cytoplasm, nucleus and nucleolus.

Early cleavage; the egg divides into two cells of equal size.

Late cleavage; the formation of a clus¬ ter of more or less regular cells.

Blastula; single layer of small cells enclosing a blastocoele.

Gastrula; showing the invagination of cells at one pole in the beginning formation of the germ layers and of the digestive tract.

Bipinnaria; early larval stage. Brachiolaria ; later larval stage show¬

ing the formation of the arms. Young starfish immediately after

metamorphosis.

Schools situated near to the sea coast may prepare cultures of eggs in the laboratory and watch the development through to the brachiolarial stage. It will be necessary to dredge in order to obtain the young starfish since they will not metamorphose in the laboratory.

In preparing a culture of eggs in order to study the various developmental stages, eggs should be taken from the ovaries of a ripe female and be placed in a culture dish containing an inch and a half of salt water. After forty-five min¬ utes the water would be changed to fresh salt water, and a drop or two of solu¬ tion from the testis of a ripe male should be added. Fertilization takes place soon afterwards, and in an hour or an hour and a half cleavage begins and proceeds rapidly. The blastulae will be formed within twelve hours, and the gastrulae may be seen swimming around the dish within eighteen or twenty hours. Bipin¬ naria will be formed within three or four days. Brachiolaria may be found after five or six weeks. The water should be changed on the culture each day.

Ascaris For a detailed study of the egg, fertili¬

zation, nuclear processes following ferti¬ lization, and the final division of the cell (mitosis), the eggs of Ascaris megalo- cephala are excellent. As these eggs are relatively small and opaque, little can be observed from the study of whole mounts. Slides showing sections through the uterus, where the eggs are fertilized and pass through the early stages of their development, should be used.

For a study of cell division, (mitosis in animal cells) nothing is better than well prepared sections of the whitefish egg. These are even clearer than Ascaris and are unexcelled for use by high school students. (Refer to slide E13.78 listed on page 4.)

Wild Fruit Fly—Drosophila The culturing and observation of the

development of the fruit fly may be car¬ ried on very easily in the laboratory. Fruit flies can be obtained around places

TURTtpROfUCTS

TURTOX Service Department Copyright, 1959, by

GENERAL BIOLOGICAL SUPPLY HOUSE (INCORPORATED)

8200 South Hoyne Avenue

Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

where over-ripe fruit is found. The sexes can be distinguished in that the male has a small, pointed and black-tipped ab¬ domen. The abdomen of the female is slightly broader, and is finely striped.

Several pairs of flies should be placed in a milk bottle containing part of a ba¬ nana. The mouth of the bottle can he plugged with cotton. Within a short time small white eggs will be seen around the sides of the bottle. With careful observa¬ tion one may see the females depositing eggs. The egg is so small that the cleav¬ ages cannot be observed, moreover, early cleavage often takes place before the egg is laid. After several days small white larvae will be seen crawling to the bot¬ tom of the bottle and feeding upon the banana. In a good culture, hundreds of larvae may be seen making their way through the fruit. As they feed, they grow rapidly and several days later full- grown larvae will crawl up the wralls of the bottle. These become more sluggish and turn a crisp brown in color. Soon they stop moving and go into the quies¬ cent pupal stage. The young emerge after twelve days. As they break through the cocoon they are of a dusky appear¬ ance and not so active, but after several hours they take on the characteristic color and actively fly around their container.

(Living cultures of wild fruit flies, as well as other strains of Drosophila may be secured at any season from Turtox at $3.50 per culture if you are not able to collect the flies locally.)

Frog The complete development of the frog

can be studied in the laboratory. If pos¬ sible it is well to let the class make a field trip some morning in early spring when the frogs are laying eggs. The eggs are laid in the early morning, but copu¬ lating pairs of frogs may be found if one is on the lookout for them. Eggs will be found in clusters lying near the surface of the water. Several “batches” should be collected in pails and taken to the laboratory. Each student should be given some eggs in order that he may follow through the development.

For culturing eggs in the laboratory, they may be placed in finger bowls half filled with spring water (or tap water which has stood overnight). Each day most of the water should be poured off and fresh water added. If one wishes to carry the development through to meta¬ morphosis, some eggs should be placed in a balanced aquarium. Three or four days after hatching tadpoles begin to feed upon algae, decaying animal tissues, or almost any type of meat.

A study of the undivided egg will show that it is enclosed in three jelly membranes. These membranes protect

the egg from dirt, bacteria and various insects which would otherwise prey upon them. The egg itself is divided into two regions: the light colored vegetal pole and the dark animal pole.

Cleavage takes place within several hours after fertilization, and should be studied carefully. Special note should be made of the planes of division. The first cleavage divides the egg into two sym¬ metrical halves, while the second cleavage forms four equal cells. Third cleavage is at right angles to the first two and is a little above the equator of the egg.

After a number of later divisions, the egg comes to consist of a layer of cells enclosing the cavity, or blastocoele. This stage is known as the blastula stage.

By the more rapid division of the cells at the animal pole the blastula becomes converted into a gastrula (or yolk plug) stage. With the formation of the gastrula, the germ layers are differentiated.

Soon after the closure of the blasto¬ pore a groove will be noticed extending over one surface of the developing egg. This is the beginning of the neural tube. Later the groove becomes more pro¬ nounced and the sides approach one an¬ other until they meet, forming an en¬ closed tube. The embryo elongates and soon becomes so differentiated that marked distinctions will designate the anterior and posterior regions. Gill slits develop, the embryo leaves the jelly and swims to the surface of the water. The external gills may best be observed in a 6 or 7 mm. tadpole.

The appearance of the legs, the growth of the tadpole and the absorption of the tail are processes which the student will thoroughly enjoy watching. If possible, carry some tadpoles through to complete metamorphosis.

Chick A form most interesting to watch in

its development is the chick. However, a bit of technique is required since the eggs must be incubated and because they are enclosed in a shell.

In studying the egg, before and during incubation, crack the shell and allow the contents to flow into a finger bowl one- half filled with saline solution. If the egg has been incubated the temperature of the solution must be 103° Fahrenheit.

For incubation, an incubator heated to 103° may be used, but if the incubator is not accessible, a sitting hen may be used as a substitute. A nest can be made in some quiet corner of the laboratory.

The following stages should be studied: Unincubated egg—membranes protect¬

ing the egg, food content, blastodisc well established (earlier develop¬ ment takes place within the oviduct of the hen.)

(43-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

FROG DEVELOPMENT A. Single cell; B. Two-celled stage; C. Four-celled stage; D. Eight-celled stage;

E. Sixteen-celled stage; F. Thirty-two-celled stage; G. Many cells; H. Blastula in section; I. Beginning blastopore; J. Yolk plug; K. Gastrula in section; L. Neural folds; M. Early embryo; N. Embryo in long, sec; O. Embryo in cross sec; P. Nine- millimeter tadpole.

A large series of Turtox Key Cards, Quiz Sheets and Charts dealing with embryology is available. Write for your free copy of the Turtox Three-Way Check¬ list of charts and biological drawings.

Incubated eggs of: 13 hours—primitive streak. 18 hours—primitive streak and head

process. 24 hours—several somites have been

formed and the nervous system is well on its way.

33 hours—heart may be seen, but circulation has not begun.

48 hours—circulation well established. 72 to 96 hours—-most organs are well

established. If it is impossible to examine the

later development day by day, at least three or four stages between the five- day and the twenty-first-day chick should be studied in order that the student may observe the diminishing of the yolk supply, the growth of the embryo, and its position within the shell.

(Refer also to Turtox Service Leaflet

No. 17, “Incubation, Fixation and Mounting of Chick Embryos”).

The development of numerous other forms may be studied either as substi¬ tutes for the above or to supplement the course. Some suggestions are: sea-urchin, crepidula, honey bee, crayfish, crab and fish. In order to get some idea as to the development of mammals, embryos of rabbit, rat, pig, cat and human are excel¬ lent. A comparison of the embryos of several of these forms is most interesting.

A number of useful texts and refer¬ ence books dealing with Embryology are suggested in Turtox Service Leaflet No. 14.

Some reference charts on this subject should be available; Turtox publishes many charts and Key Cards on em¬ bryology and these are all listed in the free Turtox Three-Way Checklist.

Turtox maintains a stock of practically all materials used in embryology courses. This includes preserved specimens, living material, lantern slides, micro¬ scope slides, models and demonstration preparations. A few of the most commonly used items are listed on the following page.

(43-3)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

PRESERVED MATERIAL FOR EMBRYOLOGY

4C4372 Asterias. Assorted Develop¬ mental Stages, front single cell through gastrula. Recommended for hurried survey by beginning classes and for quiz purposes. Per vial of mixed stages, enough for ten stu¬ dents $3.00

4C438 Asterias. Bipinnaria, larval stage. Per vial of one dozen. . 3.00

4C439 Asterias. Brachiolaria, late lar¬ val stage. Per vial of one dozen 3.00

4C4392 Asterias. Minute specimens immediately following metamorpho¬ sis. For whole mounts. Each 35 Dozen 3.50

13C451 Rana pipiens. Grass frog. Seg- mentation stages. A. 1-cell stage. Dozen . .90 B. 2-cell stage. Dozen . .90 C. 4-cell stage. Dozen . .90 D. 8-ccll stage. Dozen . .90 E. 16-cell stage. Dozen . .90 F. 32-cell stage. Dozen . .90 G. Early blastula. Dozen... . .90 H. Blastula. Dozen . .90

13C452 Rana pipiens. Gastrula stages. A. Crescent-shaped blastopore.

Dozen 90 B. Early yolk plug. Dozen. . . .90 C. Late yolk plug. Dozen 90 D. Intermediate stage between late

yolk plug and early neural plate. Dozen 90

13C453 Rana pipiens. Early embryo stages. A. Neural plate. Dozen 90 B. Neural groove. Dozen 90 C. Intermediate stage between neu¬

ral groove and neural tube. Dozen . .90

D. Neural tube. Dozen . .90

13C454 Rana pipiens. Early larval stages. A. Hatching stage. Dozen.. . .90 B. Gillridge. Dozen . .90 C. External gills. Dozen... , . .90 D. Early operculum. Dozen. . .90 E. Late operculum. Dozen.. . .90

3C455 Rana pipiens. Late larval stages. Tadpoles without legs. A. 10 to 15 mm. in length.

Dozen 90 B. 16 to 25 mm. in length.

Dozen .90

13C456 Rana pipiens. Two-legged tadpoles.

Each $0.20 Dozen 1.00

13C457 Rana pipiens. Four-legged tadpoles with long tails. Each 25 Dozen 1.25

13C458 Rana pipiens. Four-legged tadpoles with short tail. Each .35 Dozen : . . . . 1.50

13C700 Chick blastoderms. Especially fixed and flattened for mounting. Stages from twelve hours up to one hundred hours after incubation. Please specify stage wanted when ordering. Per dozen of a single stage 17.50

13C79 Chick embryos. Older ages. We can supply chicks of various days’ incubation for the entire 21-day period. Per single specimen 1.75

13C799 Chick embryos. Intermediate stages can often be supplied. As¬ sorted stages not accurately graded. Dozen 3.50

MICROSCOPE SLIDES FOR EMBRYOLOGY

Set EHS—Beginning Embryology Set Nine slides as listed below. Set $14.20

E4.13 Asterias mature eggs, w.m. .85 E4.21 Asterias polar body formation

(maturation complete before en¬ trance of spermatozoa), w.m. 1.00

E4.31 Asterias, early and late cleavage (radial). By use of high power it is possible to see cells in mitosis, w.m 1.00

E4.41 Asterias, blastula (coeloblastic). Stained to show the individual cells. w.m 1.00

E4.42 Asterias, gastrula (embolic). Stained to show the individual cells, w.m 1.00

E4.61 Asterias, bipinnaria (free-swim¬ ming larval form) w.m 1.10

E6.24 Ascaris, early cleavage, spindles in all stages of mitosis. An excellent slide to demonstrate animal mito¬ sis 2.25

E13.78 Mitosis in egg of whitefish. Every stage of mitosis is shown clearly and in abundance on each slide 2.00

E14.61 Frog, ten embryological stages, from one cell to young tadpole. Mounted entire in a depression slide. All stages on each slide.... 4.50

E16.45 Chick, 33-hour (11-14 somites). Head fold covers extreme tip of head. Head begins to turn left. First indication of cars, w.m.... . . . 2.25

All prices are f.o.b. our laboratories and are subject to change without notice.

(43-4)

TURTOX SERVICE LEAFLET No. 14

A SELECTED LIST OF BOOKS FOR THE BIOLOGY LIBRARY

We offer this brief list of books as suggestions only, admitting freely that many excellent texts have been omitted. A wider selection can be found in any good bookstore or in the catalogs of book publishers, Reference should also be made to price lists of pamphlets and books published by the United States Govern¬ ment Printing Office.

Note: General Biological Supply House does not sell books, except for the special booklets we publish. Orders for other books should be placed with the publisher or your local bookstore.

(Numbers refer Biology list on page 4.

Author Title Publisher Adell & Welton Lab. Course in Biology with Tests 13. Bacq & Alexander Fundamentals of Radio-Biology 1. Baker & Mills Dynamic Biology Today 26. Boyd Autoradiography in Biology & Med 1. Bridges & Brehme Mutants of Drosophila Melanogaster 7. Celeste Biology for Catholic High Schools 2. Colin Elements of Genetics 24. Comar Radioisotopes in Biology & Agriculture 24. Demerec & Kaufman Drosophila Guide 7. Dodge-Smallwood-et al Elements of Biology 2. Fitzpatrick & Bain Living Things 16. Goldstein Genetics is Easy 19. Goldstein How to Do an Experiment 14. Harvey Bioluminescence 1. Haupt Fundamentals of Biology 24. Heilbrun Dynamics of Living Protoplasm 1. Hunter Problems in Biology 3. Hunter & Hunter Biology in Our Lives 3. Kamen Isotopic Tracers in Biology i 1. Marsland Principles of Modern Biology 16. Needham Guide to Study of Fresh-Water Biology 9. Pauli The World of Life 17. Ritchie Biology & Human Affairs 37. Sinnott-Dunn-Dobzhansky.. Principles of Genetics 24 Spector (Ed.) Handbook of Biological Data 28 U.S. Atomic Energy Comm..,Lab. Exp. with Radioisotopes (booklet) 32 U.S. Atomic Energy Comm.. Radioisotopes—Uses, Hazards & Controls.... 32 U.S. Atomic Energy Comm.. .Isotopes in Agricultural Studies (pamph.) . . .30 Vance-Barker-Miller Biology Activities 21 Yance-Miller .Biology for You 21 Wichterman .The Biology of Paramecium 24 Williams .The Living World 23 Woodruff & Baitsell Foundations of Biology 23

to )

Price ..$2.76 .. 6.50 .. 5.20 .. 8.80 .. 1.50 .. 4.80 .. 6.50 .. 9.50 .. .25 .. 5.25 .. 4.12 .. 4.00 .. 2.60 .. 13.00 .. 5.50 .. 6.50 .. 3.60 .. 4.48 .. 9.50 .. 6.75 .. 1.00 .. 6.75 .. 4.48

... 6.75 .. 7.50 .. .30

1.72 4.80 9.50 6.25 6.75

Botany Betsey Morphology & Taxonomy of Fungi Cobb A Field Guide to the Ferns Conrad How to Know the Mosses & Liverworts. Durand .Field Book of Common Ferns Emerson Basic Botany Emerson & Shields Laboratory & Field Exercises in Botany Gray Manual of Botany Harlow Trees of the E. States & Canada Haupt Plant Morphology Henrici Molds, Yeasts, & Actinomycètes Hill-Overholtz-Popp Botany (for Colleges)

24. 17. 5.

25. 24. .24.

3. .24. .24. 35. ,24.

9.50 3.95 2.50 3.50 6.00 2.75

12.50

8.00 7.50 7.50

ftnq-

TURTOX^ROiUCTS

TURTOX Service Department

Copyright, 1958, by GENERAL BIOLOGICAL SUPPLY HOUSE

( IN CORPORATED)

8200 South Hoyne Avenue Chicago 20, Illinois

THE SIGN OF THE TURTOX PLEDGES ABSOLUTE SATISFACTION

Printed in U.S.A.

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Hough J aquès Mathews Medsger Muenscher Muenscher Pohl Platf Pool Prescott Smith Smith Thom & Raper.... Thomas Wain & Wightman

Wherry

Handbook of Trees, N.E. States 23...$8.75 Plant Families—How to Know Them 5... 2.00 Field Book of American Wild Flowers 25... 5.00

• Edible Wild Plants 23... 5.95 • Aquatic Plants of the United States 9... 5.00 • Poisonous Plants of the United States 23... 4.95 How to Know the Grasses 5... 2.25 • American Trees 10... 3.50 •Basic Course in Botany 13... 5.75 • How to Know the Fresh-Water Algae 5... 2.25

■Fresh-Water Algae of the United States... .24... 12.50 •Mushroom Hunter’s Field Guide 34... 4.95 •Manual of the Aspergilli 36... 7.00 •Field Book of Common Mushrooms 25... 5.00 •Chemistry & Mode of Action of Plant

Growth Substances 1... 9.50 .Wild Flower Guide N.E. & Mid. U.S 11... 3.95

Zoology Baker & Wharton An Introduction to Acarology 23... 10.00 Breneman Animal Form & Function 13... 6.25 Buchsbaum Animals without Backbones 33... 8.00 Demerec Biology of Drosophila 35... 13.00 Ditmars Snakes of the World 23... 5.95 Eddy How to Know Fresh-Water Fishes 5... 2.75 Elliott Zoology 4... 7.00 Hegner & Stiles College Zoology 23... 6.90 Holmes The Biology of the Frog 23... 4.90 Hutchinson A Treatise on Limnology 35... 19.50 Hyman Invertebrates; Protozoa through Ctenophora. .24... 11.00 Hyman Lab. Manual for Elementary Zoology 33... 2.75 Jahn How to Know the Protozoa 5... 2.50 Kudo Protozoology 31...10.75 MacGinitie & MacGinitie.. .Natural History of Marine Animals 24... 8.50 Moment General Zoology 17... 7.50 Nichols North American Fresh-Water Fishes 23... 1.75 Pratt Manual of Common Invertebrate Animals... .24... 9.50 Romer .Vertebrate Paleontology 33... 8.50 Stiles Lab. Explorations in General Zoology 23... 3.75 Turtox Ascaris Megalocephala 12... .75 Welch Limnology 24... 9.50 Woodruff .Animal Biology 23... 4.75 Wright Handbook of Frogs & Toads 9... 6.50

Baumgartner Bigelow. Calm Eddy-Oliver-Turner.

Horsburgh & Heath Hyman Kendall Romer Sayles Shumway Starks & Cutter....

Chu Comstock Fenton Fernald & Shepard Holland Jaques Jaques Klots Lutz Matheson

Comparative Anatomy ■Lab. Manual of the Foetal Pig 23... .Directions for Dissection of the Cat 23. ..

• The Spiny Dogfish 23... • Guide to Anatomy Study of Shark,

Necturus, & Cat 35. .. .Atlas of Cat Anatomy 29... .Comparative Vertebrate Anatomy 33... .Microscopic Anatomy of Vertebrates 20... .The Vertebrate Body 28... .Manual for Comparative Anatomy 23... .The Frog (Lab. Guide) 23... .Dissection of the Rat 29...

Entomology

.How to Know the Immature Insects 5... .An Introduction to Entomology 9... .Field Crop Insects 23... .Applied Entomology 24... .The Butterfly Book 11... , .How to Know the Beetles 5... .How to Know the Insects 5... .A Field Guide to the Butterflies 17...

. .Fieldbook of Insects 25... .Entomology for Introductory Courses 9...

2.50 2.55 2.50

2.90 1.85 5.00 6.00 7.00 4.15 2.75

.75

2.50 7.50 6.75 7.50

12.50 3.50 2.00 3.95 3.49 6.00

(14-2)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Metcalf & Flint.. Peairs Ross Swain West & Campbell

Destructive & Useful Insects 24..$12.75 Insect Pests of Farm, Garden, & Orchard... .85. .. 8.50 A Textbook of Entomology 35... 7.75 The Insect Guide 11... 3.95 DDT 8. . . 8.50

Arey Huettner

Lillie Lillie & Moore Nelsen Patten Patten Patten Rugh Weiss

Barton..... Cruickshank Peterson Peterson.... Peterson Pough

Embryology Developmental Anatomy 28... 9.50 Fundamentals of Comp. Embry, of

the Vertebrates 23... 6.00 • The Development of the Chick 16... 8.50 Lab. Outline of Embryol. (Chick & Pig) ... .33... 1.25 .Comparative Embryology of the Verteb 24... 9.00 .Early Embryology of the Chick 24... 4.25 .Embryology of the Pig 24... 5.00 .Human Embryology 24... 12.00 .The Frog (Reproduction & Development).. .24... 5.00 Principles of Development 16... 7.50

Ornithology How to Watch Birds 24... 3.50 Pocket Guide to the Birds 10... 2.95 .Birds over America 10... 6.00 A Field Guide to the Birds 17... 3.95 .Field Guide to Western Birds 17... 3.95 .Audubon Bird Guide 11... 3.95

Booth Burt & Grossenheider Carr Cockrum Comstock Gertsch Hausman Hillcourt Kaston Miner Morgan Palmer Palmer Schmidt & Davis Smith Stebbins Verrill Wright & Wright....

Nature Study How to Know the Mammals 5... A Field Guide to the Mammals 17. .. .Handbook of Turtles 9... .Laboratory Manual of Mammalogy 6... .Handbook of Nature Study 9... •The Spider Book 9... .Beginners’ Guide to Attracting Birds 25... Field Book of Nature Activities 25...

, How to Know the Spiders 5... Field Book of Seashore Life 25... .Field Book of Ponds & Streams 25... .Field Book of Natural History 24... ,The Mammal Guide 11... Field Book of Snakes (U.S. & Canada) 25... .Plandbook of Lizards (U.S. & Canada) 9... .Amphibians & Reptiles of W. North America 24... Shell Collector’s Handbook 25... .Handbook of Snakes (2 Vol.) 9...

2.50 3.75 7.50 4.00 6.75 6.00 2.50 3.95 2.50 7.00 5.00 8.50 4.50 4.50 6.00 8.50 4.00

14.75

. .28.

. .36.

..23.

. .28.

Bacteriology—Parasitology Braun Bacterial Genetics Breed-et al Bergeys’ Manual of Determ. Bacteriology. Buchanan Bacteriology Burrows Textbook of Microbiology Dubos The Bacterial Cell 15. Irving & Herrick Antibiotics 8. Kelly & Hite Microbiology 4. Levine Introd. to Lab. Tech, in Bacteriology 23. Prescott & Dunn .Industrial Microbiology 24. Rhodes & Van Rooyen Textbook of Virology 36. Sawitz Medical Parasitology 24. Smith & Conant Textbook of Bacteriology (Zinssers’) 4. Stitt-Clough-Branham Pract. Bact., Hematol., & Parasit 24. Turtox Bacteriology Booklet 12. Waksman Soil Microbiology 35.

. 6.50

.15.00 . 6.50 .11.00 . 6.00 . 6.75 . 7.50 . 4.50 . 12.50 . 8.00 . 6.00 . 12.00 . 10.00 . 1.00 . 7.50

Aquaria & Terraria Innés Exotic Aquarium Fishes 18... Innés Goldfish Varieties & Water Gardens 18... Mann .Tropical Fish 12...

9.75 5.50

.75

(14-3)

GENERAL BIOLOGICAL SUPPLY HOUSE, CHICAGO, ILLINOIS

Mellen Wonder World of Fishes Mellen & Lanier 1001 Questions Answered About Your

Aquarium

Microscope Slide Technique—Histology Conn Biological Stains Gray Handbook of Basic Microtechnique... Guyer Animal Micrology Ham Histology Maximow & Bloom Histology Stiles Handbook of Micro. Char, of Tissues

& Organs

Miscellaneous

10..

10..

36. . 24.. 33.. 21. .

28..

24. .

Bear Chemistry of the .Soil 27.. DeVries German-English Science Dictionary 24.. Ellis & Swaney Soilless Growth of Plants 27.. Hall Introduction to Electron Microscopy 24.. Hawk-Oser-Summerson .Practical Physiological Chemistry 24.. Jaeger Source Book of Biological Names & Terms. . .31. . Johnson & Bleifeld Hunting with the Microscope 12.. MacLeod & Taylor Rose’s Foundation of Nutrition 23.. Pincus The Hormones (Vol. 3) 1.. Pray Taxidermy 23.. Russell Soil Conditions & Plant Growth 22.. Saunders American Pocket Medical Dictionary 28.. Sharp Fundamentals of Cytology 24.. Shillaber Photomicrography 35. . Thimann-Scharrer-et al. ... Action of Hormones in Plants & Invertebrates 1.. Tiequet Successful Gardening Without Soil 8.. Turtox Laboratory Experiments in Nutrition 12.. Turtox Living Specimens in the School Laboratory. .12.. Turtox Microscopy Booklet 12.. Turtox (Wells) The Collection & Preservation of Animal

Forms 12.. U.S. Atomic Energy Comm...Isotopes in Industry & in Physical

& Chemical Research (pamphlet) 30.. U.S. Atomic Energy Comm.. Principle of Isotope Utilization (pamph.)... .30.. Williams .Introduction to Chromatography 8..

No, 1. 2.

3. 4. 5. 6.

7. 8.

9. 10.

11.

12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

22.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

ADDRESSES OF PUBLISHERS Academic Press, 111 Fifth Ave., New York 3, N-. Y. Allyn & Bacon, Inc., 150 Tremont St., Boston, Mass. American Book Co., 55 Fifth Ave., New York 3, N. Y. Appieton-Century-Crofts, Inc., 35 West 32nd St., New Yorkl, N. Y. Wm. C. Brown Co., 135 S. Locust, Dubuque, Iowa Burgess Publishing Co., Minneapolis, Minn. Carnegie Institution of Washington, 1530 P St., N. W., Washington 5, D.C. Chemical Publishing Co., 212 Fifth Ave., New York 10. N. Y. Comstock Publishing Co., 124 Roberts Place, Ithaca, N. Y. Dodd, Mead & Co., 432 Fourth Ave., New York 16, N. Y. Doubleday & Co., 575 Madison Ave., New York 22, N. Y. General Biological Supply House, Inc., 8200 S. Hoyne Ave., Chicago 20, 111. Gmn & Co., 205 W. Wacker Drive, Chicago 6, 111. Harcourt, Brace & Co., 750 Third Ave., New York 17 N Y Harvard University Press, 79 Garden St., Cambridge 38, Mass. Henry Holt & Co., 383 Madison Ave., New York 17 N Y Houghton Mifflin Co., 2 Park St.. Boston 7, Mass Innés Publishing Co., 210-12 N. 13th St., Philadelphia 7, Pa. Lantern Press, 257 Fourth Ave., New York 10, N Y Lea & Febiger, 600 Washington Square, Philadelphia 6 Pa J. B. Lippincott Co., 333 W. Lake St., Chicago 6 111. Longmans, Green & Co., Inc., 55 Fifth Ave., New York 3 N Y The Macmillan Co., 60 Fifth Ave., New York 11 N Y ’ McGraw-Hill Book Co., 330 W. 42nd St., New York 36 N. Y. G. P. Putnam's Sons, 210 Madison Ave., New York 16' N Y Rand McNally & Co., 124 W. Monroe St., Chicago 3 111 Reinhold Book Division, 430 Park Ave., New York 22, N Y W. B. Saunders Co., West Washington Square Philadelphia 6 Va Stanford University Press, Stanford. California P ’ ' Superintendent of Documents, U.S. Government Printing Office Wash 25 r> i ft ft. Thomas Publisher, 301 E. Lawrence Ave., Springfield 111 ’ United States Atomic Energy Commission. Washington 25 ’ D C University of Chicago Press, 5750 Ellie Ave., Chicafo 37 111 C‘ University of Michigan Press, Ann Arbor, Mich.

wu? ^ i ■ pno, 440 Fourth Ave., New York 16 N Y Williams & Wilkins Co., Baltimore 2, Md. iN' X' World Book Co., 2126 Prairie Ave., Chicago 16, 111.

(14-4)

.$3.00

. 3,75

. 5.00

. 6.00

. 5.50

.11.00

.11.00

. 3.00

. 8.75

. 6.50

. 6.50

. 9.50

. 12.00

. 5.75

. .95

. 6.00

.22.00 . 1.95 .10.00 . 3.25 . 6.00

. 7.00

. 3.50

. 1.00

. 1.00

. 1.00

. 1.00

. 4.00

CHAPTER III

SUMMARY AND CONCLUSIONS

Recapitulation of Experimental»—Experimental evidence indicates that

if there is abundant present-day life activity in the classroom, the child

is more likely to develop a lively interest in the subject field* Hence,

it becomes apparent that the development of appropriate and adequate tech¬

niques in the learning process is essential. Since one of the chief alms

of science instruction is to develop in students habits of thought and

action inherent in the methods and attitudes of science, there is need

in science courses for classroom situations which will be conducive to the

development of such habits. Perhaps the best method now known for se¬

curing such an aim is through the medium of the laboratory exercise where

problem-solving may and should be the main objective.

Principles and associated regeneration phenomena have been woven

into a resource unit for the purpose of providing for the high school

biology teacher a lively prospectus. Using the Question-Experiment approach,

experiences have been suggested for stressing several of the key ideas in

biology, while at the same time providing a stimulus for fascinating the

students and challenging their imaginations and thinking. More specifically,

questions on various aspects of regeneration have been raised; appropriate

animals mentioned as experimental material, and experiments proposed to

provide answers to the questions. Terminating the experimental activities

is a suggestive evaluation procedure* Such a procedure involves watching

$9

60

(by the teacher) the operation skills developed by the students, their

ability to observe and accurately record data, and their desire to propose

new experimental situations for verifying other biological principles.

Based on the experiences of his own classroom situation, the teacher is to

devise a factual examination to test whether the students have actually

grasped what was intended from the experiments.

Summary of Literature,—Numerous studies have been reported on animal

regeneration. Since 17U0, when the phenomenon was described in animals,

experimental morphologists have explored nearly all facets of the problem.

It is now generally agreed that:

1, Regeneration in animals is not limited to any particular group.

Some degree of repair or restoration of damaged or lost parts occurs in

all groups, from the simple protozoa to the complex mammals. There are

among both "lower" and "higher” animal groups excellent regenerators and

poor regenerators,

2, In the restoration of cellular elements in animals having lost a

portion of their body, the source of the new cells varies among different

groups. In many of the "lower" forms the cells arise from the division of

reserve cells which were already present in an undifferentiated state.

Among vertebrates ("higher" forms) most of the cells involved in restoring

the lost part result from the de-differentiation of cells originally

present in the area near the cut surface,

3, Both the quantity and quality of regeneration can be altered sig¬

nificantly by drastic changes in the environment: changes in temperature,

food supply, acidity of alkalinity of the fluid, and other environmental

factors.

61

it. The regenerated poifeion in nearly all instances re-acquired the

precise organization characteristic of the original. However, in some

cases (for example, in planarians under certain operable conditions) two

heads or two tails may form due to the presence of specific gradients of

formative materials in the animal. This means that the regenerate will be

morphologically unlike the original,

£, The stimulus for regeneration seems to be associated with the

wounding process. When the wound fails to heal in a prescribed manner, re¬

generation does not occur. Appropriate treatment of the animal to insure

normal wound healing, releases the block to regeneration. Hence, it is

now possible for adult animals that have lost their regenerative abilities

to be re-initiated in a capacity for some restoration to occur.

Resume of Findings,—The following findings are reported:

1, Regeneration mak^ specific and definite contributions to the fol¬

lowing major principles of Biology which science education agrees are

important in science in general education,

a. Growth and repair are fundamental activities for all protoplasm,

b. From the lower to the higher forms of life, there is an in¬

creasing complexity of structure, and this is accompanied

by a progressive increase in division of labor. In all

organisms, the higher the organization the greater degree of

differentiation and division of labor and of the dependency

of one part upon another*

c. Growth and development in organisms is essentially a cellular

phenomenon, a direct result of mitotic cell division. Cells

are organized into tissues, tissues into organs, and organs

62

into systems, the better to carry on the functibns of complex

organisms*

d. All cells arise through the division of previous cells* Cell

division is the essential mechanise of reproduction, of

heridity, and to a large extent, of organic evolution,

e. The environment acts upon living things, and living things

act upon their environment* Since the environment of living

things changes continually, these creatures are continually

engaged in a struggle with their environment,

f. Adult organisms that differ greatly from one another but which

show fundamental similarities in embryological development,

have originated from similar ancestors* Animals resemble each

other more and more closely the farther back we pursue them

in embryological development,

2, Regeneration experiments and demonstrations are possible in the

teaching of Biology at the Secondary School level in the following areas:

a* Protozoa

b* Coelenterates

c, FLatworms

d* Annelids

e* Arthropods (Crustaceans)

f* Echinoderms

g* Bony Fishes

h* Amphibians

i. Mammals

3* Curriculum materials are available in the literature for use by

63

the High School Teacher of Biology which will provide adequate background

information for the teaching in these areas*

U* Ample diversified materials are available from which it is possible

for an alert teacher to construct other Resource Units in this area.

Conclusions .—The following conclusions are offered from this study:

1. Certain organisms are inherently capable of regenerating the

entire body from a tiny fragment.

2. Other organisms are capable of only regenerating parts of the body.

3. There is a distinct relation between the stage of development

and regeneration in higher organisas.

U. Even higher organisms, including man, under certain conditions,

will undergo regeneration.

Recommendations.--In view of the tremendous concern about creating

new approaches to teaching courses in biology at the Secondary School

level, the writer makes the following recommendations:

1. Teachers of high school Biology should use this Resource Unit*

2. They should prepare one of their own if they should choose

not to use this one.

3. In another study growing out of this one on animal regeneration,

experiments on grafting as a regenerative process should be

conducted.

BIBLIOGRAPHY

Books

Barth, L* G, Embryology» Revised Edition, New York! Henry Holt and Company, 1953*

Bonner, J, T, The Ideas of Biology, New York: Harper and Brothers, 1962,

Hickman, C, P, Integrated Principles of Zoology, St, Louis, Missouri! C, V. Moshy Company, 1956,

Milne, L, J,, and Milne, M, J, The Biotic World and Man, 2nd edition,

Englewood Cliffs, New Jersey! Prentice-Hall, Inc,, 1958*

Waddington, C, H, Principles of Embryology, London! George Allen and

Unwin, 1957.

Weiss, P, Principles of Development, New York! Henry Holt and Company,

1939*

Weisz, P, B. The Science of Biology. New York! McGraw-Hill Book Company,

1959.

Report

Science in General Education, Report of the Committee on the Function of

Science in General Education, New York! D, Appleton-Century-Crofts

Company, 1938.

Articles

Burnett, R, W. «Vitalizing the Laboratory to Encourage Reflective

Thinking," Science Education, XXIII (March, 1939), 299-30U.

Glass, B. "Perspectives! A New High School Biology Program," American

Scientist. XLIX (December, 1961), 52U-531.

Mason, J, M., and Warrington, W, G, “An Experiment in Using Current

Scientific Articles in Classroom Teaching," Science Education, XXXIX (October, 195U), 299-30U,

McKibben, M. J, "An Analysis of Principles and Activities of Importance

for General Biology Courses in High Schoolspn Science Education,

XXXIX (April, 1955), 187-196.

65

Obourn, E. S., Darnell, E. H., Davis, G., and Weaver, E. K. “Fifth Annual Review of Research in Science Teaching,11 Science Education, XLI (Deceniber, 1957), 375-l|ll.

Simmons, M* P. "A Model Lesson in General Science,” Science Education, (March, 1939), 133-136.

Unpublished Materials

Alberty, Harold. "How to Make a Resource Unit." Unpublished Bulletin, College of Education, The Ohio State University, 19UU.

Weaver, Edward K, “How to Make a Resource Unit." Unpublished Compilation, The State Teachers College at Montgomery, Alabama, 19U6 Summer Session.