GROWTH REQUIREMENTS OF CLOSTRIDIUM TETANIl

J. HOWARD MUELLER AND PAULINE A. MILLERDepartment of Bacteriology and Immunology, Harvard Medical School and School of

Public Health, Boston, Mass.

Received for publication September 29, 1941

Information as to the chemical nature of substances concernedin the nutrition of pathogenic bacteria has been rapidly ac-cumulating in the past five years. Aerobic types, particularly,have been studied, and while there remains much to be done thegeneral scheme of affairs has been pretty well laid out. In gen-eral, the strictly anaerobic organisms have received less attention.Except for the publications of Fildes and his colleagues (Fildesand Knight, 1933) and of Pappenheimer (1935) on the "sporogenesvitamin," very little experimental work has appeared.The increasing use of tetanus toxoid in prophylactic immuniza-

tion brings this organism especially into the foreground. Suchfundamental advances have been possible in the production ofdiphtheria toxin as a result of nutritional studies (Mueller andMiller, 1941) that one may venture to hope for a similar resultin the case of tetanus. The routine production of this anaerobictoxin has until now been attended by the same difficulties as weremet in the case of diphtheria toxin. Moreover, the longerintervals recommended in the use of tetanus toxoid, and thenecessity for "recall" doses, may be expected to result in reactionsof anaphylactic type in certain individuals when toxoid preparedon peptone, particularly Witte's peptone, is employed. If itwere possible to grow the tetanus organism on a medium con-taining only chemically defined substances of low molecularweight, it should become a relatively straightforward matter to

1 Aided by a grant from the Commonwealth Fund.The writers wish to acknowledge with thanks the courtesies of various members

of the Faculty of Medicine, Dalhousie University, and of the Director of the Pub-lic Health Laboratories, Halifax, in whose laboratories a part of this work wascarried out during the summers of 1940 and 1941.

763

764 J. HOWARD MUELLER AND PAULINE A. MILLER

study and control the factors involved in toxin formation, and toobtain a uniform product free from any possible antigenic materialother than the specific substance desired.

Clostridium tetani appeared to be somewhat more complex inits growth requirements than the anaerobes of gas gangrene infec-tions. Growth of the latter usually occurs readily (underanaerobic conditions) on ordinary meat extract agar. Thetetanus bacillus may fail to multiply on such a medium, but willdo so on blood agar. Multiple requiremients of an "accessory"nature for growth were therefore assumed.

It is quite clear that the greater the number of unknownsinvolved in a problem of this sort, the more considerable are theexperimental difficulties. Proof of involvement of substance Ain growth of an organism requiring factors A, B, C and D canusually be demonstrated only by the use of a medium containingsubstances B, C, and D, and entirely free from A. In other words,it is essential to devise a basic medium containing all but one ofthe chemical substances required for growth. This compound isthen identified and added to the medium in suitable amount,and a second factor is omitted, etc. If one were certain thatall the nutrients for a particular organism were known and ob-tainable, it would become a much simpler matter. This, how-ever, is not yet the case, and with each new organism one mustfeel his way along.

It would be absurd to attempt a full description of the experi-ments which have been carried out in this study, extending nowover a period of two years. They may be summarized by thestatement that the strain which has been used (New York StateDepartment of Laboratories) apparently requires the followingmaterials for growth: an acid hydrolysate of protein (casein),tryptophane, the usual inorganic salts, adenine (or hypoxanthine),pantothenic acid, thiamine, riboflavine, "folic acid" (Snell andPeterson 1940), (Mitchell, Snell and Williams, 1941), probablybiotin, and perhaps one or more additional compounds. Themedium, as at present constituted, has the following compositionper 100 ml.:

GROWTH REQUIREMENTS OF CL. TETANI 765

Casein hydrolysate2 to supply........... .24 gram of N 1.5% caseinTryptophane............................ 5 mgm.Glucose3............................... 500 mgmAdenine............................... 0.5 mgm.Calcium pantothenate................... 0.025 mgm.Thiamine ............................... 0.001 mgm.Riboflavine.............................. 0.001 mgm.Peterson's concentrate (folic acid?)'..... 0.05 mgm.MgSO4.............................. 45 mgm.Whole commercial casein5.100 mgm.

METHODS

Choice of strain

It is probable that variations will be found in nutritional re-quirements among different strains of C. tetani. Cultures ofstrains used in routine toxin production were collected from sev-eral sources. Small quantities of the usual meat infusion brothwere inoculated, and after a week's incubation the resulting toxinswere tested in mice. The strain which gave the most potent prep-aration, coming from the New York State Bureau of Laboratories,was selected for study. This amounts practically to a randomchoice. Other strains will be examined for comparison indue time.

Anaerobic method

Since a prompt and effective method seemed essential, the mat-ter received attention at the beginning of the study, and a slightmodification of the Rosenthal (1937) chromous sulfate methodwas devised (Mueller and Miller, 1941) which has been usedthroughout. Incubation has been for 24 hours at 360.

2 The same hydrolysate used in the preparation of diphtheria toxin-hydrol-ysis by HCl, removal of excess acid by distillation, followed by PbO (Mueller andJohnson, 1941), now available from the Difco Laboratories, Inc. under the tradename "Casamino Acids."

3 Glucose may be replaced by cystine, 40 mgm. The effect of either is prob-'ably due to improvement of anaerobic conditions.

4 Kindly supplied by Professor W. H. Peterson.6 May be omitted if large inocula or prolonged incubation are used. See below.

766 J. HOWARD MUELLER AND PAULINE A. MILLER

Stock culture, inoculum, etc.The stock culture has been carried on tubes of glucose-infusion

broth by daily transfer and incubated anaerobically at 360.This is used directly as an inoculum. At various times a straightwire dipped into the culture from 8 to 4 inch has served. Morerecently, a very small (about 1 mm.) loopful has been used. Itis realized that this offers certain theoretical objections, but ingeneral it would appear that too great a refinement of this partof the technique is undesirable. Provided growth fails in controltubes with regularity, and occurs in the presence of a particularsubstance, the method is perfectly serviceable for a study of thatcompound. When all the compounds which can be studied inthis way have been identified, one may readily investigate whethersome additional factor is being added with the inoculum.The experimental media, pH 7.4 to 7.6, in 10 cc. quantities,

contained in the usual 150 x 15 mm. Pyrex test tubes and coveredwith glass caps, are autoclaved at 10 lbs. steam pressure for 5minutes. The tubes are removed promptly from the autoclave,cooled at once in water and inoculated. The glass caps arereplaced by previously dry sterilized cotton plugs and the tubesare then placed in the anaerobic jar. Dry sterilization of cotton-plugged tubes at 160°C. is attended by the distillation of a smallamount of tarry material from the cotton onto the glass tubewhich is definitely inhibitory to bacterial growth on these sim-plified media. The same fact has been observed in connectionwith diphtheria toxin production. (The tubes used are previ-ously cleaned by standing overnight in strong chromic-sulfuricacid, thoroughly rinsed and dried.)

Recording of resultsSince a uniform turbidity results, with some fine sediment, an

estimation of the relative amount of growth is made by mixingthoroughly with a glass rod after adding a trace of caprylic alcoholto control foaming from dissolved gases. This has been done ina Gates suspensiometer or with a photoelectric nephelometer.Neither is entirely satisfactory, but either one serves as a fairlyaccurate means of comparison. In the protocols presented below

GROWTH REQUIREMENTS OF CL. TETANI

an approximate simplification is introduced, and the results aregiven in per cent; i.e., 100 per cent is maximum growth, compa-rable to that obtained on a glucose meat-infusion peptone broth,and gives a direct reading of 2.2 to 2.4 cm. in the Gates sus-pensiometer.

Casein hydrolysate and tryptophane

It has not appeared worth while at this stage of the work toinvestigate the identity of the individual amino acids involvedwith the exception of tryptophane, which is absent or extremelylow in the hydrolysate. Earlier experiments on amino acids withthe diphtheria bacillus have persuaded us that the labor involvedis not repaid by the information required unless a specific purposeis to be served. Variations from one sfrain to another make thematter mainly of academic interest.The essential nature of tryptophane is shown in the following

experiment. The "base" in this case, as in the further protocols,has the composition shown above, except that the substance underinvestigation is omitted.

per cent growth

1 Base without tryptophane.................................. none2 Base without tryptophane + tryptophane 0.001 mgm. trace3 Base without tryptophane + tryptophane 0.0025 mgm....... 104 Base without tryptophane + tryptophane 0.005 mgm........ 355 Base without tryptophane + tryptophane 0.01 mgm......... 856 Base without tryptophane + tryptophane 0.025 mgm........ 1007 Base without tryptophane + tryptophane 0.05 mgm......... 1008 Base without tryptophane + tryptophane 0.1mgm.1009 Base without tryptophane + tryptophane 0.25 mgm......... 10010 Base without tryptophane + tryptophane 0.5 mgm... 10011 Base without tryptophane + tryptophane 1.0 mgm.......... 100

Adenine or hypoxanthineFrom the following experiment it will be seen that a small

quantity of adenine is necessary to obtain growth. It may bereplaced by hypoxanthine, but not by guanine, xanthine or uricacid. The quantitative relations, when hypoxanthine is sub-stituted for adenine, are identical.

767

768 J. HOWARD MUELLER AND PAULINE A. MILLER

per cent growth1 Base without adenine................................ trace2 Base without adenine + adenine 0.25Ag.................... trace3 Base without adenine + adenine 0.5og..................... 24 Base without adenine + adenine 1.0 gg..................... 55 Base without adenine + adenine 2.5&g..................... 656 Base without adenine + adenine 5.0 g..................... 1007 Base without adenine + adenine 10.0 g.1008 Base without adenine + adenine 25.0 pg.............. r....... 1009 Base without adenine + adenine 50.0 Fg..................... 10010 Base without adenine + guanine 50.0 tg..................... trace11 Base without adenine + xanthine 50.0 pg.................... trace12 Base without adenine + uric acid 50.0 g.trace13 Base without adenine + hypoxanthine 50.0,ug............... 100

Pantothenic aidThe necessity of this substance for tetanus growth is illustrated

below. It is of interest that this accessory became available insynthetic form during the progress of these experiments. Weare indebted to Professor Roger Williams for concentrates usedin the early portion of the work, and to him and to E. Merck &Company for specimens of synthetic material. We have alsoused a specimen of optically inactive pantothenic acid synthesizedin this laboratory by Mr. Everett Johnson.

per cent growth1 Base without pantothenicacid..none2 Basewithoutpantothenicacid+pantothenicacid 0.0lg....0 trace3 Base without pantothenic acid + pantothenic acid 0.025,ug... 54 Base without pantothenic acid + pantothenic acid 0.05 pg.... 155 Base without pantothenic acid + pantothenic acid 0.1pg..... 306 Base without pantothenic acid + pantothenic acid 0.25pg.... 707 Base without pantothenic acid + pantothenic acid 0.5pg..... 1008 Base without pantothenic acid + pantothenic acid 1.OAg. 1009 Base without pantothenic acid + pantothenic acid 2.5pg. 10010 Base without pantothenic acid + pantothenic acid 5pg....... 10011 Base without pantothenic acid + pantothenic acid 10,g....... 10012 Base without pantothenic acid + pantothenic acid 25,ug....... 100

Thiamine and riboflavineIt is difficult at this stage to obtain clear cut experiments

demonstrating the individual effect of these vitamins. This isprobably due to their presence in small amounts in other com-ponents of the medium, and possibly to the fact that, autoclaved

GROWTH REQUIREMENTS OF CL. TETANI

in the medium, some alteration occurs. Either vitamin alone,particularly riboflavine, exerts some stimulation, but in generalthe combination of the two is somewhat more effective. Withgreater refinement in the basic composition of the medium, thequantitative aspects of these substances will receive furtherattention. This experiment was carried out in duplicate to showthe sort of variation which is encountered.

per cent growth1 Base without thiamine and riboflavine .................... 60; 452 Base without thiamine and riboflavine + thiamine 0.1 pg.. 50; 403 Base without thiamine and riboflavine + riboflavine 0.1 g... 60; 704 Base without thiamine and riboflavine + both vitamins 0.1

pg. ea........................................... 100; 100

"Foli acid"The requirement for a factor which appeared to be new was

recognized early in the work. It occurred in liver extract in boththe 90 per cent alcohol precipitate and filtrate. As a source forattempted purification, a large lot of the 90 per cent alcoholfiltrate fraction was generously supplied us by the ConnaughtLaboratories, Toronto. The active material in this fraction wasadsorbed on norit charcoal, and the latter washed with water andeluted with hot 50 per cent alcohol. Very little active materialwas recovered, but considerable inert substance was removed bythis procedure. The charcoal was then eluted with hot 25 percent pyridine in 25 per cent alcohol. A further purification of thisactive fraction was effected, after removal of the solvent, byprecipitation with silver. Inert material separated in acidreaction and was removed, and the desired fraction came downat neutrality or slightly alkaline reaction. This silver precipitate,after freeing from Ag and Ba, served for many small-scale at-tempts at further purification. Practically every procedureresulted in loss of activity, but evidence of the participation of atleast two substances accumulated. One of these suggested by itsproperties the "eluate factor" which had been described by Snelland Peterson (1940) for certain lactic acid bacilli. A specimenof this material, kindly sent us by Professor Peterson, proved tobe highly active in the presence of the other component of the

769

770 J. HOWARD MUELLER AND PAULINE A. MILLER

silver precipitate fraction. The latter was then found to bereplaceable by thiamine and riboflavine as stated above.At this stage, Stokstad (1941) announced experiments indicat-

ing the possible dinucleotide nature of Snell and Peterson'seluate factor. A specimen of this material (from liver) generouslysupplied by Dr. Stokstad proved to have activity almost identicalwith that of Professor Peterson's. However, a second prepara-tion from Dr. Stokstad, obtained from yeast, possessed aboutfive times the activity by weight of the liver "di-nucleotide."The purity and chemical identity of the latter is therefore opento question. The growth-promoting effect is not shown bythiamine or mixtures of the latter with guanine, although Stok-stad presents evidence for partial replacement by these com-pounds in the case of his organism. Very recently Mitchell,Snell and Williams (1941) have announced a further considerablepurification of the "eluate factor" of Snell and Peterson, and havesuggested the name of "folic acid" because of its occurrence inleaves. The absolute proof of a requirement of C. tetani for thisnew substance, and the elucidation of its nature, is not possibleat the moment.The following protocol shows the requirement for the (still

relatively impure) eluate factor.pr cent grwh

1 Base without eluate factor ....................... none2 Base without eluate factor + eluate factor (Peterson) 0.025 /Ag. .faint trace3 Base without eluate factor + eluate factor (Peterson) 0.05 Ag. .faint trace4 Base without eluate factor + eluate factor (Peterson) 0.1 Mg.. 55 Base without eluate factor + eluate factor (Peterson) 0.25 jsg.. 156 Base without eluate factor + eluate factor (Peterson) 0.5 ,ug. . 307 Base without eluate factor + eluate factor (Peterson) 1.0 MAg.. 758 Base without eluate factor + eluate factor (Peterson) 2.5 ,ug.. 1009 Base without eluate factor + eluate factor (Peterson) 5 ,ug.. 10010 Base without eluate factor + eluate factor (Peterson) 10 ,ug. . 100

Biotin and whole caseinThis substance, also not yet available in synthetic form, is

possibly present as an impurity in the casein hydrolysate and theglucose used in the basic medium. It is surely present in thewhole commercial casein used to facilitate growth from smallinocula. Conclusive experiments showing its requirement for

GROWTH REQUIREMENTS OF CL. TETANI

tetanus growth have not yet been obtained but the evidence indi-cates that it is essential.

It appears that the effect of biotin can be nullified by a fractionof raw egg white, presumably protein in nature (Eakin, Snell andWilliams, 1941). This substance named variously "egg-whiteinjury factor," "antibiotin," and "avidin," is heat liable, and hasbeen prepared by the above authors in a considerably purifiedform. The union between biotin and avidin takes place uponmixing, so that a suitable amount of this neutralizing substanceadded to a sterile tube of medium will render the latter unsuitedto the growth of any organism requiring biotin. A specimen ofavidin, generously supplied by Professor Williams, appears toinhibit the growth of the tetanus bacillus. The amount requiredfor inhibition is roughly proportional to the quantity of wholecasein used in the medium when this is varied between 1 and 20mgm. per 10 ml. of medium. Purified biotin (S.M.A. Corpora-tion), however, appears not to replace commercial casein inmaking growth from small inocula possible. There is thusevidence that the casein supplies some factor other than or inaddition to biotin, and the matter is receiving further attention.

Other factorsIn addition to the possible presence of some unidentified

growth factor in the whole commercial casein mentioned above,evidence has been obtained for the presence of some other ma-terial in liver extract which leads to abnormally heavy growth oftetanus. By "abnormal" is meant growth markedly heavierthan that obtained on glucose-infusion-peptone broth. It ishoped that further work will shed some light on these matters.

Toxin productionExperiments in connection with toxin production on media

composed mainly of chemically defineable materials have beencarried out. Thus far most of these experiments have been donewith a medium in which folic acid and the B vitamins have beensupplied by means of a solution of the pyridine-alcohol eluatefraction of liver extract described above. Toxin of approximately60,000 M.L.D. per ml. for guinea pigs can be obtained with

771

772 J. HOWARD MUBLIER AND PAULINE A. MILLER

regularity. A preliminary report of this fact has been made(Mueller & Miller, 1940) and a detailed account of the experimentsis in preparation. Further simplifications of the medium will beinvestigated in relation to toxin formation as promptly as possible.

CONCLUSIONS

1. Growth of a strain of Clo8tridium tetani has been shown torequire, in addition to the usual inorganic elements, the followingsubstances: an acid hydrolysate of protein, tryptophane, adenineor hypoxanthine, pantothenic acid, thiamine, riboflavine, "folicacid," and probably biotin. One or more additional factors maybe essential.

2. A high grade of toxin has been produced on such a medium,in which the folic acid is supplied in a liver extract concentrate.

REFERENCESEAKIN, R. E., SNELL, E. E., AND WILLIAMS, R. J. 1941 The concentration and

assay of avidin, the injury-producing protein in raw egg white. J.Biol. Chem., 140, 535-543.

FILDES, P., AND KNIGHT, B. C. J. G. 1933 A vitamin necessary for the growth ofB. 8porogene8: its relation to auxin and other growth factors. Brit.J. Exptl. Path., 14,112-124.

MITCHELL, H. K., SNELL, E. E., AND WILLIAMS, R. J. 1941 The concentrationof "folic acid." J. Am. Chem. ooc., 68, 2284.

MUELLER, J. H., AND JOHNSON, E. R. 1941 Acid hydrolysates of casein to re-place peptone in the preparation of bacteriological media. J. Immu-nol., 40, 33-38.

MUELLER, J. H., AND MILLER, P. A. 1940 Tetanus toxin production on a sim-plified medium. Proc. Soc. Exptl. Biol. Med., 43, 389-390.

MUELLER, J. H., AND MILLER, P. A. 1941 A modification of Rosenthal's chro-mium-sulfuric acid method for anaerobic cultures. J. Bact., 41,301-303.

MUELLER, J. H., AND MILLER, P. A. 1941 Production of diphtheric toxin ofhigh potency (100 Lf) on a reproducible medium. J. Immunol., 40,21-32.

PAPPENHEIMER, A. M., JR. 1935 The nature of the "sporogenes vitamin," anessential growth factor for Cl. 8porogenes and related organisms.Biochem. J., 29, 2057-2063.

ROSENTHAL, L. 1937 "Chromium-sulfuric acid" method for anaerobic cultures.J. Bact., 34, 317-320.

SNELL, E. E., AND PETERSON, W. H. 1940 Growth factors for bacteria. X.Additional factors required by certain lactic acid bacteria. J. Bact.,39, 273-285.

STOKSTAD, E. L. R. 1941 Isolation of a nucleotide essential for the growth ofLactobacillus casei. J. Biol. Chem., 139, 475-476.