SPORULATION OF CLOSTRIDIUM BOTULINUM

II. EFFECT OF ARGININE AND ITS DEGRADATION PRODUCTS ON SPORULATIONIN A SYNTHETIC MEDIUM

WILLIAM E. PERKINS AND KIYOSHI TSUJINational Canners Association Research Laboratory, Berkeley, California

Received for publication January 16, 1962

ABSTRACT

PERKINS, WILLIAM E. (National Canners Asso-ciation Research Laboratory, Berkeley, Calif.)AND KIYoSHI TSUJI. Sporulation of Clostridiumbotulinum. II. Effect of arginine and its degrada-tion products on sporulation in a syntheticmedium. J. Bacteriol. 84:86-94. 1962.-Asynthetic medium which supports spore germina-tion, vegetative cell multiplication, toxin produc-tion, and sporulation of Clostridium botulinumstrain 62A is described. Arginine has been shownto play an important role in sporulation. Experi-ments involving the substitution of citrullineand ornithine for arginine, together with aminoacid analyses of culture supernatant fluids andcells, indicate that most of the arginine is brokendown by a dihydrolase enzyme system throughcitrulline to ornithine. The second step in thisreaction series, the degradation of citrulline,appears to be essential to sporulation. Theabsence of citrulline or ornithine in either growingcells or spores suggests that another product ofthe arginine dihydrolase system, adenosine tri-phosphate, may be responsible for the observedstimulation of sporulation.

Liquid media prepared from dehydrated pep-tones are satisfactory for the production ofstable, clean spores of Clostridium botulinum(Tsuji and Perkins, 1962), but being neitherdefined nor constant in composition (Lund, 1957)are unsuitable for studies on the physiology ofgrowth and sporulation.

Various investigators have reported growth ofC. botulinum or related clostridia in mixtures ofamino acids, salts, vitamins, and, usually,glucose (Burrows, 1933; Elberg and Meyer,1939; Roessler and Brewer, 1946; Shull, Thoma,and Peterson, 1949; Williams and Blair, 1950;Mager, Kindler, and Grossowicz, 1954; Camp-

bell and Frank, 1956), and in one such medi-um (Williams and Blair, 1950) sporulation wasrecorded. Prior to this study, therefore, theseformulations were evaluated.

Irregular growth of C. botulinum 62A and nosporulation occurred when a synthetic mixturesimilar to the medium described by Williamsand Blair (1950) was employed. Excellent growthwas ultimately obtained in a more complexsynthetic medium containing, with the exceptionof lysine, the same amino acids present in casein(Block and Weiss, 1956) together with glucose,the salt mixture employed by Shull et al. (1949)and Mager et al. (1954), and the vitaminsfound essential by the latter authors for thegrowth of C. botulinum type A. The amino acidconcentrations were, in most cases, similar tothose of Mager et al. (1954).

This medium, designated NCA S-1, wouldnot, however, support sporulation until thearginine concentration was increased to a levelapproximately double that employed by Shullet al. (1949) and Mager et al. (1954). This paperdeals with experiments conducted to determinethe optimal arginine concentration for sporula-tion in this synthetic medium, and with investiga-tions into the possible role of this amino acid andits breakdown products in sporogenesis.

MATERIALS AND METHODS

Cultures. The organism used in these investiga-tions is designated as C. botulinum strain 62A(from the collection of Karl F. Meyer, G. W.Hooper Foundation for Medical Research). Thespores used as primary inocula were prepared inpolypeptone broth (5%) and harvested andwashed by techniques employed by Tsuji andPerkins (1962). Deionized distilled water wasused for the last three washes and for finalsuspension.

Mlaterials. All glassware used was either Pyrex86

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or Kimax brand and was cleaned by boiling in a"Tide" detergent solution and soaking in achromic-sulfuric acid solution. Cleaning wasfollowed by ten rinses in distilled water andfinally by a deionized distilled water rinse. Thewater used for rinsing glassware and for thepreparation of all synthetic media was deionizedto a conductivity of 1.14 X 106 ohms by passagethrough freshly activated Dowex 50 WX-8 andDowex 1-X8 columns.The composition of the synthetic base medium

used in these experiments is given in Table 1.The purity of all amino acids used was testedchromatographically, and only those found to bechromatographically homogeneous were used.Modifications of this medium, in which thearginine concentration was increased, are des-ignated NCA S-2, NCA S-3, etc., the terminal

TABLE 1. Synthetic medium NCA S-i employedfor germination and growth of Clostridium

botulinum 62A

Constituent Amount Constituent (per 10 ml)

pmolesper ml

L-Alanine 4.5 Biotin 0.005 ,ugL-Arginine 13.4 Thiamine 4.0 ,ugL-Cysteine 14.0* p-Amino- 0.1 Ag

benzoic acidL-Glutamic 0.7 Glucose 0.05 g

acidGlycinet 1.6 FeSO 4-7H20 0.02mgL-Histidine 1.3 CaCl2*2H20 0.02 mgL-Isoleucinet 3.8 MnSO 4 H20 0. 10 mgL-Leucine 11 .5 MgSO4*3H2O 0.40 mgL-Lysine 8.2 NaCl 0.020

mgL-Methionine 4.0 K2HPO 4 10.0 mgL-Phenyl- 6.0 KH2PO4 10.0 mg

alanineL-Proline 4.3 Deionized dis-

tilled waterto 10 ml

L-Serine 9.5L-Threonine 8.4L-Tryptophan 0.25L-Tyrosine 1.4DL-Valine 17.0

* Media employed for other than spore germina-tion contained 7.0 ,umoles of cysteine per ml.

t Obtained from California Corp. for Bio-chemical Research; all others from NutritionalBiochemicals Corp.

number indicating the multiple by which theconcentration of arginine exceeded that in theNCA S-1 medium. Media in which citrullineor ornithine (both obtained from the CaliforniaCorp. for Biochemical Research) was substitutedfor arginine were designated NCA-C and NCA-O,respectively.

All media were prepared in 10-ml quantitiesand autoclaved in culture tubes (16 by 150 mm)at 121 C for 15 min. Glucose was added asepticallyfrom a membrane-filtered (Millipore Filter Corp.;type Ha filter) solution. The sterile media wereaseptically pipetted into the anaerobic cultureapparatus in 3.9-ml amounts.

Anaerobic culture apparatus. Figure 1 presentsa schematic diagram of the culture apparatusused. It consisted of a growth flask (A) con-structed from 15-mm Pyrex glass tubing andconnected by a cotton-plugged 9-mm side armfitted with a no. 0 rubber stopper (II) to a testtube (16 by 40 mm). The neck of the growthflask (1) was temporarily plugged with cottonand the apparatus was autoclaved at 121 C for20 min. After sterilization, 3.9 ml of culturemedium were aseptically pipetted into the growthflask A and a M6-in. rubber sleeve serum bottlestopper was substituted for the cotton plug inposition I.

Anaerobic conditions were produced in flask Aimmediately after the introduction of medium

I

A IL

FIG. 1. Anaerobic culture apparatus

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PERKINS AND TSUJI

by attaching a test tube (16 by 150 mm) con-taining 1.0 g of pyrogallic acid and 1.0 ml of 20%Na2CO3 to position II. All inoculations andtransfers were made through the serum stopperwith a hypodermic syringe fitted with a 22-gaugeneedle.

Germination and growth. Germination andoutgrowth of washed spores from the primarysuspension produced in polypeptone (5%) wereobtained in the NCA S-1 medium as follows. A5-ml portion of this stock suspension, previouslydiluted with deionized distilled water to resultin a viable spore count of 2.0 X 108 spores perml, was heat activated at 82.5 C for 10 min;0.1 ml was then inoculated into 3.9 ml of NCA S-1medium in the anaerobic culture device andincubated at 30 C. After spore germination,outgrowth, and several generations of vegetativecell division (approximately 24 hr), 0.1 ml of thegtowing culture was transferred to 3.9 ml offresh anaerobic NCA S-1 broth and incubatedfor 6 hr at 30 C.

Sporulation. After 6 hr of incubation of theculture resulting from the second transfer, a:final inoculation of 0.1 ml of growing culture into3.9 rnl of synthetic sporulation medium was madeand sporulation was allowed to proceed at 30 C.When a medium containing no arginine wasunder investigation, the final inoculation intothe sporulation medium was preceded by anadditional 6-hr incubation of growing cells inNCA S-1 medium from which arginine had beenomitted. The degree of sporulation in the varioussynthetic media was estimated microscopicallyby counting at least 300 cells and refractilespores using a dark-contrast phase microscope.The production of mature spores in all mediawas confirmed by viable counts of suspensionsheated at 82.5 C for 10 min.The thermal resistance of a spore suspension

prepared in NCA S-5 medium (67.0 ,umolesarginine per ml) was measured in 0.03 M phos-phate buffer using techniques previously de-scribed (Tsuji and Perkins, 1962). The D15.5value of these spores was 0.23 min.Amino acid analysis. The free amino acid

composition of cell-free supernatant fluids ofthe synthetic media before and after growthwas determined by ion-exchange chromatographyusing an automatic apparatus similar to that ofSpackman, Stein, and Moore (1958) as modifiedby Piez and Morris (1960). This technique per-

mits the measurement of ninhydrin-positiveconstituents in the effluent from a chromato-graphic column in amounts of 0.1 to 3.0 ,umoleswith a precision of 100 + 3%. Amounts as smallas 0.01 ,umole can be analyzed with a precisionof about 100 i 10% (Piez and Morris, 1960).A 1-ml amount of medium was deproteinized withtrichloroacetic acid (5%), made to a volume of10 ml with 0.25 N citrate buffer (pH 2.91), andcentrifuged at 10,000 X g for 10 min at 5 C inan SS-1 Servall centrifuge. The clear supernatantfluid was removed and frozen until analysis.Spore and vegetative-cell suspensions were hy-drolyzed and their amino acid composition de-termined by techniques described earlier (Tsujiand Perkins, 1962).

Toxin production. Cultures to be tested forthe presence of toxin were centrifuged for 1.5 hrat 1,000 X g. Two 20-g Webster-Swiss whitemice were injected intraperitoneally with 0.3 mlof the supernatant fluid.The immunological specificity of the toxin

was determined by passive immunization.

RESULTS

Effect of arginine concentration on sporulation.Germination of C. botulinum 62A spores andvigorous vegetative-cell growth was obtainedin NCA S-1 medium but maximal growth wasfollowed by lysis rather than sporulation. If thearginine concentration of this medium wasdoubled, however, 30% of the cells were ob-served to form mature refractile spores (Fig. 2).Further increases in arginine resulted in addi-tional sporogenesis which reached a maximumin a medium containing 67 MAmoles of arginineper ml (NCA S-5). Further increases in arginineconcentration beyond this level resulted in agradual decrease in the number of vegetative cellsforming spores.

Effect of products of arginine dihydrolase sys-tem on growth and sporulation. It has been re-ported that the clostridia contain a very activearginine dihydrolase system (Schmidt, Logan,and Tytell, 1952; Oginsky, 1955) which convertsarginine through citrulline to ornithine. There-fore, experiments were conducted to determinewhether this was the mechanism of breakdown ofarginine by C. botulinum cells.

If citrulline is an intermediate in the degrada-tion of arginine, the test organism should breakdown citrulline. This was found to be the case.

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100 r

80 F

60 1

40

20 -z0

-1

0

V5 10

8

6

4

3

0. 2

0. 1 1225 50 75 100 125

,/ M ARGININE/ ml MEDIUM

FIG. 2. Effect of arginine concentration on sporulation in NCA-S media

The replacement of arginine by 60 ;umoles ofcitrulline (NCA-C medium) resulted in abundantvegetative-cell growth and, ultimately, in theproduction of mature spores by 3 to 5% of thecells (Table 2). Contrary to the findings ofKindler and Mager (1956), ornithine (NCA-Omedium) would also support the growth ofC. botulinum. No sporulation was observed inthis medium in which arginine was replaced by70,umoles of ornithine. The test organism did notgrow when urea or creatine was substituted for

arginine in the otherwise completely syntheticmedium. Neither urea nor creatine was detectedby ion-exchange chromatography in any of thedefined media employed at any stage of growth.

Metabolism of arginine, citrulline, and ornithine.Deproteinized, cell-free filtrates of the NCA S-7medium were analyzed at intervals during growthand sporulation by ion-exchange chromatography.Results confirmed the citrulline substitutionexperiment.The large quantity of arginine initially present

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TABLE 2. Cell counts, sporulation, and pH changes during growth and sporulation of Clostridiumbotulinum 62A in various synthetic media

NCA S-7 medium NCA S-1 medium NCA-C medium NCA-O medium(arginine increased sevenfold) (base medium) (citrulline substituted (ornithine substituted

Incuba- for arginine) for arginine)tion

pH cells/mlSiporu pH cells/ml Sporou pH cells/mlsporI u pH cells/mlSiporu

days %0 6.98 -- 7.00 - 7.00 - 7.001 7.00 1.93 X 108 0 6.80 8.7 X 107 0 6.45 9.0 X 107 0 6.30 5.0 X 107 02 7.00 4.0 X 108

VOL. 84, 1962 SPORULATION OF C. BOTULINUM IN SYNTHETIC MEDIUM

TABLE 3. Amino acid composition of vegetativecells grown in NCA S-7 and NCA-O media

for 24 hr*

MediaAmino acid

NCA S-7 NCA-O

Aspartic acid 4.98 4.52Threonine 2.23 2.17Serine 2.01 2.19Glutamic acid 4.63 4.32Proline 1.29 1.41Glycine 3.94 3.69Alanine 4.84 4.58Cystine-cysteine 0.49 0.50Valine 3.37 3.07Methionine 0.12 0.14Isoleucine 3.07 4.76Leucine 3.12 3.17Tyrosine 0.50 0.55Phenylalanine 1.40 1.29Lysine 6.63 5.85Histidine 2.42 1.86Arginine 2.08 1.97Citrulline 0.10 0Ornithine 0 0xx,e-Diaminopimelic acid 0.47 0.55Glucosamine 0.13 0.24

* Tryptophan could not be detected afteracid hydrolysis. Results expressed as ,umoles ofamino acid per milligram of Kjeldahl nitrogen.

was consumed rapidly and free citrulline andornithine concomitantly appeared, indicating atransformation of arginine to citrulline, followedby an incomplete conversion of the latter toornithine (Fig. 3). The slow rate of appearanceof citrulline in the medium as compared withthe rate of arginine consumption may be theresult of the slow penetration of citrulline throughintact bacterial cells (Oginsky, 1955). The in-complete conversion of citrulline to ornithinesuggests that the citrullinase of C. botulinum is,like that of Pseudomonas ae?uginosa (Oginsky,1955), competitively inhibited by the end product,ornithine.

It is evident from analyses of C. botulinumvegetative cells grown in an arginine-free definedmedium that this organism possesses arginine-synthesizing enzymes (Table 3). Quantitativearginine-citrulline-ornithine balance sheet datacannot, therefore, be obtained with whole cellsin a complete medium. However, it is apparent,from the data presented in Fig. 3, that nearlyall of the arginine consumed by the test organism

could be accounted for on a molar basis ascitrulline and ornithine.The onset of sporulation in NCA S-7 medium

coincided with the conclusion of rapid arginineconsumption. The first sporangia were observedat the end of 2 days of incubation (Table 2),at which time the free arginine concentration inthe medium had been reduced from 90.3 to 6.7,umoles.

Deproteinized cell-free filtrates of NCA S-1medium, which contained sufficient arginine forvegetative-cell growth but not sporulation,NCA-C medium (citrulline substituted for ar-ginine), and NCA-O medium (ornithine sub-stituted for arginine) were analyzed by ion-exchange chromatography at intervals by thesame technique as the NCA S-7 filtrates. Argininewas completely consumed in NCA S-1 medium(Fig. 4), but the molar ratio of citrulline andornithine to original arginine in the medium wasless, suggesting the incorporation of propor-tionately more arginine into cell proteins. Theconversion of a higher proportion of citrullineto ornithine by the test organism in this mediumthan in the NCA S-7 medium may be a reflectionof the lower maximal pH attained in the former(Table 2). Schmidt et al. (1952) reported thatpH is an important variable in the attack oncitrulline by washed-cell suspensions of C.perfringens. Significant degradation of citrullineto ornithine by these suspension was detected bythese workers at pH 5.5 and 6.3, but virtuallynone at pH 7.2.

In the NCA-C medium, only 10 ,umoles of theoriginal 60 ,umoles of citrulline disappearedduring growth and sporulation. At the end of5 days of incubation, 1.5 ,umoles of free ornithinewere detected in the medium, and 3.2 ,moleswere found at the end of 7 days. Arginine wasnot detected in the supernatant fluid at anystage of cultural growth.

In NCA-O medium, 10.5 ,umoles of ornithinedisappeared during 7 days of growth. Neithercitrulline nor arginine was detected in thesupernatant fluid of cultures grown in thismedium at any stage of growth.Amino acid composition of vegetative cells.

Hydrolyzates of washed vegetative cells of C.botulinum 62A, harvested during the logarithmic-growth phase from NCA S-7 and NCA-O media,were analyzed by ion-exchange chromatography(Table 3). There was no significant difference inthe amino acid composition of cells growing in

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PERKINS AND TSUJI

* ARGININE

X,umuuusuumuuuussuumuuluX CITRULLINE

A-'m-mim|A ORNITHINE

2 3 4 5 6 8

INCUBATION TIME (DAYS)

FIG. 4. Arginine consumption and citrulline and ornithine synthesis in NCA-SI medium

the presence of a high concentration of arginineand those growing in the absence of preformedarginine. The small amount of citrulline detectedin cells growing in NCA S-7 medium is pre-sumed to be in the free form, since neithervegetative cells nor spores produced in complexmedia contain citrulline (Tsuji and Perkins,1962).

Toxin production. The supernatant fluid froma 24-hr and a 7-day culture grown in NCA S-7

medium was analyzed for the presence of toxin.The 7-day culture contained sufficient C.botulinum type A toxin to cause the death ofunimmunized test animals in less than 17 hr.No toxin was detected in the 24-hr culture.

DISCUSSION

Schmidt et al. (1952) reported that citrullineis an intermediate and ornithine the end productin the degradation of arginine by washed-cell

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VOL. 84, 1962 SPORULATION OF C. BOTULINUM IN SYNTHETIC MEDIUM

suspensions of C. perfringens. Oginsky andGehrig (1952a, b) obtained similar results withcell-free extracts of Streptococcusfaecalis. Knivett(1953) and, later, Oginsky and Gehrig (1953)and Slade, Doughty, and Slamp (1954) estab-lished with extracts of S. faecalis and a Pseudo-monas species, respectively, that the second stepof the arginine dihydrolase reaction series, thebreakdown of citrulline by citrullinase, results,together with the production of ornithine, NH3,and C02, in the esterification of inorganic phos-phate in high-energy phosphate, adenosine tri-phosphate (ATP).

Evidence presented here shows that arginineis also metabolized through citrulline to ornithineby growing cells of C. botulinum. Since C.botulinum appears to possess an arginine dihy-drolase system similar to that of the streptococciand pseudomonads, it is reasonable to assumethat it is also capable of generating high-energyphosphate during the citrulline-ornithine step.When C. botulinum cells were grown in a

complete but asporogenic synthetic mediumcontaining 13.4 ,umoles of arginine per ml, freearginine completely disappeared after 2 to 3 daysof incubation. In an otherwise identical, spo-rogenic medium containing seven times thisconcentration, virtually all of the arginine wasconsumed at a similar rate. This unusually largecapacity for arginine does not seem to be afunction of an increased rate of growth. The cellconcentrations in both of these media werecomparable during the period of maximal arginineconsumption (Table 2). Nor does it appear toreflect the incorporation of more arginine intothe individual cells. The amount of argininepresent in C. botulinum spores (Tsuji andPerkins, 1962) and in cells growing in the presenceof high concentrations of arginine or in theabsence of arginine (Table 3) is of the sameorder. Nearly all of the arginine consumed byC. botulinum cells in a synthetic, sporogenicmedium can be accounted for as free citrullineand ornithine in the medium, and neither is acomponent of the spore protein (Tsuji andPerkins, 1962) nor of the vegetative cell protein(Table 3). It can be reasonably concluded, then,that the requirement of substantially largerquantities of arginine for maximal sporulationthan for maximal growth in a complete syntheticmedium is neither the result of greater require-ment of this amino acid per se nor for the aminoacid products of its breakdown.

The esterification of inorganic phosphate whichaccompanies the breakdown of citrulline mayaccount for the sporogenic effect of arginine andcitrulline, since ATP could furnish a significantamount of energy for the syntheses leading tosporulation.

Evidence obtained in amino acid-replacementexperiments indicates that sporulation of C.botulinum cells can proceed in a complete syn-thetic medium in the presence of the citrullinasesubstrate, but not when ornithine is substitutedfor arginine. In the latter medium, no citrullinewas ever detected.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the tech-nical assistance of Akira Ito and Clifford L.Drake. We thank C. T. Townsend of this lab-oratory and L. E. Sacks and J. C. Lewis of theWestern Regional Research Laboratory, U.S.Department of Agriculture, for their helpfulsuggestions during the preparation of this re-port. This work was supported in part by grantEF-130, National Institutes of Health, U.S.Public Health Service.

LITERATURE CITEDBLOCK, R. J., AND K. W. WEISS. 1956. Amino acid

handbook. Charles C Thomas, Springfield,Ill.

BURROWS, W. 1933. Growth of Clostridium botu-linum on synthetic medium. J. InfectiousDiseases 52:126-137.

CAMPBELL, L. L., JR., AND H. A. FRANK. 1956.Nutritional requirements of some putrefactiveanaerobic bacteria. J. Bacteriol. 71:267-269.

ELBERG, S. S., AND K. F. MEYER. 1939. The nu-tritional requirements of Clostridium para-botulinum A. J. Bacteriol. 39:485-497.

KINDLER, S. H., AND J. MAGER. 1956. Nutritionalstudies with the Clostridium botulinum group.J. Gen. Microbiol. 15:386-393.

KNIVETT, V. A. 1953. Citrulline breakdown by acell-free extract of Streptococcus faecalis. J.Gen. Microbiol. 8:V.

LUND, A. J. 1957. Discussion of Ordal, Z. J. Theeffect of nutritional and environmentalconditions of sporulation. In H. 0. Halvorson[ed.], Spores. American Institute of Bio-logical Sciences, Washington, D.C.

MAGER, J., S. H. KINDLER, AND N. GROSSOWICZ.1954. Nutritional studies with Clostridiumparabotulinum type A. J. Gen. Microbiol.10:130-141.

OGINSKY, E. L. 1955. Mechanisms of arginine andcitrulline breakdown in microorganisms, p.

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300-308. In W. D. McElroy and H. B. Glass[ed.], Amino acid metabolism. The JohnsHopkins Press, Baltimore.

OGINSKY, E. L., AND R. F. GEHRIG. 1952a. Thearginine dihydrolase system of Streptococcusfaecalis. I. Identification of citrulline as anintermediate. J. Biol. Chem. 198:791-797.

OGINSKY, E. L., AND R. F. GEHRIG. 1952b. Thearginine dihydrolase system of Streptococcusfaecalis. II. Properties of arginine desimidase.J. Biol. Chem. 198:799-805.

OGINSKY, E. L., AND R. F. GEHRIG. 1953. Thearginine dihydrolase system of Streptococcusfaecalis. III. The decomposition of citrulline.J. Biol. Chem. 204:721-729.

PIEZ, K. A., AND L. MORRIS. 1960. A modifiedprocedure for the automatic analysis ofamino acids. Anal. Biochem. 1:187-201.

ROESSLER, W. G., AND C. R. BREWER. 1946.Nutritional studies with Clostridium botu-linum toxin types A and B. J. Bacteriol.51:571.

SCHMIDT, G. C., M. A. LOGAN, AND A. A. TYTELL.

1952. The degradation of arginine byClostridiunt perfringens (BP6K). J. Biol.Chem. 198:771-783.

SHULL, G. M., R. N. THOMA, AND W. H. PETERSON.1949. Amino acid and unsaturated fatty acidrequirements of Clostridium sporogenes. Arch.Biochem. 20:227-241.

SLADE, H. D., C. C. DOUGHTY, AND W. C. SLAMP.1954. The synthesis of high energy phosphatein the citrulline ureidase reaction by solubleenzymes of Pseudomonas. Arch. Biochem.Biophys. 48:338-346.

SPACKMAN, D. H., W. H. STEIN, AND S. MOORE.1958. Automatic recording apparatus for usein the chromatography of amino acids. Anal.Chem. 30:1190-1206.

Tsuji, K., AND W. E. PERKINS. 1962. Sporulationof Clostridium botulinum. I. Selection of anaparticulate sporulation medium. J. Bac-teriol. 84:81-85.

WILLIAMS, 0. B., AND E. BLAIR. 1950. Sporeformation in synthetic media by Clostridiumbotulinurm. Bacteriol Proc., p. 62-63.

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