JOURNAL OF BACTERIOLOGY, Dec. 1967, p. 1924-1929Copyright @ 1967 American Society for Microbiology

Vol. 94, No. 6Printed in U.S.A.

Effect of Nutrients on Physiological Properties ofClostridium botulinum Type E

B. GULLMAR AND N. MOLINSwedish Institute for Food Preservation Research (SIJK), Goteborg, Sweden

Received for publication 23 September 1967

Eight strains of Clostridium botulinum type E out of twelve tested showed goodgrowth and normal cell morphology in a synthetic medium containing choline.Growth and toxin production by a representative strain was not influenced by re-peated subculturing. In the chemically defined medium, acetylcholine, N,N-di-methylethanolamine, and lecithin could replace choline to get normal cell divisionand cell morphology of C. botulinum type E. Choline could not be replaced byethanolamine, N-methylethanolamine, or betaine. A toxigenic strain of C. botulinumtype E showed proteolytic, lipolytic, and lecithinase activity in complex media butnot in a synthetic medium. On prolonged incubation in the high temperature rangeof growth, the toxicity of the culture filtrate decreased in a complex, but not in asynthetic medium. The implications of these findings are discussed.

The uneven distribution of Clostridum botuli-num type E spores in different geographical areas(5, 7, 9, 10, 12, 13, 16, 26, 27, 28, 34) and the ob-servation that various fish products have differentcapacities to promote growth and toxin produc-tion (1, 6, 8, 35, 42) have prompted investigationsinto the behavior of this organism in differentchemical and physical environments (2, 36, 38).Lack of suitable chemically defined media hashitherto arrested the elucidation of the nutritionalrequirements of the organism. Recently, however,a few reports describing growth and toxin pro-duction of C. botulinum type E in chemically de-fined media have been published (22, 23, 41, 43).The present investigation describes the influenceof the chemical environment on some physiologi-cal properties of C. botulinum type E.

MATERIALS AND METHODSOrganisms. The following strains of C. botulinum

type E were used: strains 996/62, 1217-764/64,1537/62, 1653-1383/64, 2032/61, and 2835/61, fromA. Johanssen, Public Health Authority Laboratory,Lund, Sweden; strains E6 and E104, from C. E.Dolman, University of British Columbia, Vancouver;and strains 3 pl, 3880 Hpl, 3889 pl/1/, and 3889pl/2/, from G. Hobbs, Torry Research Station,Aberdeen, Scotland.

Culture media. The media used were proteosepeptone medium (23), Robertssons cooked meatmedium (3), chemically defined medium D-Y (22),tributyrin agar, and egg yolk agar. The latter twowere prepared by adding 2 ml of glycerol tributyrateand 10 ml of concentrated egg yolk emulsion (Oxoid),respectively, to the following base: 4 g of Proteose

Peptone No. 3 (Difco), 2 g of yeast extract (Difco),0.4 g of glucose, 60 mg of Na-thioglycolate, 2 ml ofglycerol tributyrate, 0.2 g of NaCI, 2 g of agar, anddistilled water to 100 ml, pH 7.4. Sterile stock solu-tions of glucose, Na-thioglycolate, tributyrate, andegg yolk emulsion were added aseptically to thesterile proteose peptone base. The chemically definedmedium was prepared from stock solutions of theindividual components and was sterilized by filtra-tion. The complex media were sterilized by auto-claving for 15 min at 121 C. All stock solutions werestored in the dark at 4 C. Those not used within 2weeks of preparation were discarded.

Toxin assay and neutralization tests. These wereas described earlier (23).

Proteolytic activity. Proteolytic activity was esti-mated from the casein precipitating effect of proteo-lytic enzymes in casein agar as described by Sandvik(Thesis, Vet. College of Norway, Oslo, 1962). Pre-liminary experiments had shown this method to bethe most sensitive one for estimating proteolyticactivity. The casein agar was poured into petri dishesand the agar layer had a thickness of 2 mm. Theculture filtrate to be tested was adjusted to pH 6.2before 0.025 ml was deposited in wells (6-mm di-ameter). The petri dishes were incubated at 37 C for20 hr.

Lipolytic and lecithinase activity. Reactions in eggyolk agar as described by Willis (44, 45) and byMcClung and Toabe (33) were used to demonstratelipolytic and lecithinase activity. Lipolytic activitywas also indicated by clear zones in tributyrin agar.

RESuLTSEfficiency of the synthetic medium. The chemi-

cally defined medium (D-Y) used for growth andtoxin production of C. botulinum type E was

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EFFECT OF NUTRIENTS ON C. BOTULINUM TYPE E

developed during the present investigation and itscomposition has been published elsewhere (22).The capacity of this medium to support growthand promote sporulation is further demonstratedin Fig. 1. It was also noticed that a minimal in-oculum of about 100 cells/ml was needed to pro-mote growth, and that the corresponding figurefor proteose peptone medium was about 10 cells/ml.The constancy of growth and toxin production

after repeated subculturing in D-Y is shown inFig. 2. Ten transfers did not affect growth or toxinproduction to any appreciable extent. The capac-ity of the synthetic medium D-Y to supportgrowth in a different strain of C. botulinum type Ewas also tested. It was found that 8 strains of 12tested supported growth.

Effect of choline and related substances on celldivision. Choline has been shown to have an effecton cell division of C. botulinum type E (22). Tounderstand better the mechanism of the effect ofcholine, a number of compounds structurally re-lated to choline were tested regarding their capac-ity to substitute for choline in a synthetic medium.The results are shown in Table 1. Acetylcholine,N,N-dimethylethanolamine, and lecithin couldsubstitute for choline, but not ethanolamine, N-methylethanolamine, or betaine. This indicatedthat N,N-dimethylethanolamine is the simplestcompound necessary for normal cell division inthe synthetic medium used.

Proteolytic activity. The potency of type E toxincan be greatly increased by a primary attack of

108

E0; 107=

1o6

0

x

--x

0 o

0 24- 48 72 96 120

Incubation time (hours)

FIG. 1. Growth and sporulation of Clostridiumbotulinum type E (strain 1537/62) in D-Y medium.Incubation temperature, 30 C. Symbols: X, totalnumber ofcells/ml; 0, number of sporulating cells/ml.

3(. 1 T I

t T Ir II

II

I 32i

2

O 1 2 3 4 5 6 7 8 9 10Transfers

FIG. 2. Growth and toxin yield of Clostridiumbotulinum type E (strain 1537/62) in D-Y mediumafter subsequent transfers. First inoculum: 10i sporesplus 3 ml of medium. Amount transferred: 0.1 ml ofa full-grown culture to 3 ml of medium. Growth wasestimated after 2 days at 30 C, toxin after 4 days.Symbols: X, growth; 0, toxin, mean value x ofthree mice. The standard deviation, calculated ass = \/Z(x - R)'/(n - 1), is shown by the verticallines.

TABLE 1. Effect of choline and related compoundson cell division of Clostridium botulinum type

E (strain 1537/62)

AppearanceConcn

Compound added (;Amole/ml) Chains Single

cells

Control (D-Y with cholineomitted) ............ +

Acetylcholine ............... 0.36 +Choline. . . 0.36 +Ethanolamine... 0.36 +N-methylethanolamine... 0.36 +N, N-dimethylethanolamine. 0.36 +Betaine. ... 0.36 +Lecithin.... 0.36 +

proteolytic enzymes (15, 17, 18, 39). Continuedexposure to these enzymes may bring aboutfurther fragmentation and partial inactivation ofthe toxin (14, 19, 40). This would explain thefrequent decrease in toxicity of culture filtrate ofC. botulinum type E on prolonged incubation,especially in the higher temperature range ofgrowth (2, 4, 37). Since it is possible thatproteolytic activity could be influenced by thepresence of protein in the growth medium, it wasdecided to find out whether the proteolytic ac-tivity of a toxigenic strain of C. botulinum type E

VOL. 94, 1967 1925

108 X%..X-X/*-X,.%X.-X-..X-x-x

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GULLMAR AND MOLIN

was different in the synthetic D-Y me(a complex medium, and whether suchwould be reflected by a higher stabilitformed in the synthetic medium.Table 2 shows the relative proteolyt

culture filtrate of C. botulinum type E ttion for various periods at 30 C. Thewere proteose peptone medium, c(medium, and D-Y medium. The pr(tivity could only be demonstratedfiltrates from the complex growth mlfrom the synthetic D-Y medium (Tab3 shows the effect of incubation time (ity of culture filtrate at different incuperatures. The media used were protemedium and D-Y medium. With in35 C or higher, a distinct decrease Mthe toxicity in the complex mediutoxicity in the D-Y medium was nolany appreciable extent. The toxicit:filtrates of C. botulinum type E growrmedium was higher at 35 C than at 3teose peptone medium the toxicity w30 C than at 35 C.

Lipolytic and lecithinase activilymethods used, lipase and lecithincould be demonstrated in culture fithe complex medium but not frormedium (Table 3). However, cells tranthe D-Y medium and incubated on Itest media recovered their capacitylipid- and lecithin-destructive enzymmore, when choline was replaced b!the D-Y medium, lecithinase actividemonstrated in the culture filtrate.

TABLE 2. Proteolytic activity of cultuClostridium botulinum type E (strai

grown in different media

Age of culture(days)

2468101215223050

Growth medium

D-Y

Oa0

0

0

0

0

0

0

0

0

a Results indicatezone in millimeters.

Robertssonscooked meatmedium

0

0

0

0

0

0

8

0

0

0

diameter of X

dium than inn A;4T---

DiscussIoN

ofthertoxin The chemically defined medium D-Y used inthis investigation supported growth and toxin

Lic activity of formation by C. botulinum type E. On repeatedafter incuba- subculture, the strain used did not show anymedia used degeneration phenomenon, such as restrictedooked meat growth and toxin production or abnormal celloteolytic ac- morphology. The capacity of the D-Y medium to

in culture induce sporulation is noticeable, since the strainedia but not used did not sporulate freely in complex media.)le 2). Figure In the synthetic D-Y medium, acetylcholine,on the toxic- N,N-dimethylethanolamine, and lecithin couldibation tem- replace choline without affecting normal cell3ose peptone division, but ethanolamine, N-methylethanol-icubation at amine, or betaine could not. The fact that N,N-ias found in dimethylethanolamine, but not methionine andin, but the betaine containing labile methyl groups, couldt affected to replace choline indicates that the role of choliney of culture is not that of transmethylation. The effect ofin the D-y acetylcholine could be understood if this sub-0 C. In pro- stance is hydrolyzed by the organism to get cho-(as higher at line. It is thought that choline acts as a precursor

of certain choline-containing phospholipids in theWith the cell membranes. Choline-containing phospho-

ase activity lipids have so far been demonstrated in onlyiltrates from relatively few species of bacteria (24, 30, 31, 46)il

the D-y and have not been found in the few species ofisferredfrom Clostridium tested (20, 21). The formation ofthe complex lecithin via the stepped transmethylation ofto produce ethanolamine to N-methylethanolamine, N,N-es. Further- dimethylethanolamine, and choline is known to

y lecithin in occur in lecithin-synthesizing bacteria (21, 29, 32).ty could be Our findings that ethanolamine and N-methyl-

ethanolamine did not substitute for N,N-di-methylethanolamine or choline indicate that the

re filtrate of biosynthesis of N,N-dimethylethanolamine fromtn 1537/62) ethanolamine did not occur in C. boiulinwn typeE grown in the D-Y medium. This is in agreement

with Goldfine (20, 21), who found that N,N-dimethylethanolamine and choline could not befound by transmethylation in C. butyricum. How-

Proteose ever, we found that N,N-dimethylethanolaminepeptone could replace choline. This is most easily under-

stood if one assumes that N,N-dimethylethanol-o amine is transmethylated to choline and conse-0 quently acts as a precursor of this substance.0 Another possibility is that N,N-dimethylethanol-ll amine may be a constituent of certain phospha-

tides in the cell membranes and gives rise to nor-12.5 mal cell division. This substance has been found in10 phosphatides from Agrobacterium tumefaciens0 (32) and from a choline-requiring mutant of0 Neurospora crassa unable to synthesize choline

(25), but not in C. butyricum (20).precipitation The action of choline on the cell division of C.

botulinum type E may thus be due to its role as a

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EFFECI OF NUTRIENTS ON C. BOTULINUM TYPE E

4

3

E

Oi0:

-iI/f--ii3 4 6 10 20

Incubation time (days)

2

1 -

00

B

I - , I

I

t I1II-1

3 4 6 10 20

Incubation time (days)

FIG. 3. (A)IEffect of temperature on toxicity of culture filtrate of Clostridium botulinum type E (strain1537/62) grown in proteose peptone medium. (B) Effect of temperature on toxicity of culture filtrate ofClostridium botulinum type E (strain 1537/62) grown in D-Y medium. Symbols: A, toxin strength at 35 C;0, at 37 C; and O, at 39 C.

TABLE 3. Influence ofgrowth media on the development ofenzymes

Culure filtrate Cell suspension

Growth medium Egg yolk agar Egg yolk agarClearing on Cleaning on

tributyrin tributynnLecithinase Lipolytic agar Lecithinase Lipolytic agar

activity activity activity activity

Robertssons cooked meat medium + + + + + +D-Y _ _ + + +D-Y, in which choline was sub- + _ _ + + +

stituted by lecithin

constituent of certain choline-containing phos-pholipids in the cell membranes, which are knownto be involved in the cell division of bacteria.However, if this assumption is true, further in-vestigation may show that choline really is aconstituent of the cell membranes of C. botulinumtype E grown in the D-Y medium. It is also possi-ble that the function of choline and N,N-di-methylethanolamine in the cell division of C.botulinum type E is entirely different from theirknown roles in synthesis of phosphatides in bac-teria.

Toxigenic strains of C. botulinum type Eare generally believed to be nonproteolytic. It has,however, been claimed that certain strains mighthave a weak proteolytic activity (A. Skulberg,Thesis, Vet. Collegeof Norway, Oslo, 1964). Inourexperiments, strain 1537/62 showed proteolytic

activity after 8 days in a complex medium but notin the synthetic medium. It seems that the decreasein toxicity on prolonged incubation at 35 and37 C in the complex media is due to the proteo-lytic inactivation of toxin already formed.

In our experiments, neither lipase nor lecithin-ase activity could be demonstrated in culturefiltrate from the D-Y medium. On the other hand,when cells from the D-Y medium were incubatedon the egg yolk agar and tributyrin agar, they re-covered their capacity to form lipolytic and leci-thinase activity. When choline was replaced bylecithin in the D-Y medium, lecithinase but notlipase activity could be demonstrated. Our resultsindicate that the lipid as well as the lecithin-destructive enzymes are adaptive. This seems tosupport the view expressed by Cutchins et al. (11)that bacterial lipases are adaptive in nature.

3 '

E0

x 22

Oi

0A

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1928 GULLMAR

ACKNOWLEDGMENTSWe sincerely thank Erik von Sydow, Director of

this Institute, for valuable support and useful sug-gestions. The authors also gratefully acknowledgethe skillful assistance of Anne-Marie Andersson.

This work was supported by the Swedish MedicalResearch Council (project No. 16x-700-01).

LnTRATuRE CrrED1. ABRAHAMSSON, K., N. N. DE SILVA, AND N.

MOLIN. 1965. Toxin production by Clostridiumbotulinum, type E in vacuum-packed, irradiatedfresh fish in relation to changes of the associatedmicroflora. Can. J. Microbiol. 11:523-529.

2. ABRAHAMSSON, K., B. GULLMAR, AND N. MOLIN.1966. The effect of temperature on toxin for-mation and toxin stability of Clostridiumbotulinum type E in different environments.Can. J. Microbiol. 12:385-394.

3. AMERICAN PUBLIC HEALTH ASSOCIATION. 1958.Recommended methods for the microbiologi-cal examination of foods. American PublicHealth Association, Inc., New York.

4. BARRON, A. L., AND G. B. REED. 1954. Clostridiumbotulinum type E toxin and toxoid. Can. J.Microbiol. 1:108-117.

5. BoTr, T. L., J. S. DEFFNER, AND E. M. FOSTER.1967. Occurrence of Clostridium botulinumtype E in fish from the Great Lakes withspecial reference to certain large bays. InM. Ingram and T. A. Roberts [ed.], Botulism1966. Proc. Intern. Symp. Food Microbiol,5th, Moscow, p. 21-24.

6. CANN, D. C., B. B. WILSON, G. HOBBS, ANDJ. M. SHEWAN. 1965. The growth and toxinproduction of Clostridium botulinum type Ein certain vacuum packed fish. J. Appl. Bac-teriol. 28:431-436.

7. CANN, D. C., B. B. WILSON, G. HOBBS, ANDJ. M. SHEWAN. 1967. Clostridium botulinumtype E in the marine environment of GreatBritain. In M. Ingram and T. A. Roberts[ed.], Botulism 1966. Proc. Intern. Symp.Food Microbiol. 5th, Moscow, p. 62-65.

8. CANN, D. C., B. B. WILSON, G. HOBBS, AND7 J. M. SHEWAN. 1967. Toxin production by

Clostridium botulinum type E in vacuumpacked fish. In M. Ingram and T. A. Roberts[ed.], Botulism 1966. Proc. Intern. SympFood Microbiol. 5th, Moscow, p. 202-207.

9. CANN, D. C., B. B. WILSON, G. HOBBS, J. M.SHEWAN, AND A. JOHANNSEN. 1965. The in-cidence of Clostridium botulinum type E infish and bottom deposits in the North Seaand off the coast of Scandinavia. J. Appl.Bacteriol. 28:426-430.

10. CRAIG, J. M., AND K. S. P1LCHER. 1967. Thenatural distribution of Clostridium botulinumtype E in the Pacific Coast areas of the UnitedStates. In M. Ingram and T. A. Roberts [ed.],Botulism 1966. Proc. Intern. Symp. FoodMicrobiol. 5th, Moscow, p. 56-61.

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,ND MOLIN J. BACTERIOL.

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15. DUFF, J. T., G. G. WRIGHT, AND A. YAR1NSKY.1956. Activation of Clostridum botulinumtype E toxin by trypsin. J. Bacteriol. 72:455-460.

16. EKLuND, M. W., AND F. POYSKY. 1967. Incidenceof Clostridium botulinum type E from thePacific Coast of the United States. In M.Ingram and T. A. Roberts [ed.], Botulism 1966.Proc. Intern. Symp. Food Microbiol. 5th,Moscow, p. 49-55.

17. GERWING, J., C. E. DOLMAN, AND D. A. ARNOTT.1961. Purification and activation of Clostridiumbotulinum type E toxin. J. Bacteriol. 81:819-822.

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19. GERWING, J., C. E. DOLMAN, AND A. Ko. 1965.Mechanism of tryptic activation of Clostridiumbotulinum type E toxin. J. Bacteriol. 89:1176-1179.

20. GOLDFINE, H. 1962. The characterization andbiosynthesis of an N-methylethanolaminephospholipid from Clostridium butyricum.Biochim. Biophys. Acta 59:504-506.

21. GOLDFINE, H., AND M. E. ELLIS. 1964. N-methylgroups in bacterial lipids. J. Bacteriol. 87:8-15

22. GULLMAR, B., AND N. MOLIN. 1967. Effect ofcholine on cell division of Clostridium botulinumtype E. J. Bacteriol. 93:1734.

23. GULLMAR, B., AND N. MOLIN. 1967. Some nu-tritional factors governing growth and toxinproduction by Clostridium botulinum type E.In M. Ingram and T. A. Roberts [ed.], Botu-lism 1966. Proc. Intern. Symp. Food Microbiol.5th, Moscow, p. 185-201.

24. HAGEN, P.-O., H. GOLDFINE, AND P. J. LE B.WILLIAMS. 1966. Phospholipids of bacteriawith extensive intracytoplasmic membranes.Science 151:1543-1544.

25. HALL, M. O., AND J. F. NYC. 1961. The isolationand characterization of phospholipids con-taining mono- and dimethylethanolaminefrom Neurospora crassa. J. Lipid Res. 2:321-327.

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29. KANSHmRO, T., AND J. H. LAW. 1964. Phospha-tidylcholine synthesis in Agrobacterium tume-faciens. L. Purification and properties of aphophatidylethanolamine N-methyl-transferase.J. Biol. Chem. 239:1705-1713.

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33. MCCLUNG, L. S., AND R. TOABE. 1947. The eggyolk plate reaction for the presumptive di-agnosis of Clostridium sporogenes and certainspecies of the gangrene and botulinum groups.J. Bacteriol. 53:139-147.

34. NIcKERN, J. T. R., S. A. GOLDBL1TH,G. DIGIOLJA, AND W. W. BISHOP. 1967. Thepresence of Clostridium botulinum, type E infish and mud taken from the Gulf of Main.In M. Ingram and T. A. Roberts [ed.], Botu-lism 1966. Proc. Intern. Symp. Food Microbiol.5th, Moscow, p. 25-33.

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41. SNUDDEN, B. H., AND R. V. LwcmowicH. 1967.Growth and sporulation of Clostridium bonu-linum type E in chenically defined media. InM. Ingram and T. A. Roberts [ed.], Botulism1966. Proc. Intern. Symp. Food Microbiol.5th, Moscow, p. 144-149.

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43. WARD, G. Q., AND B. J. CARROLL. 1967. Sporegerination and vegetative growth of Clostri-duin botulinum type E in synthetic media.Can. J. Microbiol. 12:1145-1156.

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