PROJECT REPORT COMMITTEE ON F000 8ESEASCN QUARTERMASTER FOOD ARO CONTAIHER INSTITUTE FOR THE ARMED FORCES CHICAGO ILLINOIS am® MTataraitinf »*#»ai MILITAIT PlAKKIlfO DZPISZOV OPPICK OP Till Q0AKTKIM4*T«« OPMXIAL [TWTffTVm SkVTiTuTrffS pCAHTY University of Texas Austin Texas I 01 VI SI0M~ ■ DEPARTMENT 4Arts and Sciences Bacteriology | 0 F PI Cl AL IMVEST1SATOR COUAiORATORS Foster £, Staten \]gfrme * no. 5” (1)* FILE NO. / [CONTRACT' MO. • M-605 / |W-ll-009-am-70190 76ft Hftitfo coTERnTs 7 initiation date f.ugust. 19A6-Maroh. 19A7 ■ . July 1, Wife-.— TITLE: [ ] PROGRESS REPORT [ j PHASE REPORT [ XJ ANNUAL REPORT [ JlERNIN AT I ON REPORT Spore Formation and Spore Germination of Anaerobic Food Spoilage Organisms, Especially Clostridium Botulinum. SUMMAIY u simple reasonably accurate method for quantitative study of spore gemination in Clostridium botulinum and other ana- erobes is described. The obstacle of dormancy has been elimi- nated, maximum counts appearing in three days. Illustrations are given of the application of the method. ♦This report is one of four, to be numbered 4-(l), 4(ll), 4(lll), and 4(IV)# The four reports will comprise the annual report on this project* ■ Continued 1 CQ«0 FORM _ 12-121 5 Apr!* ' (X*ri**4) rhysiclogical studies on spore germination, with special reference to Clostridium betulinum* I* Development of a Quantitative Method E. Staten Wynne and Jackson W, Foster Dept, of Bacteriology, University of Texas, Austin, By far th- majority of studies hitherto made on germination of bacterial the appearance of visible turbidity as the criterion of germi- nation, Obviously this technique can reveal no quantitative characteristics of the germination process and is therefore of value only in establishing that sene germination does or does not take place. Even here its value may be questioned, since it has been clearly demonstrated that gemination of spores of vari us organisms nay occur without significant subsequent vegetative proliferation* (Itane and Neill, 1919; Knight end Fildes, 1930; Xnaysi, 194-5; Knaysi and Baker, 194-7). Furthermore, various environmental conditions imposed upon geminating spores nay have no influence on the germination tine but yet may alter appreci- ably the rote of . stibsequent vegetative development (Evans and Curran, 1943). Uur own experiments confirm this finding. Direct microscopic counts have been used for quantitative studios of the germination of aerobic sp res, (Eckelmann, 1918; Curran, 1931) but such a pro- cedure is unduly wearisome and not readily adapted to use with anerobes. Also, *This project has been undertaken in cooperation with the Committee on Feed Research of the Quartermaster Food and Container Institute for the .umed Forces. The opinions or conclusions contained in this report are of the authors. They are not to be construed as necessarily reflecting the views or endorsement of the War Department, M-605 #4(1) 2 Continued with certain species, for example Bacillus anthracis. it nay be very difficult to establish microscopically a criterion of gemination, as noted by Fischoeder (1909), Swann (1927), and Cook (1932). The outstanding physiological difference between spores and vegetative cells of any one organism, namely, heat lability of the latter at a temperature innocu- ous to the former, has long been employed in quantitative approaches to spore gemination, since it is assumed traditionally that when a spore cell is so changed that it becomes heat labile, gemination has taken place (Weil, 1901 j Fischoeder, 1909; Evans and Curran, 194-3). .d. though Curran and Evans (1937, 194-5b) have indicated that the heat-labile state may actually precede rupture of the spore wall and that some morphological changes characteristic of gemination may occur prior to the loss of themal resistance of the spore, heat differen- tiation of the germinated vs, the ungeminated spore appears to be the most prac- ticable approach. The fact that a definite reproducible standard endpoint may be selected, viz,, survival at a definite temperature for a definite period of time, even though somewhat arbitrary, outweighs the overlapping between the phy- siological end morphological characters which renders the germination process an indistinct one. Consideration of other possible criteria of gemination have repeatedly brought us to the conclusion that changes in heat lability is best from every point of view, and it forms the basis of this work. The technique has been de- signed especially for Clostridium botulinun. an important organism in food poison- ing, on which apparently no quantitative gemin tion studios of any sort have been made. The method has been applied, with appropriate modification, to other anaerobic species. Experimental Some of the preliminary work was carried cut with £. botulinum Strain 115 Bj M-6Q5 #4(1) - 3 - Continued Continued Strain 62 was utilized in most of the work. Both strains were obtained from the National Conner’s association and were repurified by isolation of colonies _ ' ; I fron serial shake tubes. Toxin fcmation was demonstrated for Strain 62 u by the fact that 1,0 ml* of a Seitz filtrate of a 10-day culture was lethal for a guinea pig in less than 21 hours, while a control animal receiving 1 ml, of the filtrate inactivated at 80 degrees C. for 15 minutes survived the observation period of three weeks. Spore suspensions were prepared fron 15 day cultures in Difco brain heart infusion broth with BBL thioglycollate supplement added, *ofter four washings the cells were heated to 75 degrees C, for 30 minutes to destroy vegetative forms * and then diluted in sterile distilled water containing glass beads. The final suspensions were then shaken one hur on a rotary shaking machine to break up clumps and stored in the refrigerator. The efficacy of the honogenaticn prcce- ♦ dure was shewn by repeated comparisons of counts by plating procedures and direct microscopic counts using the Petroff-Hauser chamber. The former averaged about 50% of the latter, which nay be considered fairly good correlation. Other anaerobes used were C, chauvei. C, histolyticum. C, uerfringens. and the well-known food spoilage organism designated as putrefactive anaerobe no, 3679. The dormancy problem. The phenomenon of dormancy or delayed gemination has presented a formidable experimental difficulty which doubtless has been largely responsible for the lack of any really quantitative studies on s pore germination in C, brtulinum. Thus various authors report germination occurring only after incubation periods ranging from 53 days to 5 1/2 years I (Burke, 1919, 1923; Starin, 1924; Weiss, 1921; Sommer, 1930$ Dozier, 1924; Dickson, et, al, 1922, 1925; Dsty and Meyer, M-605 #4(1) Continued 1922; Dickson, 1928), Dormancy is net restricted to C, botulinum spores. It has been established for spores of other clostridia (McCoy and Hastings, 1928), for spores of aerobic species (Burke et. al,, 1925; hagoon, 1926; Morrison and Rettger, 1930a and 1930b), and even for cells of Bact, coli (Burke, et, al., 1925). Vegetative cells of C. botulinum have been observed to exhibit a degree of dormancy roughly comparable to that shown by the spores (Starin, 192/+), The prime requisite for systematic quantitative germination studies on nbotH spores is the complete elimination of the dormancy which has handicapped vir- tually all previous studies with this organism in this connection.. Acting on the belief that cultural environment probably conditions dormancy, we felt that the germination medium offered the best prospects for our objective. This had been shown to be the case for aerobic spore formers in which dormancy could be eliminated by supplying the correct medium (Morrison and Rettger, 1930a and 1930b; Curran and Evans, 1937). In recent years improvements have been mode in media which now give much higher counts than was possible with media formerly used and which indicates a high degree of success in eliminating the extremely long incubation period during which the spore count as measured by colony devel- opment would continue to increase. Pork infusion-*-• thioglycollate medium has been found to give considerably higher counts on a given spore suspension than other media ordinarily used for I, The following procedure for preparati n of this medium was kindly furnisl by Dr. J, Ycsair of Natirnal Canner’s Associations One pound of finely chopped lean pork is added to a liter of distilled water and boiled one hrur,. After removal of the neat and fat, the filtrate is adjusted to pH 7*4- and to each liter is added 5 gns, of peptone, 1,6 gns, tryptone, 1. .ga, dextrose, 1,25 gns, and 15 gns. of agar. It is our practice to nit the dextrose and add 5 gns, BBL thioglye:llate M-605 $4(1) Continued such studies, but no indication is available that maximum counts are obtained in short enough tine to be a useful ted for routine studje s (Brewer, 194-0$ Williams and Reed, 194-2), Even this medium culd be enhanced remarkably (30 fold) in the total count of heated spores obtainable after 21 days by the addition of 0,1 percent soluble starch directly to the gemination medium (Olsen and Scott, 194-6), Our work confirms both the superiority of pork infusion as a germination medium and the striking accentuation of starch on the germination process, and consequently on the spore counts. We have further demonstrated that starch acts primarily to adsorb and thus ronder inactive small amounts of substances present in all media which repress spore gormination. This study is the subject of a separate paper. Finally, the spore counting procedure ultimately evolved seem- ingly has eliminated dormancy as a practical obstacle in quantitative gormina- tive studies on wbottt spores and furthermore for the first time enables maximum counts to be '"btmined in an incubation period no longer than that required for sizable colony development of any anaerobe, namely 3 days. Comparison of several media popularly employed for counting C, botulinum spores-. One spore suspension heated to 7$ degrees C. for 30 minutes to destroy vegetative cells was serially diluted in triplicate in the various agar media in flat Prickett counting tubes and incubated at 37 degrees C, .n effective anaerobic seal was obtained by covering the solidified agar with 3 to 4 ml, of 2 percent agar containing BBL thioglycollate supplement, Prickett tubes are essentially flattened test tubes and, though not generally used, have the de- cided advantage of permitting colony counts to be made in a thin layer of agar ~ (cent*d,) supplement and 1 gm, soluble starch per liter,- We also adjust the final medium to pH 7,4-* The troublesome precipitate resulting on boiling the final medium may be removed by filtration under negative pressure or dis- carded after decanting the supernatant. M-608 Mil) continued instead of the entire diameter of a test tube. If maximum spore counts obtained in the pork-thieglycollate-starch medium are represented as 100, pork medium without starch gave 75, Difco brain-heext infusion 55-60, brain-heart plus 25% peptone (cf. Bristol 1925) 55, BBL anaerobic agar in Brewer anaerobic dishes as employed by Curran and Evans (194-6) 28, Difco livcr-veal 20 and Wilson and Blair'£ (1925) agar 5, Not only were counts consistently maximum in perk medium with thd glycollate-starch supplement but they occurred much earlier, reaching the peak in 4- days' incubation as compared to about 3 weeks for brain-heart infusion agar, .actually counts could be na.de on the second day but the colonies at this point are really Vo f.nall to count easily or accurately. Three-day-old colonies provide no difficulties. If dormancy exists at all in the pork-starch medium it is believed to be a negligible interference in quantitative studies. Counts, i.e,, colonies originating from germinating spores with C, botulinum have never been observed to increase appreciably on prolonged incubation up to 2-3 weeks whereas with of nor media the results arc meaningless before that time. Counts of spore suspensions have regularly been around 50+ percent of direct micro- scopic counts (Petroff-Hauscr chamber), a not too unsatisfactory correlation considering the tendency of the spores to clump in various degrees, and the fact that viability doubtless is net 100 percent, d special experiment to de- tect dormant spores (i,e., ungerminated viable spores) in this medium after 3 days' incubation failed to reveal any that germinated up to time of present writing, a period of 4. 1/2 months. Typical dormancy under these conditions manifests itself as gradually increasing counts rver the entire incubation period. Detailed proceuure for studying: gmrmduation of C, botulinum spores. The following procedures typify our approach to the quantitative study of the germination process and of factors influencing it, Une ml, portions of the appropriately diluted spore suspension were transferred to tubes containing M-605 #4(1) Continued 9 ml, of Difcc brain heart Infusion broth containing BBL thi glycollate supple- ment, This particular germination medium was chosen because the relatively moderate rate of germination occurring in it allows the study of factors both stimulatory and inhibitory to germination. The tubes were then heated to 75 degrees for 20 minutes to expel dissolved oxygen and to effect any possible Mheat activation” of the spores (cf, mvans and Curran, 194-3; Curran and Evans, 1945a), ..Ttcr appropriate intervals of incubation at 37 degrees C, in air or other at- mosphere, replicate (usually triplicate) tubes w.re re-heated to 75 degrees C. for 20 minutes tc destroy any vegetative cells which had developed as a result of germinati n. Residual spore counts were made as above in pork-thioglyccllate- starch agar, .available data indicate that germination is somewhat faster at 30 degrees than at 37 degrees; but for convenience 37 degrees was used in all these germination studies. Expression results. Most workers have utilized absolute numbers of residual spores as a basis for interpreting the effect cf a particular treatment on the gemination process. We feel that percentage gemination is tc be preferred as a more re- liable basis for interpretation of results, because of the large populations employed, and especially is this true when gemination is largely completef For example, on the comparative basis cf residual relatively small spore counts, Evans and Curran (1943) concluded that a considerable accelerati on of gemina- tion of aerobic spores had resulted from pro-heating the spores in glucose broth. If, however, the residual spores are considered as a fraction of a population and calculated as percentage of that population, the stimula- tory effect fer 4 out of the 7 positive cases w'uld be less than 6 percent and in one instance less than 0.1 percent,. Certainly the magnitude of the effect M-605 #4(1) Continued is much different when expressed percentago-wise, the cnay valid way, in our estimation, simple numerical example of this point seems worthwhile. Suppose a germination test is run under 2 treatments on a spore suspension containing 5,000 spores per ml, and residual spore count shows 100 and 200 per ml, res- pectively, While the 100 percent difference between the residual spore counts seems striking, the values for germinated spares are the design of the experi- ment and the more important data. Those would 4.,900 and 4,800 respectively, or 98 and 96 percent gemination, an insignificant difference in work of this •v nature. lieation to germination under stimulatory and under inhibitory treatments. Though germination curves may be employed for determining the effect of a given factor through'ut the time course of germination, the effect taken at any one significant incubatinn time is usually sufficient. If a stimulatory factor is being studied, the tine selected should be such that gemination is relatively small in the control in order to allow the treat- ment to manifest itself to the maximum, mn example is the effect of 0,1% soluble starch in the germination medium (brain heart bmth) shown in Table I, The spore counting medium was the usual pork-thioglycollate-starch agar. Un the '"ther hand, an inhibitory effect is best demonstrated at an incu- bation tine when gemination is nearly naxinum in the controls. Table 2 demon- strates that gemination in brain heart broth is considerably retarded by momen- tary contact with air during removal of sample tubes for counting from a desic- cator made anaerobic with on inert gas phase (natural gas, even though re-exhaustion with a Hyvac pump and replacement with inert gas is done without delay* In the unopened desiccator B7 percent of the spores geminated whereas in the desiccator opened briefly at 20 and 2A h urs only 29 percent geminati n M-605 #4(1) Continued was obtained — a striking inhibition. accuracy and reproducibility of counts. The degree of accuracy obtainable with the above method depends, of course, on the number of replicates used for determining the “average11 counts. For zero controls triplicate tubes were generally used, with triplicate dilution plated for each tube, or a total of nine counts. For other averages, triplicate tubes with duplicate or triplicate platings of dilutions were usually employed# The overall reproducibility and accuracy of counts on a C, botulinun spore suspen- sion stored in the refrigerator is illustrated in Table 3* .agreement between replicate counting tubes seems to depend on several fac- tors, including scrupulous chemical cleanliness of glassware, the presence soluble starch in the counting medium end the atmosphere in which gemination takes place. Factors conducive to variability. Considerable evidence has been accumulated that germination of “bet” spores is extremely susceptible to minute amounts of substances in the general category of impurities# As mentioned earlier these occur in all organic media and pos- sibly in tap water# **t any rate, a high of variation was experienced be- tween replicate tube counts a given dilution of the suspension until a rigor- ous cleaning procedure was adopted, .*,• marked reduction in variation fol- lowed when the cleaning was done with “Draft”, followed by thorough rinsing- vith distilled water. However, the best means of minimizing this tube to tube vari- ation proved to be the additional feature of incorporation of the starch. The adsorption effect mentioned above explains this levelling action of the starch. Finally, even though the cultivation of the gemination tubes in an atmes- M-605 #4(1) Continued phere of ordinary air gives growth, use °f an inert atmosphere of natural gas further reduced appreciably the count variation in replicate tubes. The above procedures work equally well with the four other anaerobic spore- formers tested. References Bristol, P, 1925 Growth of B, botulinus in 30% peptone. XXVII, With on Van Slyke’s amino acid method for study of bacterial metabolism, J, Infectious Diseases 26, 457-71, Burke, G; S, 1919 The effect of heat on the spores of Bacillus botulinus. Its bearing on canning methods* Part I, J, dm, Med, 22, 88-92, Burke, G. S. 1923 Studies on the thermal death tine of spores of Clostridium botulinum. 3, Dormancy or slow gemination of spores under optimum growth conditions, J, Infectious Diseases, £3, 274-284. Burke, V., Sprague, and Barnes, LaV, 1925 Dormancy in Bacteria, J, Infectious Diseases, 2*2, 555-60, Cook, R, P. 1932 Bacterial Spores, Biol, Revs,, 7, 1-23, Curran, H. R, 1931 Influence of osmotic pressure upon spore gemination, J, Bact,, 21, 197-209. Curran, H. K., and Evans, F. R. 1937 The importance of enrichments in the cultiva.ti n of bacterial spores previously exposed to lethal agencies, J. Bact j. 3J±i 179-189. Curran, H, R,, and Evans, F, R. 1945a Heat activation inducing geminati-n in the spores of therraotolerant and thermophilic aerobic bacteria, J, Bact,, 42, 335-346. Curran, H, R,, and Evans, F, R, 1945b Penicillin as a Spcmcidal Proc, See, Exptl, Biel. Med,, £8, 262-5. Curran, H. R. and Evans, F. R. 1946 The activity of penicillin in relation to bacterial spores and the preservation of milk, J, Bact,, £2, 89-98, Dickson, E. C, 1928 Dormancy or delayed gemination of spores of Clostridium botnljnum subjected to heat, proc, Soc, Exptl, Biol, Med,, 2£, 426-/, Dickson, E. C., Burke, G. S., Beck, D., Johnston,'!., and King, H. 1922 Stu .ies on the thermal death time of spores of Clostridium botulinum. Preliminary report,J.dm, Med, iissoc,, 72, 1239-1240, M-605 #4(1) Continued Dickson, E. C,, Burke, G, S., Beck, D,, and Johnston, J, 1925 Studies on the thermal death time spores of Clostridium botulinum, IV, The resis- tance of spores to heat and the dormancy or delayed germination of spores which have been subjected to heat, J, Infectious Diseases, £6> 472-483, Dozier, C, C, 1924 Resistance of spores of B. botullnus to disinfectants, XVIII. J. Infectious Diseases, 156-176. Ackelmann, E, 1918 Ueber Bnkterien, welche die frakticnerte Sterilisation lebend ueberdauern, Zentr, Bakt, Parasitenk,, II, 140-178, / Esty, J. K,, and Meyer, K, F, 1922 The heat resistance of the spores of B, botulinus and allied anaerobes, XI, J, Infectious Diseases, 31: 650-663, Evans, F, R., and Curran, H, R, 1943 The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria, J, Bact,, £6, $13-523, Fischooder, F, 1909 Beitraege zur Kenntis des Milzbrandes lentr, Bakt, Parasitenk., I, Orig,, £4, 320-417, Itano, A., and Neill, J, 1919 Influence of temperature and hydrogen-ion con- centration upon the spore cycle of Bacillus subtilis. J, Gen, Physiol*, 1, 421-428. Knaysi, G. 194$ Investigation cf the existence and nature of reserve material in the endospore of a strain of Bacillus mvcoides by an indirect method, J, Bact,, 42f 617-622, Knaysi, G,, and Baker, R. F, 1947 Demonstration,with the electron microscope, of a nucleus in Bacillus mvcoides grown in a nitrogen-free medium, J, Bact,, 52, $39-$$3. Knight, B. C. J, G., and Fildes, P, 1930 Oxidation-reduction studies in re- lation to bacterial growth. III, The positive limit of oxidation- reduction potential recuired for the germination of B, tetani spores in vitro, Biochem, J., 24, 1496-1502, Magoon, C. A, 1926 Studies upon bacterial spores, 2, Increasing resistance to heat through selection, J, Infectious Diseases, 38, 429-439, McCoy, E,, and Hastings, E. G. 1928 Dormancy of spores of Cl, acetobutyli- cum and Cl, pasteurianum. Proc, Soc. Exptl, Biol, Med,, 753-754, Morrison, E, W,, and Rettger, L, F. 1930a Bacterial spores, I* A study in heat resistance and dormancy, J, Bact,, 20, 299-311, Morrison, E, W,, and Rettger, L, F, 1930b Bacterial spores, II, A study of bacterial spore gemination in relation to environment, J, Bact., 20, 313-342. Ulsen, A. M., and Scott, W, J, 1946 Influence of starch in media used for the detection of heated bacterial spores. Nature, 1571 337* M-605 #4(1) Continued Sommer, E. W, 1930 Heat resistance of the spores of Clostridium botulinum, J,Infectious Diseases, 85-114. Starin, W. A, 1924 Puro cultures of Cl, botulinum from single cells, J, Infectious Diseases, 24, 148-158. Swann, M, B, R, 1927 On the germination period and mortality of-the spores of Bacillus anthracis. J, Path; Bact,, 27, 130-134. Weil, R, 1901 Arch, Hyg,, 36, 205 et, seq. Cited by Fischoederi Weiss, H, 1921 The heat resistance of spores with special reference to the spores of B, botulinus. J, Infectious Diseases, 28, 70-92, Williams, U. B., and Reed, J, M, 1942 The significance of the incubation temperature of recovery cultures in determining spore resistance to heat, J, Infectious Diseases, 71, 225-227, Wilson, W, J., and Blair, E, M, McV, 1925 Correlation of the sulphite reduc- tion test with other tests in the bacteriological examination of water, J. Hyg,, 24, 111-119. M-605 #4(1) 13 Continued Table 1 EFFECT UF STARCH UN GERMINATION Starch Incubation, hours Av* Count Residual Scores Germinated Scores % Germination - 0 575 - - - 2A 4.00 175 30 0,1% 2A 60 515 90 Table 2 EFFECT OF MOMENTARY CONTACT WITH .1R ON GERMINATION IN Ni.TUH.di GAS Incubation hoi rs Av. Count Residual spores Germinated snores Percent germination Desiccator 0 560 unopened 2i 74 87 Desiccator opened 0 535 .... twice to remove samples at 20 and 24 hours 28 380 155 29 Tabic 3 REPRODUCIBILITY OF SPORE COUNTS UN A SINGLE SUSPENSION Date of Count Spores per ml. 3/fo 535 3/15 560 3/27 540 4/2 575 A/9 530 A/20 560 4/20 520 A/2A 600 A/27 500 A/30 535 5/22 540 6/1 580 4v erage 548 Extreme deviation from mean: 50 = 9?o 543 M-605 #4(1) 14 PROJECT REPORT xseu&sas Atm izvzLetmm COMMITTEE ON FOOD fi£8EAtt£N oilitait plakkiho division U •>. QUARTERMASTER FOOD AKO CONTAiHER INSTITUTE FOR THE ARMED FORCES OFFIC* OF TUI CHICAGO ILLINOIS _ QOABTXXMASrSS OXMBBAX. TM?TSnPTnf4 sWstbrufiON " " " ’ |LbcaEi"tV a ——— University of Texas Austin, Texas THTsTOfl DEPARTMENT Arte and Scipmces Bacteriology OFFICIAL INVESTIGATOR COU AROR ATO RS J, W, Foster E, Staten^Ijfnne FILE NO. M-6'05 / (cORTRACf'RO. 1 _________ V W-ll-009-qm-70190 Mft c6 I *16 Ini Tl ATI OR DATE . . tec. 1$, 1946-April 15, 19A7 1946 T,Tt£t [ I RIFORT T i FNASC REPORT (x ] ANNUAL REPORT [ JteRMIRATI ON REPORT Spore Formation and Spore Germination of Anaerobic Food Spoilage Organisms, Especially Clostridium Botulinum. The germination process of spores of G, botulinum 62A is logarithmic. In air atmosphere the length of the lag period in gemination varied # i inversely with the logarithm of the number of spores per cc, in the inoculum. These relations are expressed mathematically. * This report is one of four, to be numbered 4(l), 4(ll), 4(lll), and 4(lV), The four reports will comprise the annual report on this project. 1 Continued '2-121 Physiological studios on spore gemination with special reference to Clostridium botulinum.* II* Quantitative aspects of the germination process. E, Staten Wynne and Jackson W, Foster Dept, of Bacteriology, University of Texas, Austin, Though virtually eveiy aspect of the growth and death rates of bacterial cultures has been subjected to searching kinetic analysis, the spore gemination process itself has been largely neglected. Probably this is due particularly to the failure to discriminate precisely between the actual gemination process and the subsequent vegetative development and, especially with anaerobes, to inade- quate counting methods. The only two reports really bearing on this issue, both dealing with aerobic spore formers, failed to evaluate the process other than to conclude that numbers of spores geminating increased with time (Fischoeder, 1909; Eckelmann, 1918), This paper deals quantitatively with the germination process in the anaerobic spore former Clostridium botulinum strain 62A, The germination medium was Difco brain heart infusion broth with BBL thio- glycollate supplement. The counting medium, procedures and other details are as described in the preceding paper (Wynne and Foster, 1948)* It was observed repeatedly that the logarithm of numbers of residual spores in a spore suspension in a germination medium plotted against tine gives a straight line, at least until 95 percent or more of the originally present spores *This project has been undertaken in cooperation with the Committee "'n Food Research of the Quartermaster Food and Container Institute for the Ar F'rces, The opinions or conclusions contained in this report a those of the authors. They are not to be construed as necessarily reflecting the views or endorsement % of the War Department, M-605 #4(11) 2 Continued have geminated; Data for a typical experiment are given in Table 1, and they ore plotted as Curve A in Fig; 1 together with Curves B and C, the latter two representing ' " ■ i *■! •* l ■ experiments in which the geWnination was allowed to take place in airf a« Cbn- .. f.; - * \ , j , ’ “ trasted to the natural gas atriosphote in the exjpoirimeni described by Curve A and Table 1, The general equation for a first order reaction may bo written as ,434K * 1/t log —I— (1) I - G where K « a constant t » time ©lapsing since beginning of germination (tc) I - no, spores per cc, at beginning of germination G » germinated spores at time t But since I • G » R (residual spores), wo may substitute H in the equation above giving, .OAK - 1/t log l/R (2) or K = (log I - log R) (3) ..430. The I values in Table 1 were computed assuming t0 *= 20 hours, which obviously is a minimum value, as under the experimental conditions t0 could have been any time between 20 and 22 hours. Owing to this experimental inaccuracy the K value for the 22 hour period is off, but is reasonably constant for the other periods, indicating that the germination process conforms to a first order reaction. Curve A, Fig. 1, shows also that the germination process is logarithmic, thus con- forming to the kinetic picture typical of all growth and killing rates of bac- teria, (Curves B and C) It is obvious from Fig, Ijthat the duration of the lag period depends on the concentration of the inoculum and is inversely proportional to it. By extrapo- lating the logarithmic germination curves B and C in Fig, 1, as well as others, values representing the length of the lag period were obtained according to the inoculum density as given in Table 2, The length of the lag period appears to Continued M-605 #4(11) vary as the reciprocal of tho logarithm of inoculum numbers, and obeys the follow- ing relations L = C log I M where L = length of lag phase in hours I = no, of spores per cc. in inoculum C = a constant. The validity of this expression is borne out by the fact that the values of —£— plotted against L made a straight line (Fig, 2): computed values for C at log I the different levels of inoculum fairly well, as seen in Table 2, These data were obtained from cultures incubated in ordinary air. Curiously, these relations did not apply when incubation was done in a desiccator with an atmosphere of natural gas, at least under those conditions. Discussion Neither Fischoeder (1909) nor Eckelmann (1918) drew any conclusions per- taining to the kinetics of the germination observed by them, but a plot of their data against time shows they actually were concerned with logarithmic germination processes. However, data of the former author for germination of Bacillus anthracis in goat and dog sera indicate that germination was not logarithmic un- der those conditions. It is obvious that any factor inimical to germination may at once rule out the logarithmic relation. The tentative ecnation suggested above for the length of the lag period obviously should be tested further with more data. If this equation is valid, plotting the length of the log phase against logarithm of inoculum directly should give a hyperbola, since (L)(log I) - C, Though such a plot from the values determined above is compatible with a hyperbolic curve, the points are too few to define the curve clearly. M-605 #4(11) Continued In contrast to the paucity of information on the lag period of spore ger- mination on abundance of work has been done on vegetative cells, but as spore germination does not involve actual cell multiplication we consider the spore problem one distinct from vegetative activity. The apparent failure of equation (4.) to hold in an atmosphere of natural gas (i.e, no oxygen) cannot be explained at present. Suspicion might be directed to influence of the U-R potential on germination, an effect found to apply to spores of the anaerobe Bacillus tetani (Fildes, 1929j Knight and Fildes, 1930). Rapidity of germination depended on the time required for the medium, and presumably the interior of the spores, to roach a suitable reducing intensity, and spore numbers might influence this ac- tion, Finally, natural gases may contain traces of impurities which might ac- count for this discrepancy. References Eckelnann, E. 191B Ueber Bakterien, welche die fraktionerbc Sterilisation lebend uebcrdauern, Zentr, Bait, Parasitenk,, II, 14-0-178, Fildes, P, 1929 Tetanus, VIII, The positive limit of oxidation-reduction potential required for the germination of spores of B, tetanl in vitro. Brit. J, Path., 10, 151-175. •> Fishoeder, F, 1909 Beitraegc lut Kenntnis des Milzbrandes, Zentr. Bakt, Parasitenk,, I, Urig., £1, 320-4-17, Knight, B, 0, J. G,, and Fildes, P. 1930 Oxidation-reduction studios in rela- tion to bacterial growth. III, The positive limit of oxidation-reduction potential required for the germination of B, totani spores in vitro. Biochem, J,, 14-96-1502, Wynne, £, Staten, and Foster, Jackson W, Physiological studies on spore gemination with special reference to Clostridium botulinum. I. Develop- ment of a fuantitativc method, J, Bact, In press. M-605 #4(n) Continued Table 1 Incubation hours Gemination in Natural Average Count Geminated Residual Spores Spores Gas % Log Resi- Germination dual Spores K calcu- lated for to=20 hrs 0 560 2.75 20 560 0 0 2.75 22 230 330 59 2.36 .449 24 200 360 64 2.30 .259 #28 74 485 87 1.87 .254 30 39 520 93 1.59 .267 32 28 530 95 1.45 .250 # Counts for 26 hours are not given due to accidental overheating of the tubes : in the incubator. Table 2 Relation of Duration of Log to Concentration of Inoculum in /dr Inoculum. Sporos/cc Lae Period. Hours C. calculated 2400 28 95 2100 28 93 530 36 98 340 40 101 * Equation (4-) M-605 #4(11) Continued Lo c Residue L. Spores INCUBATION TIME IN HOURS Curve At Natural gas atmosphere* Curves B and Cs Mr Atmosphere FIG, 1, GERMINATION CURVES OF C. BOTULINUM 62A LENGTH OF L~G PERIOD IN HOURS FIG* 2. RELATIONSHIP OF LENGTH OF LaG PHASE OF GERMINATION TO RECIPRUC.*L OF LOG OF NO, OF SPORES PER CC, IN INOCULUM M-605 #4(11) 7 PROJECT REPORT ksseaicv and development branch COMMITTEE 00 FOOD RESEARCH MUWWMMmHl |/<> QUARTERMASTER FOOD AMO COMTAIMER I NSTITUTE FOR THE ARMED FORCES OFFICE OF THE CHICAGO ILLINOIS . QUA IT EM AS TEE GENERAL TWPHATIIU nrsrTfutiftM — OcuTty University of Texas Austin, Texas 01 VI SION ~ DCF ARTHCMT arts and Sciences Bacteriology OFFICIAL INVESTIGATOR * — COLL ADO RATO NS “ J, W, Foster E. Staten Wynne RCFORT MO. FILE HO. / IcOH TRACT" NO. 4 (III) M-605 / W-;.l-0C9-qm-70190 INITIATION DATE A )ril-November. 194-7 . 1 July 1, 194-6 T,TLE: I ] PROGRESS REPORT [ i PH A S E RCFORT [ ] ANNUAL REPORT’ [ jTERMINATtON REPORT X •Spore Formation and Spare Germination wf Anaeorbic Spoilage Qcflnni.msM,F.apl,r.1n11,y Si Ort»itu..ni botulinum I SliMMABY Carbon dioxide has been shown to be essential for germination of the spores of Clostridium bctulinum 62 A in a synthetic medium; CU2 could not be replaced by a mixture of malate, fumaratc, succin- ate, glutarate, aspartate, glutamate and cis- aconitate; 1% yeast extract was found to replace CUo qualitatively, at least; It is concluded that hitherto unrecognised substances of unknown nature are necessary;for bypassing the COp requirement of germination id this organism, / In a co mplex medium Ce-0 was only stimuJ atory, and could be re- placed completely by oxalacetate, as well as partially by a mixture Of the stable dicarboxylic acids, GOr could not be shown to exert any effect on germination of the spores of A other anaerobes or A aerobic species in complex media, but termination in one of the lat- ter was significantly stimulated by the stable C/ dicarboxylic acids. 1 CQM0 FORM |G«|G| 5 Ap P I I 46 •*•1*1 Continued Physiological studies on spore germination, with special reference to Clostridium botulinum* III. Carbon dioxide and germination. Also a note on CU2 and aerobic spores £• Staten Wynne and Jackson W, Foster From the Department of Bacteriology, University of Texas, Austin The requirement of CU2 for vegetative cell development of b? ctcria is com- mon knowledge and needs no review here, but scarcely anything is known of the relation of to the process of bacterial spore germination as considered distinct from subsequent vegetative development. One might consider the latter in the sense of transition from heat stable to heat labile form. (Wynne & Foster, 1948a), Relevant is the incidental observation that spores of one cut of 3 strains of C. botulifaum failed to produce colonies in 72 hours when incubated in a vacuum (Morrison & Retiger, 1930). The experience in a rather common one that special efforts to eliminate CO2 from the culture system, and to minimize the formation of CO by the cells in the inoculum by supplying a low nutrition level medium a retardation of growth which may extend indefinitely. our study of factors doterm: nant in the germination process itself (as dis- tinct from subseque t vegetative development) of Clostridium botulinum begun in two previous papers (Wynne t. Foster, 1948a and b) has included examination of the CO ef-.cct. This stems from the finding that anaerobic sis secured by alka- line pyrogallcl seems to delay germination of ubotM spores. Background informa- tion and general methodology are covered in the first of these papers and need not be reiterated here. To secure anaerobic conditions free of vacuum desic- cators containing the culture tubes were evacuated with a Cenco Hyvac pump and refilled with natural (illuminating) gas (CH,) cleansed of CO2 by slow passage through a gas washing train consisting of 3 pottles of NaOH and one of N/10 Ba(UH)2* The latter was second last in the chain, functioning as a CU2 indica- tor, As an added precaution normal NauH was always placed in the bottom of the desiccator. Where a CCu atmosphere was required, it was added from a cylinder or generated in the desiccator by mixing excess acid with the calculated amount of solid NaHCO-3, Unless otherwise specified germinal ‘on always took place in Difco brain-hcort infusion broth with BBL thioglycollate supplement, and always the inoculum was about 500 spores per ml, of medium. Table 1 compares the sp 're gemination in atmospheres c staining 0, 1 and 5 percent Clu respectively. The CO effect is striking. Germination is negligible in the absence of GO2$ whereas almost all the spores germinated in the presence 'f COq* the higher tension buing somewhat better. The difference between the two GO2 treatments actually was greater than it appears; turbidity developed in 15 h-urs in the 5% GO2 desic- cator and in 19 hours in 1% (X>2* No turbidity appeared in the zero C02 control *This 'project has boon undertaken in cooperation with the Committee on Food Research of the uartermaster Food and Container Institute for the Armed Forces, The opinions or conclusions contained in this report are those of the authors. They are not to be construed as necessarily reflecting the views or endorsement of the War Department, M-6Q5 M (IIX) 2 Continued at 22 hours, the teminatic.. cf the experiment. However, a CU2 effect cculd net be obtained for f°ur other species cf ana- erobic spore formers tested similarly: Clostridium ch alive i. C. hlstolyticum. C, perfringena; and the well known food spoilage organism designated as putre- factive No, 3679. This was true even at pH 6,0, chosen to reduce the solubility of CU2 in the medium and being the 1'west pH supporting germination of these anaerobes. Thus under identical conditions germination of C. botulinun spores is inhibited by lack of CU , and germination of the other four anaerobes is not., A possible interpretation of this is given in the discussion below. The Cj3 xperijneafdescribed above (else Table l) demonstrates only a rate eifect under the conditions used, for whereas only 7 percent germination occurred in the dm free control at the 22 b ur peried, prolongation of incubation always resulted in high germination and pronounced turbidity. Failure to demonstrate an absolute CO2 effect in brain-heart medium, oven with several painstaking ex- periments involving modification of pH, exhaustive pumping, omission of the col- loidal agar cf the anaerobic supplement, continuous gassing with N2, etc,, was considered on two counts as bein- most likely tied up with the complex nature of the brain-hwart mediums (l) the effect con be accentuated by eliminating complex media in favor of synthetic (Gladstone, Fileos and Richardson, 1935) or employing complex media at a minimal nutritional concentration, i,cf, with re- spect to carbohydrate and protein content (Rockwell and Highberger, l926 and 1927), Count (1) may really be considered to anticipate the issue of count (2) which states that CO2 should be dispensable so long as certain organic substances are present in whose synthesis CU2 participates. The presence of such substances is likely in complex media of biological origin and in such media therefore the need for should be obviated. While our attack on these lines was under way other reports have appea ed which confirm the 1-gic of this approach, (C4 dicarboxy- lic acids, White and Workman, 194-7; aspartic acid, Lardy, ot cl, 1947 and Lardy, 1947.) A synthetic medium similar to that devised by Roessler-** (1946) for growth of 0. botulinum was used as a starting point, but with only 1/10 the regular concentration of amino acid & • (Results were similar, however, with the full me- dium, which contains 1% amine acids.) This media supported abundant vegetative development of our strain of C. botulinum and spore germination was much slower than in complex media, seemingly opening an approach t- factors essential for germination, including those involving CP2 &nd not, *cr good anaerobic growth it was expeditious to add 0,2% glucose V the synthetic media, as gcrmimtl-n is negligible in its absence. With an inoculum of around 500 spores per ml,, clear cut turbidity devel- oped in the presence of CO 2 at about 72 hrurs but counts at 87 hours showed that only abmit 15% of the sp- res had germinated. In subsequent work a 5-day incubation period was employed for positive CU2 controls, for in this time ger- mination counts were well ever 50 percent. In such a medium it is possible to approach an absolute Cu2 requirement fr-r germination, Thus in an experiment 1 This medium had the f lie wing n: dl-leucine, ,008314.; dl-ph-jnyl~ alanine, .0132M; 1-arginine, .0065M; dl-valine, -.008314; ctl-isolcucD.no, ,00AM; 1-tryptophane, 0.11M; 1-tyrcsine, .0003M; dl-ni'6thi'nine. ,002M; dl-throcnino, .OObTM; ,Q1M; l-histidino, ,0C13M; bi tin, 5 nX/cc; PABA, .02 X/cc; nicotinamide, 1 X/cc; thiamin, 0,2 X/cc; yeast nuclcjic acid, ,01Na thiogly- ccllate ,05%, .0002M; MnSU/, .000114 CaCl2, ,0001M; FoSU,, .00005M; K2HP0;, ,015M; KH2 #015M. M*605 #4(III) Continued whore the pc sitivo (1%) C02 control showed 61% gcrminati n in 5 days, the C02 free treatment showed only 11% germination and no turbidity even after 17 days (Table 2),. Indeed, thw figure of 11% nay not bo significant at all owing to the fact that the spore counting method has an overall accuracy of + 9% and occasionally with spreads wider than this, (Wynne & Foster, 1948a), Under those conditions clear cut turbidity always follows gemination within a few hours. Thus probably no gemination at oil occurred in the C02 free tube, the 11% value doubtless being an experimental counting errr, It scons safe, therefore, to c ncludo that CW2 is absolutely essential for spore germination of C, betulinum in a medium otherwise adequate for that ; re- cess, This apparently is the first demonstration of Co2 requirement specifically for the germination process, and apart from subsequent vegetative development* By-passing COo, Uxalacetic acid — /long the lines discussed in count (2) ab- vo the dicarboxylic acids wore tested for their ability to permit gemination in the absence f CUp, as the universality of the W'"d-Werki":nn reaction via pyruvate fixation of CO indicates the likelihood of their being involved hero. The pri- mary'- fixation product, cxalacetic acid (Oil), is generally in biological equi- librium with malic, fumrric and succinic acids, all vital catalysts or intermedi- ates in cells, O/i in brain-heart media definitely promotes the gemination rate of bot spores in the absence of gaseous CO*-, (See Table 3) and apparently bypasses Experiment 1 in Table 3 shrws that the oil induced spore ger- mination at a rate appreciably faster than a 1% CO. gas tension, and in Experi- ment B it was equal to the CO in promoting gemination. In experiment B the chances arc that 0**** also would have shown up a faster germination had the counts been made at a shorter incub ticn period. , The 04u effect night, to a certain extent, be ascribed tc CO2 resulting from the spontaneous decomposition of in*** tc G0o and pyruvic acid (Krampitz and Work- man, 1941| Krebs, 1942), in solution at 37° has a very short half life and its decomposition is catalyzed by amine groups and by traces of cationic metals. However, since Oi*u gives a gemination rate, exceeding that of CO2 the effect seemingly is due tc the U-u* per so, though 0U9 may contribute tc the rate par- tially, Gemination by (J.U wr-s n-1 retarded when the medium was continuously exhausted with a Hyvac pump for 4,5 burrs after addition, the idea being tc remove quickly any CO2 generated fre u*U, (hxp. B, Table 3) * no lessening of the effect by this continuous CO2 removal was observed the probability of a direct 0**4 participation seems good,. Maybe a brief contact with such as would ccur in the above pumping ex- periment, would suffice for germination, but other experiments sh wed that con- tact with a 1% C02 atmosphere for the initial 4 h urs followed by removal (Hyvac) and replacement with C02 free gas had an insignificant effect on gemir^tion. Stable C/ dicarboxylic acids.— .* rixture ' f 1-malio, funaric and succinic acids (Na salts), each at a c naant ration of 3,3 x M was shown repeatedly t have a definite acceleration on gcminati-'n rate in the absence of CUp, Those acids wore nr't as effective as C02 ( r 0*1) in promoting go min tin, T'hc efficacy'- of these acids in promoting germin'tin was ro ughly abrnt 1/3 that of a 1% C02 gas phase. The inability of the acids t substitute fully for o.*._ has been encountered provi usly (Shiva ana R ;rs, 1947, and there) and probably relates to membrane penetration at pH values in physiological range, in which these acids Continued M-605 #4 (III) are almost 100 percent dissociated. It will be recalled that U,*u itself decs net penetrate unaltered cells of Micro coccus (Kramptiz and Wcrk- nann, xeu), end several "'ther examples c°uld bo. given. If these acids diffuse in the n locular (undissociated) forn as the fme acids, it would be ejected that diffusion w'ued be greatest at pH 3 to 4 as the acids are elm st entirely in in locular fr m in this range as c-ntratted to a negligible percentage nt i'H 6 "r above. It was not possible to test this; with botulinum as gemina- tion is inhibited at pH values below 6, It will be recalled that White and Workman (1947) found that the aicarboxylic ? cids r their respiratory pre- cursors bypassed the C0o requirements for colifom bacteria. The specificity rf the effect for the 0/ dicarboxylic acids on bot gemina- tion is exemplified by the fact that no demonstrable acti-n was given by glutarate, glutar te, valerate, butyrate, propionate, lactate or pyruvate, Un •the other hand a striking stimulation in vegetative development was induced by all of these acids (except pyruvic) at 10" 3 M, So narked was this that cultures with well advanced turbidities showed surprisingly snail germination percentages. This is a fine example of the fallacy of judging germination rates by the inten- sity of vegetative turbidity. *.sp rtic acid, — .*s u.U .is converted to aspartic acid by transaninati n, one night expect this ml no acid also wr uld bypass the C0p requirement, the latter participating in the synthesis cf aspartate.. This has indeed been demonstrated for Lactobacillus arabin; sis (Lardy et al, 1947s and Lardy, 1947) in which case aspartate is apparently the only constituent of cell material in whose synthesis CUp participates, excepting perhap s for relatively insignificant amounts of other components. This was proved by isotopic CXp, substantially the entire con- tent of the labelled C in the cells being in the carboxyl groups of the cellular aspartate. It is likely that the other dicarboxylic acids are c averted to aspartate via luA. xhe b°t germination tost was conducted in Rcessler's synthetic medium (1/10 strength amino acids)which, as a basal medium, lacked NaHCUo, biotin, and aspar- tic acid. The f Hewing treatments were set up in triplicate tubes of the basal medium: (1) CU2 free gas phase (zero control) (2) 1% gas phase (3) $ m% biotin/nl in CU2 free gas phase (4) 5 ma bi'tin/ ml. in 1% CU2 gas phase (5) 10” 3 and 10”Caspar!ate respectively in CUp free gas phase (6) 10“ 3 and 10” sp art at e respectively plus 5 rj( biotin/nl* in GU2 free gas phase - (7) 10-3 M and M 0(-koto glut arat c respectively plus 5 biotin/nl, in CU2 free g s phase (B) 10”3 M aspartate lus 1 mid 10 nj cleato/ml. respectively in CU2 free gas phase . pleate was touted because of its known bi tin sparing acti n, andO(-kotoglutar- ate because conceivably it could generate dicarbnxylic acid precursors of aspartate. ..ft. r 4 bo 5 days incubation, turbidity commenced in only treatments (2) .nd (4), namely those with 01*2 in the gas phase. Spore counts :.iade of these on the 7th day s hr wed 601 gemination. The remaining 6 treatments shewed no tur- bidity up to the 17th day, when they were removed for spore counts. In every case spore gemination was nil nr negligible and it nay be concluded that the substances used, and in the c robin tion tested, could not by-pass the requirement for CUo, M>605 #4 (III) Crntinued In the expcrim.-nt biotin, aspartate, cleate were tested all in ~nc mix- ture, in the or nesntration ranges as before, except that 100 n tf/cc of ole at a was also tried, and again gemination took place only in those treatments with a c nt pining gas phase, both with and without the tost supplements, whereas gemination was insignificant in the absence of Cug even after prolonged incuba- tion (14 days)* Finally, the following known or suspected brassing substances and available participants in the tricarboxylic acid respiratory system were tested in combi- nation, all at in basal synthetic medium, in the ; rosencc and in the ab- sence of as: art at o, mediate, furmrafco, succinate, glut orate, glutamic acid, glutaratc, and cis-aconitato. These w:.re entirely unsuccessful in by- passing OU2. When was present, the gorr.iinr.ticn rate was unaffected in this medium, indicating nr* toxicity caused by the supplements. Complex Sue, laments <— .Use, the following complex organic supplements wore tested in triplicate tubes at 0,1 and 1.0% levels in^the basal synthetic medium, gain in the absence and in the presence of CU?; brain ho aid infusion, liver and yeast extract, all Difco, The free yeast and liver treatments incub ted in 'no desiccat r, the CU2 free brain-heart in another, and the CU2 free synthetic medium in another. Within 40 hours in the absence of CU2 oil the yeast tubes developed rked turbidity and a single 1% liver and a single 1% brain heart. To av id c ntaminating the other tubes with fermentation CO2, those turbid tubes were removed, pasteurized and held for spore counts, **11 the tubes of synthetic medium in GU showed turbidity at 3 to 4 days and were removed for counting on the 5th day, a he C0o free synthetic medium showed no turbidity even after 15 days, the terra:notion of the experiment, and the remaining liver and brain-heart tubes in free atrmsphmre behaved similarly. Residual spore counts for this experiment arc in Table 4* It is clo2 free treatments were conducted in desiccators with air as the gas phase. In no case was it possible to retard germination of these organisms in a Cu0 free atmosphere. Evidently this is due to the presence in the nutrient broth of organic substances bypassing the CU2, although no at- tempt was made to confirm this with synthetic media. Interestingly enough, though CU2 did not enhance the germination of any of these four aerobes, in one, B. mesentericust the stable dicarboxylic acids mixture (3,3 x M each) 2 extract pf 0.1 and dried liver M-CG5 #4 (III) Continued distinctly accelerated the germination. Thus in a typical experiment with an in- oculum or 3CIO spores per ml,, 29 percent germination was obtained in the GL»2 free treatment :fter 2$ hours, and 71 percent in the C, treatment. This organism presumably was inefficient in the conversion of CO2 dicarhoxylic acids. The behavior of vegetative development of each cf these a-;;robes in relation to Cup is in decided contrast to the spores, for in each case Gu induced a marked acceleration. This again emphasizes the distinction between tne gemi'.-ti-m pro- ccyf and subsequent vegetative activity of sp or oforifling bacteria. ■Discussion The:- germination process and vegetative cells are not affected alike by CX»2 and the dicarbcxylic acids. The fact that germination in four out of five anaerobes tested failed to respond to Cu2, whereas C. bet- linum did, indicates species or strain d, ffercnces, This applies also to the differences described for the four aerobic spore formers. .1 clue to the nature, of these effects comes from the fact that in complex media (i.e., brain-he-art infusion) Cup deprivation on„y slowed d wn the rate of germination but die not step it, whereas in synthetic medium germination could be entirely suppressed Indefinitely without CGp, Judging from the evi once avail- able, this could mean that present in complex media are substances as yet unknown which can bypass CUp, Some such substances are known (vide supra, G, dicarboxylic aqids and asp artic acid) but these c'uld not substitute for in the germination cf bet spores in a synthetic medium which is otherwise ad quote for gemination and growth. Does tbi; moon, then, that present in complex natural, material s are additional new substances capable cf bypassing the OU2 requirement, and in whose synth et is CU„ part icy at os when they are net supplied artificially to the medium? Seemingly the positive germirrtion results obtained in a Cup free system upon ad- dition of small amounts of yeast extract to t <. basal synthetic medium, speak in this behalf, fter t'is vrk was completed the recent r.port f -nd lonod (1947) was received. Those ruth?re, working with cfli#found -end Cr% dicer- bcxylic acids ; nd their amino dcriv t; vos t" be effective OUp bypassing agents, but that they lai. did net suffice, and they came to exactly the seme oonclus ns as above; namely, 'ther essential GUp bypassing agents ar present in complex naturai.mat rialt, uno m y cosily visualize thot Gu:, is involved in synthesis of bi*logical substances oth r tnan 0, and Cc acids aid t.b.„ derived aspartic and glutamic acids, and, indeed, at l.a.st no ot]~Xif system already is known, viz,, crrb'u-.yl t?'n of C>4-ketcglutaric acid to oxalsuccinie read (echoa, 1945), others are under sus- picion, and new '\ics .ot only are a distinct possibility but on the basis of the above evidence must exist. Variati'ns in rcspouse t■* 0, dicarboxylic acids mean tint there arc required to different degrees by different organises. Thus, the response by e, b tulinum in the complex . edium to adeed G. acids indicates teesc were limiting op near limiting in gGrirj.inr.ticn, Complete lack cf response to Cu... b; the other Clcstri ;..ia indicates that whatever bypassing agents (presumably including the acids) were present were sufficient to bypass kX>2 entirely. Similar difcrences shrwod up in the aerobes; though removal of Gup did not retard y,mination in any cf the four species, C, acids significantly stimulated gomin tion in h. ms ntgrievs and were therefore limiting oven in the presence of It is possible tht the acids a ay fully b 'pass Dtp in this organism. If acids play a r 'lc in the by- 7 Continued M-*6}5 #4 (XI I) passing cf in the other three aorcbes, the concentration present in nutrient broth must be adequate, though other substances m y be involved. The main cone vision obtainable from all these observations is th -t organisms differ widely in the extent t* which medium components enable them to bypass their Co2 requirements and that some hitherto unrecognized (Xm bypassing substances ex- ist. A corollary is that a complete diet cf organic compounds renders dispen- sible for germination and initiation of growth. References ijl, S, J., white, 4, G, C., and Werkman, e. H. 194-7 Replacement of Carbon dioxide in hot ere trophic metabolism, J. Bact., £4, 23, Gladst ne, G. P,, Fildes, P,, and Richardson, G, M. 1935 Carbon dioxide as an essential factor in the growth of bacteria, Brit, J. Lxptl, Path., 16, 335-348. Kramptiz, L. u„, and Werkmann, G. H. 1941 The enzymatic decarb xylation of oxa- lacetate, Birchom, J,, 595-602, Krebs, H. 194-2 The effect of inorganic salts on the ketone do imposition of oxaloacetic acid, J., 36 , 303-305. H. a,. Potter, R. L, and Uvejchm, C. 1947 The rrle of biotin in bicarbonate utiliaati n, J. Chem,, 169, 451-452'. Lardy, H. ... 194-7 The role ~f biotin in the bi chemical fixation of carbo dioxide, of papers, 112th Meeting of .jp, Chcm. S^c., 43c, Lvrff, , and Mo nod, j, 1947 Essai d’analyse du role de 11 .'anhydride carbcnique dans la croissancc jaicrobieimc, .nn, inst, Pasteur, 73 , 323-347, Irrrison, B, W,, .and Rettger, ... F, 1930 Bacterial Spores. 11, .. Study of bac- terial spore germination in relation to environment, 4. Bact, 20; 311-342, y uohoa, S. 1945 Isocitric dehydrogenase -.rid carbon dioxide fixation, J. Biol, Chcmt, 159, 243-244. Rockwell, G. B,, and H; ghberger, J, H, 1926 Carbon dioxide as a factor in the growth of the tubercle bacillus and of other acid fast organisms. J. Infec- tious Diseases, 38, 9.2-100, Rockwell, G. n,, and Highberger, J, H, 1927 The Necessity of carb~n dioxide for the growth of bacteria, yeasts and molds, J, Infectious Diseases, 4Qj 438-446. Roessler, W. G. 1946 Person:! communication, Shive, W,, and Rogers, L, L, 1947 Involvement of biotin in the biosynthesis of ox lacotic andOC-ketoglutaric acids, J, Bi' 1, Chcm., 169. 453-454. Wynne, B. S., and Foster, J. W, 1948a Physiological Studies on Spore Germination with special reference to Clostridium betulinum. I, Development of a quan- titative method, J, Bact, In press. Wynne, p.- S, and F' star, J. W. 1948b Physiological Studies on spore- germination, with special reference to Clostridium betulinum. II. Quantitative ..spects of the germ: qation process, J. Bact, In press.. Mf605 #4'III) C ntinuod Table 1 EFFECT uF CU2 CONCENTRATION ON GERMINATION Incu- bation Hrs. GU Tension %. Resi- dual Spores Germinated Spores % Gemination 0 560 —~ -— 22 0 520 4© 7 22 I 90 470 84 22 $ 18 540 97 Table 2 quantitative indefinite inhibition of germination of C. botulinum spores due to the absence of Co^. Cu in atmos- .phereT % Incubation nv. count Germinated days residual spores spores 1 Germination - 0 560 - *0 17 500 60 11 1 5 220 3>U 0 61 * Counts corrected for volume loss of % during prolonged incubation over NaUH, No turbidity developed in any of the tubes in this series. M**605 $4 (ill) 9 Continued Table 3 Effect of oxalacetate on germination in Co^free gas phase Experiment A Incubation, hours CO in atmos- phere . % u , 10~3 M iiv, count residual spores Germinated. % Germination 0 - - $35 - - 20 0 - 4-60 . 75 14 20 1 340 19$ 36 20 0 + 74 460 86 Experiment B Incu- bation, hours in at- mosphere, ai h _ __ Uxala- cetate added Exhaustion period, minutes Av, Count residual sdores Germinated Spores /J Germination 0 - - 520 - - 23 0 0 30 470 50 10 23 0 f—* o i eo S 30 35 405 93 23 0 icr3 m 270 65 455 88 23 1 0 30 21 500 96 M-6Q5 #4 (III) 10 Continued Table 4 Effect of Complex Supplements on Germination Incubation Days % CO. _ Supplement Added Re Avg. Count sidual Spores Germinated % Spores Germ i n at io n 0 1 z + 1% Yeast 450 15 435 97 1 + extract 1% Liver 230 220 49 1 + extract 1% Brain 35 415 92 2 heart 1% Yeast t 45 405 90 2 extract 0,1% Yeast 120 330 73 2 extract l/o river 385 65 14 2 extract* 1% Brain 50 400 89 5 + heart* ■ None 160 290 64 15 - None 425 25 5 15 - Ip liver 475 0 0 15 ext r act. f 0.1/b Liver 475 0 0 15 extract 1% Brain 430 20 4 15 heartt 0.1/b Brain 410 40 9 One out heart of three tubes of which repre sents remaining two. M*605 #4(III) 11 PROJECT REPORT mbvbascw and mmuatum iiihci COMMITTEE OR FOOD RESEARCH himtaat flahkm Division C .QUARTERMASTER FOOD AND COHTAIMER fiSTITUTE V 1 ' FOR THE ARMED FORCES 0FF,C* 0r ™ CHICAGO ILLINOIS 9»A«TB»MA«TM OBMIRAL TOFTTFATThs iHstrtu-rlbk" ■ 1 ' - 1 ' ■" 11 1"'J 1 1 1 University of Texas Austin 12, Texas ITvTsTos T ' ' ' OIF ARTS 1ST ' Arts and Sciences Bacteriology 'official Ilf VEST! GATOR ' COLL ABO RATO S3 J. Wo Foster . Eo Staten ’Wynne REPORT HO. FI LI HO. . / ICOKTRACf'HO, 4 (IV) M-605 / Wll-009«qm«70190 TETTIircreffVgMlI mmatioh oat* August 1946 - April 1947 1 ,uuly 1946 TITLE! t ] FROORESS REPORT ( J FNAll RIFORT [ X J AM MU At RfFO RT [ ]t ERKIN ATI OH • 1*0117 Spore Formation and Spore Germination of Anaerobic Food Spoilage Organisms, Especially Clostridium Botulinum summary The effects of concentration of medium, salts, pH, surface tension, temperature, visible light, and oxygen on sporulation in Co botulinum have been studied® Over the concentration ranges tested",- 'relationship appeared to exist between the molar concentration of the salts used (NaCl, KC1, and NagSO^) and total spores or percentage spores resulting® M-605 #4 (IV) 1 Continued Pgrfl1** l2~121 Physiological Studies on Spore Formdilo'.h .Ip Clostridium botulinum* K, Staten Wynne Department of Bacteriology, University of Texas, Austin, Texas The voluminous literature on the physiology of spore formation in bacteria has been reviewed b several workers, including Dozier (1924-), Brunstetter and Ma- goon (1932), Cook (1932), and Knaysi (194-5), Comparatively little work has boon done on the sporulation process in Clostridium botulinum. the most extensive oe- ing probably that of Leifson (1931). As part of a larger program on sporesl bf C. botulinum in relation to the importance of this organism as a hazard in ioods a systematic study has been made of factors Influential in formation of its spores. Methods Cultures The strains of G, botulinum were with one exception (Texas No, 29) obtained from the National Canners* Association. Strain 62A was employed for most of the work. The others were numbered 78, 6B, 116B and 213B, Toxicity was demonstrated for 62A by the fact that 1.0 ml of a Seitz filtrate of a 10-day broth culture was lethal for a guinea pig in less than 21 hours, while a control animal receiving 1 ml of the filtrate inactivated at 80 C for 15 minutes survived the observation period of 3 weeks. Medium Comparison of spore formation by all six strains above in veal infusion pep- tone, liver infusion peptone, liver-liver infusion, BBL fluid thioglycolate and Difco brain heart infusion, resulted in the choice of brain heart infusion broth with BBL thioglycolate supplement as a basal medium for these studies. Ordinarily no anaerobic device was needed, as deep tubes of the medium, preferably boiled just prior to inoculation, gave excellent growth from loop inocula in less than 24. hours, Sporulation was not significant for any strain in the synthetic medium ■of Roessler (Wynne and Foster, 194-8) with 0.1 percent agar added to improve ana- erobiosis. Staining and Microscopic Counting Comparison of several spore staining methods resulted in the adoption of the following procedure, v/hich represen s a combination of May's (1926) use of a chromic acid mordant with Conklin1 s (1931) stein: 1. Treat heat-fixed films with $ percent chromic acid for two minutes. Wash. 2. Cover with 5 percent aqueous malachite green and steam 5 minutes. 3. Destain with HJJ $ to 10 seconds. 4. Counterstain with 5 percent aoucrus mercurochrome 20 to 30 seconds. Wash. • This method has consistently given beautiful preparations readily allowing quantitative differentialien of spores and vegetative cells, Clostridial_iqrms_ * This project has been undertaken in cooperation with the Committee on Food Re- search of the Quartermaster Food and Container Institute for the .armed Forces, The opinions or conclusions contained in this report are these of tne author. They are not to be construed as necessarily reflecting the views or endorsement of the War Department. M-605 #4 (IV) Continued retaining the green dye in the primordium hatfe arbitrarily been considered spores. Forms taking the pink counterstain with no green bodies within have been counted as vegetative cells*’ The total of spores and vegetative cells counted for each determination was at least,200 or more. These figures permitted computation of percentage of spores out of the total population. Tot el spore end vegetative cell count per ml of broth was determined in the Petroff-Hauser chamber. Staining was unnecessary for this purpose as the refmo- tile spores arc easily distinguished witl. the 43X lens and subdued illumination. Duplicate or triplicate counts were ordina.—/ made on each of triplicate tubes for each dotemins.tion, with 50 to 200 small squares counted, representing a total of 200 or more cells per count. Series five counts each on two cultures showed the deviation from the mean to be around «12 percent. Good correlation was con- sistently obtained between percent spores obtained from Petroff-Hauser counts and the staining method above. For example, an average of four determinations on a culture by the Petroff-Hauser method gave 16.8 percent spores, as compered to 13.8 percent by stained smears. The staining method is superior when the percentage of spores is low, and it was routinely used to check results obtained by the Petroff- Hauser counts where the spore percentage was 10 or losi. It should bo emphasized that zero spores in the tables simply means too few to detect by staining, i, o., loss than 0,5 percent. Effect of various environmental factors on sporulaticn Concentrate n of Medium and Medium Components Conflicting reports exist in the literature dealing with the of ect of con- centrated media versus dilute media on rate and relative amount of sporulation with organisms in the genus Bacillus. The more recent studies seem to indicate that percentage of spores is increased in more dilute media. This particular factor appears not to have been studies in C. botulinum. Concentratims of brain heart infusion broth (Difco) from 1 /4 to 5 times the normal strength have been tested, each with the usual BBL supplement added, A striking inhibition of sporulation occurred in concentrations above that nor- mally used. (Table 1), This applied not only to total number of spores but percentage of spores. This depression of sporulation occurred despite the fact that in the 2X end 4X medium strengths the total cell counts were n"'t strikingly different from the count ir the normal (IX) medium. The percentage of spares was not changed appreciably in the X/2 and X/4 media though the total counts were reduced, The ingredients of the medium wore tested individually by addition to regular strength broth so that the final concentration of the component was 5X that in normal brotn. Four percent nBacto,! peptone was also tested since it has been re- ported that Bactc-peptcne, but not proteose-peptone, contains a factor inhibiting sporulaticn in B, subtilis (Heberts and Balwin, 194-2), Uf all the individual substances tested (Table l) Nad was the on„.y one which specifically diminished, spore formation, without any significant effect on the vegetative population. Other experiments have sh~'wn, in fact, that 2 percent added Nad generally gives less than 0.5 percent spores. The failure of 1 percent added to depress percent spores (though it did decrease total spores somewhat) is not surprising in view of Leifson’s (1931) finding that the phosphate ion stimulates sporulation in C. botulinum. M-605 #4(IV) 3 Continued It is tempting to postulate the presence of a factor in the untested ”infu- sionu portion of the brain heart medium which was responsible for the absence of detectable spores in the two most concentrated media. The slowness with which turbidity developed indicates toxicity of some sort. While visible growth oc- curred overnight in the control medium plus 2 percent NaCl, it developed only af- ter 2 days with quadruple, and 3 days with quintuple, strength medium. The ex- istence of a sporulation-inhibiting factor is rendered plausble by the reduction of ab ut 70 percent in both total and percentage spores in double strength medium. IVen more drastic reduction (over 90 percent) has been observed in double strength medium, while other experiments have repeatedly shown added 0,5 percent NaCl to effect a reduction in total spores of only 25 to 30 percent, with still less ef- fect on spore ©storage, The failure of 0.8 percent added glucose to affect sporulation signif icantly may seem surprising, particularly since the final pH was reduced to 6,4. Rele- vant is the observation by Matzuchita (1902) that 5 to 10 percent glucose was op- timum for spore formation in this species. Salts It has been reported that proper c ncontrations of inorganic salts selec- tively inhibit sporulation in C. botulinum (Leifson. 1931), Chloride and nitrate ions were effective in inhibition, while ammonium and phosphate, and to some ex- tent sulfate, ions stimulated spore formation. In extensive physiological studies on spcrulation in certain Bacillus forms, Schreiber (1896) concluded that salts such as NaCl, and in concentrations which inhibit gr wth also delay the development of spores, ' Tho effect of varying concentrations of WaCl, KC1, and added to brain heart infusion are given in Table 2, Values for log of total spores v. log molar concentration plottec in Figure 2 seem to give a straight lino in each case, con- sidering the estimated *5.2 percent accuracy of the spore counting method used. Such would be expected if the relationship between concentrrtion of these salts and the resulting absolute number of spores is an exponential one. Thus in the equation Cn.s = K (1) if C ~ molar (or ionic) concentration n = dilution coefficient - constant for the parti- S = no. spcros/co cular salt u80a- and K = a constant*!' . the particular salt used. Then n log C + log S = log K = K* (2) It obviously follows that log C v, log S should give a straight line. Simi- lar curves were obtained by plotting log C v, log percent spores. It will be observed that although lower molar concentrations nf are more tcxic to sporulation than NaCl or KC1, higher concentrations arc less effec- tive. This is a perfect illustration of Fulmer’s (1925) contention that, in gen- eral, the relative action of two factors ■ n a bacterial species cannot be deter- mined from a study of/single equimolecular concentration, since their curves of concentration v. effect may cross, as in Figure 1, M-605 #4 (IV) Continued £H The optimum pH for spore formation in C, botulinum was found by Leifson(193l) to be 6,2 to 6,3, with both grrwth and spoliation ceasing at ab'ut pH 6,0, It has been suggested that fermentable carbohydrate inhibits speculation mainly by increasing acid production (leifson, 1931). This w uld be true particularly in a poorly buffered medium, Sporulati n in brain heart infusion broth with initial pH values of 6,0 to 10,0 is given in Table 3. Though spore formation practically ceased at pH 6,0, in agreement with Leifson’s (1931) findings, the 70 spcres/cc found by plating may well represent a significant number from the standpoint of practical food bacteriology. The point of minimum spore counts was the most favorable for maxi- mum vegetative cell numbers. Vegetative development has been fairly good at pH values as low as 5,0. Surface Tension Though Larson, et al (1925) observed a marked depression of in B, subtilis at surface tension values of less than 45 dynes/cm, this effect may well have been due to a diminution of oxygen supply, since the usual pellicle was not formed. To cur knowledge no previous studies have been made on the effect of surface tension on spore formation in anaerobic bacteria. It is recognized that changes in film pressure at the medium/air interface represented by surface ten- sion, may not necessarily parallel variations in interfacial tension at cell mem- brane/medium, but even approximations of the latter require rather intricate apparatus (Davis,1927), Measurements of surface tension were made with a Du Nouy tensiometer on stan- dard size surfaces at equilibrium. The fundamental equation for determining sur- face tension by this method has been represented (Harkins and Jordan, 1930) as CX = _FP_ x 4r.R where * surface tension in dynes/cm R = neon diambtor of elatinum ring P = pull in dynes (determined from scale re dings on a calibrated instrument) F = correction factor The values of F wer.- obtained from tables (Harkins and Jordan, 1930), Though no claim is made for absolute accuracy of 0( values listed in Table 4, the values of F calculated from measurements of p and R and the known values of of for ben- zene and H2<-» checker the literature values within *1,4 percent. With sodium, lauryl sulfonate as the surface tension depressant, spore forma- tion was not affected at values above 35 dynes/cm (Table 4), but appeared to decrease logarithmically at surface tensi 'ns below this value, as seen in Figure 2, The nature of the depressant appears more important, however, than the actual surface tension. Thus, an o{ value f 36 dynes/cm obtained by means of sodium lauryl sulfonate or s'dium ricinoleate gave no significant depression of tion, while values of 3b,6 and 37 dynus/cm produced by zephiron chloride and ltHyaminvtl, respectively, completely inhibited growth. Nature of the surface M-605 $4 (IV) Continued active agents in relation to the inhibition of tact trial growth they cause has been described by several authors, (Gibbs, ct al, 1926; Frobischer, 1927; Pizarro, 1927; Day and Gibbs, 1928), Temperature- Reports on the optimum temperature for sporotation of C. botulinum shew wide divergence. While sp're formation was not observed above 22 C by Roemer (1900), and was claimed virtually absent above 35 C by Von Ermengem (1897), ether workers have found the optimum to be 37 C (Landman, 1904; Orr, 1922). Though our studies have not been extensive, we have observed no appreciable difference fc*spore formation at 30 and 37 C, However, at 24 C speculation was depressed as much as 50 percent. Visible Light No,effect was observed on formation by this factor. Oxygen Tension It has be.n reported that broth cultures of C. brtulinum exp- sed to the air speculate faster than th se sealed with vaseline (Senior, 1930). Traces of oxy- gen have also been found beneficial tc sp're formation in other anaerobes (kigula, 1897; Matzuchita, 1902), With B. tetani and the bacillus of symptomatic anthrax, speculation was wh*lly lacking in the complete absence ~f oxygen, (Zinsser, 1906). In limited studies at 13 days’ incubation, we have been unable tc detect dif- ferences in spore percentages between atm'spheres 'f ordinary air and natural gas. Discussion Behring (1889a and 1889b) proposed the general thesis that spcrulati-n is an intermediate stage in normal development and may be partially or completely sup- pressed by proper concentrate ns of gr wth-inhibiting substances, which exert a partial physiological damage to the cell shrt 'f total prevention of growth. Though Behring has not received proper credit, a tremendous amount of literature has sh wn the correctness of his general concept with such diverse agents as un- favorable temperature (Pasteur, et al, 1881; Phisalix, 1892; Migula, 1897; Matzu- chita, 1902; Bongcrt, 1903-1904; Drranyi, 1930); concentration of nutrients (Matzuchita, 1902); pH (Behring, 1889a* Daranyi, 1930); pressure (Matzuchita, 1902); disinfectants (Behring, 1889a and 1889b; Inux, 1890); gr'wth products of other organisms (Bnger, 1903-1904; Mellon, 1927); and ‘xygen (Leifs<~n, 1931), Uur wn investigations are in c nplete accord with Behring's hypothesis. Referenc es Behring, E. 1889a Bcitraege zur Aeti'lcgie des Milzbrandes, I-III, Z, Hyg, Infekti^nskrankh,, 6, 117-144. Behring, E. lB89b Bcitraege zur des . JLlzbrandes. VI-VII. Z. Hyg. Infokti'nskrankh,, 7, 171-185. B ngert, J. 1903-1904 Bcitraege zur Biologic des Milzbrandbacillus und scin Nrchweis im Krdaver der grossen H ustiere. Zentr. Bakt. farasitenk,, I, Oris.. 34 497-507, 023-630. 772-792; 35, 14-24, 168-201. M-605 #4 (IV) Continued Brunstetter, C,, r.nd Magorn, C. 1932 Studies cn bacterial spares. Ill, C ntributicn'to the physiology of spare production in Bacillus myccides. J. Bact., 2J±y 85-122* / Conklin, M. E, 1934- Morourochrome as a bacteriological stain, J. Bact,, 27, 30-31. ‘ R. P, 1932 Bacterial Spores, Bixl* Revs,, 7, 1-23, Daranyl, J, V. 1930 Das Wesen der Bakteriensporenbildung und ihre Stellung in Fcrtpflahzungssystem* gentr, .Bakt, Parasitenk*, I Urig., 117, 543?-547* Davis, N, 1927 Interfacial Tension and Bacterial Growth, J, Bact,, 13, 381-386, •' : i ; ( } ) ..V 7* Day, xv. a., and Gibbs, W. M 1938 Surface Tension in relation to bacterial gr-wth, with special reference*to Lactobacillus acidophilus and Lactobacillus bul- garicus. J. Infectious Diseases, 97-107, ' ,1 Y: VA": ■■■?. ' • ■ Dozier, C, C* 1924 Resistance of spores of B. to disinfectants, XVIII. :. J* Infections D1 eases, 35, 156-176. ~ M, 1927 Studies up-n the rolati nship between surface tension and the action of disinfectants,, with specical reference to hexylres- rcinol,. J. Bact., 13, 163-182* Fulmer, j6, I., 192o The ef oct -f ammonium salts upon the swelling of colloids and upon the'growth of yeast at various temperatures. Colloid Symposium Mon graph, 2, 204-208, Cited in Buchanan, R. L., and Fulmer, E. I, 1930 physiology aiid Bi chemistry of jBacteria, Williams and Wilkins Cot, Baltimore, Md, Refer YoL. II, p. 284. Gibbs, h, Mt, Batchelor, H, W., and Sic.kcls, T, N# 1926 Surfo.ce tension and bacterial growth. J. Bact,, 11, 393-406, o' t - | : • ' ' ; v - ■' * Harkins, W. D., and Jordan, E. F. 1930 A method fr the determinati n of surface and interracial tension from the maximum pull ' n a ring,. J, An, Chem, Soc.,-, 1751-1772. Knaysi, G* 1945 A study of some environmental factors which control endospore formation by a strain of Bacillus mycoides. J, Bact,, /$, 473-493., Landmann, G, 1904 Ueber die Ursache der Darmstaedter Bohnen Vergiftung, Hyg. Rundschau. 14: 449* (Cited by Tanner, F. W., and Oglesby, E. Wt 1936 Pood Research, 1, 481-494. parson, W., P,., Cantwell, W, F., and Hartzell, T, B. 1919, The influence of the surface tension of'the culture medium on the growth of bacteria., J. Infec- tious Diseases, 25, 0.-46. Leifson, a, 1931 Bacterial Spores. J, Bact,, 21, 331-356, Matzuschita, T._ 1902 Zur Physiologic der Sporenbildung der Bocillen, nebst Bemerkungeh zum Wachstum einiger Anaercben., Arph.Hyg, Bakt,. 43. 267, abstracted in Zentr. Bakt. Parasitenk.,,' I, Ref., 32, 137-139. M-605 $4 (IV) Continued May, H, G* 1926 A safe spore stain for class use. Stain Tech,, 1, 105-106, Mellon, R. R, 1926 Studies in microbic heredity*!. The agglutinin-absorption react, on as related to the newer biology of bacteria, with special reference to the nature of spore formation, J. Immunol,, 12, 355-375, Migula, W, 1897 System der Bekerien, Band.I, .dlgemeiner Teil, Gustav Fischer, Jena, Urr, p, F. 1922 The pathogenicity of Bacillus botulinus. J. Infectious Diseases, 30, 118-127. Pasteur, L., Chamberland, Ch., and Roux, E, 1881 De 1’attenuation des virus ot de leur retour la virulence, Gompt, rend., 429-435, Phisalix, C,, 1892 Etat asporogene hercditaire du bacillus Lc Bull. m£d,, 21f 293, eited by Migula. Pizarro, U, R, 1927 The relation of surface tension to bacterial development, J. Bact., 13, 387-408. Roberts, J, L., end Baldwin, I, L. 1942 Spore formation by Bacillus subtilis in peptone solution altered by treatment with activated charcoal, J, Bact,, 653-659. Roemer, P. 1900 Ein Beitrag zur Aetiologie des Botulismus. Zcntr. Bakt, P-r^sitcnk,, I, urig., 27, 857-862. Roux, E, I89r Bacteridie charbonneuse asporogene, .um. inst, Pastuer, 4, 25. Cited by Migula. 0, 1896 Uebor die physiologischen Bedingungon der endogenen Spo- renbildung bci Baa llus anthracis, subtilis, und tumesdieJIS# Zentr. Bakt, Sommer, L. W. 1930 Heat resistance of the spores of Clostridium botulinum., J, Infectiv'us Diseases, £6, 85-114. Van Ermcngem, E, 1897 Ueber einer neuen Bacillus und seine Bezie- hungen zuim Botulisms, S. Hyg. Infektionskrankh., £6, 1-56, TJynne, E, Staten, and Foster, Jackson W, 1948 Physiological Studies on spore germination, wita special reference to Clostridium botullnum, III, Carbon dioxide and germination, *J.so a note on CO2 and aerobic spores, J, Bact,, £5. I_ press, Zinsser, H, 1906 simple method for the plating of anaerobic organisms, J. Exptl, Pied,, 8, 542-546, 8 Continued M-605 #4 (IV) Table 1 Lffect of Concentration of Difco Brain Heart Infusion and its Components (BBL thioglycolate supplement present trhoughout.) Brain heart infusion .Uded com- ponent , XX Final PH Spores x 10~6 Vegetative Cells x 10“b Total, x 10-6 % Spores 5X 7.X 0 7 7 0 a \ 7.2 0 37 37 0 2X 7.0 8 62 70 11 X (Control) 6.8 28 xs 76 37 X/2 7.6 21 25 X6 X6 xA 7.2 9 13 22 XI X Nc.Cj. (2/.) 7.0 2.5 55.5 58 X X Na2 (IJb) 7.X 16 25.5 a.5 39 X Glucose (0.8a) 6.X 26 xo 66 39 X Difco Proteose peptone (Xa) 6.8 82 95 177 X6 X uifco Bacto peptone (Xa) 7.6 66 156 222 30 -Incubation, 5 days. X = normal or usual strength M-605 #4 (IV) 9 Continued Table 2 Effect of added salts on sporulation in brain heart infusion broth. Salt Percent con- centration Molar con- centration Spores x 10“6 Vegetative cells x 10' Total. “6 x 10“6 Percent Spores — — — 44 46 90 49 NaCl 0.5 a .086 29.5 39.5 69 43 u 1.0 ,171 2 51 53 4 tt 1.5 .256 0.5 44.5 45 1 it 2.0 .342 <0.2 45 45 Negligible it 2.5 - 5.0 .428 - .855 Good growth in 24 hours. No spores seen. KC1 0.5 .067 21 36 57 37 it 1.0 .134 1.5 34.5 36 4 ii 1.5 .201 0,6 31 32 2 tt 2.0 .268 0.3 21 21 1.5 1) 2.5 .336 <0,1 21 21 Negligible II 3-5 .403— .671 Good growth overnight. No spores seen. Na2SU^ 0,5 .035 6.5 23.5 30 22 it 1.0 .071 4.0 22 26 15 tt 1.5 .106 4.0 22 26 15 n 2.0 ♦ 141 3.0' 24 27 11 it 2.5 .176 2.5 27 29.5 9 ti 3.0 .211 1*8 24 26 7 n 3.5 .246 0,8 22 23 3 n 4.0 .282 1.4 12.5 14 10 ii 4.5 .316 <,.04 7.0 7 Negligible 1; 5.0 ,352 Growth delayed and scanty - - - - M-605 #4 (IV) ... - 10 - Continued _Incubation- 11 days at 37 C Table 3 Effect of pH on Sporulation Initial ES. Spores/cc x 10” Vegetative , cells/cc x 10”^ Total/cc x 10-6 % Spores 6,0 ,00007* 540 540 0.00001* 6.5 3.5 180 184 2 7.0 57 67 124 46 7.5 57 66 123 46 'r.O 61 47 108 56 8.5 26 22 4B 54 9.0 20 14 34 59 9.5 20.5 21.5 42 49 10.0 6 29 35 17 * Though nc ) spores were detectable by microscropic examination, spores/cc. , plating in Yesair 1s medium showed 70 M-605 #4 (IV) 11 Cont inued Table 4 Effect cf Surface Tension on Sporulation Concentration sodium 0(, dynes/ lauryl sulfonrte x 10" 5 'cm Spores x 10“b Vegetative Cells x 10“6 Total Cells x 10-6 % Spores None 45.3 34 27 61 56 1 38.6 33 41 74 45 2 36.7 32 39 71 45 3 3-4.7 36 50 86 42 *4 34.5 16 50 66 24 +5 33.8 4 71 75 5 #6 33.2 0.3 33 33 1 * Two of throe replicates grew. + Unc of throe replies ites grew. M-605 #4 (IV) 12 Continued L-oC ''v ,CC- NapSU/ ». NaCl jiKCl LUG MUL*R CONCENTRE! IoN FIG. I EFFECT OF S XTS ON SPORUL ETION OF C. BOTULINUM 62^, DYNES PER CM FIG. 2. EFFECT UF SURFACE TENSluN OF DEPRESS ANT USED: SODIUM L.JJRYL SULFONETE. M-605 #4 (IV) 13