project acp&ai COMMITTEE 0# E00® RElEAS'C'i QUARTERMASTER FOOD AND CONTAINS* INSTITUTE FOR THE ARMED FORCES CHICAGO ILLINOIS BB8RA&0* Aim BBTWbVIinir »RMC8 NlfclTAftT VLAtCStim 017111 ox QVWICM OF TNB QUA«TS«MASTSS -OWWM& COOF ERAT1R6 INSTI TUT! D« jmTvT University of Texas Austin,o Texas bl V) SI on Arts and Scienc e s loirTtfcrJTcMT 0FFI Cl Al INVESTIGATOR Jo Wo Foster coiXacokators Bacteriology- S,> So' Wynne RCROAT RO. 6 Ft LI KO. M-605 For Ftluqtt ddvcAIN(5 ,r" 1 July 1947 “31 December 194,7 I nFmll OR 6ATC .1. July 1546 T» Tt Ef . 1 F*9§*E5S AfFORT FHAAC RlftfRT ‘ANNUAL RCFCRT jrSMtNAViaN Ai-PORT Spore formation and,spore germination of anaerobic food spoilage organismsi0 especially Clostridium botu irnim , -JULMiil— Data obtained: in this study,, together with information in • the literature lead to the conclusion that the classical '*dormancy®’ observed in anaerobic as -well as aerobic spores is not a biologi- cal character of the spore itself,, but is rather a function of the mediumo ,e Dormancy" may be considered as a manifestation of. the presence of anti-germination substances in normal mediao "Dormancy” can be eliminated with varying degrees of success<, depending on the medium and the nature of the inhibitivs substances by the- incerpo~ ration of various J^sppIements.0.• in particular soluble starch ( ‘ol percent)„ which absorbs some,, but not all,, the inhibiiy>fs0 . The term. 0dormancy” would appear to be of dubious value in connection with bacterial sporeso M=605 $6 1 WjTforw1"1 5 Af»r11 H I 2** f-2 ) The Problem of nDormancy11 in Bacterial Spores^ 2 Jackson W. Foster and E. Staten Wynne Department of Bacteriology, University of Texas Our experience with germination of spores of various species of Clostridia, especially C. botulinum. has led to conclusions which we believe warrant the con- sideration of investigators of this group of organisms and of the aerobic spore formers as well. Pertinent observations have been made by previous authors, but it appears that all the recent available evidence and various aspects of the dor- mancy problem have not hitherto been crystallized into one comprehensive concept. The essential points already established (Morrison and Rettger, 1930a and 1930b for review of previous literature; Curran and Evans, 1937; Olsen and Scott, 1946; Wynne and Foster, 1948a; Foster and Wynne, 1948) are these: (1) Colony counts from a spore suspension may be submaximum in a few days incubation, and increase with time over prolonged periods. This indicates delayed germination and is a well-known phenomenon for both aerobic arri anaerobic spores, This is the characteristic feature of the classical dormancy, (2) This effect depends on the medium used, it being marked in some, and non-existent in others. 1 The research which this paper reports was undertaken in cooperation with the Committee on Food Research of the Quartermaster Food and Container Institute for the Armed Forces. The views or conclusions contained in this report are those of the authors4 They are not to be construed as necessarily reflecting the views or endorsement of the War Department. 2 Present address: Dept, of Plant Sciences, University cf Oklahoma, Norman, Oklahoma, M-605 $6 (3) The final spore counts of a given suspension depend greatly upon the medium employed, though in each medium counts are at the maximum obtainable in it. (4) Certain substances, notably soluble starch (0.1 per cent), eliminate the delayed germination in some media. In others the starch increases the final maximum count, whereas in still others it has no apparent effect. In general, all media containing complex nitrogenous materials preferred for counting spores, show the starch effect. Data exemplifying all those points may be found in Table 1„ From the in- formation in the literature, as well as in Table 1, the following points may be mades (a) The so-called "dormancy" in these organisms is not an inherent function of the spores, but rather of the medium in which the spores are placed to germi- nate. (b) Even where "dormancy" occurred, or in me ia with low final counts, the large size of colonies speaks against a nutritional inadequacy. (c) Accordingly, a plausible conclusion is that "dormancy" i.c., delayed germination, and also submaximal counts, are due to substances in normal media inhibitory to spore germination. "Dormancy" w'mld then be only a manifestation of anti-germination substances in the medium and seemingly is, therefore, merely an artifact, (d) In the case of the characteristic delayed rate of spore germination during "dormancy" one may consider this to be a function of sub-inhibitory concentrations of certain substances in the normal medium* (e) In the case of submaximal counts, the inhibitory substances are of such nature and concentration that the germination rate of a portion of the spore population is zero, i.e,, they never germinate, for all practical purposes. (f) The inhibitors evidently are of different types, but one is the unsaturatod fatty acids (oleic, linoleic, lenolenic) which are present in almost M-605 #6 (g) The appreciable increase in germination rate, occasioned by the soluble starch in some media, is due to absorbtion of certain inhibitory substances, in- cluding the Cl8 fatty acids. In some media starch has little or no effect in enhancing counts known to be low, (h) The different degrees of inactivation of germination inhibitors by starch indicate that different media contain varying amounts of different in- libitors. Solvent extraction of some media indicates some of these naturally ccurring inhibitors are non-lipid, (i) The significance of these substances inhibitory to germination is evidenced by the fact that the fresh infusions of beef, pork, and liver, or- linarily considered ideal nutrients, contain significant amounts of germination inhibitors, as demonstrated by the considerable enhancement of germination caused qy addition of starch. Incidentally, fresh beef or liver infusion with starch is just as suitable as the widely used (for anaerobes) pork infusion medium of £esair, with added starch, (j) Effects similar to that of starch have been observed in the past when supplements containing native proteins and polysaccharides are added to the usual eactericlogical media. Though this action has been arbitrarily ascribed to un- specified nutritional factors, an equally plausible idea is that they function physically in the manner of st~rch, through colloid absorbtion of inhibitory sub- stances, and in some cases this has been proved definitely, (k) Unless shown that no effect is obtained by starch, the above data and ideas make it imperative that this substance be an invariable component of all ledia in work on anaerobic as well as aerobic spores where maximum counts in the shortest time are desired. Even though starch apparently does not counteract all known cases of retarded germination, it is the simplest overall treatment avail- able without altering the nutritional status of the basal medium. M-605 #" 4 (1) The term ’’dormancy’1 in the above connection no longer has the degree ?f validity it formerly enjoyed, and there now appears to be some question as to the justification of its continued use, unless in cases where the effect is shown be be independent of the germination medium. M-605 $6 Table 1 C. botulinun 624 Spore Recoveries in Various Agar Media^ Basal Medium 1 Per Cent Soluble Starch Incubation? Days 3 ' 6 17 33 Yesair’s plus 0,1 percent starch - 1 3 1 41 362 Difco nutrient - 28 30 22 - Difco nutrient + 35 42 35 - Difco brain heart infusion - 4 6 6 - Difco brain heart infusion + 12 23 30 30 BBL anaerobic - 0 0 2 *T BBL anaerobic 4* 9 1 6 8 Difco yeast extract, 1,0 percent - 19 17 15 - Difco yeast extract, + 15 12 11 mm lo0 per cent Corn steep liquor solids, 8 10 8 1.0 per cent Corn steep liquor solids, 1*0 percent + 7 9 11 14 Fresh beef infusion^ - 43 50 47 r Fresh beef infusion + 60 60 53 ~ Fresh liver infusion-^ - 24 24 23 - Fresh liver infusion + 46 52 49 — All media contained 0,2 per cent glucose and 0.2 per cent thioglycolate. The somewliat lower counts in this column as compared to the 6-day values is due to the obscuring of small colonies by- development of gas bubbles and cloudiness in the media* 3 Prepared exactly like Yesair’s medium (cf. Wynne and Foster, 194&.) M-605 #6 References Curran, H. R., and Evens, F# R. 1937 The importance of enrichments in the cultivation of bacterial spores previously exposed to lethal agencies0 J. Bact,, 2if 179-189* Foster, J. W,, and Wynne, E. S. 1948 Physiological studies on spore germina- tion, with special reference to Clostridium botullnum. IV. Inhibition of germination by unsaturated fatty acids# J. Bact., 55. In press# Morrison, E, W#, and Rettger, L# F. 1930a Bacterial spores. 1# A study in heat resistance and dormancy. J. Bact., 20, 299-311# Morrison, E. W#, and Rettger, L, W. 1930b Bacterial spores. II# A study of bacterial spore germination in relation to environment# J. Bact., 20, 313-342. Olsen, A, M. and Scott, W. J. 1946. Influence of starch in media used for the detection of heated bacterial spores. Nature, 1J57, 337. Wynne, E, S,, and Fester, J. W. 1948 Physiological studies on spore germina- tion, with special reference to Clostridium botulinum. I# Development of a quantitative method, J. Bact., j>5P In press. M-B05 #6 7