$TUDEES GM THE S9QRULATlQN AND GERMWATEGN OF P‘ETREEFACTEVE ANAERGEE 3679 (PA 3579) ‘fhasis hr 92% Dear» a? Ph. 9. (IMECHPGAN STATE UN‘E‘.’ERS§?¥ Syed‘ ifichammsé Shamsuzsfiaha 1957 J HESIE‘. This is to certify that the thesis entitled "Studies on the Sporulation and Germination of Putrefactive Anaerobe 3679” presented by Syed Mohammad Shamsuz-Zoha has been accepted towards fulfillment of the requirements for _P_h_._D_degree inmogy 8.: Public Health Major professor Date June 7; 195? 0-169 STUDIES ON THE SPORULATION AND GERMINATION OF PUTREFACTIVE ANAEROBE 3679 (PA 3679) by Syed Mohammad Shamsuz-Zoha An Abstract of A Thesis Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 ABSTRACT A study of the sporulation and germination of spores of a Putrefactive Anaerobe has been initiated. This strain is desig- nated as PA 3679 by the National Canners Association and is used as a test organism in safe processing of medium acid canned foods. PA 3679 does not sporulate rapidly in usual laboratory media. In addition these infusion media contain particles of proteinaceous material which are difficult to separate from spores. An aparticulate medium and a technique has been deve10ped for the rapid production of clean spores of PA 3679. The sporula- tion medium contained trypticase, l.5%; peptone, 1%; glucose, 0.2%; sodium chloride, 0.5% and dipotassium phosphate, 0.25%. Synchron- ization and stirring of the cultures had a pronounced effect on the Sporulation and complete sporulation was obtained in ## hours. Af- ter sporulation, the cultures were aerated briefly which resulted in the freeing of the spores from the sporangia and caused lysis of the few remaining vegetative cells. The Spores were harvested and washed by centrifugation and could be stored at h°C. in water or phosphate buffer without germinating. The thermal prOperties of the spores obtained by the above procedure were compared to those prepared from brain heart infusion. It was observed that PA 3679 spores which had been freed of growth medium and vegetative cells have Z values of 17.1-17.9 and ngalues of 0.83-0.95, regardless of the medium in which they have been pro- duced. 3 re & 3. -\ ).u- r- Spores of PA 3679 germinate in 10-15 minutes in 4% yeast ex- tract solution. Yeast extract could be partially replaced by ala- nine, adenosine, glucose and phosphate solution, in which complete germination was observed in 45-55 minutes. Attempts were made to characterize the germination acceler- ating factor present in yeast extract. The component is dialysable, heat stable and stable toward mild hydrolysis with N/lO acid or base, but loses its activity when hydrolyzed with 4N Hasop. STUDIES ON THE SPORULATION AND GERMINATION OF PUTREFACTIVE ANAEROBE 3679 (PA 3679) by Syed Mohammad Shamsuz-Zoha A Thesis Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 {f H E. ii ACKNOWLEDGMENTS The author wishes to eXpress his gratitude to Dr. H. L. Sadoff, Department of Microbiology, for his ever ready help, valuable crit- icism, suggestions and constant encouragement throughout the course of these investigations. The author also wishes to express his sincere thanks to Dr. R. I. Costilow for his valuable criticism and suggestions in these studies. Acknowledgments are also due to Dr. J. L. Fairley and Dr. C. I. HOppert, of the Department of Biochemistry, for their generous sug- gestions and supply of biochemicals used in these investigations. LIST TABLE OF CONTENTS OF TABLES O O O O O O O O O O O O O O 0 O O O O O I O O 0 ' LIST OF ILLUSTRATIONS o o o o o o o o o o o o o o o o o o o o 0 Vi I. II. III. IV. INTRODUCTION . . . . . . . . . . . . . . . . . . .‘. . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 5 A. General B. Factors Affecting Sporulation Temperature pH Surface tension Available moisture Visible light Oxygen availability Medium composition Nutritional factors Antisporulation factors C. Germination General Quantitative methods of study of germination Physical requirement for germination Effect of temperature on germination Role of metals on germination Role of organic substances stimulating germination Effect of antibiotics on germination EXPERIMENTAL O O O O O O O O O O O O O Q 0 O O O O O O O 22 A. Sporulation Organism Media Synchronization technique Cleaning and harvesting of spores B. Heat Resistance Studies C. Germination Studies D. POpulation Studies RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . 3o E11 Page A. Sporulation Effect of various media Sporulation in stirred and unstirred cultures Synchronization technique Effect of various medium components on sporulation B. POpulation Studies C. Heat Resistance Studies D. Germination Studies Preliminary studies Replacement of yeast extract by adenosine Effect of amino acids on germination Effect of carbohydrate and other carbon sources on germination Characterization of germination accelerating factor, present in yeast extract. v. DISCUSSION 0 O O O O O O I O O O O O O O O O I 0 O O O O O 57 VI 0 SUWY O O O O O O O O O O O O O O O O O O O O 0 O O O 0 65 VII 0 BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O O O O O 67 9. 10. ll. 12. LIST OF TABLES Page Growth and Sporulation of PA 3679 in Various Media . . . . 31 Effect of Peptone Concentration on Growth and Sporula- tiOn Of PA 3679 o o o o o o o o o o o o o o o o o o o o o 32 D Values as Calculated from the Formula of Stumbo and COChrane (1950) I O O O O O I O O O O O O I O C C O O O O 0 1+0 Germination of Spores of PA 3679 Obtained without Lysis of Sporangia as Observed by Light Transmission . . . . . . Al Germination of Spores of PA 3679 Obtained without Lysis of Sporangia as Observed by Staining . . . . . . . . . . . 42 Germination of Spores of PA 3679 Obtained after Aeration Treatment of Culture and Cell Lysis . . . . . . . . . . . . 4h Germination of Spores of PA 3679 in Casamino Acid and Yeast Extract Solution . . . . . . . . . . . . . . . . . . 45 Replacement of Yeast Extract by Purines and Pyrimidines, and Nucleosides in Germination of Spores of PA 3679 . . . . 46 Germination of Spores of PA 3679 in .wlmino Acid Mixture . .48 Germination of Spores of PA 3679 in L-alanine, L-arginine, L-phenylalanine Mixtures in Presence of 1 and 2% Yeast Extract or AdenOSine . C . C 0 O O . C O . O O C C . O O O 50 Germination of Spores of PA 3679 in Alanine, Adenosine, Glucose and Phosphate (AAGP) with Addition of Yeast ExtraCt o o o o o o o o o o o o o o o o o o o. e o o o o o O 51 Effect of Carbohydrates and Other Carbon Sources in Germination or PA 3679 Spores o o o o o o o o o o o o o o O 53 Figure II III IV VI LIST OF ILLUSTRATIONS Page Diagram Showing the Synchronization and Stirring Technique for Growing and Sporulating PA 3679 in Anaerobic Condition . . . . . . . . . . . . . . . . . . 24 Total Viable Count and Spore Count in Stirred and Unstirred Culture . . . . . . . . . . . . . . . . . . . 33 Total Viable Count and Spore Count in Stirred and Unstirred Culture Initiated with Synchronized IflOCUlumoooooooooooooooooooocoo 35 Thermal Resistance Curve Drawn by Method of Stumbo (191+8) O O O O O O O O O O O O O O 0 O O O O O O O O O 37 Thermal Resistance Curve Drawn by Method of Stumbo (191+8) O O O O O O O O O O O O O O O O O O O O O O O O 38 Rate of Germination of PA 3679 Spores in Alanine, Adenosine, Glucose and Phosphate (AAGP) and Yeast Extract SOlution . O O C O O . O . . O C O O C . . . . 0 5h JHE‘. INTRODUCTION A bacterial endospore is considered as "a structure that is a veritable fortress against most of the detrimental effects of the environment."1 These endospores are resting bodies that are formed within the bacterial cell. Their reproductive role is limited since each sporulating bacterial cell is capable of forming only a single spore, and this in turn gives rise to a single vegetative cell. The spore's biological role is not clearly understood and at the present stage of inconclusive studies, it appears to serve a protective func- tion. However the spores' peculiar nature and particularly their great tolerance to extremely unfavorable conditions have afforded grounds for speculation and experimentation as to their mode of formation and their germination processes. Spores have drawn the attention of microbiologists for more than seventy years, and have been of interest to investigators in all phases of bacteriology. As Lamana points out, "the endospore has had a major role in historical develOpment of bacteriology, for only after their discovery and rec- ognition as exceptionally resistant bodies did it become possible to devise crucial experiments that decisively disposed of theories of spontaneous generation."2 Structurally endospores appear as spheroidal, dense, refrac- tile bodies and they contain nuclear material, carbohydrate, fat, lOginsky, Bacterial Physiology (San Francisco: W.H. Freeman & Co., 1959), p. 365. 2Lamana and Mallette, Basic Bacteriology (Baltimore: Willi- ams and Wilkins & Co., 1953), p. 169. 2 protein, and relatively large amounts of calcium and 2,6-dicar- boxy pyridine. With the exception of some species of Vibrio, Spirillium, and Sarcina, only two bacterial genera are known to form spores. These are Bacillus and Clostridium. Some of the spore forming bacteria are of much importance in pathogenic bacteriology because they pro- duce diseases in humans and animals while others are of main concern in industrial processes. The spores' extreme resistance to all fac- tors known to be lethal to vegetative cells of bacteria, e.g., heat, dessication and toxic chemicals, have posed a great problem in ster- ilization processes used in various food industries. During the past decade a number of investigators have initi- ated basic studies into various phases of physiology of bacterial spores. Preceding this work most of the investigations of spores had been confined to an evaluation of their heat tolerance to supply data needed in the canning industry. These data were of empirical nature since the very nature of the heat resistance phenomenon is not clearly understood. This has led to the general realization that more basic information is needed regarding the nature of sporo- genesis and the germination process of bacterial spores. Many food microbiologists feel that the degree of heat re- sistance of Spores is a function of the medium in which they are produced. There are early reports that infusion media produce spores of higher heat resistance than "clear" broths. These obser- vations may have been due to the lack of correct methodology needed for the study of resistant properties of the spore. Extraneous proteinaceous material present in spore suspen- sions that are used for heat tolerance studies may have an affect on the apparent heat resistance of spores. It is well known that the materials which lower the oxidation-reduction potential of a medium, enhance the recovery of heat or radiation damaged cells. Further- more, in the biochemical studies of Spores it is desirable to have them uncontaminated with non-spore material. With this view in sight, studies were undertaken to deve10p a process for the produc- tion of "clean spores" in a clear liquid medium which would facili- tate the study of the germination process. As mentioned before, limited studies appear in the literature with regard to the physiology of Sporulation and germination of bac- terial spores. Although there is general agreement with Knaysi's view that Sporulation is a normal process in organisms which char- acteristically form spores, Opinions seem to differ with regard to the fundamental nature of the process. Knaysi (1948) assumes that Sporulation occurs in the members of family Bacillaceae when healthy cells face starvation. Foster and Heiligman (1949) have prOposed that Sporulation is a sequence of integrated biochemical reactions which are independent of vegetative growth. Schmidt (1950) believes that Sporulation is a function both of environment and of "cellular factors" determining reaction to the environment. Wynne (1948), on the other hand, has drawn attention to an essentially correct con- cept, the "Behring hypothesis," which suggests that spore formation can be conSidered as an intermediate stage in normal cellular deve1- Opment. Spore develOpment may be partially or completely inhibited by any physiological damage short of total prevention of growth. The transition from the vegetative cell to the Spore probably 3 requires more stringent environmental conditions than comparable growth requirements for a given species. Many reagents which have no effect on growth, inhibit or stimulate sporulation of bacterial cells. Variations in pH, which may not have appreciable effects on vegetative multiplication, may stOp sporulation. While the range of temperature for vegetative growth may vary from 20-40°C., tempera- ture ranges for sporulation are generally much narrower. In addi- tion, the sporulation temperature is usually lower than that for Optimum growth. During the process of germination, which may be described as the transition from heat stable spore to a heat labile form, there is an initiation of measurable metabolic activity in the spores. At no time during the germination is this activity comparable to that of a vegetative cell. From this it may be inferred that there is a fundamental difference between the process of spore germination and vegetative growth. In fact the findings described in the succeed- ing pages lead to the need for a visualization of some process by which the protein and enzymatic structure of the spore is trans- formed from the state of inactive and heat resisting protoplasm, to the enzymatically active protein of the vegetative cell. The purpose of the present studies was to deve10p a process for the production of clean spores which could be used in the study of physiology of germination. In addition a comparison was made between the thermal prOperties of the spores obtained from clear medium and those from infusion media. LITERATURE REVIEW General. Although spores may represent a stage in the normal develOpment Of certain bacteria, the production of a spore is con- trolled by the environment in which the cell is grown. Under cer- tain conditions, it is possible to produce only vegetative cells, from members of those genera which will normally form spores. Collier (1956) has shown that by reducing the concentration of tryp- ticase and phosphate in a complete medium, cultures Of Clostridium roseum can be maintained in a vegetative stage. Similarly Green- berg (1954) observed that the omission Of manganese from a semi— synthetic medium resulted in complete inhibition Of sporulation Of Bacillus cereus (terminalis). There are also reports in literature of the develOpment of permanently sporeless strains by a process of mutation (Knaysi, 1938). Despite the above examples, there is general agreement among the workers in this field that Sporulation is a normal physiological process of cells of those organisms which characteristically form spores. Sporulation. Studies in the last seventy years seem to in- dicate that the conditions required for sporulation may often by quite different from those considered Optimal for growth, and the metabolic activity of vegetative cells may not be at their maximum at the time spores are produced. The effect Of physical and chemical factors on sporulation is difficult to interpret since the classical method of single fac- 17rd.“ 'pk 6 tor determinants is inadequate. Too little is known Of the effect Of any factor on the sporulation process or of the interaction of two or more factors. The methodology for the quantitative study of sporulation em- ploys either microsc0pic counts of total cells and spores or viable counts of the number Of heat stable Spores. The first method is limited in its precision by sampling errors and by cell autolysis. Therefore, viable counts are more desirable. The effect 3: temperature 22 sporulation. Cook (1932) Knaysi (1948) and Schmidt (1955), who have reviewed the earlier works of German authors, have reported that Cohn (1876) and Koch (1888) studied the effect of temperature on the sporulation Of Bacillus subtilis and Bacillus anthracis respectively. Cohn (1876) reported the growth and sporulation Of B; subtilis at 47-50°C. Koch (1888) showed that cultures of B; anthracis deve10p rapidly at 35°C. and that sporulation starts after less than 20 hours Of growth. Schriber (1896) concluded from his studies on B; anthracis, anéEE: tilig, and Bacillus tumesans that the effect Of temperature on sporu- lation is slight and is due to the extension of vegetative growth. Similarly in Migula‘s (1897) studies with B; subtilisj the most rapid sporulation occurred at temperatures Of most rapid vegetative growth. On the other hand, spores are not formed at all temperatures that al- low growth. More intensive and apparently exact studies on the effect of temperature on vegetative growth and spore formation were made by Holzmeuller (1909). He showed that germination took place within narrower intervals Of temperature than did germination. From these studies as well as those Of Knaysi (1945, 1946, 1948) it can be con- 5L3: 1‘: T‘s-mi. 7 cluded that the Optimum temperature for sporulation is close to that Of vegetative cell growth, but the range is generally narrower. Sim- ilar results are reported by Esty and Meyer (1922) and Curran and Evans (1937, 1945). Later workers observed that a temperature slight- ly lower than Optimum for vegetative growth was most favorable for the sporulation of anaerobes (Hitzman, 1954; Zoha, 1954; Collier, 1955; and Sugiyama, 1951). The effect 2; pg’gg sporulation. The Optimum pH for sporula- tion in aerobes has been Observed to be in the range of pH 7.0-7.5 (Knaysi, 1945; Foster £3 21., 1948; and Curran, 1934). Spore forma- tion is very sensitive to the develOpment Of acidity in the media. (Fitzjames, 1955) In case Of the anaerobic spore formers, the Optimum pH for sporulation was reported to be in the range of 6.9-7.9 (Wynne and Foster, 1948; Esty and Meyer, 1922). Schmidt (1952) observed that the pH for sporulation of Clostridium spprogenes is 7.7-7.9. A pH Of 7.0-7.2 was Observed Optimum for sporulation in 91. roseum, Clostridium botulinum, Clostridium tetani and Clostridium putrificnm (Hitzman, 1954; Collier, 1955; Zoha, 1955; Mohrke, 1926). The opti- mum pH for sporulation of Putrefactive Anaerobe 3679 (PA 3679) is re- ported tO be 7.5-7.8 (Brown, 1956). The sporulation Of anaerobic bacteria has not been reported below pH Of 6.1. The effect 2; surface tension 22 sporulation. The effect of surface tension on the sporulation Of 91. botulinum was studied by Wynne (1948). He showed that the lowering of surface tension by means Of lauryl sulfate causes no significant depression Of sporula- tion at tensions above 35 dynes/cm. At values lower than 35 dynes/cm, a logarithmic decrease in sporulation occurred. The nature of depres- . ., , “u. gal-W 8 sant however seemed more important than the actual surface tension. This is in contradiction to the Observation made by Larson (1919) who showed marked depression of sporulation in B; subtilis at surface ten- sion values of less than 45 dynes/cm. Wynne (1948) stated that Lar- son's results may have been due to a diminution of oxygen supply. Effect 3f available moisture 23 sporulation. No direct rela- tionship has been established between the availability Of water and the process of sporulation since the effect Of added salts varyeso Migula (1897) has stated that moisture is necessary for sporulation. On the other hand, Holzmeuller (1909) observed that sporulation was hastened when material containing aerobic spore forming bacteria was allowed to dry on a cover glass. Other authors (Leifson, 1931; Williams and Purnell, 1955) have demonstrated that spore formation is more sensitive to conditions of low water availability than is vege- tative growth. Effect 2: visible light 2g sporulation. Holzmeuller (1909) found that diffuse light had no effect on sporulation of Bacillus mycoides, and similar results were reported by Schreiber for certain other bacilli. Schreiber (1896) claimed that sunlight exerted a deleterious effect on sporulation of Bacillus spp. Wynne (1948) did not find any significant difference in the extent Of sporulation Of 91. botulinum in presence or absence of light. Effect 2; oxygen availability 2g sporulation. Aerobic spore formers require Oxygen for Sporulation. However Knaysi (1948) be- lieves that the absolute necessity of molecular oxygen for sporula- tion Of Bacillus Spp. has not been conclusively proven, even though earlier workers (Migula, 1897; Holzmeuller, 1909; Bayne-Jones, 1933) 9 showed definite increases in sporulation in Bacillus spp. on aeration. The need for oxygen was also confirmed by the studies of Hardwick and Foster (1952) with washed cells of B; cereus. Roth £3 31; (1955) working with B; anthracis and Bacillus globigii, showed that 0.7-1.0 mMol. Of 02/L/min was required to complete the sporulation of 24 hour old culture, which had been initiated with a heat shocked spore inoc- ulum. When cultures were inoculated with vegetative cells at their maximum growth levels (just prior to sporulation), only 0.1-0.2 mMol Of OZ/L/min was required for complete sporulation. Leifson (1931) showed that anaerobes exhibit considerable difference in their ability to sporulate under increased oxygen tension. The sporulation of Cl. tetani and Clostridium novii was inhibited at a partial pressure of oxygen equivalent to 1 cm of Hg, while Cl. sporogenes and Clostridium chauveri were inhibited at slightly higher levels. Somer (1930) has reported that broth cul- tures of C1. botulinum, when exposed to air,sporulate more rapidly than control cultures. Traces of oxygen in the medium have been re- ported to be beneficial to spore formation in other anaerobes (Esty and Meyer, 1926). Wynne (1948) however Observed no difference in the Spore yields of £1; botulinum in an atmosphere Of air or natural gas. Collier (1956) has shown that oxygen will inhibit sporulation in C1. roseum. Effect 2: medium composition 2; sporulation. Since sporula- tion is considered as a normal phenomenon in the life processes of . spore forming bacteria, the nutritional requirements for the growth and multiplication of vegetative forms must play a role in their metamorphosis to spore forms. Conflicting reports exist in 10 the literature dealing with the effect of nutrient concentration of the growth medium on the rate and relative amount of sporulation that will occur in a given culture. Recent studies with organisms of the genus Bacillus show that the spore yield is increased when nitrogen and carbon sources are not in extreme excess (Knaysi, 1945). This has also been found true in case of Cl. botulinum (Wynne, 1948). Kaplan and Williams (1941) who studied Cl. sporogenes showed that an increase in the concentration Of peptone beyond certain concentra- tion levels inhibited sporulation. Williams (1931) was unable to get satisfactory yields of spores with B; subtilis in several synthetic media. Recently some chemically defined media containing growth factOrs and amino acids have been described by various authors (Williams and Harper, 1949; Frank and Campbell, 1953). These media gave satisfactory yields of spores Of B; cereus and other aerobes, as well as some anaerobes. In general, the extent of sporulation in dilute media is prOportion- al to the concentration of the nutrients while in 'concentrated' media the accumulation of inhibitors like fatty acids reduce the number of spores (Hardwick and Foster, 1949). Very few studies have been carried out regarding chemically identified nutritional factors required for sporulation. Foster and Heiligman (1949) have stated that nutritional factors favoring spor- ulation do not necessarily "stimulate" the onset of sporulation per se, but instead provide a metabolic shift leading to the transforma- tion Of vegetative cells to spores. Hardwick and Foster (1952) in studies of the sporulation process in B; cereus showed that sporo- genesis could take place in the absence of exogenous nutrients. 11 They labeled the process as "endotrOphic sporulation" and recognized that the exogenous nutrition of the sporulating cells had been com- pleted prior to actual sporulation. Cells are literally "committed" to sporulation! Stimulatory and inhibitory effects of some factors on sporu- lation are reported in literature. The recent work of Grelet (1950, 1952) suggests that a complex balance of suitable ions and energy source is required for sporulation; e.g., sporulation of B; megather- Egg occurs when concentrations of nitrate, sulphate and iron are at low levels, while calcium, sodium and chlorine have no effect. With low levels of glucose, potassium, magnesium, and manganese are re- quired for sporulation. Leifson (1931) reported that phosphate and ammonium ions, and to some extent sulphate ions, increased Spore yields in cultures of Cl. botulinum. Univalent cations such as sod- ium, potassium and lithium stimulate sporulation in Bacillus spp. (Fabian and Bryan, 1933). Foster and Heiligman (1949) found that the addition of potassium to an enzymatically hydrolyzed casein medium resulted in an increase of more than ten-fold in the spore yield Of B; cereus, while the effect on vegetative growth was slight. In the case of Cl. roseum, ferrous iron stimulates spore formation (Hitzman, 1955), and ammonium ions and sulphur in various forms are necessary for spore formation in the National Canners' Association (NCA) strain of PA 3679 (Brown, 1956). Few investigations of amino acid requirement for sporulation have been made. DL-alanine inhibits spore formation in a strain of B; cereus as reported by Foster and Heiligman (1949). These authors found that only leucine and isoleucine, of nineteen amino acids 12 tested, were active in the reversal of the inhibitory effect of DL- alanine. The beneficial effect of leucine in the sporulation of B; cereus has been confirmed by Williams and Harper (1951). Blair (1950) reported that the omission of methionine from a synthetic medium suppressed Spore formation in C1. botulinum, but it should be noted that vegetative develOpment was also lessened. Lysine was found to replace arginine for growth Of C1. botulinum, but no spores were noted. A somewhat similar relationship was found when an in- creased tyrosine concentration was used to fulfill a phenylalanine requirement. Glucose, in low concentrations, is known to stimulate the sporulation of aerobic spore formers (Foster 23 21;, 1949; Grelet, 1952). Oxalate seems to be required specifically for the formation of heat stable spores of B; megatharium (Powell, 1951). Virtually no confirmed knowledge exists concerning the role of growth factors in sporulation. Hayward (1943) noted a slight beneficial effect of inositol for a strain of B; subtilis, and Wil- liams and Harper (1951) have noted a similar effect with p-aminoben- zoic acid with two strains of B; cereus. Mellon (1926) reported that sporulation in a species of B; cereus occurred earlierand more abundantly when a filtrate of a culture of symbiotic strain was add- ed. Increased sporulation has been reported by other authors in mixed cultures (Powell 33 31;, 1955). Schmidt (1952) suggested that there are factors present in medium in which cells have been grown .which have an important effect on spore formation. Lund (1954) has used a "spent media" for the sporulation of a mutant strain of PA 3679. 13 Effect 2; antisporulation factors 23 sporulation. Knaysi (1948) believes that bacterial spores are formed when nutritive substances are depleted from.growth medium. This hypothesis has been used to explain poor sporulation results in concentrated media. However, recent investigations have indicated that the pres- ence of antisporulation factors may account for poor spore crOps. Baldwin and Roberts (1942) reported that peptone concentrations a1- lowing growth but not sporulation gave good yields of Spores follow- ing adsorption of the medium with charcoal. Similarly Foster 23 51; (1950) found that a treatment of complex media with charcoal or sol- uble starch gave substantial increases in growth and percentages of spores in Bacillus larvae. Later Hardwick at 31. (1951) showed that the nOn-volatile saturated fatty acids (010-014) and the unsaturated fatty acids (oleic, linoleic, and linolenic) were strongly inhibi- tory to Sporulation at concentrations of 50 mg/ml, while concentra- tions three times this value were inhibitory for vegetative multi- plication. Alanine and. Q-alanine in concentrations of 1-2 mg/ml, prevent the stimulation of sporulation of B; cereus by glucose (Foster and Heiligman, 1949). Much earlier, Tarr (1932) found that addition of asparagine to casein hydrolysate prevents spore forma- tion of several Bacillus species. Krask (1953) showed that methio- nine sulfoxide, which is a specific antagonist of the conversion of glutamic acid to glutamine, inhibited spore formation in B; subtilis in simple glucose-salts-glutamate medium. The effect of antibiotics on sporulation has not been studied extensively; however, Collier (1956) reported that penicillin will inhibit sporulation of anaero- bic bacteria. 3.4 I r E Swimmer“.- 14 Often the presence in the growth medium of nutritional factors which are known to stimulate germination, may inhibit sporulation by a process analagous to the competitive inhibition by methionine sul- foxide. Powell (1950) has reported high yields Of spore-like heat labile cells when B_._ megatherium was grown in muslin. containing glu- cose, glycerOphosphate, pyruvate and lactate. These compounds are also germination initiators for B; megatherium spores. Germination 22 bacterial spores. When spores are stored under suitable conditions, they remain viable for many years without appre- ciable loss of heat stability. But if they are transferred to favor- able environment, the spores will become permeable to stain and lose their heat stability. This change in the spore's property, followed by its transformation into a vegetative form, is usually referred to as germination. With most organisms, at least an hour elapses before the first vegetative forms appear. The germination process is slow compared to vegetative growth, but faster than spore formation. The gradual process of change of a dormant, inactive, heat stable spore to an actively respiring heat labile cell has been visualized to in- volve intermediate stages of transition. These intermediate stages show varying biochemical properties. However, when spores become permeable to basic stains, they also lose heat stability, and quan- titative measurements of germination by either process are quite well related. Quantitative methods 2f study 23 germination. In earlier studies, the outgrowth of cells following the incubation of spores in a nutrient medium was used to indicate germination. However this method is not quantitative and therefore not suitable for the present 15 study. In older literature, (Knaysi, 1933) the period of germina- tion was defined as the time which elapses between the beginning of incubation of the spores in growth medium and first division of the germ cells. However, modern studies utilize the spore's loss of heat stability, the permeability to basic stains, and change in Op- tical prOperties as criteria for germination. Powell (1950), Levinson (1955), Stewart and Halvorson (1954) have measured the rate of germination of spores by staining pro- cedures. After treatment with nutrient solutions, the germinated spores were able to take up simple dyes like methylene blue while the ungerminated spores did not. The most accurate method of determining the germination rate is to follow the loss of heat stability of the spores being tested. Yet this method depends considerably on the various conditions under which the spores are heated and also on the choice of recovery media. Germinating spores undergo changes in refractive index and therefore their germination can be followed by phase contrast micro- sc0py (Pulvertaft and Haynes, 1951; Brown, 1956). Powell (1950) Ob- served that the turbidity of spore:suspensions decreased markedly on incubation with germinating nutrients. These measurements in the change in light transmission can be observed in any photoelectric colorimeter over most of the visible spectrum. This procedure has been found to be very effective in measuring the rate Of germination at short intervals. The method correlates with either staining or heat lability techniques as described above. Resting spores do not respire at a rate detectable by common- ly used manometric techniques. However, germinated spores show res- 17 Sugiyama (1951) also found lower temperatures of incubation to be more favorable for the growth of Cl. botulinum. Exposure to sublethal temperatures often stimulates both the rate Of germination and num- ber of spores that germinate (Evans and Curran, 1934; Curran and Evans, 1937, 1945; Murrell, Olsen and Scott, 1950). Mefferd and Campbell (1951) found that furfural at a concentration of l p.p.m. increased the percentage of germination and replaced the effect of preheating or heat shocking in thermOphilic bacilli. The eXposure of spores to low or high pH is reported to have effect similar to heat shocking. (Murrell, 195A; Fitzjames, 1954; Zoha, 1955) Heat activation of spores of PA 3679 has been reported by Reynolds (1941) and Stumbo 33 El; (1950). Such heat treatment has been reported to reduce the resistance of spores to chemical agents like phenol and formaldehyde (Reddish, 1950). 32l2.2£ metals i3 germination. The effect of heavy metals on the growth, morphology, and metabolism Of the vegetative form of micro-organisms has been studied in some detail, but not much work has been done on the influence of heavy metals on the germination of bacterial endospores. Keilin and Hartree (1947) reported that 20 mM 8-hydroxyquinoline (oxine) inhibited the germination Of B; subtilis spores in glucose-yeast extract media. The inhibition could be re- versed by washing and resuspending spores in fresh medium. Powell (1950) studied the effect of oxine on the germination Of g; subtilis spores in a synthetic media consisting of L-alanine, glucose and phosphate and found complete inhibitions in presence of 10 mM oxine, British anti-lewisite, 2-3 dimercaptoprOpanol, (B.A.L.) inhibits germination partially at 4 mM and completely at 10 mM/mli _A partial l6 piratory activity which can be easily detected. This initiation of measurable respiration can also be utilized as a measurement for the rate of germination. Requirements for spore germination. Little work is reported in earlier literature on the nutrition of spore germination. A com- prehensive list of various compounds that initiate germination is presented by Stedman (1956) and Schmidt (1955) has also made a survey of the subject. The physical environment which is necessary for germination is usually very similar to that for vegetative growth (Holzmeuller, 1909; Cook, 1932; Knaysi, 1948; Wynne, 1952). Effect 22 temperature 23 germination. Temperature changes af- fect the rate Of spore germination and vegetative growth to different degrees. Mehl and Wynne (1951) reported the rate of germination of spores of PA 3679 to be a function of temperature over the range of 20-45°C., when thermal lability was used as a criterion for germina- tion. Using the Arrhenius equation, they calculated a value of 10,300 calories for the activation energy Of that reaction which was rate limiting in the germination of spores of PA 3679. Similar ex- periments performed in our laboratory by Sadoff (1957) showed a value of 15,900 calories as an activation.energy for the germination of spores of PA 3679 (NCA strain). It should be pointed out that in the latter case the germination was followed turbidimetrically and obviously a different reaction was rate limiting. Reed (1942) found that incubation temperatures lower than 37°C. were more favor- able for the maximum recovery of spores of Cl. botulinum when colony counts were used to determine survivors from heat processing. 18 reversal of inhibition was observed by the addition of soluble salts of zinc, magnesium, OOpper and iron. The effect Of mercuric chloride was also studied. It was found to inhibit germination and this in- hibition could be overcome by washing and inclusion of thiolactate or B.A.L. in the germination medium. Powell (1951) found that 10 mM/ml also completely inhibited the germination of B; megatherium spores. Levinson and Sevag (1953) found that iron and COpper inhib- ited germination and respiration in spores of B; megatherium. They also noted a stimulation of germination and respiration by manganese, cobalt, and zinc. Although manganese stimulated germination of B; megatherium spores, it had no effect on spores of B; subtilis or B; cereus. The effect on spore germination of several inorganic ions inhibitory to glycolysis or respiration has also received some atten- tion. Powell (1951) incubated Spores of B; megatherium in buffer with test substances for 30 minutes before adding glucose and followed the germination by staining. Cyanide, fluoride, iodoacetate and azide in concentrations of 1.0-10 mM had no effect. Levinson and Sevag (1953) noticed an inhibition of germina- tion and respiration of B; megatherium spores by a high phosphate concentration (0.05 mM) which could be overcome by chloride or other univalent ions. Hachisuka 33 El; (1935) observed during their studies on B; subtilis that M/lO iodoacetate, arsenite, fluoride and cyanide did not inhibit germination but did inhibit vegetative growth of the germinated spore. Harrell and Halvorson (1955) found that arsenate and aside at levels which completely inhibit vegetative cells (lo-ZM) 19 have no effect on the germination of spores of B; cereus (terminalis). Krishnamurty (1957) had difficulty in germinating spores of ‘B; cereus (terminalis) which had been grown in semi-production equip- ment. These spores would germinate in L-alanine and adenosine after repeated washing with pH 7 phosphate buffer or dialysis in versene (ethylene diamine tetracetic acid). Manganese and magnesium ions stimulated the rate of germination in versene-washed spores. Copper, chromium, iron and mercury inhibited germination of versene or phos- phate treated spores. Using arsenate to overcome the inhibition of germination by the metals, he observed that iron would also inhibit germination. Similarly Brown (1956) Observed that Spores of PA 3679 germinated rapidly in versene and that beryllium was found to be in- hibitory. Orggnic substances stimulating germination. Some 95 to 100 organic compounds including amino acids, carbohydrates, purines, pyrimidines, nucleic acids, nucleotides, nucleosides, and di- and tri-carboxylic acids have been used as stimulatory and inhibitory agents in the study of germination of bacterial spores (Keilin and Hartree, 1947; Hills, 1949, 1950; Powell, 1950; Powell and Hunter, 1953; Stewart and Halvorson, 1953; Pulvertaft and Haynes, 1951; Levinson and Sevag, 1953; Wynne and Mehl, 1956; Hachisuka, 1955; Hitzman and Halvorson, 1955; Lawrence, 1955; Church and Halvorson, 1956). Most of these studies were concerned with the germination of spores of aerobic bacteria and relatively few studies appear on the role of nutrients in the germination of spores of anaerobic bacter- ia. Wynne and Foster (1948) using heat sensitivity as a criterion 20 of germination, studied the factors affecting germination of spores of C1. botulinum and other clostridia. Germination of Spores of C1. botulinum in brain heart infusion medium was markedly delayed when CO2 was not present. Carbon dioxide could be replaced by oxalo- acetate in the germination of Cl. botulinum spores. Although yeast extract could also replace carbon dioxide in synthetic media, Ander- son (1951) reported that the addition of NaHCO3 (0.10 to 0.15%) to medium containing spores Of C1. botulinum was essential for the prompt development of colonies. Reynolds (1952) Obtained higher spore counts of PA 3679 when NaHCO was added to tryptone-yeast- 3 extract-thioglycollate medium. Similar effects of the addition of NaHCO3 to Yessir pork infusion media is reported by Wynne gt El; (1955) for Cl. botulinum types A and B. Long chain fatty acids (oleic, linoleic, linolenic) were found by Foster and Wynne (1948) to inhibit germination of spores of Cl. botulinum but not spores of aerobic bacilli. This inhibition could be relieved by the addition of starch in the germination med- ium. Roth and Halvorson (1952) found that unsaturated fatty acids do not inhibit spore germination unless they are rancid. Benzoyl peroxide produced similar inhibition of germination but its effect could be partially overcome by catalase. The formation of colonies on solid media was used as the index of germination and organisms tested in the study were PA 3679, 9;; botulinum, B; subtilis, and B; stearothermOphilus. Wynne 3; a1. (1953) have reportedsthe ger- mination of spores of several clostridial species in buffered glu- cose pH 7.0 under anaerobic conditions in 48 hours. This germina- tion was inhibited by 1 mg/ml oleate of pH 4.8. 21 Mundt 23 a1. (1954) have studied germination of spores of a strain of Cl. Sporogenes and observed rapid germination in 60% glu- cose and 8% sodium chloride, conditions which do not allow the germin- ated spores to mature into vegetative cells. Wynne and Gaylen (1955) have reported rapid germination of spores of several clostridial species in phosphate buffered glucose at 75°C. This effect was more pronounced when glucose was auto- claved with buffer. Hitzman, Zoha and Halvorson (1955) reported that spores of 9;; roseum and Cl. botulinum types A and B will germinate rapidly in a 5-10 minute period, when incubated with L-alanine, L-arginine and L-phenylalanine at room temperature. Alanine could be replaced by pyruvate with a decrease in the rate of germination. Arginine was essential to the germination process (Zoha, 1955). Effect 2: antibiotics 22 germination. A number of studies have been conducted in recent years concerning the effect of anti- biotics on the heat resistance of bacterial spores (Curran and Evans, 1940, 1946; Anderson g;_gB;, 1950; Adams, Hyeres and Tischer, 1951; Cameron and Bohun, 1951; Williams and Campbell, 1951; Kaufman, Ordal and E1 B151, 1954; wynne g; g;;, 1953). The results have been some- what variable but in general antibiotics are neither sporostatic nor Sporocidal. EXPERIMENTAL Studies 22 the Sporulation 2f Putrefactive Anaerobe BBZQ Materials and Methods: 1. Organism. A National Canners Association strain of Putre- factive Anaerobe 3679, obtained from our laboratory culture collec- tion, was used during this investigation. The requirement for serine for growth in a defined synthetic media (Frank and Campbell, 1955) and blackening of meat particles was used as a criterion for the authen- ticity of the strain. 2. Mggig. Considerable difficulty is encountered in Obtain- ing good yields of spores of some spore forming anaerobes in common laboratory media. Putrefactive Anaerobe 3679 (PA 3679) is known to form spores only after a considerable period of time of incubation. In the general practice of cultivating this organism, media contain- ing various amounts of solids and tissues are employed. These media include thioglycollate broth (B.B.L.), brain-heart infusion (Difco), veal infusion, peptone-liver infusion with added particles of liver, and pork-pea infusion with added starch. All these media increase the difficulty of Obtaining clean spore suspensions since they con- tain particles of solids, tissues or agar which are difficult to separate from Spores. The clear liquid medium described by Hitzman, Zoha, and Halvorson (1955) for the sporulation of 9;. botulinum, and other anaerobes, hereto referred to as 'TSZ' medium, was used in these studies. It contained trypticase 1.5%, sodium chloride 0.5%, dipotassium phosphate 0.25% and dextrose 0.2%. Several modifications of TSZ medium, with decreases and increases in each component and the 23 addition of peptone were also used. The TSZ medium containing pep- tone will be referred to as TSP. In the inoculation Of the culture medium, a heavy cell suspension amounting to 10% Of the total culture volume was introduced into a freshly prepared flask of the medium. In TSP medium, 0.1% sodium thioglycollate was added if no anaerobic precautions were used during the cultivation of PA 3679. Synchronization technique. Since a heavy inoculum is used in cultivation of this anaerobe, the culture resulting is not of a uni- form physiological age, and cells will sporulate at various times during the course of the growth of the culture. A portion of the spores formed earlier during the period of incubation will germinate and probably start another life cycle. This naturally would result in a poor spore crOp. To overcome this difficulty,a procedure de- scribed by Collier (1956) for the synchronization of sporogenesis was used. This technique was used for BB. roseum and consisted of 4-6 transfers of an actively growing culture at 4 hour intervals. The procedure used in this study was slightly modified and carried out as follows: A spore inoculum of PA 3679 was introduced into a test tube of TSP medium. At the end of twelve hours, 10 ml of the culture was transferred into a 250 m1 Erlenmyer flask containing 200 m1 of fresh TSP medium. Transfers were made at 6 hour intervals and the entire contents of the third flask were introduced into the sporulation flask (Fig. I). This vessel contained three litres of TSP medium and was agitated by means of a magnetic stirrer. Anaer- obic conditions were initiated in stirred cultures by bubbling city gas through the medium for 30-60 minutes after inoculation. Growth flasks which were used for inoculum were maintained at 24 FIG. I SYNCHRONIZATION AND STIRRING TECHNIQUE URMH. hvak v3) 060 EDLUW ‘3‘; $3003 uth m m KNKAxXW Max beams}. n «a \« \kx Q0»: homo N x /M.... m m , 53 use . from 1. Wilma fl new \SQHN mo \thhN 25 37°C. while sporulation was always carried out at approximately 33- 35°C. The initial pH of the medium was 7.2-7.3 and this was main- tained all through the period of sporulation. POpulation studies. These studies were directed toward the determination of the growth rate of vegetative cells and the rate of spore formation in stirred and unstirred cultures of PA 3679. A synchronized inoculum was used for both the stirred and unstirred culture. Counts were taken at two hour intervals over 24 hours of incubation and then at every 4 hours until the fiftieth hour. A final count was taken at the end of one week period. Total counts and spore counts were made in Pricket tubes which were prepared as follows: The counting medium, which consisted .of TSP and 0.2% starch with 2% agar, was heated and poured into Pricket tubes. Methylene blue was also added as an oxygen indicator in the tubes. These tubes were autoclaved at 121°C. for 30 minutes. Samples were withdrawn from stirred culture flask by means of a sterile syringe, and dilutions were made in distilled water blanks. One-half m1 of the dilutions was poured into melted TSP agar in Pricket tubes and shaken thoroughly to insure prOper mixing. After the tubes cooled and the agar solidified, 3 ml of plain agar con- taining 1% sodium thioglycollate were poured over the TSP agar in the tube to insure an anaerobic seal. This "capping agar" removed traces of oxygen in the tube and kept it anaerobic. The tubes were then incubated for 48 hours at 37°C. and colonies counted. For spore counts,samp1es withdrawn from the culture were heated in sterile tubes for 15-20 minutes at 90°C. This treatment was sufficient to destroy the vegetative cells. Microsc0pic exam- 26 inations were also made to detect the onset of sporulation in the cultures. Cleaning and harvesting 2i spores. Small quantities of the medium could be directly centrifuged in a Serval Super Centrifuge and the few vegetative cells and cell debris (which appear black or gray) could be mechanically separated from the white compact layer of the spores. This method was too cumbersome for harvesting spores from 6-12 litres of culture. Besides, even though 95-99% sporulation was obtained from the TSP medium, it was desirable to destroy the remain- ing vegetative cells. Lysozyme was effective against these cells but, unfortunately, the spores germinated at the conditions necessary for enzyme activity. A 15-20 minute aeration of the culture at the end of 44 hours incubation period was found to be a more effective treatment since it resulted in the lysis of nearly all vegetative cells. The spores were harvested by centrifugation in an electrically driven Sharples Super Centrifuge which was placed in a hood. This precaution was necessary to maintain the habitability of the labora- tory and to prevent contamination of the laboratory with the spore- 1aden aerosol. The spores were washed 8 to 10 times with distilled water and stored at 4°C. Spores obtained from brain heart infusion, after two weeks' incubation at 37°C., were filtered through cheese cloth padded with glass wool and then washed several times in the water. Microsc0pic examination showed that the spore suspension contained a considerable amount of extraneous material. These spores were further purified by gradient centrifugation at 4°C. This technique consists of centri- fuging spores in a density and viscosity gradient established by lay- ering various concentrations Of sucrose in the centrifuge tube. The clear band of clean spores which was formed was withdrawn from the 27 sucrose solution by means Of a hypodermic needle and finally washed free of sugar. Heat resistance studies. The heat resistance of the cleaned spores originally grown in infusion medium was compared to that of spores produced by the synchronization culture technique. The follow- ing procedure based on that of Bigelow and Esty's method was used for thermal death time determination. Aliquots of the stock spore sus- pension were diluted in sterile medium and M/250 phosphate buffer separately to give suspensions containing 10,000 spores/m1. Two ml of suspension was put in each thermal death time tube (TDT) by means of a 5 m1 sterile syringe. The TDT tubes consisted of 9 ml pyrex glass tubes, 15 cm in length. Before use they were cleaned in di- chromate cleaning solution, thoroughly rinsed in water, and steril- ized. The tubes containing spore suspension were sealed and sets of 5 tubes were placed in metal holders. The tubes were heated by total immersion in an electrically heated and constantly stirred constant temperature Oil bath (:0.15°C.). The sets were heated for various times, at each of 4 temperatures in the range of 230 F.-250 F. A time correction of 2.5 minutes, which had been previously determined, was allowed for the time lag involved in heating the contents of the tube to bath temperature. At the end of the specified heating time, the holder and tubes were removed from the Oil bath and rapidly im- mersed in a cold water bath. The cold tubes were wiped clean, flamed, and tubes containing buffer suspension were Opened and the contents transferred to freshly prepared and exhausted B.B.L. fluid thioglycollate tubes. The tubes containing the medium suspension were kept sealed. Growth was observed after 36 hours of incubation. .ma *i— W‘ 28 Treatment 2: Data. A logarithmic order of death was assumed and the data obtained were treated according to the conventional method of Townsend, Esty and Baselt (1938) to construct a thermal death time curve. The following formula as suggested by Stumbo (1948) was also used to calculate D values, which is applied in the con- struction of thermal resistance curve. D = U (1) log a - log b D = time in minutes to accomplish 90% reduction in the number of spores. U = time of heating in minutes a = number of spores subjected to one time-temperature relationship (the number of spores present per sample multiplied by the number of replicate samples). b = number of Spores surviving at the end of heating time U. The value of b was derived by applying the equation of Halvor- son and Zeigler (1932) to data obtained for samples subjected to time-temperature conditions that destroyed only a portion of the total number of organisms in the sample. x = 2.3 log E (2) where X = most probable number (MPN) of spores surviving per replicate sample. :3 ll total number of replicate samples. number of sterile samples as evidenced by lack of growth in subculture tubes. .0 II then b = X multiplied by the number of replicates. D values calculated from the data obtained correSponding to each time temperature relationship were used to construct a thermal 29 resistance curve. Germination studies - (methods). Germination of apspore‘sus- pension was followed in a Cenco-Sheard photometer by the change in Optical density which occurred when they germinated. The procedure was correlated with the uptake of dye by the germinated spores. Ali- quots measuring 0.5 ml of 10;2/m1 spore suSpension were pipetted into 15x100 pyrex test tubes and heated to 80°C. for 10 minutes. The tubes were removed from the water bath and allowed to cool down to room temperature. The germinating solution was then boiled with 0.01% sodium-thioglycollate and 4.5 ml of this suspension was added to the heat shocked spores while the solution was still warm. A spore con- trol was similarly made with water and 0.01% sodium thioglycollate. Readings were taken at 5-minute intervals. Slides were made 50 min- utes after the addition of the nutrients using crystal violet stain. This procedure was followed all through the germination studies. 3H: RESULTS Studies on Sporulation g: £_ 2679 Effect 2; various media. Of those media which are reputed to induce sporulation of anaerobes, only TSZ medium and Brewers thio- glycollate medium showed a reasonable response (Table 1). Further- more, the addition of peptone markedly increased the percentage of sporulation in cultures of PA 5679 grown in TSZ medium. In Table 2 it is seen that the use of TSZ plus 1% peptone (TSP) yielded 90-99% sporulation. This medium, (TSP), was therefore used during further studies on growth or sporulation of PA 5679. Cultures of the test organism in this medium sporulated in 72 hours. Sporulation in stirred and unstirred culture. Since spores germinate rapidly in TSP medium there was always a problem of har- vesting the spores from still cultures without germination. This difficulty could be overcome by chilling the sporulated culture for 24-48 hours and harvesting the spores at #°C. However it was ob- served that, if the medium was constantly stirred under anaerobic conditions, the percentage of sporulation was increased and the spores produced could be harvested at room temperature. The total viable counts and spore counts in stirred cultures remained constant while the unstirred culture showed a rise in total count and decrease in spore count after one week of incubation (Fig. II). This undoubtedly was due to the germination of a considerable number of the spores. Synchronization Technique. Spores of PA 5679 can be obtained 31 m &N.o omoosaw monogm on u I masonw voom n +++ Rmm.0 :omm M *m.o Homz :vkonw Auschw n ++ *m.a owmoaummuaeo voow muo> m ++++ museum soon a + o §OmIos ++n &om ++++ &oaIm ++++ XNIH ++++ I ++++ Koa ammo Iflpmhnv + &m adepmmm RmIN ++ KMIN ++ I ++ I + I + omouoma &H + Rm encumom &wmIom +++ Row ++++ RmHIOH ++++ KNIH ++++ I ++++ ocoumom Rm + Nme Rom +++ &mmIom +++ $OmIo: ++++ $m ++++ I ++++ odo»mmm &H + Nme §mHIOH +++ $OH ++ I ++ I + I + gunman wannaom &H + Nma amIm ++ amIm ++ I ++ I + I + amassed ad + Nma &om ++++ &OMION ++++ I ++++ I ++++ I +++ AooMflQv mpmaaoohHmOfisp mawsmnm wpmnmmonm aswcom KOHIm +++ ROHIm +++ I ++ I + I + was Edwmmqwma .omo Icquma spas szflcos Nma defipmupcmonoo madam Rm ++ $NIH ++ I ++ I ++ I + Imonm Hmdflwflho mo mam: wsflnflmpcoo assume Nwa RUMION w+++ &OHIm ++++ I +++ I ++ I ++ adfivms Nae.» fiomImH +++ RNIH +++ I +++ I ++ I + ROOMHchmpmuvmnmc cow Imsmsfi nudes uamnm monomm nuBOHG mmuomw masonw monomm spaoao monomm npzonu monomm gasonwo ease: Moms a mhsom Nu mhzom w: soapmnsosfl mo mafia mazom :m mnsom NH " dHsz mDOHm¢> zH whom 4m ho ZOHedflbmomm 92¢ meaomw H flgmda 32 monomm on RON.O mmoosmw u I Rmm.o :omm M npwoam ooow ham> n ++++ Rm.o Homz museum coom n +++ Km snowmen nvsonw u ++ «ESHUmE mfiovgmm we anOHw Moon R + o &oaIm +++ I +++ I +++ I +++ I ++ oaOpmom Rea spas Efifipma enoummm &OHIm +++ I +++ I +++ I +++ I ++ asfiuoa m=0pmomoo ROHIm ++++ I ++++ I ++++ +++ ++ encumom &H was mmdo Issasss mos seen any gow ++++ Rowlom ++++ $OMION ++++ ++++ +++ odoummm Rm + Nms &mm ++++ RmmIom ++++ &omIo: ++++ ++++ ++++ encumom mm + Nma &onIom ++++ KohIow ++++ &OHIm ++++ ++++ ++++ mnoumom Km.o + Nma *omIom +++ Roamm +++ I +++ +++ ++ Nma monomm museum mmnomm masons monomm npaouu monomw npsouw monomw npsoawe dwvmz Moon H mason Nu mason mg mnzon :N mhdon NH deflumnnoqw mo mafia ZOHadquomm zo onadezmozoo mzoemmm ho BOMhhm N mqmda 33 FIG. II TOTAL VIABLE COUNT AND SPORE COUNT IN STIRRED AND UNSTIRRED CULTURE x32, 25 N5 om 3:525 2:50 2on Id notzmca 2:50 .28. Id gauzw 2:60 325 II. BEE 2:50 _2o._.IIo AmmDOIV m2; O¢ Om _ d 0 ON _ l N SHHBWHN :lO 90'l 54 from stirred TSP medium in 72 hours while cultures of Cl. roseum and Cl. botulinum will Sporulate completely in 8-12 hours. The spores of the latter organisms were obtained by synchronization technique. Application of the same procedure resulted in the sporulation of PA 5679 in #4 hours. It was necessary to modify Collier's (1956) pro- cedure for use with the PA 5679 because more than # transfers at intervals of less than 6 hours resulted in lysis of the culture. Gas- sing was observed in the sporulation flask in 1 hour, after the trans- fer of the inoculum, and sporulation began at the 16th hour of incu- bation. It was interesting to note that the exponential growth of vegetative cells had not quite ceased at this time. Sporulation occurred exponentially in the stirred culture and was completed after 44 hours of incubation. The total viable counts and spore counts of the stirred, synchronized cultures were approximately 10 times those of the unstirred cultures (Fig. III). These results are in accord with those obtained from unsynchronized cultures. The synchronized culture technique for the production of spores of PA 5679 has been successful with final culture volumes of 10-12 litres. In TSP or TSZ medium, various levels of glucose, lactose and starch were tested for their effect on sporulation of PA 5679. Of the three carbohydrates, only glucose affected sporulation. High concentrations of glucose, which have been found inhibitory for the sporulation of Bacillus spp., also inhibited sporulation in PA 5679. A glucose concentration of 0.2% was critical because lower levels resulted in poor cell growth and higher levels in poor sporulation. Similarly, the phosphate and sodium chloride concentration J ‘Ii —...3. 35 FIG. III TOTAL VIABLE COUNT AND SPORE COUNT IN STIRRED AND UNSTIRRED CULTURE INITIATED WITH SYNCHRONIZED INOCULUM 74, ,x . 3 NM / ‘3” I 35b“; 0 O ' 4 ’ 5.2:” 6 ID 59-0): 0‘ :‘De; l (0.3... Q “Co-C V 4 :33: 30:0 0000 0000' o -h-& .- ‘ saga * Isaak-m oo< r ZOHmDWZH amdmm szmm 924 SDHQMS mma ZH whom 4% ho mmmomm ho WHDA4> Q n mqm¢e _ A1 TABLE A GERMINATION OF SPORES OF PA 5679, OBTAINED WITHOUT LYSIS OF SPORANGIA‘ As Observed by Light Transmission Per cent Transmittance Media Time in Minutes 0 5 10 15 20 25 #50 60 120 12 hr 24 hr 1. Thiotone 5% 1A 14 1h 14 1A 1A 1A 1A 1A gg 29 2. Peptone 5% 15 15 15 15 15 15 15 16 18 g2 55 5. Yeast ex- tract 5% 18 18 18 18 18 18 18 18 .20 gg 52 A. TSZ 21 21 21 21 21 21 21 22 26 growth growth 5. TSP 20 20 21 21 21 21 21 21 25 growth growth 6. Yeast ex- tract 5% 18 18 18 18 18 l8 l8 l8 l9 gé 22 l""‘CONTROL 25 25 25 25 25 25 25 25 25 25 25 ‘ Spores heat shocked for 10 minutes at 80°C. 0.01% sodium thioglycollate added to each germinating mixture. Temperature 25-26°C. pH of suspension 6.9-7.1 "Control - spore suspension in distilled water with 0.01% sodium thioglycollate. TABLE 5 GERMINATION OF SPORES OF PA 5679 OBTAINED WITHOUT LYSIS OF SPORANGIA‘ As Observed by Staining Reaction _— _: Per cent Spores Stained Mediau Time - 2 hr 4 hr 12 hr 2E‘hr Control ‘ 3-4% 5-h% 3-h% 5-4% 1. 5-4% 5-10% 10-20% 20-50% 2. 5-4% 5-10% 10-20% 25-55% 5. 5-4% 5-4% 10-20% 20% 4. 3-4% 20% 70-80% Growth 5. 3-4% 25% Growth Growth 6. 5-h% 5-10% 10% 30-40% ‘.01% Sodium thioglycollate added to all mixtures including control. Spores were heat shocked for 10 minutes at 80°C. Temperature 25-26°C. pH of suspension 6.9-7.1 “See Table 4. 43 When spores of the test organism were Obtained after complete lysis of the sporangium and vegetative cells, they germinated rapid- ly in casamino acid, tryptone, thiotone, peptone, beef extract, and yeast extract solutions. The results,which are summarized in Table 6,indicate that spores of PA 5679 germinate in 60 minutes at room temperature in a complex nutrient solution. Yeast extract solution initiated more rapid and complete germination than any other complex medium. Since casamino acid in combination with 2% yeast extract was as good a germinating agent as 5% yeast extract, further experi- ments were performed with yeast extract and casamino acid combina- tions. The results of these studies are summarized in Table 7 and show that something more than amino acidS(Casamino acids) are re- quired for the rapid germination of PA 5679 spores. Replacement g: yeast extract by adenosine. Hills (1949) had shown that yeast extract could be replaced by adenosine in the ger- mination of spores of aerobic bacilli, and a similar situation could be envisaged in spores of anaerobic bacteria. Experiments were then planned with purines, pyrimidines, and nucleosides, in combination with casamino acids. The following compounds were used in M/250 concentration with 5% casamino acids: adenosine, thymidine, inosine, adenine, guanadine, creatinine, xanthine, uracil, and guanine. Only adenosine produced complete germination after 24 hours and partial germination in 50 minutes. Partial germination was also observed in other mixtures, after 24 hours, but this effect was probably due to the casamino acid itself. However, further experiments with de- fined mixtures of amino acids confirmed that adenosine could partial- ly replace yeast extract (Table 8) as a germinating agent. 44 .s oases com .. H.5Im.m ma .Uoow pm mopsswfi OH pom vexoonm use: mouomm .oomNImN waspmhmmaoe .GOHpsaow wwflpmnwauow some on cmvum oumaaoohawownp Esfivom RHO.O . amIH mm mm mm mm mm mm mm mm mm mm mm ..qomezoo aomImw sssoso mm mm mm ww mm mm mm am am am somssxo sumo» + Rm uflom osfiammmo .m Rmmwom nuaouw M“ MM. NM MM. flfl mfl .flw mm ON Rm pomppxo pose» .: IRQAIm spaces mm mm om ma ma as EH as us ma Rm ososoana .m amHIoa. mesons mm mm mm mm mm ma ma ma as as an odosaoa .m amIm mesons Hm mm mm mm. mm mm ea ea ma as an possess soom .H .cfia om noose mes rm mes ma oma ow on mm om ma oa m o ,soassaom msaomssasow meassosm l! mopasflz as GBHB ooqmupfismsmpa acme hem ‘III’ [III III III mcHnHMHm cum scammfiamsmha pnmfiq an uo>Aomno m4 amHmMH Aqmo Q24 mmDBADD ho Bzmzadmma onedmm< mmehd szHhmmpo md QZOHBDAom Bopomno m4 canon «m ho mmmomm ho ZOHedszmmw zH mmaHmOMQODz wmzHQHZHmNm Qz< wmszDm wm Budmexm Emdmw ho Ezmzmodqmmm w wqm¢e The effect gf amino acids 23 germination. In an effort to 47 characterize the amino acids or acid that were necessary for the ger- mination of spores of PA 5679, experiments were performed with six- teen amino acids which are known to be present in yeast extract, as well as casamino acids. pared so that concentrations were in the same prOportions as are found in yeast extract. present in the yeast extract. companying Table 9. 10. ll. 12. 15. 14. 15. Amino Acids Alanine Arginine Phenylalanine Aspartic Acid Glutamic Acid Histidine Isoleucine Leucine Lysine Methionine TryptOphane Threonine Tyrosine Valine Glycine iixtures of amino acid solutions were pre- Concentration 0.50 % 0.78 % 2.2 % 5.1 % 6.5 % 0.94 % 2.90 % 3.60 % 4.00 % 0.79 % 0.88 % 5.40 % 0.60 % 3.4 % 4.4 % Each mixture lacked one amino acid normally The following key is used for the ac- 48 TABLE 9 GERMINATION OF SPORES OF PA 5679 IN AMINO ACID SOLUTIONS AS OBSERVED BY LIGHT TRANSMISSION‘ Per cent transmittance Amino Acid Time in Minutes Omitted 0 5 10 15 20 25 30 60 120 24 hours 1. None 25 25 25 25 25 26 28 28 28 40 2. None + 1% Yeast Extract 24 26 28 34 36 37 38 42 45 59 5. None + 2% Yeast Extract 25 25 27 55 40 46 55 56 57 4. None + Adenosine 26 26 26 26 26 27 29 56 59 48 5. Alanine Omitted 29 29 29 29 29 29 29 29 29 51 6. Other amino acid omitted“ 28 28 28 28 28 29 31 33 36 41 7. Yeast Extract 2% 24 24 24 24 25 26 50 55 56 46 Control"‘ (Aqueous spore suspension) 30 30 30 30 30 30 30 30 30 32 ‘Spores heat shocked for 10 min. at 80°C. Sodium thioglycollate 0.01% added to each germinating solution. pH of the germin- ating solution 6.9. Temperature incubation 25-27°C. “0miBSionr of any of the amino acids e.g., arginine, aspartic acid, glutamic acid, glycine, histidine, iso-leucine, leucine, lysine, methionine, threonine, tryptOphane, tyrosine and valine, showed the same rate of germination. ‘*'Control - see Table 4. 49 The pH of the mixtures wane brought to neutrality. Table 9 indicates that the amino acid mixture which did not contain alanine did not germinate the spores. One per cent yeast extract solution would not replace alanine in the germination of PA 5679 spores. Two per cent yeast extract solution in the absence of alanine germinated spores at a lower rate than 4% yeast extract. However 2% yeast extract in a complete mix- ture of amino acids showed the same rate Of germination as a 4% yeast extract solution. These results indicate that the spore re- quired alanine for germination. Experiments were then planned with L—alanine, L-arginine and L-phenylalanine mixtures which had been shown to bring about germina- tion of spores of Cl. botulinum types A & B. The data given in Table 10 show that only L-alanine with the addition of 1% yeast extract produced some germination. The yeast extract in these experiments could be replaced with adenoSine. Further experiments with alanine, J adenosine mixture with addition of l and 2% yeast extract resulted in germination responses comparable to 4% yeast extract as is shown in Table 11. Effect 23 carbohydrates and other carbon sources 23 germina- tigg 2; 2A 5622. Various carbohydrates have been known to initiate and stimulate the germination of Spores of aerobic bacteria. Some of the best known are glucose, maltose, sucrose, mannose, ribose, and various caramelized preparations. Similarly acetate, succinate, fumarate, malate, and pyruvate are known to stimulate the germina- tion of Spores of B; subtilis. The effect of these compounds was Observed in the presence or absence of alanine and adenosine. Table GERMINATION 0F SPORES or PA 3679 IN ALANINE ARGININE AND PHENYLALANINE IN PRESENCE 0F 1 AND 2% YEAST EXTRACT OR ADENOSINEO TABLE 10 50 Per cent Transmittance Time in Minutes Germinating Solution 0 5 10 15 20 25 50 40 60 120 24 hrs Control“ 24 24 24 24 24 24 24 24 24 24 26 L-Alanine,L-Argin- ine, L-Phenylala— nine 24 24 24 25 25 25 26 26 28 28 40 L-Alanine, L-Argin- ine and yeast ex- tract 1% 24 26 28 28 30 30 36 40 42 44 58 L-Alanine, L-Argin- ine, L-Phenylala— nine and yeast ex- tract 2% 25 22 29 52 54 40 41 44 46 57 Growth L-Alanine, L-Argin— ine, L-Phenylalanine and Adenosine 25 25 25 26 26 26 28 28 34 40 61 L—Alanine and yeast extract 1% 22 25 26 26 27 28 52 41 44 49 69 L-Alanine and yeast extract 2% 21 26 28 39 40 42 46 48 51 60 Growth L-Alanine and Aden- osine 24 24 24 26 27 29 54 55 41 44 46 5% Yeast extract 20 26 30 38 46 54 66 66 67 67 Growth *Spores heat shocked for 10 minutes at 80°C. 0.01% Sodium thioglycollate added to the mixture. pH 7.2 Temperature of incubation 25 ‘* See Table 4 -270 C o 51 TABLE 11 GERMINATION OF SPORES OF PA 5679 IN ALANINE, ADENOSINE, GLUCOSE AND PHOSPHATE (AAGP) WITH ADDITION OF YEAST EXTRACT- Per cent Transmittance Time in Minutes Germinating Solution 0 5 10 15 20 25 50 6O 24 hrs. AAGP + 1% yeast extract 18 24 50 58 59 40 41 42 +*" AAGP + 2% yeast extract 16 20 27 29 52 54 59 40 + AAGP 19 21 24 26 28 34 35 41 44 Yeast extract 4% 12 20 31 35 37 38 38 38 + Control'twater) 20 20 20 20 20 20 21 22 24 * Spores heat shocked for 10 minutes at 80°C. 0.01% Sodium thioglycollate added to the mixture. pH 7.2 Temperature 25-26°C. "See Table 4 *“ + = growth of cells into vegetative cells 52 12 shows the effect of some of the above mentioned compounds on the germination of PA 5679 spores. Glucose or caramelized glucose stim- ulated the germination of spores of the test organism. This confirms a previous Observation by Wynne (1954) who reported the germination of PA 5679 spores in buffered glucose in 48 hours. Germination in the above case was noted in buffered glucose in 48 hours._ Solutions containing alanine, adenosine, glucose and phosphate effected complete germination in 50-45 minutes. Various levels of each component were tried to accomplish a rate comparable to 4% yeast extract. The Optimal level of the nutrients induced complete germination in 20-45 minutes and initiated measurable germination as early as 5 minutes after the addition of the reagents to the spore. The concentrationswarte as follows: L-Alanine 0.001% Adenosine 0.00025% Glucose 0.05% KZHPO4 0.005% If 1% yeast extract was added to the above mixtures, the rate of germination was comparable to 4% yeast extract. The process was complete in 20 to 50 minutes (Fig. VI). Characterization g: germination accelerating factor present 13 yeast extract. When 1% yeast extract was added to the germinat- ing solution Of alanine, adenosine, glucose and phosphate (AAGP), the rate of germination was comparable to that Obtained in 4% yeast extract solution. The characterization of the accelerating compon- ent was attempted in the following manner. Yeast ribonucleic acid (RNA) is known to stimulate the ger- mination of spores of some aerobic bacilli (Hills, 1949) after mild tit-u.vi 4. rum; “‘1' .- w 53 .3 OHnt mom... ..Opmnmmonm cam mcsmonovm .mqsando. .OcamImN I sOHpmpsosH no eunumsomaoa .N.m mm: mm cum manprs waspmansmm some on wound oumHHoohHmOHSp assvom RH0.0 .Ooow pm mopsdHa OH sow vmxoonn amen monomm. amIs ms as as ms ms ms ms ms ms ...somazoo aosIm or om ms ms ms ms ms ms ms omocsm amo.o + .a.<.< aosIm mm mm as as ms as as ms ms osssoos am.o + .a.<.< somIms or mm ms ms ms as es as as sossem amo.o + .m.<.< aomIms or mm mm as as as ms ms as cmososs amo.o + ...a.s.< amana m: mm mm mm mm om ms as as osssamonm + amo.o omoo ISHO .ossmosocd .oanmH4 aoanw + spaces on mm rm mm HM. mm. NH a an someone nudes aoanm + mesons sm am am as ms ss 0s m sosmsm Rm0.0 + Rm pomspxo ammo» aoanm + mesons on mm sm as as as ms os cmosoms amo.o + an somssxo shoes asana ++ masons mm mm mm mm .wm mm ms a omoosso Rm0.0 + Rm pomspxo ammo» movaqHa on sense eossmsm mosoam meson rm ow on mm om ms 0s m o sossssom mnseossasow mopssHE sH oaHe OosmppHsmqmsa usoo pom I .20HmmH2mzdma emeH Mm Qm>mmmmo m4 mmmomm whom «m 20 20HB¢2HEmmO 2O mmomsom 20mm R. + do<< l4 do<< In 83.5 sec» $4 lo Bantam snow» o\o_ lo .838 II x .II.“ I.) It!) «'8'- 0.? on BONVIIIWSNVHI .LHOI'1 .LNBOHBd 55 hydrolysis with N/lO NaOH. Therefore, r hydrolysis by both 1N KOH and 1N H230,+ was performed and the hydrolysate neutralized and tested for its ability to germinate the spores Of PA 5679. These hydroly- sates did not compare with 4% yeast extract or AAGP solution in their germinating ability. Furthermore, the addition of these hydrolysates to AAGP solution did not enhance the rate of germina- tion of PA 5679 spores. It was assumed that yeast extract could act as either chelat- ing agent and thus suppress the inhibitory role of the metal on germination or act as metal donor to enhance the rate of germination. Four grams of yeast extract were dissolved in 100 ml of 5% versene solution and tested for germination. This solution did not show any increase or decrease in its germinating capacity. This indicated that if the yeast extract was supplying a metal it was not possible to remove it by chelation. The reverse control did not increase the ability of AAGP to germinate spores and from this it was concluded that the yeast extract was not acting as a simple chelating agent. The fact that the yeast extract was not a metal donor Was confirmed by the addition of ash of yeast extract to AAGP solution which had no effect on the rate of germination by that solution. Yeast extract was first boiled and then autoclaved for one hour at 121°C. and the suspensions prior to and after autoclaving were tested. Neither prolonged heating nor autoclaving of yeast extract affected the rate of germination of PA 5679 spores in yeast extract solution. One hundred m1 of 4% yeast extract solution (1) freshly pre- pared and (2) autoclaved were each treated with animal charcoal and 56 filtered. This was repeated three times. The filtrate after three treatments with animal charcoal retained the original color of yeast extract and no change in its germinating capacity was noted. Dialysis gf yeast extract. Fifty m1 Of 4% yeast extract wage dialysed against 500 ml of distilled water. The dialysate was con- centrated to 50 ml and tested for germination along with the material in the dialysis tube. Both fractions showed activity approximately comparable to 2% yeast extract. Further dialysis of the fraction that was retained in the tube resulted in a solution of little activ- ity and germination comparable to 5-4% yeast extract was Observed in the mixture of first and second dialysate upon concentration to the original volume. Hydrolysis g: ygggt extract. Hydrolysis of yeast extract with either N/lO base or acid at 121°C. had no effect on its ability to germinate spores. When yeast extract was treated with 6N HZSO4 and autoclaved for 1 hour, a black, sirupy liquid was obtained. This, on precipitation withB'tCflHHor the removal of sulfate, gave deep red, clear liquid which was neutralized with NH#OH. The liquor was then treated with animal charcoal, resulting in a straw-colored suspen- sion. The suspension was adjusted to original volume and tested for germination. The suspension, either alone or in combination with the components of AAGP mixture, failed to stimulate germination. re Hartm— “flu-731.. a. ‘ I a 7"? Y"? ' DISCUSSION Putrefactive anaerobe 5679 has been widely used as a test organ- ism in thermal resistance studies. Various workers have used a wide variety of media for spore production, spore count determinations, and thermal resistance studies. However, very few authors have re- ported the use Of a liquid medium without particles for the growth and sporulation of PA 5679. Brown (1956) and Lund (1954) attempted to use a "spent media" or simple trypticase media, but failed to ob- tain good sporulation. The TSP medium described in this thesis was effective in supporting good growth as well as a high percentage of sporulation Of the test organism. NO attempts were made to charac- terize the nutritional factors stimulating sporulation. The effect of peptone however was pronounced and although peptone could not re- place trypticase in the medium, trypticase alone failed to yield good sporulation. Further studies on the effect of peptone fractions on stimulation of sporulation are needed. I The higher yield of spores in stirred cultures may be due to more complete utilization of the substrate under these conditions "- . 'l'.In:'\|'nh Kin-n..- V“! ia.-s. than in standing culture. PA 5679 is a strict anaerobe and grows at the bottom of the culture flask where utmost anaerobic conditions prevail. Although gassing in the initial stages will bring about circulation of the nutrients through the culture medium, cells tend to settle down as soon as the gassing stOps (4-6 hours in unstirred cultures). Diffusion of the nutrients to the cells at the bottom of the flask may become the growth limiting factor. Since one cell 58 forms one spore, limiting growth will also limit the spore crOp. More- over a sporulating organism may produce some activators for sporula- tion, since "spent media" has been known to increase sporulation in anaerobes. However stirring alone does not yield as high a pOpulation of spores as does synchronization plus stirring. The process of sporu- lation in synchronized culture is completed in 44 hours as compared t0 6 days in unsynchronized cultures. The exponential rate of sporulation Which was observed in the stirred cultures lends an interesting insight into the problem of the formation of spores. Spores are formed after the exponential growth of the organism has ceased. We have thought in the past that the metabolic activity of the organism in this period was at a low ebb and yet it is capable of the protcplasmic rearrangement that leads to a spore. Sporulation appears to be autocatalytic. It must be necessary to build up levels of key intermediates in the cells or in the cul- f ture medium prior to sporulation. This is borne out by the observa— tion that the cells which have "settled out," as in unstirred cul- tures, sporulate at a greater initial rate than those of stirred cul— rcvs—v- . tures. The cells which have "settled out" are at higher unit concen- tration than correSponding stirred cultures. The exponential rate of spore formation argues against deple- tion of nutrients as a cause of sporulation. During logarithmic growth, the substances are disappearing from the medium at an expon- ential rate. On the other hand, spores are only produced in the sta- tionary phase at which time the metabolic activity of a constant pOp- 59 ulation of cells is assumed to be constant. These cells would pro- duce a linear decrease in nutrient concentration which is difficult to reconcile with exponential spore production. The study of the mechanism of sporulation in the absence of growth media, "endotrOphic sporulation," has not been possible with the anaerobes. Handling of the vegetative cells exposes them to oxy- gen and they lyse. Moreover, although the sporulation of Bacillus mycoides in absence of exogenous nutrients is well established, such phenomenon may not be general. In absence of any study of the pro- cess and nature of sporogenesis in anaerobes, one may conclude in words of Cook (1952) that "sporulating bacteria form spores because they form spores." Oginsky (1955) has described Sporogenesis as "defensive mech- anism." However such "defense mechanisms" are not to be compared with the cyst formation in protozoa, where an organism develops a resistant coat over its body to protect itself from adverse condi- tions. Since spores are so radically different from the vegetative p. cells in their biochemical activity, they must be separate and dis- E tinguishable entities that could be pictured as an alternating gen- eration in the life cycle of bacteria. The following diagram illus- trates the above statement. ? 7 Vegetative é——Vegetative -‘-:?'—:' PreSpore —=-,-’-v Spore Cell Cell ' 1 I I Phase I Pregerminated , Spore , II Phase 17 Germ Cell “\Germinated Spore 6O Spores grown in TSP medium were found to have the same heat resistance properties as of those Obtained from infusion media pro- vided the latter were freed from vegetative cells and debris. This however is in apparent contradiction to previous observations made by several authors that the heat resistance prOperties of the spores are dependent on their cultural history. Most of the workers in food microbiology feel that the spores produced in liquid medium free from particles have a lower heat resistance than those prepared in partic- ulate media. But the one prime factor which has been neglected by almost all workers in their heat resistance studies is use of "clean spore" suspensions. It is important that the spores which are used in heat resist- ance studies be free of debris which might aid in their recovery af- ter heat damage. Heat resistance studies with highly purified spores of PA 5679 indicate that heat resistance is an inherent prOperty of the spore despite its cultural history. Substances inhibitory or stimulatory to germination present in a recovery media will affect the results in heat resistance studies. The nature of the heat resistance of spores is unknown, and has been attributed to the impermeability of the Spore coat, the lipoidal components Of the spore, the state Of the enzyme and pro- tein components in the spore and possibly the spore's bound water content. None of the above theories are adequate and fundamental study of the mechanism of heat resistance must be initiated. The cleaning procedure as noted before involves aeration of the culture which results in the lysis of vegetative cells. The ef- fect of oxygen may be two-fold. The anaerobes,which possess flavo- 1— _-_w umm'. 0. 61 protein system of hydrogen transport,will form H20 (hydrogen per- 2 oxide) in presence of oxygen. Because of the absence of catalase activity in these organisms,peroxide might accumulate, and may cause lysis. In addition, the presence of peroxide may "Lwofo the arginism and stimulate the release of bacteriOphage. Since most clostridia are lysogenic for phage, this would result in mass lysis. It was also noted that rapid transfers of an actively growing culture for more than four times resulted in mass lysis. This could be due to carryover of enough dissolved oxygen during the transfer, since no strict anaerobic precautions were mentioned. Further stud- ies are necessary to prove the exact role of oxygen in lysis of cul- tures of PA 5679. Preliminary experiments to show rapid germination of spores of PA 5679 in either complex or defined nutrients were unsuccessful. These Spores as noted before had been harvested and heated to 80°C. for 15 minutes in an effort to accelerate their release from spor- angia. This may have resulted in the shrinkage of the vegetative cell around the sporangium rather than its rupture with the freeing of spores. There appeared to be a physical barrier to the uptake of germinating nutrients by the spores. This was indicated when the germination of the spores in nutritive media was studied by staining. In later experiments when "clean Spores" were obtained after the aeration treatment, and heat shocked for 10 minutes at 80°C., they germinated rapidly at room temperature in solution of peptone, thio- tone, beef extract and yeast extract. At pH 7.2 rapid germination of PA 5679 spores in yeast extract solution could not be observed at temperatures above 45°C., although Wynne (1956, 1957) has reported 62 germination of PA 5679 and other meSOphillic clostridia at 75°C. Wynne has based his report of germination on his inability to recover spores incubated at 75°C., a temperature which is lethal to germin- ated spores after 10-15 minutes exposure. Buffered glucose solution without the addition of yeast extract did not bring about germination, even at the end of 24 hours incubation at room temperature. A solution containing alanine, adenosine, glucose, and phos- phate has been reported to germinate spores of the thermophillic bac- teria B. coagilii, B; stearothermOphillus and B; thermoacidurans in 5 to 24 hours. The same compounds caused the germination of spores of PA 5679 in 20-45 minutes. However yeast extract (4%) will germin- ate these spores in 10-15 minutes. This action of yeast extract at 4% concentration, which resulted in complete germination in 10-15 minutes, could not be achieved even after changing the levels of con- centration of alanine, adenosine, glucose and phosphate (AAGP) mix- ture. Although no exhaustive attempts to characterize the acceler- ating factor in yeast extract have been made, the preliminary stud- F ies indicate that the compound may be a small peptide. It is de- stroyed by hydrolysis with 4 normal acid and not by autoclaving at 121°C. for 15 minutes in the presence of N/lO acid or base. It is KIT "0; .~ dialysable and soluble in 70% alcohol, but insoluble in other organic solvents. The compound must be needed in a very small quantity, since the germination rate of AAGP mixture plus 1% yeast extract is equivalent to that of 4% yeast extract solution. One per cent yeast extract does not induce germination in less than 24 hours. The possibility that metallic ions were stimulatory or inhibitory to germination could not be demonstrated. Spores washed with versene 63 or the inclusion of versene in yeast extract solution had no effect on the rate or percentage of germination. Moreover, the addition of yeast extract ash to AAGP mixture had no effect on rate of germina- tion. Oginsky (1955) has described germination as a reversal Of sporulation, but has put forward no evidence for her statement. The only work describing intracellular changes during the process of spore germination is reported by Fitzjames (1955). He Observed changes in the distribution Of phosphorus content of the spores dur- ing germination. The initial stages of spore germination or "awaken- ing" proceed with a sudden rise in detectable respiratory activity, a loss in heat resistance, and permeability to stains. Fitzjames noted that there was an increase in cold-acid soluble phosphate fraction and a decrease in hot trichloroacetic acid soluble phosphate fraction. This effect was found to be independent of the phosphate supplied in the germinating solution. This indicates that during this initial phase of germination, protein bound phosphorus of the dormant spore becomes acid soluble. The other changes that have been noted during the process Of spore germination are loss in weight, re- lease Of solids (dipicolinic acids), uptake of water, and alteration in response to acid hydrolysis. (Powell, 1955; Robinnow, 1955; Foster, 1956) The exact mechanism by which the dormant spores change into the actively metabolising form is still not understood. The role of the simple organic compounds which initiate ger- mination is unknown. These compounds perhaps supply energy to bring about unlocking of the occlusion structure of the Spore coat and thus expose an active enzyme site to start the sequence of reactions (?) leading to the germinated spore. Although no conclusive studies are 64 available on the enzyme nature of the germination process in spores Of bacteria, such an assumption could be accepted on the basis of the initiation of metabolic processes observed during the germination. The cleavage of adenosine by germinating spore (Lawrence, 1955), the presence of ribosidase enzyme in the spore (Powell, 1956), the oxi— dation of glucose by heat shocked spores (Church, 1955) and alanine deaminating activity of the spores (Levinson, 1956) are proof that the "awakening" process is accompanied by enzyme catalysed reactions. The "awakening" or pre-germination is reversible, and can be achieved by heat shocking 0r exposing the spore to sub-Optimal concentration of the germinating solutions. ' The philosOphical question that then presents itself is, whether the spores' "awakening" is the first step in the germination process or whether germination is the sum total of the enzymic re- actions leading to a germinated spore. The question will only be resolved when considerably more critical studies have been made on spore germination. F~ ”w _-.m M‘u._lm r O. SUMMARY The study of the physiology of spore formation and germination in a spore forming anaerobic bacterium has been initiated. A medium, TSP, and a technique was devised by which spores of Putrefactive Anaerobe 5679 (PA 5679) could be obtained as 'clean' preparations in short time. This medium contained trypticase but the addition of peptone had a pronounced effect on rate and extent of spore forma- tion. PA 5679 sporulated in TSP medium in 44 hours, as compared to the conventional two week period Of incubation in particulate medium. Spores were Obtained in concentrations of log/ml of the medium. Stir— ring and synchronization of the cultures were effective in increasing the Spore yield. A 15-20 minute aeration of the sporulated cultures resulted in the lysis of Sporangia and vegetative cells, facilitating the harvesting of 'clean' spores: Further cleaning was performed by re- I peated washings with distilled water. Spores obtained from TSP medium were compared for their heat- resistance prOperties to those obtained from particulate medium. If 'Clean' spores from either source were found to have the same heat resistance. Z values were of the order of 18.1-18.5, D250 values were 0.85-0.95. and F250 values were 4.9-5.1. NO previous studies have appeared on the rapid germination of the spores of PA 5679. Brown (1956) showed germination of PA 5679 spores in versene after the spores had been stored for nine months. 66 Brown's freshly prepared spores, however, did not germinate rapidly. On the other hand, PA 3679 spores obtained from TSP medium germinated rapidly in 4% yeast extract, and germination was completed in 10-15 minutes. A mixture of alanine, adenosine, glucose and phosphate solution germinated the spores of PA 3679 completely in 40-50 minutes. The rate of germination in alanine, adenosine, glucose and phosphate mixture was comparable to those of #% yeast extract when 1.0% yeast extract was added to the mixture. Attempts were made to characterize the accelerating factor present in yeast extract. It was dialysable, heat stable, and stable toward hydrolysis with N/lO acid or base, but labile to strong hydrolysis with #N HZSO#. Metal- lic ions did not seem to effect spore germination. The studies described in the thesis have afforded an insight into the process of sporulation and have demonstrated that sporula- tion is an autocatalytic process. .«y B.“ .‘J'Oo'iaOI‘Lh-‘h? "w..'w.\ O", "-' BIBLIOGRAPHY Allen, M. B., The dynamic nature of theromOphily. J. Gen. Physiol. 52; 205-24. 1950. Adams, A.T., Ayers, J. C., and Tischer, R. B., The effect of subtilin and heat in preventing spoilage of meat. Food Technol. 5; 82-86. 1951. Andersen, A. A., Michener, H. D., Preservation of food with antibiot- ics. Food Technol. fl; 188-189. 1950. 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