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JIIIII'Q “(A W ,|" "' ,.I.I,III.III'I' In“.an .II‘ In H I .I,".,I'I“‘-II,,I' ”Evy, “I“tzfi‘tafi EELS! LI B R A R Y Michigan :8 rate University This is to certify that the thesis entitled V J Germination of Spores of Clostridium perfringens FD] presented by y Cristina Vaqueiro has been accepted towards fulfillment of the requirements for Ph.D. d . Food Science and ’egreem Human Nutfition flax/(mm Major professor Date W 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. --“—~. ‘_ - -_~_ _—___ .__.- _____ GERMINATION OF SPORES OF Clostridium perfringens FDl By Cristina Vaqueiro A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1979 x m I/ a ABSTRACT GERMINATION OF SPORES OF Clostridium perfringens FDl By Cristina Vaqueiro The purpose of this investigation was to design a chemically defined medium for the germination of spores of Clostridium perfringens FDl and to determine the effect of several factors. Factors studied were heat shock, pH, nutrients and inhibitors. Initial experiments demonstrated that a chemically defined medium containing 18 amino acids, glucose and some mineral salts, promoted full germination of the spores. A heat shock at 80 C for 10 minutes was necessary to activate the spores. Tests with individual amino acids indicated that alanine (30 mg/l), glutamic acid (224 mg/l) and leucine (92 mg/l) were the most effective germinants. Spores of C. perfringens FDl also required a pH of 6.0-6.5 and the presence of sodium chloride, dibasic potassium phosphate and L-cystine for rapid germination. The addition of glucose or fructose (or a sugar containing a terminal glucose or fructose moiety) was required for germination. Sodium cthride had a stimulatory effect on germination, and its effect was only partially substituted by sodium or potassium nitrite or sodium bicarbonate. Cristina Vaqueiro The pH had a marked effect on germination rate and the extent of germination. Satisfactory germination occurred in chemically defined media from pH 5.5 to 7.0, or in complex media from pH 5.5 to 9.0. Inhibitors of vegetative growth usually permitted spore germination at concentrations that were inhibitory for vegetative growth. Inhibitors of germination of aerobic spores, that act by blocking the L-alanine-induced germination system in spores of bacilli, were ineffective or only slightly effective in inhibiting germination of spores of C. perfringens FDl. The chemically defined germination system designed for spores of C. perfriggens FDl was also adequate for spores of g. pgrfringens strains ATCC 3624 and NCTC 8238, but failed to support germination of spores of C. sporogenes (PA 3679) and Q. perfringens strain ATCC 12195. A mis padres ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Kenneth E. Stevenson for his guidance and patient encouragement during the course of the investigation and preparation of this dissertation. Appreciation is also expressed to the members of my graduate guidance committee: Dr. L.G. Harmon, Dr. P. Markakis, Dr. E.S. Beneke, and Dr. R.N. Costilow. The assistance and cooperation of Ms. Marguerite Dynnik and my fellow graduate students is gratefully acknowledged. Finally, I wish to express my appreciation to the Department of Food Science and Human Nutrition for provi- ding the assistance and facilities to make this study possible. TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION. LITERATURE REVIEW . Clostridium perfringens Transformation of Spores Into Vegetative Cells: Activation. . . . Methods of Activation Germination . . Outgrowth . Germination Requirements of Bacterial Spores. Other Factors that Affect the Germination of Spores of Clostridia. . . . Other Ways to Promote Germination Alkali-Induced Germination. Nitrites and Sodium Chloride. Inhibitors. . . . Inhibitors of Vegetative Growth . Specific Inhibitors of Germination. MATERIALS AND METHODS . Organisms Spore Production and. Preparation of Spore Sus-. pensions. Heat Shock. Media . . . Sporulation Medium. Plating Medium. Germination Medium. . Carbohydrates Used as Germinants. Nitrogen Sources Used as Germinants Mineral Salts Inhibitors. . . . Inhibitors of Vegetative Growth . . Inhibitors of Germination of Aerobic Spores. . iv Page vii ix -J ._i._a._a_a_a—J_a Q (DNOO‘U'IUWOJ somumcna-w w pH. . . . Sterilization Inoculation Incubation. . Measurement of Germination. . . Calculation of Percent Germination and Extent of Germination (Y). . . . . . . RESULTS . Germination in Complex Media. . . Germination in Chemically Defined Media . . Effect of Alanine, Glutamic Acid and Leucine on Germination of Spores of C. perfringens FDl Effect of a Heat Shock on the Germination of Spores of C. p_rfringens. . . Effect of pH on Germination of C. perfringens FDl in Complex and Chemically Defined Media . Effect of Ingredients of the Basal Medium on Germination of Spores of C. perfringens FDl Effect of Different Mineral Salts as Substi- tutes of Sodium Chloride. Effect of Different Sugars on Germination of Spores of C. perfringens FDl. . . . Effect of Inhibitors on Germination of Spores of C. perfringens FDl . . . Germination of Other C. perfringens strains and PA 3679 . . . . . . . . . . . . DISCUSSION. Considerations on Amino Acids and Germination . Considerations on the Germination Medium and Other Compounds on the Germination of Spores of C. perfringens . . Enzymes Related to the Germination of Bacterial Spores. . . Effect of pH on Germination of Spores of C. erfrin ens FDl . . . Metanlic Considerations on Germination of. Spores of C. perfringens FDl. . . . Minimal Requ1rements for Germination. Anaerobic Conditions in Germination of Spores of C. perfringens FDl . . Effects of Inhib1tors of Vegetative Growth on Germination . . Effect of Specific Inhibitors of Germination. Responses of Other.§. p_rfringens Strains to the Germination Conditions of c. perfringens FDl . . . . . . . . . . . 61 63 65 67 68 69 7O 72 72 Page CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . 74 REFERENCES . . . . . . . . . . . . . . . . . . . . . . 75 vi Table 10 LIST OF TABLES Concentrations of amino acids used as germinants in chemically defined media. . Classification of amino acids based on the characteristics of the R group . Percent change in optical density of a spore suspension of Clostridium perfringens FDl in various chemically defined media after 5 and 24 hours . . . . . . Effect of the nonpolar and polar- charged groups of amino acids on germination of spores of C. perfringens FDl after 24 hours at 45 C . Effect of alanine, glutamic acid and leucine on the germination of spores of C. perfringens FDl after 5, TO and 24 hours at 45 C . . Effect of D-isomers of alanine, glutamic acid and leucine on the germination of spores of C. perfringens FDl after 2, 6 and 24 hours at 45 C. Effect of various heat treatments on the germi- nation of spores of C. perfringens FDl in chemically defined media after 24 hours at 45 C. Effect of single ingredients and combinations on germination of spores of C. perfringens FDl after 1, 4 and 24 hours at 45 C. Effect of different salts on germination of spores of C. perfringens FDl after 24 hours at 45 C in media with or without amino acids. Effect of different sugars on germination of spores of C. perfringens FDl after l and 24 hours at 45 C. . . . . . . . . . Page 22 28 30 31 32 34 37 41 43 45 Table Page ll Time (in hours) needed to obtain 50% germination of C. Ferfrin ens FDl spores in BHI + YE (pH 7. OT, pH 7.0) and chemically defined medium (pH 6.0) containing various concentrations of potassium sorbate. . . . . . . . . . . . . . . . 50 l2 Minimum concentration of potassium sorbate (%) needed to limit germination of spores of C. per- frin ens FDl to s 50% in three media at pH 5. O- 7. D after 24 hours at 45 C . . . . . . 5T 13 Effect of Lauricidin plusTM on germination of spores of C. perfringens FDl in three media after 1, 5 and 24 hours at 45 C. . . . . . . . . 52 l4 Effect of Veronal (V) and Methylanthranilate (MA) on germination of C. perfringens FDl after 1, 5 and 24 hours at 45 C (pH 6.0) . . . . . . . 53 viii LIST OF FIGURES Figure ~ Page l Effect of a heat shock at 70 C for 20 min on germination and outgrowth of Clostridium perfringens FDl at 45 C in FTG or FTG-base plus 18 amino acids. . . . . . . . . . . . . . . 35 2 Effect of pH on germination of spores of c. perfringens F0] in complex and chemically defined media (complex media after l hour and chemically defined medium after 2 hours at 45 C. 38 3 Effect of pH on germination of spores of C. perfringens FDl in chemically defined media after 5 hours at 45 C. . . . . . . . . . . . . . 40 4 Germination of spores of C. perfringens FDl in BHI + YE with different concentrations of potassium sorbate (pH 7.0) . . . . . . . . . . . 46 5 Germination of spores of g. perfringens FDl in FTG with different concentrations of potassium sorbate (pH 7.0) . . . . . . . . . . . . . . . . 47 6 Germination of spores of C. perfringens FDl in chemically defined medium with different concentrations of potassium sorbate (pH 6.0) . . 49 7 Germination of different strains of C. perfrin- gens and PA 3679 after 24 hours at 45 C in the chemically defined germination medium containing glutamic acid and leucine adjusted to pH 6.0 . . 55 ix INTRODUCTION Studies on germination of spores of Clostridium were limited for a long time because of the need for tedious anaerobic techniques and a lack of adequate media for the production of spores of some clostridia. However, during the last fifteen years new culture media were developed thus permitting the production of considerable amounts of spores. Consequently, an increase in investigations concer- ning germination of spores of anaerobes has been observed in the last ten years. The germination requirements of several species of clostridia have been investigated. Extensive studies have been conducted on germination of C. bifermentans, C. roseum, g. sporogenes and Q. botulinum, but specific knowledge about the germination systems in these spores is quite limited. With regards to C. perfringens, almost nothing is known concerning its germination requirements. Germination responses of spores of C. perfringens have primarily been studied in complex media, and little information is available on germination of these spores in media of known chemical composition. The mechanism of germination in spores of bacilli has been the subject of several investigations. However, for clostridial spores there is little information concerning the mechanism of action of germinants, the characteristics of the receptor sites and the effect of inhibitors on the germination process. The objectives of this project were: l) to design a chemically defined medium for the germination of spores of g. perfringens F0]; 2) to determine the minimum nutritional requirements for germination; and 3) to investigate the effects of various compounds on germination. LITERATURE REVIEW Clostridium perfringens Clostridium perfringens is a nonmotile, gram-positive, anaerobic rod with blunt ends which forms spores, but many strains form few spores in normal laboratory media. Dis— tinctive characteristics are a stormy fermentation of milk at 46 C and nonmotility (Breed gt 11., l957). It is more widely spread than any other anaerobic pathogenic bacterium, and its principal habitats are soil and the intestinal contents of man and animals. Since the late l800$ it has been linked with food poisoning, but it was not until 1959 that g. perfringens foodborne illness was oficially recog- nized (Smith, l968; Duncan, 1970; Ladiges gt _l., l974; Matches gt 11., l974). Clostridium perfringens causes a disease characterized by acute abdominal pain and diarrhea, accompanied by little or no nausea and vomiting. The incubation period is usually 9-l5 hours, the illness is of short duration, and fever or other signs of infection are rarely observed (Hall 33 31., 1963; Strong gt al., 1963; CDC, l977). Food frequently becomes contaminated from the natural sources where C. perfringens can be found. The main problem is the presence of spores which survive cooking, and then germinate and grow in the contaminated food (Ladiges £3.ll-: 1974). Because of the complex media and tedious anaerobic techniques used for the production of spores of anae- robes, studies on germination were consequently limited. In 1965 a review by Perkins (1965) indicated that 224 references cited regarding bacterial spores included only 15 dealing with clostridia. However, after 1965 improved media were designed for the production of spores of clos- tridia and the interest in germination of clostridial spores increased. Currently, several Sporulation media are available, permitting the production of large numbers of spores of C. perfringens. Among these media are those designed by Ellner (1957), Kim gt 11. (1967), Duncan and Strong (1968), Ting and Fung (1972), Goodenough and $01- berg (1972), and Sacks and Thompson (1975, 1977, 1978). Transformation of Spores Into Vegetative Cells The bacterial spore is a dormant structure that enables the cell to persist under unfavorable conditions, with little or no metabolism. A new cycle of growth and multi- plication may start when the environmental conditions are adequate for these purposes (Lewis, 1969). Dormant spores germinate poorly or not at all under conditions that permit germination of aged or heat-activated spores. It has been suggested that the high concentration of cystine present in spore coat proteins is responsible for the dormant state. Disulfide bonds present in these proteins need to be broken to promote changes in tertiary structure resulting in activation of spores and changes in permeability (Keynan and Halvorson, 1965; Keynan gt_al,, 1965). The release of spores from the dormant state is under precise control (Lewis, 1969). The whole process of trans- formation of a dormant bacterial spore into a vegetative cell can be divided into three stages: activation, germina- tion and outgrowth. Each stage must correspond to one or more macromolecular processes which are slowly beginning to be understood (Freese and Cashel, 1965; Keynan and Halvor- son, 1965; Lewis, 1969). Activation. Spores of freshly harvested cultures usually do not germinate or do so only slowly. Often some germination can be induced by placing the spores into rich media, but a long lag period will preceed germination, which will by unsynchronized and incomplete (Keynan and Evenchik, 1969). Activation is a reversible process based on a change in the marcomolecular structure of the spore coats. It is a nonmetabolic change and terminates the dormant state temporarily without ending cryptobiosis. When activated or aged spores are placed under germinative conditions the cryptobiotic state is lost irreversibly (Keynan and Halvor- son, 1965; Keynan gt_al,, 1965; Keynan and Evenchik, 1969). An activation phase may not be recognizable for all spore preparations or strains, or for all germination conditions (Lewis, 1969). Roberts (1968) demonstrated heat activation of spores of clostridia by heating at 70 C for 30 minutes. The presence of calcium dipicolinate also activates spores, presumably due to its chelating abilities (Freese and Cashel, 1965). Methods of Activation. Three common methods used for activation of spores are: exposure to sublethal heat, pH treatments and aging. Among these, the simplest is heat activation which helps condition spores for physiological germination. Some bacterial spores are extremely dormant and require temperatures as high as 100 C or more for activation. The activation energy required is high, similar to that required for heat denaturation of macro- molecules (Keynan and Halvorson, 1965; Keynan gt gt., 1965). Heat activation induces three measurable changes in a spore suspension: 1) increases the germination rate, 2) activates some enzymes and 3) reduces the requirements for the induction of germination. Heat activation can be influenced by pH (the optimum for spores of bacilli is pH 2-3), adsorption of salts and media in which spores are activated (Keynan and Halvorson, 1965; Keynan t al., 1965; Keynan and Evenchik, 1969). When changes in pH are used to promote activation, the chemical composition of the spore is changed because of the release of dipicolinic acid, which is associated with a change in permeability of the spore coat (Keynan and Evenchik, 1969).:1 Treatments at low pH may also affect the heat resistance of the spores (Alderton and Snell, 1969). Finally, natural activation can be achieved by aging. Upon storage spores behave as if they have been heat- activated. Aging and heat activation seem to be similar phenomena, and thermodynamically there seems to be no difference between heat activation and aging. Both end. the state of dormancy and the only difference is that during heat activation dormancy is lost temporarily while during aging dormancy is lost irreversibly (Keynan and Evenchik, 1969). Germination. Germination is a well-defined stage in the developmental cycle of sporeforming bacteria. It is essentially the conversion of a resistant and dormant spore into a sensitive and metabolically active form (Gould, 1969). Germination is a process of hydrolysis, depoly- merization and exudation of low and high molecular weight substances (Lewis, 1969). The resulting cell has lost the characteristics of a bacterial spore, is metabolically active, heat-labile and non-refractile, yet readily distinct from a vegetative cell (Keynan and Halvorson, 1965). At the same time that depolymerization of the spore coat is taking place, biosynthesis of the new cell wall is occurring (Lewis, 1969). The breakdown products can be utilized in the synthesis of the new wall in the outgrowing cell (Vinter, 1965). Several changes are associated with germination, such as the loss of resistance to dehydration, pressure, vacuum, UV and ionizing radiation, antibiotics, chemicals and . extremes of pH. There are also some cytological changes, such as the disintegration of the cortex and the appearance of some of the elements of the cytoplasm. The optical density of a spore suspension also decreases by about 60% due to the excretion of dry matter from the germinating spore. Phase darkening is the result of the loss of refrac- tive index of spores during germination as a result of the combined effects of excretion of dry matter, swelling and redistribution of water inside the spore (Gould, 1969).' These changes, together with the release of calcium, loss of heat resistance and onset of stainability, have been used as criteria to follow germination of spores (Treadwell _£._l-: 1958). Different conditions are required for germination and growth, indicating that there are two different processes involved. Special nutritional requirements are needed for germination and outgrowth to transform the spore into a normal vegetative cell. One important fact is that spores can germinate in the presence of inhibitors of macromolecular synthesis, indicating that production of new compounds is not essential for germination (Keynan and Halvorson, 1965). Outgrowth. Outgrowth is a process of synthesis of new macromolecules, particularly those not present in the resting spore. The result is the emergence of a new vege— tative cell (Keynan and Halvorson, 1965; Keynan and Even- chik, 1969). Germination Requirements of Bacterial Spores The germination requirements for bacterial spores can be simple and specific. Amino acids, sugars, organic acids, ribosides and even mineral salts have been reported as germinants for bacterial spores. Germinants can be metabolizable and nonmetabolizable compounds. However, the specific metabolic effects of metabolizable germinants is controversial, some authors claim germination is a metabolic process while others believe germination is non- metabolic. Germination requirements for spores of bacilli have been widely studied. Among the most common germinants, L-alanine has been the most effective. Germination by L-alanine can be competitively inhibited by its D-isomer. The mechanism of action for germination by L-alanine has been explained as a conversion of the amino acid into some compound, such as pyruvate, that can be used as an energy source (Heiligman gt gl., 1955; Gould, 1969). Freese and 10 Cashel (1965) proposed that L~a1anine and its analogs act as germinants because they are deaminated by alanine dehy- drogenase and, simultaneously, NAD is reduced to NADH. However, since mutants lacking the enzyme germinate in the same manner as spores of the wild type, this effect seems to be caused by some reaction other than the NAD-dependent enzymatic reaction of alanine dehydrogenase (Freese and Cashel, 1965). Ribosides, particularly adenosine and inosine, are also excellent germinants. These compounds promote germi- nation almost instantaneously when used at concentrations as low as ca. 10'5M, with higher concentrations being inhibitory (Pulvertaft and Haynes, 1951). The main function of ribosides is probably as a source of utilizable phos- phates or ribose (Gould, 1969). Little information is available on the effect of different sugars on germination of bacterial spores. Hachisuka t l. (1955) reported that germination of e spores of B . subtilis in the presence of asparagine iso- leucine, serine and valine was stimulated by the presence of glucose. Heiligman gt 1. (1955) also reported that a mixture of glucose, L-alanine and adenosine permitted rapid germination of spores of bacilli. Germination requirements for clostridial spores are more complex than those of Bacillus spores (Holland gt gl., 1969; Haites and Wyatt, 1971). L-alanine was reported to 11 induce 100% germination of t. bifermentans (Waites and Wyatt, 1971). In the absence of L-alanine a mixture of L-arginine, L-phenylalanine and L-lactate also induced rapid germination (Waites and Wyatt, 1971). Germination induced by L-alanine requires the presence of sodium chloride and sodium phosphate, since omission of these compounds prevented or reduced the germination rate (Naites and Hyatt, 1971). For another strain of t. bifermentans spores germinated in a mixture of L-alanine, L-phenylala- nine and L-1actate (Gibbs, 1971). Spores of t. sporogenes germinated in the presence of L-alanine and sodium ions, but germination was faster in the presence of L-phenylalanine and L-arginine. D-alanine was neither inhibitory nor induced germination alone, although it induced germination in the presence of other amino acids (Holland gt gl., 1969; Haites and Hyatt, 1971). Under alkaline conditions t. sporqgenes strain PA 3679h was able to germinate in a medium containing L-alanine and pyrophosphate (Uehara and Frank, 1965). For t. roseum, a mixture of alanine and arginine promoted germination. However, the same mixture was not effective for t. botulinum which required yeast extract and bicarbonate to induce rapid germination (Hitzman gt gl., 1957). Treadwell gt g1. (1958) reported that it is difficult to attain germination of t. botulinum in synthetic medium. 12 With respect to the effect of ribosides on germination of clostridia, apparently these spores do not require the presence of ribosides for germination. This fact confirms that the germination requirements for spores of clostridia are strikingly different when compared with spores of bacilli (Gould, 1969). There are few publications concerning germination of t. perfringens and most of them are related to germination in complex media. Busta t al. (1973) tested several com- plex nitrogen sources on germination of t. perfringens, among them isolated soy protein, sodium caseinate, trypti- case and casaminoacids, as well as mixtures of all amino acids. Without heat activation less than 50% of the spores germinated in 12 hours in the complex nitrogen sources, but if spores were heat activated, more than 90% of the spores germinated in one hour. A combination of 18 amino acids also permitted considerable germination (Busta ££.11-: 1973). When soy proteins were used they had both stimula- tory and inhibitory effects. Furthermore, there is an apparent relationship between treatment given to the soy proteins and their effect on germination (Busta and Schroeder, 1971; Schroeder and Busta, 1973). Ahmed and Walker (1971) determined the optimum condi- tions for germination of spores of t. perfringens S45. They reported excellent germination in vitamin-free casami- noacids, trypticase or yeast extract. Germination in 1% 13 yeast extract was enhanced by L-cystine, L-cysteine, L- tryptophan and L-tyrosine, as well as the addition of sodium bicatbonate, glucose, lactate, thioglycollate or sodium chloride. The previous single amino acids in the presence of glucose and sodium chloride permitted good rates of germination. However, one of the best germinant solutions was a mixture of L-cystine and sodium chloride. This system promoted minimal germination without an oxygen scavenger (Ahmed and Walker, 1971). Nonmetabolizable germinants have also been used for germination of spores of clostridia. Sodium and manganese ions apparently enhance germination by their effect on some structure protective to the core of the spore, which depends on associated ions for stability. Surfactants that alter the permeability, as well as chelates, also act as germinants (Gould, 1969). Ando (1978) reported germina- tion of E. perfringens spores in a mixture of potassium chloride and potassium phosphate at pH 7.0. Other Factors that Affect the Germination of Spores of Clostridia Among other factors that can affect the rate and extent of germination, the effect of carbon dioxide in germination media has received considerable attention. Some anaerobic bacterial spores, such as those 0f.E- botu- linum, have an absolute requirement for carbon dioxide and 14 fail to germinate under vacuum (Treadwell t al., 1958). Spores of other clostridia such as t. chauvei, Q. hystoly- ticum and t. perfriggens do not show a requirement for carbon dioxide (Wynne and Foster, 1948; Holland gt gl., 1969). The requirement for carbon dioxide can be provided by sodium bicarbonate. Even though a stimulatory role for sodium ions has been postulated when sodium bicarbonate is used in germination media, the stimulatory effect of sodium bicarbonate is due to the bicarbonate ion. Recently, Enfors and Molin (1978) determined that the stimulatory effect of carbon dioxide on spore germination was more pronounced at low pH (5.2-6.0) where the primary molecular species is 002. Clostridium perfringens germinated in the presence of 5% carbon dioxide, and the rate of germination was faster than that observed for t. sporqgenes. Oxidation-reduction potential and water activity have been widely studied as factors which affect the growth of IQ. perfringens. However, little information is available concerning their effects on germination (Kliger and Guggen- heim, 1938; Hanke and Katz, 1943; Reed and Orr, 1943; Mead, 1969; Ades and Pierson, 1973). For t. tgtggt, germi- nation is retarded if the Eh of the medium is increased, but no conclusive remarks were made about inhibition caused by Eh or gaseous oxygen (Knight and Fidles, 1930). It seems that oxygen itself, and not Eh, is the decisive factor in the inhibition of germination of spores of t. butyricum 15 (Douglas _t_gl,, 1973). Other Ways to Promote Germination Alkali-Induced Germination. Alkaline treatment of spores removes a protein fraction from the spore coat of IQ. bifermentans, increasing the germination rate. Although Waites gt gt. (1972) reported that alkaline treatments do not affect the viability of the treated spores, Duncan gt gt. (1972) stated that alkaline treatments reduced the apparent viability of heat-sensitive strains of t. perfringens to about 0.0005% recovery. The alkaline treatment only affected the normal germination system and viabile spore recovery was increased to 90-95% in the presence of lyso— zyme (Duncan gt gt., 1972). Lysozyme and other 1ytic enzymes act on the murein of the spore coat, but the spores must be treated with reducing agents or low pH to break the disulfide bonds to facilitate the enzymatic attack. These 1ytic enzymes are effective in inducing germination of heat~injured,1acid treated or alkali-treated spores of heat-sensitive or heat-resistant strains of t. perfringens. This enzymatic action is dependent upon incubation time, pH and temperature (Duncan gt gl., 1972; Adams, 1973; Ando, 1975). Treatment with EDTA at pH 9.5 permitted lysozyme- induced germination of spores of Q. perfringens by sensi- tizing them to the action of lysozyme. The spores are not 16 sensitized by breaking disulfide bonds, but rather by chelation of cations which seem to play an important role in resistance of spores to lysozyme (Adams, 1973). Nitrites and Sodium Chloride. The effects of common food additives, such as sodium chloride and sodium nitrite, have been evaluated in the germination of spores of clos- tridia. Nitrite actually stimulates germination, especially under acidic conditions at elevated temperatures, but out- growth is completely blocked (Duncan and Foster, 1968a). Germination of PA 3679 with sodium nitrite, has been achieved at temperatures as high as 90 C. Ionic germination apparently takes place by induction of a volume change per- mitting core hydration or by activation of some enzymes (Duncan and Foster, 1968b). The conditions needed for nitrite-induced germination are different from those pro- moting physiological germination or growth. This type of germination is dependent on high temperature, low pH and relatively high concentrations (2.0%) of nitrite (Labbe and Duncan, 1970; Rhia and Solberg, 1975). Concentrations of sodium chloride as high as 4.0% did not interfere with germination of spores of PA 3679 (Duncan and Foster, 1968b). Inhibitors Two types of inhibitors affecting the life cycle of sporeforming microorganisms are those affecting vegetative development and those inhibiting germination of spores. 17 The inhibitors of interest in this study are potassium sorbate and Lauricidin p1usTM which inhibit vegetative growth, and methylantranilate and sodium 5-5 diethylbar- biturate which inhibit germination of aerobic spores. Inhibitors of Vegetative Growth. Potassium sorbate has been utilized mainly for its fungistatic and bacterio- static effects. The effective inhibitory species in the undissociated form of sorbic acid which apparently inhibits the oxidative assimilation of carbon (Frazier, 1967). A wealth of information is available on its action on vege- tative growth; however little is known about the effect of potassium sorbate on germination of bacterial spores except that germination of bacterial spores takes place at concentrations which would inhibit growth (Gould, 1964). All the published information on lauricidin concerns the effect of this compound on the vegetative growth of bacteria. The mechanism of action of lauricidin is asso- ciated with the hydrophilic and hydrophobic parts of the molecule. The hydrophobic part of the fatty acid is apparently the most important portion of the molecule and Kabara and coworkers postulate that this compound affect the fluidity of the cell membranes (Kabara, 1978; Kabara and Vrable, l977). Lauricidin plusTM, a mixture of lauri- cidin and sorbic acid, was recently patented as an anti- microbial agent which has activity against a wide spectrum of microorganisms. 18 Specific Inhibitors of Germination. Methylanthra- nilate inhibits germination of spores of Bacillus species. The inhibition of germination by this compound is irre- versible and is not eliminated by subsequent washing of the treated spores with the inhibitor (Prasad and Srini- vasan, 1969). Methylanthranilate interfers with the L- alanine-induced germination system as a competitive inhi- bitor. The effect can be counteracted using a combination - munwa ' I of D-glucose, D-fructose and potassium ions, presumably to activate a separate germination system (Prasad, 1974). Sodium 5-5 diethylbarbiturate or veronal is a well F known inhibitor of germination of aerobic spores. Sierra (1968) tested the effect of veronal on the germination of spores of g. subtilis in a complex medium. Germination of heat-activated and nonheat-activated spores was prevented in the presence of 10 mM veronal. The mechanism of inhibi- tion is reversible and seems to be associated with L-alanine induced germination. The activity of the inhibitor is increased by decreasing the pH or increasing the concentra- tion of veronal at a constant pH. Thus the undissociated molecule is the active species. The inhibitory action of veronal is also counteracted by the combined addition of D-glucose, D-fructose and potassium ions (Sierra and Bowman, 1969). MATERIALS AND METHODS Organisms Clostridium perfringens FDl from the culture collec- tion of the Michigan State University Food Microbiology Laboratory was used in all experiments. Also, t. perfrin- gggg strains NCTC 8238, ATCC 3624, ATCC 12195 and t. sporo- ggggg PA 3679, obtained from the same collection, were used in some experiments. Spore Production and Preparation of Spore Suspensions Spores were produced by a modification of the method described by Duncan and Strong (1968). An active culture was obtained by three subsequent transfers of t. perfrin- gggg vegetative cells in Fluid Thioglycollate medium (FTG). The first culture was grown for 18 hours at 37 C, 1 ml was transferred to 9 m1 of fresh FTG, incubated for 4 hours at 37 C, and finally 1 ml of this culture was inoculated into 9 ml of FTG and grown for 4 hours at 37 C. This last FTG culture (10 ml) was transferred into 100 ml of Duncan- Strong Sporulation medium (05) and incubated for 18 to 24 hours at 37 C. Spores were harvested by centrifugation in a Sorvall refrigerated centrifuge at 14,600xg for 30 19 20 minutes at 4 C. Spores were washed with chilled deionized, sterile water 5 to 7 times and stored at 4 C. Heat Shock Several heat shock treatments were given to the spore suspension to promote activation. Spores were heat shocked in distilled deionized water at 70 C, 75 C and 80 C in a temperature controlled (t1 C) water bath for 20, 15 and 10 minutes, respectively. U933. Sporulation Medium. Duncan-Strong Sporulation medium was prepared using the following formula: yeast extract, 0.4%; peptone, 1.5%; soluble starch, 0.4%; sodium thio- glycollate, 0.1%; dibasic sodium phosphate, 1.0% (Duncan and Strong, 1968). PlatinggMedium. Tryptose—Sulfite-Cycloserine basal medium (TSC); Tryptose, 1.5%; soytone, 0.5%; yeast extract, 0.5%; sodium metabisulfite, 0.1%; ferric ammonium citrate, 0.1%; agar, 2.0% (Harmon _t _l., 1971). No cycloserine was added. Germination Media. Brain Heart Infusion, 2.5%; yeast extract 0.27% (BHI + YE; Duncan gt_gl., 1972). Fluid thioglycollate (FTG) broth (Difco). FTG prepared in the laboratory (LFTG): trypticase, 2.0%; sodium chloride, 0.25%; dibasic potassium phosphate, 0.1%; sodium sulfite, .. '1‘”... w ‘nair. .- - ' 21 0.02%; sodium thioglycollate, 0.06%; L-cystine, 0.04%; glucose, 1.0%. Chemically defined media were prepared utilizing the inorganic ingredients of LFTG plus L-cystine. Na thioglycollate and glucose (this medium was designated FTG-base). The nitrogen source was represented by mixtures of amino acids or by a single amino acid. Carbohydrates Used as Germinants Ribose, xylose, fructose, lactose, sucrose, cello- biose, melibiose, melezitose, and raffinose were substi- tuted for glucose in the germination system. Sugars were L present at a final concentration of 1.0%. Non-metabolizable analogs of glucose were also used. These were represented by«- andfi-methylglucopyranosides which were tested at a final concentration of 1.0%. Nitrogen Sources Used as Germinants Complex and simple nitrogen sources were used as ger— minants, trypticase and casaminoacids were used at a final concentration of 2.0%. Also individual amino acids were tested, in different combinations and concentrations. The concentrations used were based on the ones reported by Busta gt gt. (1973) and are listed in Table l. D-isomers of L-alanine, L-glutamic acid and L-leucine were tested. They were added at the same concentration as the corres— ponding L—isomer. Table 1. Concentrations of amino acids used as germinants in chemically defined media Amino Acid mg/l Amino Acid mg/l Alanine 30 Serine 63 Valine 72 Threonine 49 Leucine 92 Cysteine 3 Isoleucine 61 Tyrosine 63 T Proline 113 Aspartic 71 i Phenylalanine 50 Glutamic 224 Tryptophan 17 Lysine 82 g Methionine 28 Arginine 41 Glycine 27 Histidine 31 23 Mineral Salts Several mineral salts were tested as substitutes for sodium chloride. They were tested at a concentration of 0.25% in the presence of glucose, 1.0%; glutamic acid, 0.022%; and leucine 0.018% at a final pH of 6.0. The mineral salts used were the following: potassium nitrite, potassium nitrate, sodium nitrite, sodium nitrate, potas- sium chloride and sodium bicarbonate. Inhibitors Two types of inhibitors were tested in the germina- tion system: 1) inhibitors of vegetative growth and 2) inhibitors of germination of aerobic spores. Inhibitors of Vegetative Growth. Potassium sorbate US) was tested in complex (BHI + YE and FTG) and chemically defined media, over a pH range from 5.0 to 7.0 at concen- trations ranging from 0.1 to 2.0%. Lauricidin a known inhibitor of growth of gram-posi- tive bacteria has been combined with KS to produce an inhibitor called Lauricidin plusTM. It was tested over a concentration range from 0.03 to 0.5 mg/ml in complex and chemically defined media. This compound was first dis- solved in fresh media tempered to approximately 60 C and then added aseptically to fresh media tempered to the same temperature. 24 Inhibitors of Germination of Aerobic Spores. Sodium 5,5-diethylbarbiturate or Veronal (V) was tested at a final concentration of 10 mM (a concentration which inhibits germination of aerobic spores). It was only tested in chemically defined media. (FTG-base + L-alanine, FTG-base + L-glutamic acid, FTG-base + L-leucine and FTG-base + L-glutamic acid + L-1eucine). Methylanthranilate (MA) was tested in FTG and in FTG- base + L-glutamic acid + L-leucine at a final concentra- tion of 0.5 mM. p_H_ Adjustment of pH was carried out with 0.1N hydro- chloric acid and 0.lllsodium hydroxide. The pH range used was from 4.0 to 10.0 with intervals of 0.5. A Corning model 7 pH meter was used for measurement of pH. Sterilization When media with unadjusted pH were used, sterilization was performed by autoclaving at 121 C for 15 minutes. However for the chemically defined media, only some ingre- dients were autoclaved. Sugars and amino acids were filter sterilized and added to the media aseptically. When media with adjusted pH were used, filter sterilization with Gelman membrane filters (0.45/0) was used to avoid changes in pH which occur during autoclaving. 25 Inoculation Screw-cap tubes, 13 x 10-mm, were used as germination containers. Each tube contained 5 m1 of culture media and 0.5 ml of a spore suspension were added to give an initial optical density of 0.32 t 0.04. Incubation All germination experiments were conducted at 45 C in a forced air incubator. Measurement of Germination Three criteria were used to follow germination of the spores: a) Decrease in optical density (00) of a spore suspension at 625 nm on a Spectronic 20 (Bausch and Lomb) with a red filter. Readings were taken every 30 minutes during the first two hours and subsequently every one hour up to 6 to 10 hours. A final reading was taken after 24 hours; b) Observation under an American Optical Series 10 phase-contrast microscope for darkening of individual spores; c) Loss of heat resistance after heat shock at 80 C for 5 minutes, followed by plating on TSC. Incubation of plates was performed under anaerobic conditions at 37 C for 24 hours. 26 Calculation of Percent Germination and Extent of Germi- nation (Y) The percent change in 00 was calculated as follows: % change in on = 9111:991me =90—Dtx 100 ODi 001 Where: 001 = initial 00 ODt = 00 at time t The extent of germination (Y) was calculated as follows: _ ODi-ODt _ gout Y ‘ ——ooi-oof ' ODi-ODf Where: ODf = fina1 OD AODt= Difference of ODi-ODt at time t The percent germination was calculated considering the relationship between decrease in 00 as a percent of the initial 00, and the extent of germination measured by loss of heat resistance of the spore suspension. A 55% decrease in the initial 00 corresponded to 99.99% germination. Greater decreased in 00 may occur due to loss of cell constituents and cell lysis, particu- larly after incubation for 24 hours. RESULTS Germination in Complex Media Spores germinated relatively rapidly in BHI + YE, FTG and FTG-base + Trypticase. During the first 0.5 to 1.5 hours there was a marked drop in 00525 after which the spores were phase dark when viewed under phase microscopy. These complex media supported substantial outgrowth after germination. Germination in Chemically Defined Media Since spores of t. perfringens F01 germinated in FTG, FTG-base plus all 18 amino acids present in trypticase was used as the basis for a chemically defined medium. As expected, this medium promoted germination of the spores and also supported some outgrowth. In comparison to FTG, there was a relatively long lag phase before the onset of germination. There was a drop in 00625 of 50% after 2.5 hours and by 6 hours outgrowth was also observed. However, the nutrients present in this medium were not enough to support luxuriant growth. The 18 amino acids that supported germination were split into four groups (Table 2) based on the characteristics 27 28 Table 2. Classification of amino acids based on the characteristics of the R group Nonpolar Polar Polar-charged Basic Alanine Glycine Aspartic Lysine Valine Serine Glutamic Arginine Leucine Threonine Histidine Isoleucine Cysteine k Proline Tyrosine f Phenylalanine Tryptophan Methionine 29 of their R groups (Lehninger, 1975). When media were prepared with two or more groups of amino acids, the most efficient media for germination were those having the nonpolar and polar-charged groups. The nonpolar group and the polar-charged group were able to support germina- tion, while the polar and basic groups were unable to promote germination (Table 3). Interestingly, the nonpolar P and polar-charged groups were more effective alone than in combinations containing the other two groups of amino acids. For example, the polar group completely blocked i the germinative action of the nonpolar group when the two L were in combination. The effect of single amino acids in the nonpolar and polar-charged groups on germination is shown in Table 4. Only alanine, glutamic acid and leucine supported germina- tion with a decrease in 00625 ‘>50% within 24 hours at 45 C. Effect of Alanine, Glutamic Acid and Leucine on Germination of Spores of C. gerfringens FDl During the initial stages of germination, the fastest germination rate among single amino acids added to FTG-base was obtained with leucine, followed by glutamic acid and alanine (Table 5). However, after 24 hours no marked differences were observed in the extent of germination for each of these three amino acids. When added at twice the normal concentrations, the rates of germination 30 Table 3. Percent change in optical density of a spore suspension of Clostridium pgrfringens FDl in various chemically defined media after 5 and 24 hours Change in OD (%) Mediaa Time 5 hours 24 hours None 0 0 All amino acids 4 59 All except polar 6 40 All except polar-charged 0 5 All except nonpolar 3 45 All except basic 3 41 Nonpolar + polar-charged 50 53 Nonpolar + polar ' O 0 Nonpolar + basic 7 63 Polar + polar-charged 3 48 Polar-charged + basic 7 48 Nonpolar 18 71 Polar-charged 13 47 Polar O 3 Basic 0 7 “Km. 1 A aA11 media contained FTG-base plus the amino acid group(s) indicated (pH 6.8) 31 Table 4. Effect of the nonpolar and polar-charged groups of amino acids on germination of spores of t. perfringens FDl after 24 hours at 45 C Amino acida Dficggasg) Amino acida ?:cggaf§) Nonpolar group 70 Leucine 59 Polar-charged group 67 Isoleucine 19 Aspartic 24 Proline 13 F Glutamic 71 Phenylalanine 16 Alanine 53 Tryptophan 10 Valine 20 Methionine 10 h ; aA11 media contained FTG-base (pH 6.8) plus the amino acids indicated Am.o :av mmmnuwhd op umuuw mm: umamuwucw Amvuwom ocPEm been 32 cos me go, mm me mm x» ea; + ep< oe_ am < co. am me me x» ems + ape cop ee em Nm me am xx ap< + ape cop _L as ea am mp XN accuses co. Nu _m .we mm _N xm aeeee_< co. _c an me we mm xm easeaepe ooF mm _m we NM mp x2 ae_e=es Do, me me am «N m_ X? aeaeep< ooF mm mm on NN mp x_ e_Eaa=~e use ARV no at Agm Nab no ea Neel Lev go as ‘11 cowumcwEwa mmmmsumo cowumctzgmu mmmmLumc :owumaEwa mmmmgumo em op m muwu< ocws< mgzog cw «EFF 8 me be mesa; am can op .m Lance _na hemmewcccea .m co mmcoam mo cowumcwEme mg» co mcwuamp new uvum qumpspm .mcwcmpm co pumeem .m mpnmh 33 increased for glutamic acid and alanine while the rate for leucine was essentially unchanged. When combinations of two of the three amino acids were used at half the original concentration, the mixture of glutamic acid and leucine gave the best results regarding rates of germination. The extent of germination after 24 hours was similar for single amino acids or any of the mixtures (Table 5). I I D-glutamic acid and D-leucine supported rapid germina- tion when used alone or in combination with their L-isomers. However,D-alanine caused a delay in the onset of germination, even when L-glutamic acid and/or L-1eucine were used as $7 germinants. This inhibitory action affected only the initial stages of germination (Table 6). Effect of a Heat Shock on the Germination of Spores of C. perfringens Initially a heat shock of 70 C for 20 minutes was given to spore suspensions. With this activation treatment spores germinated at a satisfactory rate and extent, either in FTG or in a chemically defined medium supplemented with 18 amino acids. However, germination was more rapid in FTG than in the synthetic medium. Nonheat-shocked spores germinated in an asychronous manner in FTG and did not germinate in the chemically defined medium (Fig. 1). From the previous results it was apparent that a heat treatment at 70 C for 20 minutes modified the germination 34 Table 6. Effect of D-isomers of alanine, glutamic acid and leucine on the germination of spores of g. per- fringens FDl after 2, 6 and 24 hours at 45 C. Decrease in OD (%) Amino acida 2 hours 6 hours 24 hours L-ala 3 16 65 D-ala O 10 58 LD-ala 0 7 59 L-glu 46 50 50 D-glu 46 46 50 LD-glu 46 so 54 L-leu 48 48 56 D-leu 46 50 50 LD-leu 48 48 52 L-glu + L-leu 56 56 63 D-glu + D—leu 48 52 52 LD-glu + LD-leu 44 43 43 L-glu + D-ala 3 23 50 L-leu + D-ala 3 16 61 L-glu + L-leu + D-ala 29 50 51 L-glu + L-leu + L-ala + D-ala 33 52 55 aThe amino acid(s) indicated was added to FTG-base adjusted to pH 6.0. 35 I‘Dr- OHeat Shocked (FTG) ONonheat Shocked (FTG) inHeat Shocked (FTG-base + 18 amino acids) . INonheat Shocked (FTG- base + 18 amino .acids) OD625 0.1 l l l A- l C) .2. 4' 16 124' TIME (h) 1. Effect of a heat shock at 70 C for 20 min on ger- mination and outgrowth of Clostridium perfringens FDl at 45 C in FTG or FTG-base plus 18 amino acids 36 requirements. Using different time/temperature relation- ships, as the temperature used during the heat shock increased, the germination response in a chemically defined medium also increased. However, in some instances, no improvement in the ability to promote germination in media lacking a specific group of amino acids was obtained (Table 7). This was the case when the polar group was omitted, E where a slight decrease in the germination extent was obtained as the heat-shock temperature was increased. Since a heat shock at 80 C for 10 minutes generally resulted in the best germination in these chemically defined media, it E‘ was selected for use in further experiments. Data presented in the following Tables and Figures was obtained using spore suspensions which had been heat shocked at 80 C for 10 minutes. Effect of pH on Germination of C.tperfringens FDl in Complex and Chemically Defined Media Changes in pH dramatically affected germination induced by FTG-base containing single amino acids or combinations of amino acids which promoted germination. Germination in the complex media took place over a much broader pH range. On the acid side BHI + YE lost its ability to promote rapid germination at pH 5.5, FTG at pH 5.0 and the chemically defined medium with glutamic acid and leucine at pH 4.5 (Fig. 2). 0n the alkaline side, the complex media supported 37 Table 7. Effect of various heat treatments on the germina- tion of spores of t. pgrfringens F01 in chemically defined media after 24 hours at 45 C. Change in OD (%) Mediaa 70 C/20 min 75 C/15 min 80 C/10 min All except nonpolar 11 37 52 All except polar-charged 9 10 7 All except polar 51 42 40 . All except basic 9 28 47 I aThe groups of amino acids indicated were added to FTG-base, pH 6.8. 38 Au me am meson m cmumm Eamume umcwwmu appwqumgo new Lao; P cmuwo ovums xm—quuv menus umcwwmu prmuPEmco use xmpasoo cw you mcmmcwgecma .0 mo mucoam we cowumcwsgmm :0 :a co uomeeu .N .mwm O I“ O 3 0.0.. 0.0 0.0 OK 0.0 O..." O.v . . er . . . . TLH. Yd . a . O 11, ~ 4. \. .. x .. a; s . um i \ s / I’ ~ WP o I x . u" z , . v lo \ s. N / — ~ he I w \ N a. /. u s \ an .I / a ‘s\ “N ¢ \ nu / an ‘II-III \* a ll. \\ ./ \\k‘ n" .1"\\ ./. \\\\ 1 mv G. . K \ \\ amazon \1I1... \ 3.8.53 «111.1 .\ or. 1....-- .t my>.+ 2&0 IIIIII. Om 39 relatively rapid germination at pH 9.0, but not at pH 10.0. Outgrowth was observed in the complex media even at pH 8.5. For the chemically defined media containing alanine, glu- tamic acid or leucine, there was a narrow optimum pH for germination around pH 5.5 to 6.5. When glutamic acid and leucine were both present a slightly wider optimum pH range was observed (Fig. 3). a Effect of Iggredients of the Basal Medium on Germination of Spores of C. perfringgns F01 Spores were washed 10 times in chilled, distilled, deionized water in an attempt to completely remove any adsorbed substances from the spores and experiments were conducted by adding ingredients to media containing glucose, glutamic acid, and leucine. When the pH was not adjusted, the extent of germination was low if only one other ingre- dient was added. When the pH was adjusted to 6.0, partial germination was promoted only by the addition of sodium chloride or L-cystine; after 4 hours of incubation decreases in 00625 of 35 and 32% were obtained with sodium chloride or L-cystine, respectively. The other ingredients did not cause any noticeable change id 00625 even after incubation for 24 hours at 45 C (Table 8). When two ingredients were added to the medium contain- ing glucose, glutamic acid and leucine, the most effective combinations contained sodium chloride. The media containing 4O .0 me no mczm; m Loam» emcee cmcwwoo zFPmumEmcu cw Pom mcmmcwcccma .0 mo mmcoam co zowuchEgmm :o :a mo aummwm .m .mwa 92900 N1 ss0'1 v. 2.. 03 o.» o.» ow on o... o 7 _ _ _ _ _ O i o. - o~ :m._+:._o-1 - on =4o-- D l m... - oe - on - om 41 Table 8. Effect of single ingredients and combinations on germination of spores of t. perfringens FDl after 1, 4 and 24 hours at 45 C Ingredienta Change in 00 (%) 1 hour 2 hour 3 hour NaCl (Na) 15 35 38 HZHPO4(K) 0 0 0 NaZSO2 (NaS) 0 3 6 Na-thioglycollate (NaT) 0 0 0 L-cystine (C) 27 32 32 Na + K 21 41 50 Na + NaS 3 27 41 Na + NaT 0 22 38 Na + C 35 44 47 K + NaS O 0 6 K + NaT 0 0 6 K + C 36 42 47 NaS + NaT 0 O 3 NaS + C 18 29 30 NaT + C 17 34 46 Na + K + C 50 54 54 Na + NaS + C 13 30 35 Na + NaT + C 39 44 48 Na + K + NaS + C 46 46 50 Na + K + NaT + C 46 50 50 Na + NaS + NaT + C 32 44 44 Basal Complete 51 56 56 aMedia contained 224 mg/l L-glutamic acid, 92 mg/l L- leucine and 10 g/l glucose adjusted to pH 6.0. 42 sodium chloride combined with dibasic potassium phosphate, sodium sulfite or L-cystine had decreases in 00625 of 50, 41 and 47%, respectively after 24 hours. Intermediate changes in 00625 were obtained with sodium chloride + sodium thioglycollate, and sodium sulfite + L-cystine, with 39 and 31% decrease in 00625, respectively. The remaining combinations gave negligible changes after 24 hours at g 45 C. The changes in 00625 obtained with any of the I mixtures of two ingredients of the basal medium were usually less than and occurred more slowly than those obtained with the complete basal medium supplemented with Bl glucose and amino acids. When combinations of three or more ingredients were used, combinations containing sodium chloride, L-cystine and dibasic potassium phosphate were most effective in inducing germination (Table 8). Effect of Different Mineral Salts as Substitutes of Sodium Chloride When a series of mineral salts were tested to determine their ability to substitute for sodium chloride in a chemi- cally defined germination system, only sodium nitrite, sodium nitrate and potassium nitrite induced some germina- tion in media without glutamic acid and leucine. All of the mineral salts tested induced partial germination in the presence of glutamic acid and leucine (Table 9). 43 .mcwosz F\mE mm use cwom owsmpafim P\ms cum umcmmucou cmp + Him e? Pod memuewcccmm EV 86:. .mnxu 0.. .i... 05 0 oo- ..... G ”we a a .. 0N6 l..- . 9 Au .-- m_o. 22:on axe L 8.0 47 -mcp:mocou ucmgmwmwv saw: and cw Fad mcmmcwgmcwm .0 mo museum mo cowumcwscmw m m @9599 coo—— ’ ” I, I l ’I ’[I ’ ’ o o I [loll-Vlkuu‘\ ... ...H V / .\. \ _ . W 9 .7... .nb.m Iav mumncom Sawmmmuoa mo mcowp .m .9: a: 85% fiancee-v. .x. L 8.0 48 concentrations tested at each pH level. At the optimum pH (6.0-6.5) germination in the chemically defined medium was only 60% in the presence of 0.25% potassium sorbate, and higher concentrations completely inhibited germination (Fig. 6). Germination rates decreased in complex and chemically defined media as the potassium sorbate concentration increased. Table 11 shows the time needed to obtain 50% germination in media containing various concentrations of potassium sorbate. The minimum potassium sorbate concen- trations needed to limit germination to 50% are presented in Table 12. Concentrations of Lauricidin plusTM ranging from 0.03 to 0.5 mg/ml did not markedly affect the extent of germina- tion in complex media; even at the highest concentration used more than 70% of the spores germinated within 24 hours at 45 C. However, the rate of germination decreased when media contained 2 0.12 mg/ml Lauricidin plusTM. In the chemically defined medium, the extent of germination and germination rate were substantially affected by the presence of 0.5 mg/ml Lauricidin plusTM (Table 13). Methylanthranilate did not inhibit germination in complex media. In chemically defined media the germination rate was decreased by the presence of the inhibitor and only 62% germination was observed after 24 hours at 45 C (Table 14). Sodium 5,5-diethylbarbiturate (veronal) at a 49 .Ao.o :av mumncom E=_mmmuoa co mcowumcpcmocou “cmgmmmwc saw: anvms umcwmmu x—_momEm;o cw pom mcmmcmcmcwm .0 mo mmsoam we cowumcwscmw EV 88:. 00.0 11. ONO -II o I 233mb. o\o ONO 50 Table 11. Time (in hours) needed to obtain 50% germination of t. perfringens FDl spores in BHI + YE (pH 7.0), FTG (pH 7.0) and chemically defined medium (pH 6.0) containing various concentrations of potassium sorbate. Chemically K-sorbate (%) BHI + YE FTG Defined 0.0 24 1 0.75 24 1.0 2.0 >5 >24 1.5 24 >24 >24 . D. 2.0 <24 >24 >24 aReadings were taken at 0.5 hours intervals during the first two hours and at one hour intervals from 2 to 10 hours. A final reading was taken after 24 hours. 51 Table 12. Minimum concentration of potassium sorbate (%) needed to limit germination of spores of C. pgr; ininggns FDI to s 50% in three media at pH 5.0- 7.0 after 24 hours at 45 C Chemically pH BHI + YE FTG Defined 5 0 -a 0 1b 0.1b 5.5 0.3 0.5 0.2 6.0 1.5 1.5 0.2 I; 6 5 2.0 1 5 0.25b i 7.0 2.0 1.5 0.25b ‘ aInhibition by pH ; b L Lowest concentration used 52 Table 13. Effect of Lauricidin plusTM on germination of spores of t. pgrfringens FDl in three media after 1, 5 and 24 hours at 45 C Decrease in OD (%) Media Time (hrs) 1 5 24 BHI + Ye (pH 7.5) Control 44 _a - 0.03 mg/ml 22 24 41 0.12 mg/ml 14 16 37 0.50 mg/ml 34 . 37 46 FTG (pH 7.0) Control 40 - - 0.03 mg/ml 29 32 44 0.12 mg/ml 22 33 39 0.50 mg/ml 22 32 43 Chemically defined (pH 6.0) Control 42 52 52 0.03 mg/ml 41 49 49 0.12 mg/ml 17 43 47 0.50 mg/ml 2 18 28 aOutgrowth occurred resulting in increases in OD. 53 Table 14. Effect of Veronal (V) and Methylanthranilate (MA) on germination of t. perfrin ens F01 after 1, 5 and 24 hours at 45 C (pH 6.0 Media and Decrease in OD (%) Inhibitors Time (hrs)' 1 5 ~24 FTG Control 50a -b I - Chemically defined Controla 46 49 53 P L-alanine 9 46 51 I L-glutamic acid 42 50 50 L-leucine 44 52 57 L-glutamic + L-leucine (MA) 6 31 34 F L-alanine (V) 11 37 39 L-glutamic (V) 44 47 37 L-leucine (V) 44 50 53 L-glutamic + L-leucine (V) 46 49 51 aDetermined at 0.5 hours bOutgrowth 54 final concentration of 10 mM had no effect on germination rate or extent of germination in complex or chemically defined media (Table 14). Germination of Other C. perfringens strains and PA 3679 For three different batches of spores of Q. perfringens FDl germination varied from 87 to 91% after 24 hours at 45 C in the chemically defined medium containing glutamic acid and leucine. Clostridium pgrfringens strains NCTC 8238 and ATCC 3624 reached a maximum germination of about 70%. However, only 13 and 27% germination occurred for PA 3679 and Q. perfringens strain ATCC 12195, respectively (Fig. 7). 55 VNOO OH0< m m N o 0» oz 100 F 80 _ 60 40 - 20 20.._.