ROOM USE ONLY ABSTRACT RECOVERY PATTERNS OF SPORES OF PUTREFACTIVE ANAEROBE NO. 3679 IN VARIOUS SUBCULTURE MEDIA FOLLOWING MOIST AND DRY HEAT TREATMENT by Jorg A. L. Augustin The purpose of this investigation was to study the pattern of re- covery of spores of Putrefactive Anaerobe No. 3679 (P.A. 3679) in various recovery media following moist and dry heat treatment. Two spore crops were produced in beef heart infusion and trypti— case-yeast extract broth respectively. The spores produced from these media after having been freed from their vegetative sporangia and any foreign material were dispensed into small metal cups in amounts of O. 01 m1 and dried under vacuum at room temperature. Heating these spores in moiSt and dry heat was accomplished by subjecting them to saturated steam in the thermoresistometer or in miniature retorts, and to hot air enclosed in thermal death time (TDT) cans in miniature retorts using saturated steam as a heating medium for the hot air en- trapped in the TDT cans. After the heat treatments, the spores were transferred into the various subculture media which included infusion type media, formulated media and dehydrated media. The effectiveness of the subculture media in recovering surviving spores was established by determining D. values on the basis of endpoint determinations over a temperature range of 230017 to 2800F in moist heat and 255OF to 2 Jorg A. L. Augustin to 320°F in dry heat. D. Values were calculated by the modified Schmidt method. The 2 values were obtained graphically. The results revealed significant differences in decimal reduction times (D values) as affected by the heating medium, the sporulation medium and the subculture medium. The fact that increased D values were obtained when the synthetic medium was supplemented with DNA, RNA and/or vitamin BIZ indicated that at least in part nutritional defi- ciencies of some of the media might be responsible for the differences in the D values obtained with the various subculture media. D values were consistently and considerably higher for beef-heart infusion spores and for those spores which were subjected to dry heat treatment. The 2 values did not vary greatly with the sporulation medium in which the Spores were produced. The largest differences in 2 values re- sulted from the use of the two different heating media. Dry heat treat- ment of the spores resulted in 2 values of 36°F and 33°F respectively. Moist heat treated spores which were produced in beef-heart infusion showed 2 values of 19. 50F with the exception of those spores which were subcultured in trypticase or eugonbroth. In those cases the 2 values were 18. 2013‘ and 17. 5017‘ respectively. Moist heat treated Spores which were produced in trypticase-yeast extract broth gave z values ranging from 15. 50F to 17. 00F depending on the type of subculture medium. In general highest D values were obtained when the heated spores were subcultured in beef infusion. Subculturing of the heated spores in liver infusion, pea infusion or yeast extract resulted in slightly lower D 3 Jorg A. L. Augustin values. Even lower D values were obtained when eugonbroth or trypti- case were used as subculture media. The lowest D values resulted from the use of a synthetic medium. In spite of these similarities found in the recovery of the heated spores regardless of the type of heating medium used, the relative magnitudes of the D values as well as of the 2 values with reference to the subculture media used were different with moist heat than with dry heat. This led to the conclusion that the requirements for germination and outgrowth are different for spores which were subjected to moist heat than for those that were subjected to dry heat. The above results and the conclusions drawn therefore lend support to the theory that the logarithmic order of death of bacterial spores is the result of the inactivation of an essential gene and that the differences in D values obtained are the result of random injury of the genetic material of the spores which forms the basis for the nutritional require- ments of these spores for germination and outgrowth. RECOVERY PATTERNS OF SPORES OF PUTREFACTIVE ANAEROBE NO. 3679 INVARIOUS SUBCULTURE MEDIA FOLLOWING MOIST AND DRY HEAT TREATMENT BY Jorg A. L. Augustin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1964 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. I. J. Pflug, chairman of the thesis committee, for his guidance and patience during the course of this study and during the prepara- tion of this manuscript. The author also wishes to thank the other members of the thesis committee, Dr. R. L. Anderson, Dr. L. Dugan, Jr., Dr. R. C. Nicholas and Dr. H. L. Sadoff for their suggestions and criticism during the preparation of this manuscript. The author is indebted to the National Institute of Health for their financial support of this study through Grant No. AI-03708-03. ii TAB LE OF CONTENTS Page INTRODUCTION ............................ 1 REVIEW OF LITERATURE ...................... 4 1. Death of bacterial spores .................... 5 a. The order of death of bacterial spores ........... 6 b. Thermal destruction curves . ............... 8 c. Nature and mechanism of death .............. 9 2. Methods for studying heat resistance of bacterial spores . . . 13 a. Apparatus .......................... 13 b. Experimental methods ................... 16 c. Methods of calculating D values .............. l7 3. Some factors affecting the thermal resistance of bacterial Spores. . . . . ......................... 20 a. The effect of the sporulation medium ........... 21 b. The effect of the initial spore concentration ........ 24 c. The effect of the medium supporting the spores during heating ........................... 24 d. The effect of the heating medium ............. 26 e. The effect of the composition of the subculture medium. . 29 MATERIALS AND METHODS ..................... 33 1. Preparation of the spore suspensions ............. 33 a. Spores grown in beef-heart infusion . . . ......... 33 b. Spores grown in trypticase medium ............ 34 c. Harvesting and cleaning of spores ............. 35 iii Page 2. Media for standardization and recovery ........... . . 37 a. Infusion type media ...................... 37 b. Formulated media . ..................... 38 c. Dehydrated media ...................... 39 , 3. Enumeration of initial spore count ................ 42 4. Preparation of the spore suspensions for thermal treatment . . 42 5. Heat treatment .......................... 43 a. Moist heat ..................... . . . . . 44 b. Dry heat ........................... 46 6. Calculation of results ...................... 48 3.. Calculation of D values ................... 48 b. Determination of z values .................. 50 c. Lag corrections ....................... 51 RESULTS ................................ 55 1. Preliminary studies ....................... 55 2. Resistance of Spores to saturated steam ............. 6O 3. Resistance of spores to dry heat ................ 79 4. Statistical methods of analysis .................. 82 5. Rearrangement of heat resistance data ............. 87 GENERAL DISCUSSION ......................... 95 SUMMARY AND CONCLUSIONS . . . . . . .I . ............. 104 BIB LIOGRAPHY .......... . . . . . ........ . . . . . I 0 7 APPENDIX . . . . ........... . . . . . . . . ........ 114 iv 10. ll. 12. 13. TAB LES Heat penetration data for cups in TDT cans heated in saturated steam in miniature retorts .................... Average lag correction factors ............... The effect of the type of cup used as a container for spores during heat treatment . . ................... .. Preliminary studies of the effect of several additions to the synthetic medium . . . . ..................... The effect of the addition of NaHCO3 to the synthetic medium on the recovery of beef-heart spores following their heat treatment ............................. D250, and 2 values and thermodynamic constants for moist heat resistance of P.A. 3679 spores ............... D values and their corresponding 95 percent confidence limits in minutes for moist heat resistance of beef-heart infusion Spores of P.A. 3679, obtained in various recovery media D values and their corresponding 95 percent confidence limits in minutes for moist heat resistance of trypticase-yeast extract spores of P. A. 3679 obtained with various recovery . media ............................... Comparative initial counts of P. A. 3679 spores in various subculture media ....... . . ................ The effect of the addition of RNA, DNA, and Vitamin B12 to the synthetic medium on the recovery of moist heat treated beef heart spores . . . . ..................... D300 and 2 values and corresponding heats of inactivation for dry heat resistance of P.A. 3679 spores ....... D values and their corresponding 95 percent confidence limits in minutes for dry heat resistance of beef-heart infusion Spores of P. A. 3679 obtained in various recovery media . . . . . D values and their corresponding 95 percent confidence limits in minutes for dry heat resistance of trypticase-yeast extract Spores of I- . A. 3679 obtained in various recovery media . Page 61 62 80 81 83 Page 14. D values and their correSponding 95 percent confidence limits in minutes obtained by four different methods of calculation . . . . 84 15. Numbers of surviving beef-heart infusion spores of P.A. 3679 recovered in various subculture media following heat exposure at constant times . .......................... 88 16. Number of surviving trypticase-yeast extract spores of P.A. 3679 recovered in various subculture media following heat exposure at constant times . . . . ....... . ....... 89 1A Calculation of data for lethal rates . . . .............. 114 2A Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in beef infusion following moist heat treatment at various temperatures ........................ 116 3A Results of tests of beef-heart infusion spores of P.A.. 3679 subcultured in liver infusion following moist heat treatment at various temperatures ........................ 117 4A Results of tests in beef-heart infusion Spores of P.A. 3679 subcultured in pea infusion following moist heat treatment at various temperatures ........................ 118 5A Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in yeast extract following moist heat treatment at various temperatures ........................ 119 6A Results of tests of beef-heart infusion Spores of P.A. 3679 subcultured in trypticase following moist heat treatment at various temperatures ........................ 120 7A Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in eugonbroth following moist heat treatment at various temperatures ........ . ............... 121 8A Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in a synthetic medium following moist heat treatment at various temperatures ................ 122 9A Results of tests of beef-heart infusion spores of P. A. 3679 subcultured in beef infusion following dry heat treatment at various temperatures ...... . ................. 123 vi 10A 11A 12A 13A 14A 15A 16A 17A 18A 19A 20A 21A Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in liver infusion following dry heat treatment at various temperatures ..................... Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in pea infusion following dry heat treatment at various temperatures ..................... Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in yeast extract following dry heat treatment at various temperatures ..................... Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in trypticase following dry heat treatment at various temperatures ..................... Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in eugonbroth following dry heat treatment at various temperatures ..................... Results of tests of beef-heart infusion spores of P.A. 3679 subcultured in a synthetic medium following dry heat treatment at various temperatures ............... Number of positive tubes recovered from trypticase spores of P.A. 3679 subcultured in beef infusion following moist heat treatment .......................... Results of tests of trypticase spores of P.A. 3679 subcultured in yeast extract following moist heat treatment ......... Results of tests of trypticase spores of P.A'. 3679 subcultured in eugonbroth following moist heat treatment ......... . Results of tests of trypticase Spores of P.A. 3679 subcultured in beef infusion following dry heat treatment .......... Results of tests of trypticase spores of P.A. 3679 subcultured in yeast extract following dry heat treatment . . . . . . . . . . Results of tests of trypticase Spores of P.A. 3679 subcultured in eugonbroth following dry heat treatment ..... . ..... vii Page 124 125 126 127 128 131 133 134 135 FIGURES 10. ll. 12. 13. 14. 15. Page Stratification of tubes with paraffin-mineral oil mixture. . . . . 41 The Thermoresistometer . . . . . 45 The Miniature Retorts . . . . . . . . . . . . . . 47 Thermal resistance curves of beef-heart infusion spores subcultured in beef infusion. . . . . . . . . . . . . 64 Thermal resistance curves of beef-heart infusion spores subcultured in liver infusion . . . . . . . 65 Thermal resistance curves of beef-heart infusion spores subcultured in pea infusion . . . . . . . . . . . . . 66 Thermal resistance curves of beef-heart infusion spores subcultured in yeast extract . . . . . . . . . . . . . . 67 Thermal resistance curves of beef-heart infusion spores subcultured in trypticase . . . . . . . . . . . . . . . . 68 Thermal resistance curves of beef-heart infusion spores subcultured in eugonbroth. . . . . . . . . . . . . . . 69 Thermal resistance curves of beef-heart infusion spores subcultured in synthetic medium . . . . . . . 70 Thermal resistance curves of trypticase-yeast extract Spores subcultured in beef infusion . . . . . . . . 71 Thermal resistance curves of trypticase-yeast extract spores subcultured in yeast extract . . . . . . . . 72 Thermal resistance curves of trypticase-yeast extract spores subcultured in eugonbroth. . . . . . . . . . . . . 73 Thermal resistance curves obtained from D values calculated by four different methods from moist heat resistance data of beef-heart infusion spores subcultured in yeast extract . . . 85 Thermal resistance curves obtained from D values calculated by four different methods from dry heat resistance data of beef-heart infusion spores subcultured in beef infusion . 86 viii 16. 17. 18. 1A Logarithms of the number of surviving, beef-heart spores in various subculture media followin moist heat treatment with heating times chosen to yield 10 survivors with beef infusion as the subculture medium . . Logarithms of the numbers of surviving beef-heart spores in various subculture media followin ,dry heat treatment with heating times chosen to yield 10 survivOrs with beef infusion as the subculture medium . Logarithms of the number of surviving trypticase spores in various subculture media following heat treatment with . times chosen to yield 103 survivors. with beef infusion as the subculture medium . Heat penetrations curve for cups in TDT cans heated in saturated steam ix Page 90 91 92 115 INTRODUCTION Different food products require different amounts of heat for sterilization. Possible explanations for this are differences in nutri- tional composition of and/or presence or absence of inhibitory sub— stances. Little is known about the nutritional requirements for germination and outgrowth of spores of food spoilage organisms following moist or dry heat treatment. What little information is available refers to moist heat only, and in most cases the experimental arrangements do not allow definite conclusions as to which of the subculture media fulfill best the nutritional requirements of these spores. It is the purpose of this investigation to obtain information regard- ing the resistance of spores of Putrefactive Anaerobe No. 3679 (P.A. 3679) to moist and dry heat. Of specific interest are answers to ques- tions as to the nutritional requirements of these spores for germination and outgrowth following heat treatments, and as to the nature and mech- anism that leads to their destruction. P.A. 3679 is used in the food canning industry as one'of several test organisms for the evaluation of thermal processes for low acid type foods. The goal in designing thermal processes is to destroy all microorganisms which are either of public health significance and/or might cause spoilage during normal procedures of handling, storage or distribution. In low acid type foods, chief attention is paid to bacterial 2 spores which exhibit considerably higher heat resistance than their vege- tative cells. Canning sterilization procedures are designed according to the moist heat resistance of bacterial spores. However, where the separ- ately sterilized food product is filled aseptically into pre-sterilized con- tainers, sterilization of the containers by moist heat has proved to be impractical. Instead, dry heat in the form of dry air, inert gases, or superheated steam, or in combination with gaseous agents which are known to be sporicidal have been used in the past. Infusion type media for both sporulation of P. A. 3679 as well as for its recovery following the heat treatment, have been unsurpassed so far by any formulated, dehydrated and synthetic media, from the cri- terion of sporulation media producing spores of greater heat resistance and as subculture media with reference to efficiency in recovery of the heated spores are concerned. The exact composition of these infusion type media is not known, and are thus difficult to simulate in a syn- thetic medium. Therefore, the experimental design will be primarily geared to answer questions as to whether the nutritional requirements are the same for the germination and outgrowth of spores which have been produced in different sporulation media, as well as for Spores which were subjected to treatment in moist heat compared to those which survived dry heat treatment. Information of this nature will be sought by subjecting dried spores of known concentrations to dry and moist heat for various times and at various temperatures followed by subculturing in several media. Determination of 90 percent destruc- tion times (D values) at various temperatures, and rates of changes of these D values with temperature with various media will serve as com- parative criteria in evaluating this problem. It is believed that some of the differences in the recovery of the spores in various subculture media following the heat treatments are due to nutritional deficiencies in the subculture media. An effort will be made to prove this theory by supplementing the subculture medium giving lowest recovery with certain compounds which are believed to be contained by the subculture medium giving highest recovery. REVIEW OF LITERATURE A detailed review of all aspects of spore bacteriology is obviously beyond the sc0pe of this investigation. Therefore, this discussion will be limited to those studies which are believed to be essential and help- ful for the understanding of this investigation. The t0pics reviewed are: bacterial endospores with particular emphasis on the spores of the test organism used in this study, thermal destruction of bacterial spores, methods of studying heat resistance of bacterial spores and factors affecting the thermal resistance of bacterial spores. The main sporeforming bacteria are found in the genera Bacillus and Clostridium. In a few instances, the ability to form bacterial endo- spores has also been observed with certain cocci and spirilla. Bacterial spores are formed inside the vegetative cells. Normally one spore is formed per mother cell. The endospores become free when the mother cells lyse. Some of the main characteristics which distinguish bacterial spores from their corresponding vegetative cells are: high refractility, greatly reduced metabolic activity, high density, presence of dipicolinic acid (DPA) and increased amounts of calcium, resistance to usual staining techniques, resistance to heat, and to ultra- violet and gamma ray irradiation. P.A. 3679 was first isolated by E. J. CAMERON in 1927 (TOWN- SEND, ESTY AND BASELT, 1938) from a spoiled can of corn. Some controversy exists as to whether this organism is to be considered as a strain of Clostridium sporogenes, or as a separate species. Accord- ing to GROSS, VINTON AND STUMBO (1946) the former is serologically different from the latter. However, since the cultural differences re- ported by these workers, are few, and based upon only one strain of each organism, it is doubtful whether these differences are significant enough to warrant P.A. 3679 to be considered as a separate species. CAMPBELL, JR. AND FRANK (1956) reported P.A. 3679 to have a nutritional difference from C. Sporogenes since P.A. 3679 requires serine for growth whereas the latter does not. These organisms are alike, however, in that they both form spores under pr0per conditions, their vegetative cells are mesoPhilic, anaerobic, use the Stickland reaction to obtain energy for growth, and produce gas and a typical putrefactive odor in meat products and in most laboratory media. 1. Death of Bacterial Spores The only practical criterion for death of bacterial spores is their failure to germinate and reproduce under conditions satisfactory for germination and outgrowth of the normal live spore. The study of the rate of destruction of bacterial spores as well as the changes in rates of destruction with changing temperatures have not only opened the path for mathematical treatment of this phenomenon which, in turn, has proven to be extremely valuable in the design of industrial sterilization pro- cesses, but also has provided valuable information as to the possible biochemical nature of death and the mechanisms causing it. A brief dis- cussion of some of the literature that has been published in this field of study, appears to be an integral part of this investigation. a. The Order of Death of Bacterial Spores: The mathematical expression for the logarithmic order of death of unicellular organisms is dN N = -kdt (1) where N = number of organisms k 2 rate of destruction t = time Integrating this equation gives log N -kt + constant, or (2) log N = -kt + log N0 (3) where NO N initial numbers of spores number of spores surviving Solving equation (3) for k gives k : l/t(log No - log N) (4) KATZIN, SANHOLZER AND STRONG (1943) assuming a 90 percent de- struction of the original pOpulation arrived at the following equation: k l/txlog NO/0.1No : l/t (5) or k l/t The time “t” in this case was defined as the decimal reduction time, that is the time required to reduce the original p0pulation by 90 per- cent, or the time required for the survivor curve to traverse one logarithmic cycle if the logarithm of the number of survivors is plotted against time. Originally the term Z was used to designate decimal re- duction time. Later SCHMIDT (1950) suggested the term D because of possible confusion with z the reciprocal of the slope of the thermal death time curve. The term D is now generally accepted as the desig— nation of decimal reduction time. Replacing 1/k by D the following equation is obtained: D = t/(log N0 - log N) (6) The assumption of the above mathematical relationship of spore destruc- tion has been the subject of controversy for a number of years. Reports describing deviations from the logarithmic order of death in the forms of curves ranging from concave up to concave down and to sigmoidal have been as numerous as those describing straight-line relationships. The subject has been extensively reviewed by RAHN (1929, 1930, 1943) and later by SCHMIDT (1957). Since then, FRANK AND CAMPBELL (1957), and WALKER, MATCHES AND AYRES (1961) reported survival curves which were concave up. Curves with a shape of concave down were reported by ORDAL AND LECHOWICH (1958), WALKER, MATCHES AND AYRES (1961), EL-BISI ET AL. (1962), LECHOWICH AND ORDAL (1962), and LICCIARDELLO AND NICKERSON (1963). Some of these workers reported the occurrence of a logarithmic survival pattern with various deviations from it in the same publication, and in some cases even with the same organisms, all depending on the experimental cir- cumstances. The various shapes of survival curves reported and the possible reasons for deviations from the straight-line relationship have been discussed by EL-BISI AND ORDAL (1956b), AMAHA AND ORDAL (1957), and BALL AND OLSON (1957). SCHMIDT (1957) while giving 8 full recognition to the existence of non—logarithmic survival patterns in some instances, accepts the logarithmic order of death as a convenient working hypothesis. For, it is only by making this assumption that sur- vival data can be subjected to any kind of mathematical treatment. b. Thermal Destruction Curves A similar controversy appears to exist with reference to the semi- logarithmic relationship between rate of destruction and temperature. BIGELOW (1921) was the first to demonstrate this relationship. He named this curve the thermal death time (TDT) curve. Similar findings were reported later by STUMBO (1948), STUMBO, MURPHY AND COCHRANE (1952), PFLUG AND ESSELEN (1953, 1954), and ANDERSON (1959), PHEIL, NICHOLAS AND PFLUG (1963). Actually BIGELOW (1921) obtained a sigmoidal curve, but he explained the deviations from the straight-line relationship at both ends of the curve as being due to experimental errors at the higher temperatures, and accepted the initial lag at the lower temperatures as an experimental fact. HALVERSEN AND HAYS (1936) reported also a lag in the thermal resistance curve at the lower end of the temperature range studied. GILLESPY (1954) showed that if a logarithmic order of thermal reduction is assumed, then by applying the Arrhenius equation, the thermal resistance curve, although straight over limited temperature ranges, would run asymp- totically to both the ordinate as well as to the abscissa. ESSELEN AND PFLUG (1956) reported a less steep slope of the thermal resistance curve at the higher temperatures, and explained this as 9 being due to possible changes occuring in the spore supporting media during heating. LICCIARDELLO AND NICKERSON (1963) reported a concave down curve with Clostridium Sporogenes but a more or less straight line with Spores of Bacillus subtilis and Salmonelia senftenberg 775W over temperature ranges of 40°C and 20°C respectively. PHEIL AND PFLUG (1964) reported a break in the thermal resistance curves: at the lower temperatures, 172-2300F they obtained a z value of 25°F and at the higher temperatures of 235°F to 250°F, one of 15°F. c. Nature and Mechanism of Death LEWITH (1890) postulated that coagulation of protein was the cause of death of bacterial Spores for both moist and dry heat. He attributed the extremely higher resistance of bacterial spores to dry heat as com- pared to moist heat to a change in sensitivity of proteins toward coagu- lation in dependence to its water content, and was able to demonstrate this experimentally by heat treating egg albumen containing various amounts of moisture. The protein coagulation theory was substantiated by WILLIAMS (1929) by the facts that conditions inhibiting coagulation result in increased heat resistance. Furthermore, it was shown by RAHN (1945a) that the temperature coefficients for thermal death of Spores and protein coagulation are of the same magnitude. VIRTANEN (1934) agreed with this theory at least as far as the moist heat sensitivity was concerned; however, he extended it to the inactivation of some vitally important enzymes as being the major event leading to the destruction of a cell. RAHN AND SCHROEDER 10 (1941) demonstrated that bacterial cells can be inactivated and still re- tain enzyme activity. On the basis of logarithmic order of death, they postulated that death was due to heat inactivation or heat coagulation or a critical and rare molecule in the cell. Further RAHN (1945b) upon analyzing the logarithmic order of death on the basis of probabilities of destruction of bacterial cells, demonstrated that death must be the result of thermal inactivation of one or only a few critical molecules of the cell. Since the probability that two or more molecules within a cell are inactivated is the product of the probabilities of the destruction of each molecule, he showed that, if the destruction of four or five or more molecules were directly associated with thermal death of uni— cellular organisms, their order of death would cease to be logarithmic and would eventually follow that of multicellular organisms. Since the bacterial cell contains more than several thousand molecules of each enzyme, he rules out enzyme inactivation as the critical event leading to the death of bacterial or sporal cells. On the basis of the bacteriolo- gists' definition of death as the loss of the ability of bacterial cells to reproduce, as well as on the basis that cell division is under genetic control, he suggests that the denaturation of a critical gene molecule be considered as the critical event causing death. With reference to dry heat destruction, RAHN (1945a) postulates the cause to be oxidation, because dry cells display no life function, that enzymes of Spores are not active in the absence of moisture, that all endogenous catabolism was absent in dried spores and finally that ll dried proteins would not coagulate even when heated to 100°C. Increased death rate, according to RAHN, is attributable to increased rate of oxi- dation. INGRAHAM (1962) without furnishing any experimental evidence tries to explain the heat killing of bacterial cells as being the result of either heat inactivation of an essential gene or the destruction of a certain structure such as the cell membrane. Experimental evidence as to the former possibility has been furnished at least in part by ZAMENHOF (1960) who demonstrated that dried Spores of Bacillus subtilis, and dried cells of Escherichia coli when subjected to heating in vacuo, showed an increase in rate of mutation, that the carrier of the mutational injury was deoxyribonucleic acid (DNA), and that with some exceptions the occurrence of a larger percentage of mutants was not the result of higher heat resistance among the mutant cells. On the basis of these results, it is quite conceivable that certain lethal mutations might be induced by the application of heat. Some experimental evidence as to the possibility of structural disruption of the cells as the lethal result of heat treatment has been furnished by HUNNELL AND ORDAL (1961) who followed the structural changes occuring in spores of Bacillus coagculans during heating at 121°C in M/15 phosphate buffer. Micro- scopic examination of the spores revealed the disappearance of the cortex as the first phenomenon which was followed by a filling of the Spore coat by the core. At this stage the spores had lost their viability. During further heating, disintegration of the core occurred, and eventually it was only the spore coat that retained some structural integrity. 12 Heat has been shown to be an agent lethal to bacterial Spores, if imposed upon the latter in sufficient quantity. Its possible mode of action on the sporal cell may aid in explaining the mechanism and possible also the nature of death of bacterial spores. A brief outline of the theories which have been advanced as an explanation for the logarithmic order of death appears, therefore, to be apprOpriate. TISCHER AND HURWICZ (1955) reason that in view of the fact of the monomolecular type of mechanism involved in bacterial destruction, heat cannot be visualized as a continuous and homogenous wave front. If it were, the probability of survival would only have two values: zero and one. However, since the probability for survival gradually decreases with increasing time of heat application, the above authors conclude, that the logarithmic order of death of bacterial spores can be explained only by assuming that heat energy consists of discontinuous, homo- genous or nonhomogenous quanta of energy. BALL AND OLSON (1957) visualize the "bullets" causing death as molecules in a medium which greatly exceed the energies, momenta and velocity of the average molecules. On the basis of this theory they are able to explain the effect of pH, the presence of sodium chloride, fats and oils on the rate of destruction of bacterial spores. CHARM (1958) in his ”kinetic effect theory" compares bacterial destruction rates to chemical reaction rates. He visualizes the bac- terial cell as being composed of a number of sensitive volumes surrounded by a number of water molecules, all enclosed within the cell wall. When the water molecule next to the sensitive volume exerts sufficient energy 13 onto this sensitive volume, inactivation of the cell occurs. Due to the distribution of energy among the molecules in the cell, not all cells would be inactivated at the same time. 2. Methods for Studying Heat Resistance of Bacterial Spores In designing experimental arrangements for thermal destruction studies, it is important to select the type of apparatus most feasible to carry out such studies, the prOper experimental procedure and the type of statistical evaluation of the data to be obtained. These three aspects of the experimental design of this thesis will be discussed in the follow- ing sections. a. Apparatus The early studies of heat resistance of bacterial spores were mostly carried out using test tubes or capillary tubes as containers and a water bath or an oil bath as the heating medium. BIGELOW AND ESTY (1920), and ESTY AND MEYER (1922) placed the spore suspensions into tubes which were subsequently sealed in an oxygen blast flame and then sub- jected to the desired heat treatment in an oil bath. WILLIAMS, MERRILL AND CAMERON (1937) developed the "tank" method where the spores are heated in some food stuff (mostly pureed food materials) in an agi- tated tank under pressure. Samples are removed at various intervals and tested for surviving spores. SOGNEFEST AND BENJAMIN (1944) first reported the use of thermal death time (TDT) cans and outlined a method for inserting a thermocouple into the can for the purpose of 14 obtaining information about the time-temperature relationship during retorting. In this method too, the spores are suspended in some pureed food material, filled into the cans which are then sealed and subjected to the desired heat treatment. Subculturing is not necessary in this case if spores of gas producing organisms are employed in the experi- ments. The cans are merely placed in an incubator following the heat treatment and examined periodically for gas formation which exhibits itself in the bulging of the cans. MAGOON (1926) heated spores in capil- lary tubes in an oil bath. This method was later improved by STERN AND PROCTOR (1954) who designed an apparatus with an automatic transfer device for exposing biological suspensions in capillary tubes to high temperatures in an oil bath. FARKAS (1962) developed a modified version of this machine which was made to hold 20 or more capillary tubes and which would be adOpted to hold various sizes of tubes and other containers. While the rate of heat transfer in capillary tubes is greatly im- proved over that found in test tubes and TDT cans, it is far from in- stantaneous and therefore correction calculations have to be made for the heating and cooling lag in order to obtain the actual exposure time at the desired temperature. The first apparatus where this problem is eliminated to the point where the times necessary for the heated spores to reach the desired temperature and for cooling are negligible provided the total exposure time exceeds three or four seconds, was de- signed by STUMBO (1948). This machine which is commonly called a 15 thermoresistometer, consists of a heating chamber and a sliding plate onto which small metal cups containing the spore suspension are placed, and on which these samples are moved into and out of the heating cham- ber. ESSELEN AND PFLUG (1956) describe a similar apparatus using pistons with rings to transport the metal cups instead of the sliding plate. PFLUG (1960), in order to obtain a more positive seal, deve10ped a modified version of this thermoresistometer by constructing a double seal Operation. This machine has the further advantage that it can be used for both moist and dry heat. Studies on thermal resistance of spores in dry heat involve rather long exposure times. The number of replicates that can be treated in the thermoresistometer at any one time is limited to five cups. In studies of this kind ten to thirty or more replicates are run for each time of exposure. PFLUG, AUGUSTIN AND JACOBS (1961), in order to eliminate such time consuming procedures, placed up to ten tin- plated metal cups into TDT cans, sealed the cans, and subjected them to moist heat in miniature retorts. Obviously, inside the cans dry heat conditions exist. Ten or twelve cans can be heat treated at any one time with this method. KOESTERER AND BRUCH (1962) in their studies of dry heat resis- tance of bacterial spores, inserted test tubes containing the dried spores into holes of heated cylindrical aluminum blocks. The spores were either dried directly in the tubes, or they were first placed onto 16 filter strips or mixed into sterile sand, dried and then placed into the tubes. b . Experimental Methods: BIGELOW AND ESTY (1921) and ESTY AND MEYER (1922) ex- pressed heat resistance of bacterial spores in terms of thermal death points which they defined as the number of minutes required at a given temperature to destroy a spore suspension of a known concentration. The physical arrangement of their tests was such that they placed a series of tubes, all containing an equal number of spores, into a heated oil bath. At various time intervals one tube was removed and checked for survival. ESTY AND WILLIAMS (1924) reported the occurence of ”skips, " meaning that spores in single tubes which are exposed to heat for several consecutive time intervals show destruction at one time and survival at a longer time of exposure. As a solution to this problem they suggested the use of replicate tubes for each exposure time. In doing so, they found that at a certain exposure time only a certain percentage of the tubes showed growth, and that plot of the logarithms of the per- centages of survivors against time on a linear scale resulted in an approximate straight line relationship. Although their suggested method of plotting "survivor" curves did not find acceptance, their findings introduced the use of replicate samples in most similar studies carried out thereafter. While these early studies did not involve any calculations and were only concerned with the establishment of the thermal destruction time, 17 more emphasis has been placed recently on establishing rates of destruc- tion of bacterial spores expressed as D values. Such values can be cal- culated from survivor data or determined graphically from survivor curves. Such procedures however involve much time consuming pro- cesses as dilution of the heated spores, plating and counting. Further- more, it has the disadvantage that the accuracy of the counts becomes questionable if the number of survivors fall below a certain minimum. For these reasons, several workers, among them STUMBO (1948), STUMBO, MURPHY AND COCHRANE (1950) and SCHMIDT (1957) prefer to establish D values on the basis of end—point determinations. In this method, multiple samples are run at successive time intervals with apprOpriate spacing so that with the shortest time most or all replicate samples show growth and with the longest time of exposure either a very small fraction of the tubes or none exhibit growth. Details on the various methods of calculation will be outlined in the following paragraph. c. Methods of Calculating DValues: STUMBO (1948) used equation (6) replacing t by U, No by A and N by B, where A designated the total number of samples heated multiplied by the number of spores per sample, and B was calculated by assuming one surviving spore per container when less than the total number of containers showed survival. STUMBO, MURPHY AND COCHRANE (1950) modified this method by calculating B by means of the ”most probable number” technique which was deve10ped by HALVORSON AND ZIEGLER (1933). In this case B equals the most probable number of 18 spores per replicate times the number of replicates. This is calculated from the following equation: § = 2. 303 log n/q (7) the most probable number of survivors per replicate the total number of replicates the total number of sterile replicates where "x n q A number of exposure times are chosen at a certain temperature. For each exposure time the corresponding D value is calculated. The final D value is the average of all these D values. This method is often called the unweighted average method. The probability method developed by SCHMIDT (1957) is a modifi- cation of the method which was first deve10ped by REED (1936), and has been worked out on the basis of the following two assumptions: First, any sample that does not show survivors at a given exposure time, does not show survivors at a longer time of exposure at the same temperature, and second, any sample showing survivors at a given ex- posure time will also show survivors at a shorter exposure time at the same temperature. These assumptions permit the data of all process times of a given temperature to be combined for the computation of the D value. The probability of sterility is first calculated on the basis of the cumulative number of samples showing growth and the cumulative number of sterile samples, according to the following equation: P = (n+l)/(m+n+2) (8) the probability of sterility at a given time the cumulative samples not surviving each exposure time obtained by adding the where P n 19 negative samples downward from the shortest exposure time to the longest m : the cumulative samples surviving each exposure time obtained by adding the positive samples upward from the longest exposure time to the shortest The P values corresponding to the various exposure times are plotted on probability paper. A line is drawn between these points. From this curve the time correSponding to P = 0. 50 is determined. This time point is called the L. D. 50 value which is the time at which 50 percent sterility occurs. This time corresponds to 0. 69 organisms per tube according to HALVORSON AND ZIEGLER (1933). D is then calculated according to the following equation: D = L.D. 50/(log A - log 0.69) = L.D. 50/(logA+ 0.16) (9) where A : number of spores per sample prior to the heat treatment. LEWIS (1956) in reviewing the various methods for calculating D sharply criticized the STUMBO, MURPHY AND COCHRANE method for its bias. With reference to the SCHMIDT method, his only criticism is the fact that no detailed analysis is available for it. He suggests the use of the Spearman-Karber method or the maximum likelihood method, often called the loglog method, both described by FINNEY (1952). The Spearman-Karber method uses a different procedure to calculate the mean sterility time Vt and the variance of this time. Equation (9) is used to calculate D with the modification the L. D. 50 is replaced by Vt’ The loglog method is suggested by LEWIS (1956) as an alternative of the Spearman-Karber method mainly for reasons of saving in time 20 consuming experimentation. Mainly as a result of the criticism by LEWIS, SCHMIDT (1957) added to his original procedure a method for estimating the precision of the D values. He suggested the following method to calculate the 95 percent confidence limits for D: 95% c. L.D = L.D. 50 t 95% c. L. (10) logA + 0.16 where 95% C. L. =1. 96 x ZS/ZN where 25 = the time difference in heat dosage between P0. 84 and P0. 16 the total number of tubes in the time groups showing partial survival. N Equation (10) was later modified by PFLUG (1962) to the point where N was taken as the total number of replicates between P0. 16 and P0_ 84' Several workers compared the D values obtained by various methods of calculations. ESSELEN AND PFLUG (1956) compared the results obtained by the STUMBO, MURPHY AND COCHRANE method and the SCHMIDT method. The values obtained by these two methods were generally in good agreement. LEWIS (1956) compared D values obtained by the methods of STUMBO, MURPHY AND COCHRANE, Spear- man-Karber and maximum likelihood did not find very substantial dif- ferences. Also ANDERSON (1959) compared D values calculated by the STUMBO method, the STUMBO, MURPHY AND COCHRANE method and the SCHMIDT method and the probit method. 3. Some Factors Affecting the Heat Resistance of Bacterial Spores SCHMIDT (1957) classifies these factors into three general groups: inherent resistance, environmental influences active during the growth 21 and formation of the spores, and environmental influences active dur- ing the time of heating of the Spores. The latter two factors being closely associated with this investigation will be discussed in the following. a. Effect of the Sporulation Medium ESTY AND MEYER (1922) reported that the type of sporulation .medium used has a significant effect on the heat resistance of bacterial spores. Furthermore they observed some variation in the heat resis- tance of Spores which were grown in different batches of identical media. It was indicated by the authors that this effect might be attributed to variations in the pH of the media at the end of the sporulation process. Recently, WHEATON AND PRATT (1961) found differences in heat resistance of spores of one strain of Putrefactive Anaerobe 3679 pro- duced in four different media. The highest degree of heat resistance was found in those spores which were produced in beef heart infusion, the lowest in those grown in trypticase medium. The spores with an intermediate degree of heat resistance were those grown either in liver infusion or pork infusion. Numerous efforts have been made in the past to determine the identity of those components in the sporulation medium that affect the heat re- sistance of the spores. SUGYIAMA (1951) found increased heat resis- tance of spores of Clostridium botulinum grown in casitone medium with increasing amounts of various fatty acids, increased degree of unsatura- tion of these fatty acids, and up to a certain concentration level, in- creasing amounts of iron, calcium and magnesium. The effect of the 22 calcium ions was especially apparent when the sporulation medium was deprived of its calcium ions. EL-BISI AND ORDAL (1956a) found a decrease in heat resistance as well as in the calcium content of spores of Bacillus coagulans var. thermoacidurans with increasing phosphate concentration in the sporulation medium. It was concluded from these experiments that the phosphate in the sporulation medium interfered with the availability of bivalent cations to the sporulating cells. AMAHA AND ORDAL (1957) found that heat resistance of spores of Bacillus coagulans, var. thermoacidurans could be increased by the addi- tion of calcium and manganese to the sporulation medium. The addition of magnesium was without any effect. SLEPECKY AND FOSTER (1958) demonstrated an increasing heat resistance of spores of Bacillus magi- terium, when the sporulation medium was supplemented with calcium ions, but a decrease in heat resistance when increasing amounts of man- ganese and zinc ions were added to the sporulation medium. LEVINSON AND HYATT (1964) working with spores of Bacillus megaterium reported an increase in heat resistance of these Spores when a liver-type sporula- tion medium was supplemented with calcium, and a decrease in heat resistance when the phosphate concentration of this medium was increased, or supplemented with either L-proline or L-glutamic acid. An increased heat resistance was the result of the addition of L-glutamic acid to a synthetic medium. The addition of manganese, L-alanine, L-leucine, L-valine or D-glucose did not appear to affect the heat resistance of the Spores. 23 As a result of the above findings, efforts have been made to deter- mine any possible relationship between the concentration of certain components of the Spores that are a characteristic part of the spores, such as calcium and dipicolinic acid (DPA), and their heat resistance. LECHOWICH AND ORDAL (1962) found an increase in heat resis- tance with increasing amounts of calcium, magnesium, manganese and DPA in the spores of Bacillus subtilis. This relationship however did not hold for spores of Bacillus coagulans. In this case the DPA content was lower in spores which exhibited a higher degree of heat resistance. CHURCH AND HALVORSON (1959) reported a decrease in heat resistance of spores of Bacillus cereus with decreasing amounts of DPA, whereas BYRNE, BURTON AND KOCH (1960) found the Opposite to be true for spores of Clostridium roseum. The heat resistance of bacterial spores appears not only to be de— pendent on the composition of the sporulation medium, but also on the temperature of sporulation. SUGIYAMA (1951) found highest heat re- sistance when spores of Clostridium botulinum were produced at 370C. Both higher and lower sporulation temperatures resulted in decreased heat resistance. WILLIAMS AND ROBERTSON (1954), EL-BISI AND ORDAL (1956b), ORDAL AND LECHOWICH (1958) and LECHOWICH AND ORDAL (1962) all reported an increased heat resistance with increasing temperatures of sporulation up to 37°C. 24 b. The Effect of the Initial Spore Concentration Reports about this effect on the heat resistance of bacterial spores are rather conflicting. ESTY AND MEYER (1922) and WILLIAMS (1929) indicated that the increase in survival time with increasing spore con- centration is somewhat different from that expected from the logarith- mic order of death. AMAHA (1961) reported an increase in survival time with increasing initial spore concentration with spores of C. sporo- genes, B. natto, and B. megaterium. He and his co-workers established the following relationship between the heat resistance of these Spores and change in their initial Spore concentration: logt = a+blogN where t is the survival time, N is the initial number of spores per sample, and a and b are constants depending on bacterial genus, species and strain, the temperature and the nature of the heating medium. SISLER (1961) reported very little change in heat resistance of spores of B. subtilis CCC 5230 with changing spore concentration in moist heat. In superheated steam however, there was an increase in D values at the lowest spore concentration used. c. Effect of the Medium Supporting the Spores During Heating ESSELEN AND PFLUG (1956) reported that with spores of Putre- factive Anaerobe 3679 a lower heat resistance was found when the spores were heated in previously canned vegetable purees than when heated in purees which were prepared either from fresh or previously frozen vegetables. The highest resistance was found in those cases where 25 the Spores were suspended in M/15 phosphate buffer during heating. REYNOLDS ET AL. (1952) also reported that the heat resistance of the same spores varies according to the medium in which they were sus- pended during heating. STUMBO, MURPHY AND COCHRANE (1950) reported highest heat resistance of spores of Putrefactive Anaerobe 3679 and Clostridium botulinum which were heated in a suspension of M/15 phosphate buffer, and lowest heat resistance when heated in dis- tilled water. Heating of these spores in various vegetable and meat purees resulted in an intermediate heat resistance. ORDAL AND LECHOWICH (1958) showed that Spores of Bacillus coagulans, var. thermoacidurans exhibited highest heat resistance when heated in M/40 phosphate buffer as compared to heating in phosphate buffers of higher or lower concentrations, M/lOO glycylglycine, M/100 trihydroxy- methylaminemethane buffer, M/lOO ethylenediaminetetraacetic acid solution or distilled water. ANDERSON (1959) observed no significant difference in heat resistance of spores of Bacillus subtilis 5230 which were heated either in M/lS phosphate buffer as a free suspension or on filter paper strips, or dried from a M/15 phosphate buffer solution. As already pointed out, the effect of the presence of other compo- nents in the spores suspension during heating on the heat resistance of the spores has been investigated. SUGYIAMA (1951) noticed that the heat resistance of Spores of Clostridium botulinum Type A and Type B suspended in M/15 phosphate buffer could be considerably increased by the addition of increasing amounts of sucrose, up to a concentration of 26 50 percent. Supplementation of the suspension containing 0. 15M sodium chloride with 0. 001M calcium chloride or 0. 001 M magnesium chloride greatly decreased the heat resistance of the spores. AMAHA (1961) reported that with Clostridium sporoienes the presence of sucrose or glycerol in concentrations ranging from 10 to 50 percent in the spore suspension did not affect the survivor pattern of the Spores. The addi- tion of starch also showed no effect. However, when serum albumen, ovalbumen or either two different types of laboratory peptones were added in concentrations of up to 0. 5 percent, the heat resistance of the Spores was significantly increased. This held true when the Spore sus- pension was supplemented with yeast nucleic acid in concentrations of up to 1 percent, or heat coagulated serum albumen or ovalbumen. With reference to the dry heat resistance of spores, the only work reported in reference to the effect of the supporting medium during heating comes from BRUCH, KOESTERER AND BRUCH (1963) who observed that several Species of bacilli and clostridia showed a higher heat resistance when heated in sand than when heated on a filter strip or on a glass wall. (1. The Effect of the Heating Medium Most of the studies on heat resistance of bacterial spores were carried out using moist heat in the form of saturated steam or aqueous solutions as the heating medium. One of the first reports regarding the relatively high heat resistance of bacterial spores in dry heat stems from LEWITH (1890). In 1922, TANNER AND DACK mentioned the extremely high resistance of five strains of Clostridium botulinum 27 spores to dry heat. OAG (1940) studied the dry heat resistance of Spores of Bacillus anthracis, Bacillus welchii, and Bacillus anthracoides. These spores were heated on glass slides in an electric oven. The tem- peratures applied ranged from 120°C to 400°C. He reported a sharp change in heat resistance of these spores at 1600C. Above this tempera- ture, the Spores were killed significantly more rapidly than at tempera- tures below 1600C. The study of dry heat resistance of bacterial spores gained momen- tum with the advent of aseptic canning and space craft sterilization. COLLIER AND TOWNSEND (1956) studied the resistance of spores of Bacillus stearotherm0philus, Bacillus polymyxa, and of Putrefactive Anaerobe 3679 to saturated and superheated steam. All Spores showed considerably more resistance to superheated steam than to saturated steam. However, no correlation was found between the relative heat resistance of these Spores in superheated steam and in saturated steam. PFLUG (1960) repeated these experiments with spores of Bacillus subtilis strain 5230 with essentially the same results. SISLER (1961) reported a threefold increase in the 2 value of spores of Bacillus subtilis strain 2223 in superheated steam as compared to the z value of saturated steam. Similar results were reported by PFLUG AND AUGUSTIN (1961), and KOESTERER AND BRUCH (1962) who were subjecting spores to hot air instead of superheated steam. The latter further reported that no correlation existed between the relative heat resistance of various 28 genera of spores in dry and in moist heat. MURRELL AND SCOTT (1957) studied the heat resistance of spores of Bacillus stearotherm0- phious, Bacillus megaterium, and Clostridium botulinum in heating media of various water activities (aw). Contrary to all expectations, the heat resistance of all these Spores was not found to be highest at an aw of or near zero (absence of water, complete dryness), but at an aW of approximately 0. 8. By far the lowest degree of heat resistance was found to exist with spores heated in a suspension of M/15 phosphate buffer (aW = 1. 0). The authors concluded that dry heat resistance of bacterial spores under conditions of low water activities was largely dependent on the maintenance of some part of the Spore contents in a relatively dry state. JACOBS (1963) and JACOBS, NICHOLAS AND PFLUG (1964) studied the heat resistance of Spores of Bacillus subtilis strain 5230-10 by subjecting them to steam of various water contents. They found that the decimal reduction time was a function of the water vapor content of the heating atmOSphere within the temperature range studied. The slope of the thermal reducation time curve (l/z) was found to be lowest at 50 percent water vapor content, and nearly as high in a completely dry at- mosphere. The respective values of 2 were 44 and 41°F. At 25 per- cent water vapor content of the atmosphere a z of 25°F was reported, and at a complete water vapor saturation of the atmosphere, 2 was found to be 17°F. PHEIL, NICHOLAS AND PFLUG (1963) studied the effect of various gases used as heating media on the thermal resistance of Bacillus 29 subtilis strain 5230. All the gases used appeared to exert a similar effect on the spores. The slopes of all thermal resistance curves were identical. However, the decimal reduction times obtained increased in the order of helim, nitrogen, oxygen, air and carbon dioxide. The values obtained with the first three gases were almost identical, but clearly distinct from the last two gases. e. The Effect of the Composition of the Subculture Medium SUPFLE AND DENGLER (1916) reported that spores of Bacillus anthracis after being subjected to heating showed survival times of 4 minutes when subcultured in nutrient broth, and 30 minutes when suba- cultured in the same broth which had been supplemented with 3 percent glucose and 5 percent horse serum. MORRISON AND RETTGER (1930a, 1930b) reported similar findings. CURRAN AND EVANS (1937) found only minor differences in survival with different enrichments added to the nutrient agar with spores of Bacillus cohaerens, Bacillus subtilis and Bacillus albolactis that were not heated prior to counting. However, differences were found after the same spores had been subjected either to ultraviolet irradiation or moist heat. The conclusion was made from these studies, that spores become more exacting in their nutrient re- quirements following heating or irradiation. REYNOLDS ET AL. (1952) were able to increase the extent of recovery of Spores of Putrefactive Anaerobe 3679 which had been subjected to moist heat in various low acid type foods such as meat and vegetable purees, if following the heat treatment, the mixtures were supplemented with certain enrichments. 30 Several media have been reported to give maximum counts for heated spore of clostridia. WHEATON AND PRATT (1961) made an extensive comparative study of several recovery media, infusion type, dehydrated and formulated media. The differences reported in counts of spores of Putrefactive Anaerobe 3679 which were heat treated were quite drastic. Fresh meat infusions generally yielded the highest sur- vival counts. With the exception of yeast extract-starch-bicarbonate agar, as described by WYNNE, SCHMIEDING AND DAYE (1955), the formulated media generally resulted in somewhat lower counts. By far the lowest number of survivors were obtained with the so-called de- hydrated media such as eugonagar. Infusion-type media are time consuming to prepare. Therefore, FRANK AND CAMPBELL (1955), in an effort to find a formulated medium which would give comparable results of heated bacterial spores to infusion-type media screen tested a number of media. Spores of Putrefactive Anaerobe 3679 were used in these tests. The results re- vealed the highest number of survivors with Yesair's pork infusion which had been fortified with 0. 05 percent sodium thioglycollate. Recovery was, however, almost as high with the yeast-extract-starch-sodium thioglycollate-sodium bicarbonate agar as described by WYNNE, SCHMIEDING AND DAYE (1955), if the last two components were omitted from the medium. Eugonagar with or without supplements, such as starch, malt extract or liver infusion resulted in significantly lower results. These results were more or less in accordance with those 11— i 31 reported by WHEATON AND PRATT (1961) with the exception that the latter reported an even higher count with beef infusion and pork-pea infusion agar. AMAHA (1961) reports an increase in survival time with heat treated spores of Bacillus natto when glucose was added to nutrient broth. When other carbohydrates were added, he found only those sugars to be effec- tive in extending the survival time that were also able to support good growth of the organism in a synthetic medium when added as the sole source of carbon. It was further demonstrated that the survival time of the spores in moist heat varied greatly with the addition or omission of certain vitamins and amino acids in the recovery medium. ESTY AND MEYER (1922) reported that Spores often remain dor- mant for considerable periods of time--up to 378 days--following a heat treatment. Since very few workers exposed their heat treated Spores to such prolonged incubation times, it is often not known whether the failure of the spores to germinate and outgrow in a certain medium is actually the result of the absence of essential nutrients in the recovery medium or the presence of some compound which retards or inhibits germination and outgrowth. The study made by AMAHA (1961) indicates that the addi- tion of glucose, certain amino acids and vitamins serve the purpose of satisfying the nutritional requirements of the spores. On the other hand, FOSTER AND WYNNE (1948) clearly demonstrated that the germination of spores of several species of clostridia was inhibited by the presence of unsaturated fatty acids such as oleic acid, linoleic and linolenic acids. 32 This inhibitory effect could be overcome by the addition of 0. 1 percent starch to the recovery medium. The growth of the vegetative cells of these organisms was not inhibited by these acids. Similar studies were carried out with a number of strains of Clostridium botulinum by OLSON AND SCOTT (1950) and with a number of bacillus species by MURRELL, OLSON AND SCOTT (1950). They also demonstrated that the addition of starch, but also that of charcoal, or serum albumen, was efficient in overcoming inhibitory factors present in the recovery media. AMAHA AND ORDAL (1957), however, reported that the addition of starch to nutrient agar recovery medium failed to alter the survivor counts of heat treated spores of Bacillus coagulans, var. thermoacidurans. MAT ERIALS AND METHODS The experimental procedure employed in this investigation is de- signed to evaluate the differences in recovery in various subculture media of moist and dry heat treated spores of P. A. 3679 and to investi- gate the mechanisms involved in the thermal destruction of these spores. 1. Preparation of Spore Suspension Spores of P. A. 3679 were prepared in two different sporulation media which in the past have been shown to produce Spores of different moist heat resistance. The organism of known history was obtained from Dr. C. F. Schmidt of Continental Can Company in Chicago, Illinois. a. Spores Grown in a Beef Heart Infusion Medium: A large number of spores were grown in beef heart infusion accord- ing to a procedure described by WHEATON AND PRATT (1961) and WHEATON (1963). The medium was prepared as follows: Boil 15 lbs of cleaned, defatted, chopped beef heart from freshly slaughtered animals for one hour in 15 liters of distilled water. - adjust pH to 7. 4 with sodium hydroxide, - filter material through cheese cloth and cotton, - air dry solid meat particles, and make fluid up to 15 liters with distilled water, - add 150 grams gelatin and 150 grams Bacto-tryptone, - adjust pH to 8. 5 with sodium hydroxide, - add 75 grams isoelectric casein (DIFCO) and dissolve completely in broth, then add 4. 5 grams glucose, 4. 0 grams dipotassium phosphate and 45 grams sodium citrate, 33 ('1 . 34 readjust pH to 7. 2 with hydrochloric acid, - dispense and autoclave for 30 minutes at 1210C, just prior to inoculation, exhaust medium for 20 to 30 minutes in free flowing steam and cool to 320C. Using this medium, Spores of P. A. 3679 were prepared according to the following 5 chedule: out Transfer 1 ml of a heat shocked stock Spore suspension into each of five 16 x 150 mm screw cap test tubes containing approximately a 1 cm deep layer of dried beef heart particles, a pinch of elec- trolytically reduced iron powder, and 15 m1 of the beef heart ex- tract. Stratify this mixture with a 1:1 mixture of mineral oil and paraffin, and incubate at 320C until the tubes show good growth as indicated by gas formation. Transfer the entire content of each of these tubes into each of five 1" x 8” test tubes containing approximately a 1 cm deep layer of beef heart particles, a pinch of iron and 25 ml of beef heart extract, stratify and incubate as outlined above. Transfer the entire content of each of these tubes into each of five 500 ml Erlenmeyer flasks containing a small layer of dried beef heart particles, a inch of iron and 400 ml of bee heart extract, and incubate at 32 C for a period of 24 hours. Transfer the entire contents of each of the above flasks into each of five 2000 m1 Erlenmeyer flasks each containing a small layer of dried beef heart particles, 3 pinches of iron and 1500 m1 of the beef . 0 heart extract. Incubate for approx1mately three weeks at 32 C. Spores grown in Trypticase Medium: This Sporulation medium which was recommended by BROWN (1963) was prepared as follows: Dissolve 30. 0 g trypticase (BBL), 1. 0 g yeast extract (BBL) and l. 0 gram ammonium sulphate in 1000 ml distilled water, adjust pH to 7. 3 with sodium hydroxide, 35 - dispense in 16 x 150 mm screw cap test tubes, 250 ml Erlenmeyer flasks and 2000 ml Erlenmeyer flasks and autoclave for 15, 20 and 30 minutes respectively at 1210C. Using this medium, 8000 m1 of spores of P.A. 3679 were prepared according to the following: - Transfer 1 ml of a heat shocked stock spore suspension into each of four 16 x 150 mm screw cap test tubes containing 20 ml of the above medium, stratify with a 1:1 paraffin mineral oil mixture, and incubate at 300C until good growth has developed as evidenced by , gas formation. - Transfer the contents of each of these tubes into each of four 250 ml Erlenmeyer flasks containing 200 ml of the above medium and incu- bate at 300C for 12 hours. - Transfer the entire contents of each of these flasks into each of four 2000 ml Erlenmeyer flasks containing 1700 m1 of the above medium and incubate at 30°C until maximum Sporulation is attained. The time for the cultures to reach maximum sporulation was 96 hours. c. Harvesting and Cleaning of Spores The beef heart grown spores were harvested and cleaned according to the method described by TOWNSEND ET AL. (1956) as follows: - Filter the entire Sporulation mixture through a sterile layer of fiber glass with cheese cloth on both sides in order to remove the meat particles. - Place the separated broth containing the Spores into sterile 250 ml screw cap centrifuge bottles, and subject it to centrifugation in an International Centrifuge for 30 minutes at 1600 rpm. - After the entire crude spore suspension has been subjected to this treatment, add some sterile water and some sterile glass beads to the precipitated spores, shake this mixture vigorously for several minutes and recentrifuge. Repeat this step several times until the supernatant appears relatively clear. - Resuspend the Spores in neutral phosphate buffer (equal amounts of M/15 KHZPO4 and M/15 KZHPO4), shake for several minutes and 36 centrifuge. Repeat this step three times, except do not centrifuge last time. - Store the concentrated spore suspension in a screw cap bottle in a refrigerator at 40°F. MicroscoPic examination of this spore suspension revealed the presence of debris as well as sporangia. In order to free the spores from their sporangia and to achieve further removal of the debris material the spore suspension was subjected to an enzymatic treatment that was des- cribed by GRECZ, ANELLIS AND SCHNEIDER (1963) and to further cleaning as follows: - Prepare an aqueous M/15 phosphate buffer solution containing 80 mg lysozyme* and 40 mg trypsin*. - Sterilize this solution by filtration, — Add this solution to 200 ml of the spore suspension and incubate it . o . . for 81X hours at 45 C under constant stirring, - Pass through a sterile glass filter, centrifuge and wash with M/15 phosPhate buffer ten times, - Store the cleacped Spore suspension in a screw cap bottle in a refri- gerator at 40 F. Microscopic examination revealed complete removal of sporangia from the spore suspension, and also of most of the debris material. The spore crOp obtained from the trypticase-yeast extract medium was cleaned in the same manner with the omission of the initial glass fiber-cheese cloth filtration. Also in this case NaZHPO4 was used instead of KZHPO4 in the preparation of the phosphate buffer. This *California Corporation for Biochemical Research, Los Angeles 63, California. appeared to facilitate drying of the diluted spore suspension. 37 The cleaned spore suspensions were finally diluted to the desired concentra- tion, and stored in screw cap bottles under refrigeration. 2. Media for Standardization and Recovery a. Infusion type media: - Beef Infusion: - Pea Infusion: - Liver Infusion: Pregaration of infusion: Mix 500 g lean ground beef with 1000 ml distilled water in a Waring Blender, boil for 1 hour, cool and filter through 2 layers of cheese cloth. Formulation: beef infusion Preparation of infusion: 1000 ml Bacto-peptone 5. 0 g Bacto-tryptone 1. 6 g soluble starch 1. 0 g KZHPO4 l 25 g Final pH 7. 4 Mix 100 g of frozen peas with 1000 ml distilled water in a Waring Blender, boil for 30 minutes, coal and filter through 2 layers of cheese cloth. Formulation: Preparation of infusion: pea infusion 1000 m1 Bacto-tryptone 10. 0 g Final pH 7. 4 Mix 500 g of fresh chopped beef liver with 1000 ml of distilled water in a Waring Blender, boil for 1 hour, cool and filter through 2 layers of cheese cloth. Formulation: liver infusion 1000 m1 Bacto-peptone 10. 0 g KZHPO4 l. 0 g Final pH 7. 4 b. 38 Formulated Media: Yeast Extract: Trypticase: Synthetic: 1. 2. 3. Yeast Extract (BBL) starch (BBL) distilled water Final pH 7. 2 Trypticase distilled water Final pH 7. 2 amino acides: L-arginine L-aspartic L-cysteine glycine L- glutamic L-histidine DL-isoleucine L-leucine L-lysine, HCL DL-methionine DL-phenylalanine L-proline DL- threonine L-tryptOphane L-tyrosine DL -valine DL-serine DL-alanine biotin folic acid niacin DL- Ca-pan- thothenate vitamins: 0. 05 mg 0. 20 mg 4. 00 mg l. 00 mg mineral salts: KZHPO4 KHZPO4 MgSO4x7HZO NaCl FeSo4x2HzO MnSO4xHZO 10.0g 1.0 g 2.0 g 1000 ml 50.0 g 2.0 g 1000 ml 3.00 0.45 0.25 0.20 0.50 0.20 0. 50 1. 50 0.85 0.60 2.00 0.45 1.00 0. 10 0.40 2.00 0.25 OQUQOQUQUQUQOQO'QUQOQUQOQOQOQGQOOO'Q o 4:. N 00 riboflavin 1 . 00 mg pyridoxal, HCl 0. 50 mg thiamine, HCl 1. 00 mg p-aminobenzoic acid, K-salt 0. 24 mg 1 . 00 g 1. 00 g .Omg .Omg .Omg .5mg HNNO 39 4. distilled water 1000 ml Final pH 7. 2 c. Dehydrated Media: - eugonbroth: eugonbroth (BBL) 30. 0 g distilled water 1000 ml Final pH 7. 2 The 30 g eugonbroth contain: Trypticase 15. 0 g .Phytone 5. 0 g NaCl 4. 0 g cystine 0. 2 g NazSO3 0. 2 g Na-citrate l. 0 g dextrose 5. 0 g Media which were to be used to study the recovery of heat treated spores were filled into 16 x 125 mm disposable culture tubes in amounts of 8 ml per tube. After the tubes were filled and st0ppered, they were sterilized according to the following procedure: 0 - infusion type media 25 minutes at 1210C - formulated media 15 minutes at 1210C - synthetic medium 12 minutes at 116 C The eugonbroth medium, prior to being filled into the culture tubes, was boiled for 2 minutes, then filled into the tubes. The tubes were then st0ppered, and the medium was sterilized for 15 minutes at 118°C. Im- mediately following sterilization, all media were cooled by immersion in cold water. Innoculation of the media with spores normally occured within less than 12 hours following the preparation of the media. Immediately prior to innoculation, the following additions were aseptically made to each tube: 40 0. 1 m1 of a 10 percent sodium thioglycollate solution 0. 1 ml of a 40 percent dextrose solution 0. 2 ml of a 4 percent sodium bicarbonate solution Dextrose was not added to eugonbroth media. While dextrose was steri- lized by filtration, the other two compounds were sterilized by heat. Following innoculation with the heat treated spores, the tubes were stratified with a 1:1 mixture of pre-sterilized paraffin-mineral oil. (See Figure 1) Media which were used for the determination of initial Spore counts were supplied with 15 g agar per 1000 ml of medium. The sterilization procedures for these media were the same as outlined above with the exception that the time of sterilization was prolonged for 5 minutes in this case. All media were sterilized in amounts of 300 ml in 500 ml Erlenmeyer flasks. Prior to being poured into Prickett tubes containing the spore suspension, each flask was supplied with 4 ml of a 10 percent sodium thioglycollate solution and 8 m1 of a 4 percent sodium bicarbonate solution. Following solidification of the agar, a small overlayer of the basal medium was added to each tube. The criteria for growth were colony formation in the case of agar counts, and gas formation and/or turbidity development with the broth type media used for end-point determinations. All inoculated tubes were stored at 370C. The storage times ranged from 24 to 48 hours for the agar samples, and two weeks after no more positive tubes developed, which amounted to approximately six weeks, for the samples containing the broth media. 41 6.2.5638 30 3.2238 - 5:28 £3 cosmoasfifi . H MMDOHM 42 3. Enumeration of Initial Spore Count Comparative Spore counts were made to determine the initial spore concentration with each medium used as a subculturing medium. . Also periodic determinations of the initial spore count were carried out throughout the period of experimentation using the. yeast extract medium only. These periodic checks served to note any possible changes in ini- tial spore concentration during storage. Prior to innoculation, the Spore samples were heated shocked in a boiling water bath for 8 minutes and cooled in ice water. ”Following this treatment, the samples were diluted and then pipetted into the Prickett tubes in such amounts as to yield a spore count of approximately 20 to 80 per tube. A 0. 85 percent aqueous saline solution was used as the dilu- tion menstruum. The spores grown in trypticase-yeast extract germinated spon- taneously in the various culture media. Therefore no heat shock treat- ment was applied to them prior to innoculation. 4. Preparation of the Spore Suspensions for Thermal Treatment Sample cups (11 mm outside diameter by 8 mm deep and formed from tinplate 0. 008 in. thick or from aluminum plate 0. 02 in. thick) were first washed in butanone, then in absolute alcohol, dried, placed into petri dishes and sterilized at 3200F for 2 hours. The flask contain- ing the standardized Spore suspension was placed on a magnetic stirrer which kept the suspension constantly agitated. The sample cups were loaded with spores using a micro syringe burette which held 0. 25 ml 43 calibrated in 0. 0001 ml. The micro syringe burette was filled from the agitated flask containing the spore suspension. A volume of 0. 01 ml of the spore suspension was measured into each cup. Twenty-five cups could be loaded each time the syringe was filled. The petri dishes con- taining the loaded cups were placed in a vacuum drying chamber and held there under reduced pressure for at least 24 hours at room temperature. The dried samples were stored in a desiccator at room temperature until used for the heat treatment. This storage time amounted to no longer than 48 hours. 5 . Heat Treatment The thermal resistance of the spores was determined by exposing the spore samples in the cups to different heat treatments either in the thermoresistometer, or Open or enclosed in TDT cans placed in miniature retorts, depending on the type of heating medium the spores were to be exposed to as well as the time of exposure, and then subcultured in various media. The results, in numbers of positive and negative tubes at each time-temperature combination were used to calculate D values which in turn were used to plot the thermal resistance curves. D values were established on the basis of the recovery of the heat treated beef'heart‘ grown spores in all subculture media listed in paragraph 2 of this chapter. Identical studies were performed with the trypticase spores limiting however the number of subculture media to three, namely beef infusion broth, yeast extract, and eugonbroth. 44 a. Moist Heat: Heat treatments lasting less than three minutes were carried out in the thermoresistometer described by PFLUG (1960). This apparatus is shown in Figure 2. It can be used for dry heat up to 3800F and for moist heat to 310°F, and insures immediate heating and cooling of the Spore samples. The exposure chamber is connected to a steam reser- - voir where the temperature is accurately controlled by a thermocouple temperature sensing controller and an apprOpriate air Operated pressure control valve. The temperature of the steam reservoir was checked by an industrial thermometer. In order to insure agreement in tempera- ture between the thermoresistometer heating chamber and the steam reservoir periodic checks were made through thermocouples located at both locations. The desired exposure time was set on two automatic timers, 1 and a Precision timer recorder2 was used to measure the actual exposure time of the spore samples. Upon activation of the time controlling device, the cups were introduced into the steam chamber, at the end of the pre- set treatment period, the cups were released automatically from the chamber, and dr0pped into the subculture medium. Five cups were treated at one time. 1Manufactured by Eagle Signal Corp. , Moline, Ill. and Dimco- Gray Co. , Dayton, Ohio. 2Manufactured by The Standard Electric Time Co. , Springfield, Mass. 45 usuoEoumfimOHthofifi ssh. . [I x . ll‘. . 1,: .34? v D. .N MMDDHM A 46 All long time heat treatments (longer than 3 minutes) were carried out in miniature retorts. These heating devices which have been demon- strated to produce identical results to those obtained with the thermo- resistometer by PFLUG AND AUGUSTIN (1961) were used because of the larger number of replicates that can be treated at one time. These retorts are shown in Figure 3. They are supplied with saturated steam from the same reservoir as the thermoresistometer. The temperature controlling system is the same as described for the thermoresistometer. Ten cups containing Spores were placed into a sterile Open TDT can in a petri dish. Immediately prior to the heat treatment, the TDT can was put onto a perforated plate of a holding rack, put into one of the re- torts and Subjected to the desired heat treatment. At the end of the pre- determined exposure time, the TDT can was removed from the retorts and from the holding rack and placed into a sterile petri dish. From there the cups were transferred aseptically into the subculture medium. b. Dry Heat: Dry heat is defined in this investigation as heated air of the normal surrounding atmosphere. In order to obtain this condition 10 metal cups containing the dried spores were placed into each TDT can. The cans were sealed with a Dixie Automatic Can Sealer, 1 and subjected to the desired heat treatment in one of the miniature retorts using saturated steam as the heating medium. Upon completion of the heat treatment, 1Manufactured by the Dixie Canner Co. museum: whdumwfifie 63H. .m HMDUHM 48 the TDT cans were cooled in running cold water in the retort, removed from the retorts and Opened aseptically. Their content, the cups con- taining the spores were placed aseptically into the test tubes containing the recovery medium. In this type of heat treatment, heating and cooling was not immediate. In order to evaluate the extent of the heating and cooling lag, heat pene- tration studies were made in which the time-temperature pattern inside the TDT cans was followed by a means of a c0pper constantant thermo- couple inside the cans, and a Honeywell -Brown temperature recorder. The results of this study are shown in the Appendix and the calculations necessary for the lag corrections are outlined in paragraph 6c. 6. Calculation of Results The heat resistance of the spores was evaluated by determining their D and 2 values, which were defined previously. a. Calculation of D Values: All D values were calculated using the modified Schmidt method described by PFLUG (1962). This method has been outlined in the litera- ture review chapter. Four different methods of calculations were used to determine D values from heat resistance data obtained with beef heart grown spores which were heated in moist and dry heat, and recovered in yeast extract medium. They were: the modified Schmidt method, the Stumbo, Murphy and Cochrane method, often called the unweighted average method, the 49 Spearman-Karber method and the loglog or maximum likelihood method. The first two methods have been outlined in the literature review chapter. The Spearman-Karber method which has been described by LEWIS (1956) is outlined in the following: D is calculated from equation (6). Just as this was the case with the modified Schmidt method, N represents the number of surviving spores per tube corresponding to the mean sterility time, namely 0. 69 organisms. The mean sterility time t is cal- culated as follows: 1:: tu+ d/Z '- dri/ni where tu : the highest exposure time included in the calculation (1 = the time interval between two exposure times ri = the number of sterile samples ni : the number of replicates. The 95 percent confidence limits for D were computed as follows: 95% C. L‘D = (t i 1. 96 s)/(logNO - log 0. 69) where s : v1/2, and V d?- runs. - rimn,2 - 1) The loglog method has been outlined in detail by FINNEY (1952) and LEWIS (1956). In this method the rate constant is calculated as k according to the following equation: k : (logeNo) 2wi - Ewiyi ZWiti where ti = the time of exposure Yi = Yi - 771 w. : 1 the weighing coefficient obtained from Yi 771 the working deviate obtained from Yi at} 65 Ia: 50 Yi is obtained from the following equation: Y1 = logeNO - kti Wi is obtained by interpolation from FINNEY"S appendix Table XVII. 771 is obtained from either one of the following equations: :3770 +13: -1 -q -= owe-m, 77., , and A are all obtained from the above Table. p and q H. H. H refer to the fractions of negative and positive samples respectively. This method consists of several successive cycles of computations which give estimates of k that near the theoretical maximum likelihood estimate asymptotically. In order to eliminate possibly several cycles of computations, k is first calculated by any of the other methods of cal- culating D. Then k is calculated from the equation D = 2. 303/k. From this k, Yi is calculated as described above. The entire cycle of compu- tation is then carried out resulting in a new k value. This new k is used in the next cycle. The series of calculations is repeated until the k values are in agreement. D is then calculated according to the above equation . b. Determination of 2 Values: The 2 values were determined graphically by plotting the logarithms of the D values against the linear values of temperature. The z value is defined as the negative reciprocal sloPe of this line commonly expressed as the number of degrees Fahrenheit necessary for a tenfold change in D value 3 . 51 c. Lag Correction: The corrected heating time of the samples was obtained from the following equation: tC = t-(u) = t'-tm+te where tC the corrected time of exposure of the samples t = the time of exposure of the samples in the retort tm : the time required for the can interior to reach maximum temperature te : the time equivalent of tm at maximum temperature. u : lag correction The term te was determined according to the general method as outlined by BALL AND OLSON (1957). The characteristics of the heat penetration curves which were established on the basis of three deter- minations in each of tiavo TDT cans with temperature measurements taken at five second intervals and are compiled for each temperature at which lag correction might be necessary in Table l. The heat penetration curve for 3200F is shown in Figure 1A. The destruction rates L for any value of T associated with a particu- lar thermal reduction curve were calculated from the following equation: L = log'l(T - T1)/z where T : selected temperatures corresponding to certain times on the heat penetration curve T1 Z the retort temperature the time required for the thermal resistance curve to traverse one logarithmic cycle. L values were computed for T values which were taken from the heating curve at two seconds' intervals. The sum of these L represent te. U values are for each temperature and z values are compiled in Table 2. A sample calculation is shown in Table 1A. 52 Lag corrections were only included in the calculation of the D values if the former amounted to less than 1 percent of the L. D. 50 values. It was found that such corrections were only necessary in a few cases at temperatures of 315°F and higher. 53 TABLE 1. Heat penetration data for cups in TDT cans heated in steam in miniature retort, * m 1 ___=L— T1 T0 j f1 tp f2 0F 0F sec. sec. sec. 285 67 1.0 6.04(3.3-l3.5)a 7.9 22.2 300 65 1.0 4.3 (3.5-5.3) 6.0 21.7 315 65 1.0 4.25(3.6-4.6) 5.7 23.6 320 66 1.0 4.2 (3.7-4.8) 6.4 25.2 T1: retort temperature, 0F To: initial temperature in TDT can j : Lag factor = (T1 - TA)/(T1 - To) f1 : slope of the first straight line of the heating curve, expressed in seconds required for this line to traverse one logarithmic cycle f2 : slope of the second straight line of the heating curve expressed as for fl tp : Time point of intersection of the two straight lines of the heating curve in seconds * data are average of 6 runs arange is listed after each average ratio 54 TABLE 2. Lag correction factors __ w zoF U, min. 285°F 300°F 315°]? 320°]? 33 -- -— 0.18 0.17 36 o 19 o 15 0.17 0.17 39 -- -- 0.15 0.16 RESULTS 1. Preliminary Studie s Two types of cup materials were available as containers for spores during heat treatment: aluminum and tinplate. In anticipation of possi- ble effects of corrosion products derived from these materials during drying of the spore suspension and during the heat treatment and as well, and probably above all during incubation in the subculture medium, a preliminary comparative study was undertaken with both kinds of cups. The results, as shown in Table 3, reveal considerably higher D values with tinplate cups as compared to aluminum cups regardless of the type of heating medium. The reason for this difference is not known. However, since both yeast extract medium and pea infusion contain suf- fficient iron to satisfy the nutritional requirements of the germinating and outgrowing spores, the supply of iron-type corrosion products from the tinplate cups appear hardly to be the reason for this difference in D values. Rather, it seems that some inhibitory factor associated with the aluminum cups might account for the low D values obtained with these cups. Tinplate cups were used throughout the remaining experiments. When the synthetic medium was first used for the subculturing of heated Spores, zero survival was obtained. As a result of this a com- parative study was undertaken using two compounds which were known to stimulate the germination and possibly the outgrowth of spores: Ethylene-diaminotetraacetic acid (EDTA) and sodium bicarbonate. The 55 TABLE 3. The effect of the type of cup used as a container for spores during heat treatment Heating Temperature D values Medium oF Tinplate cups Aluminum cups Dry Heat 255 37. 5 minutes 30. 1 minutes Moist Heat 250 88. 0 seconds 50. 0 seconds Recovery media: Dry Heat : Yeast Extract Moist Heat : Pea Infusion Spore suspension: Dry Heat : Beef heart spores, crude suspension Moist Heat : Beef heart spores, clean suspension 57 results of these experiments in which the addition of minerals was in- cluded as another variable are compiled in Table 4. It is obvious from these data, that only sodium bicarbonate enhanced the recovery of the spores following the heat treatment. EDTA had no effect whatsoever at the concentration in which it was used. However, RIEMANN (1963) and BROWN (1956) noted that the germinating effect of EDTA is lost, if it is added to the medium in too high concentrations. In such a case, EDTA not only removes metals inhibitory to germination but possibly also some of the essential minerals. This possibility might be without any effect on germination, but could seriously limit outgrowth. That EDTA is acting as an inhibitor is indicated by the fact that recovery is somewhat reduced in the case where EDTA is added to the medium con- taining sodium bicarbonate with or without minerals. A somewhat more extensive experiment was carried out comparing the effect of sodium bicarbonate on the recovery of spores in the syn- thetic medium following both moist and dry heat treatments. The results which are shown in Table 5 confirm the above findings, and those re- ported by WYNNE, SCHMIEDING AND DAYE (1955) and RIEMANN (1963)' with reference to P.A. 3679. WYNNE AND FOSTER (1948) reported carbon dioxide only to have a favorable germinating effect on spores of Clostridium botulinum but not on P. A. 3679. From these findings, it appears that the germination- stimulatory effect of sodium bicarbonate varies not only with the organism, but also with the type of culturing medium used. Since sodium bicarbonate has never been reported to have 58 TABLE 4. Preliminary studies of the effect of several additions to the synthetic medium on the recovery of spores of P.A. 3679 following dry heat treatment at 300°F. 1 Recovery Media r l INC. of Tubes Showing Growth No. of Replicates 5 minutes 15 minutes Synthetic w/o minerals 5 0 0 Synthetic with minerals 5 0 0 Synthetic w/o minerals, with NaHCO3 5 5+ 2+ Synthetic with minerals and NaHCO3 5 5+ 5+ Synthetic w/o minerals, with EDTA 5 0 0 Synthetic with minerals and EDTA 5 0 0 Synthetic w/o minerals, with NaHCO3 5 5+ 1+ and EDTA Synthetic with minerals, NaHCO3 and 5 5+ 4+ ED TA -minerals refer to all salts added to the medium as outlined in the experimental part of this thesis with the exception of phosphates which were added in all cases -concentration of NaHCO3 : 0. 5 percent -concentration of EDTA : 10 M 59 TABLE 5. The effect of the addition of NaHCO3 to the synthetic medium on the recovery of beef heart spores following their heat treatment Heating Tempearature D values Medium F Control + 0.5% NaHCO3 Moist Heat 260 7.5 sec. 9. 50 sec. 275 1.4 sec. 1.60 sec. Dry Heat 315 1. 39 min. 2.12 min. 60 any inhibitory effect on either germination or outgrowth of spores, it was added to all the subculturing media used in this work. 2. Resistance of Spores to Saturated Steam A summary of the D250 values and the z values as well as their corresponding energies of inactivation of beef heart grown spores is given in Table 6. All D values obtained with these spores at various temperatures are compiled in Table 7. The thermal resistance curves are shown in Figures 4 through 10. The 2 values, and therefore also the energies of inactivation are the same for all media, namely 19. 50F and 64, 700 calories with the exception of eugonbroth with a z of 17. 50F and an E of 70, 500 calories, and trypticase with a z of 18. 2°]? and an E of 68, 200 calories. Due to the differences in z values, the relative magnitude of the D values ob- tained with the last three subculturing media changes with increasing temperatures. At 280°F the D values decrease in the following order: synthetic medium, trypticase, eugonbroth. This points out clearly that comparative heat resistance studies are of little value unless D values are established at least at two different preferably extreme temperatures. At 2300F the D values obtained with the various subculture media can be grouped into four classes of decreasing distinctly different magni- tudes: The highest D values are obtained with either beef infusion or pea infusion. The second class includes liver infusion yeast extract and eugonbroth. To the third and fourth class belong trypticase and the syn- thetic medium respectively. At 2750F again beef infusion gives highest 61 Meagan on. pomnmno on. :3, 63m> m Himmaqd. oom* -- -- -- .. ma 2:. .3 m .2 me .o osefiasm 33 com .8 m. 2 2 o .w: 8... .3 m .2 E .o gaoaneomam .. -- -- -- 2: com .3 N .3 3 .o 8339»; 02 com .2 o 2 mm .o 3a 2: .3 m .2 N .a 82:5 the» -- -- .. -- 3a 2:. .3 m .3 mm a .8353 «mm -- -- -- -- 3a 2: .3 m .S a .o .8335 .834 42 813 o .2 mm .o as 2:. .3 m .3 a. .a .8335 seem am am hon has .83 *m *m .mo .9. £8 .83 monomm endofinguh. 5.93an a: o>ooom monomm unmom moo m ”I“ H monfimfl cw “H3053 mm oonmumwmou «do: 3on 90m Qoflm>flomcw mo mods: mafipnommonuou pad manage N Odd omNQ .0 Hdméflb 62 1: 11 :1 Tmmo.1mmmo. mmmo.noomo. mmmo. 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D values and their corre5ponding 95 percent confidence limits in minutes for moist heat resistance of trypticase- yeast extract spores and various recovery media (M T— mm Temp. oF Beef Infusion Yeast Extract Eugonbroth 230 6.74 5.27 3.77 6.26-7.21 -- 3.50-4.05 5. 12 -- -"" 4. 75-5. 37 240 0. 841 __ __ 0.809-0.878 250 0. 329 0. 337 0.166 0.298-0.361 0.313-0.360 0.157-0.l75 260 0.0805 0.0792 0. 0378 0.0748-0.0863 0.0690-0.0893 0.0347-0.041 270 0. 0273 0. 0225 0.0259-0.0290 0.0707-0.0245 -- 64 200.0 I i 1 ‘ Curve 0 : moist heat 1 Curve b-dr-j heat, up to 3 months storage of unheated Spare SUSpension Carve c1 dry heat, up to 9710mm storage at spore sus- pension |00.0 600 40.0 30.0 ,1 _._-p_.....—-._ 20.0 10.0 ’ 6.0, 0.6 0.4 I 0.3 ._ .__.-_ _. -- , , , ‘ ' ' ' V 0 - v . . . . . , . . . . . . . ‘ * u - . 1 . . A . . 1 ....... 002 ‘ ' ‘ . - . . 1 - ~ 0 . . , .4 . , .. . r . < - . - L . . . . . . , r 9 4 ..... 0.| 0.94 0.03 0.02‘ *1: : *3 .5?“ W . -‘ :,-f ;—-i 230 250 270 290 3|O Temperature, °F. FIGURE 4. Thermal resistance curves of beef-heart infusion Spores subcultured in beef infusion 65 2000 . Curve 0 = maist heat 1‘ "f4? Curve 0 ; dry heat, up to 3 months . . _ , . , ,_ :‘. " . storage of unheated I000 : _ ~ 7 .. spore suspension 7 ' Curve ; dry heat, up to 9mmths 60.0 ; Q ? ‘ i f V - > . ‘ ' ‘ f storage of spare sus- “ - ' ‘ pensmn 40.0 30.0 200 «it u .. 1 C l0.0 6.0 Log D, min. 0.6 0.4 ’ '0 0.3 ‘ 0.! 0.06 0.04~ 0.03 0.02 230 250 270 290 MO Temperature, 0F. FIGURE 5. Thermal resistance curves of beef-heart infusion spores subcultured in liver infusion 66 = moist heat :dry heat, up to 3 months storage of unheated Spore suspensron . dry heat, up to 9 months 50.0? ;_ _ ii.» storage ofspore sus- ‘ - ' ' ~ ‘ ‘ -' ' pensron |000 40. .300 200 I00 I g . ,. . I E E Q“ _;_ armor. o __J 003 02 '" . 2‘. - .‘_r.. 51.. O 230 250 270 290 3|O Temperature, °F. FIGURE 6. Thermal resistance curves of beef-heart infusion spores subcultured in pea infusion 67 200.0 . , , , , f ‘ ' storage or' unheated I000 57-,- ; a s = ~ - g . i *j _ spore suspension >*~I+—~- , " , _ V r", \g - A t ; ., Curve c: dry heat, up to 9monthsI ' ; - ‘ ' storage of spare 505- I pension : ' I V . . x 6‘13 ; ; jCiifve c : moist heat "“" ‘” 't .'""" *t‘f: ~: . K" ”1" T T Curve 0 : dry heat, up to 3 months I I . . ; ‘ ; L.' ' ' 60.0 I _.__._._.. __.. - .1 i r - ' .. .- s .- Q-.-— .— . v 1 5‘“--. - h.— . . - - .. . . ... .. ----- ”W..T-. ._ ,__ - ... f ...,..- A .Tmsr - ' ‘ , I L I - 2 ' ‘ ' . ' .4“ ...-. ‘ , ‘ I .._.__k--; _. h- _; .1?» . --.; f‘ ‘ ..7 -r. 7...:fi-h f .- . . b b “I I ' """ “-"1‘ A? . ' ‘ If T'4 o -——-——‘.-v—.—o-——o -————.—.L—— . ' I s , ‘ .1 I ‘ ‘ _s --.- --....,._r .-——— __g , s , kt -., - ”r- I _ _ _ ,_ ' ‘ - ”—u—d -— ——.- as; *- W-.- ' I 1 T. - t V "'Y"‘.“' ' .. ,_ l u r .' —--+. {~—-—-—-~o—-———r—-<‘I-- a.-- .. - fit-.. -.— -.—- #1 ' ' I -..- - i a “I ,- 7;... _ ,, I . I . ' I . u , L I I digits“- Q. _ s w. I I I. I L I i {. I I I r3“? hIné" a-.;,_,_-.: -,_.-.V._. f“? --..; I V, rrn T r a 4 ~- _ .. r. ._.-~ —-I <-; -—.‘_ },-._< , 1 T ._._r-- ...... - . - -.,;MI--._.- '-;_ L .._, is.--,,L gnawinwn ..-. .--_‘..:__s -- -__ ”Us- .7-..‘ Eh-.. I i * . y . ~ , I 2.: . A : I r z . 002 A ' - , A I ~ ‘ 4 - . r . 230 250 E70 290 310 Temperature, °F FIGURE 7. Thermal resistance curves of beef-heart infusion spores subcultured in yeast extract 200.0 100.0 1 60.0 68 ~ rve 0 = moist heat Curve I) = dry heat, up to 3 months storage of unheated Spore suspensron rve or dry heat, up to 9 months storage of Spore sus- pensmn 400 30.0 200 I00 230 250 270 290 3|O Temperature, °F. FIGURE 8. Thermal resistance curves of beef-heart infusion spores subcultured in trypticase 200.0 |00.0 60.0 40.0 f, 30.0 ‘ 200 I00 0| 0. 0. 0.0 0. 230 FIGURE 9. 69 moist heat :dry heat, up to 3 months SIOIIch of unheated soore suspension dry heat, up to 9months strrcge of spare sus- petsion 250 270 290 3|0 Temperature, °F. Thermal resistance curves of beef—heart infusion spores subcultured in eugonbroth |00 80. 60. 70 ,1, ..,_ _ ... , e~~ i ,Curve 0 ~rhapist heat ' i ICurve b dr heat . . . . .. ~8._.._ -__.~l _.-- o- _,.. 40.0 30.0 20.0 8.0 ' 6.0 i 4.0 0. I 0.08 FIGURE 10. 0 ———. . 250 270 290 3'0 Temperature, °F Thermal resistance curves of beef-heart infusion Spores subcultured in synthetic medium Log .0, min. ' 0.02 71 §---Curve a moist heat . i Curve b dry heat |00 . I 80.0 60.0 40.0 30.0 20.0 V o , . . . . . . t . . . . 1 . . . . - - .—~ - — 7—- '- -o——¢—-.- —-—.— v- ‘o- — —» i . . . . . . - D . u .. , _ . I0.0 8.0 6.0 4.0 3.0 2.0 to 0.80 0.60 0.40 0.30 Hf 0.20 E 0.10 ‘ 0.08 006 0.04 _ 0.03 ‘ 230 250 270 290 3|0 Temperature, °F. FIGURE 11. Thermal resistance curves of trypticase-yeast extract spores subcultured in beef infusion 200 I00 80.0 60.0 40.0 , f 30.0 . L, 200 I00 8.0 630 0.80 060 0.40 0.30 _ 0.20 CI 0.08 004 ' 0.03 002 FIGURE 12. 72 Curve a moist heat Curve b d heat 250‘ ' V .270 i 290 Vista Temperature, °F. Thermal resistance curves of trypticase-yeast extract spores subcultured in yeast extract Log 0, min. 200 I00 800 60.0 40. . 30.0 é. 20.0 . I00 0.02 230 FIGURE 13. 73 p u c .f“. : .Curve a moist heat I .f '1 ICu’rve b .dry heat 250 270 i 290 .3I0. Temperature, °F. Thermal resistance curves of trypticase-yeast extract spores subcultured in eugonbroth 74 D values. Distinctly different and decreasing D values in the following order were obtained with pea infusion, yeast extract and liver infusion. The lowest D values that were all of the same magnitude were found when either trypticase, eugonbroth or the synthetic medium were used for sub- culturing. The fact that the D values obtained with the infusion type media and yeast extract are changed in their relative magnitude at the two tempera- tures is explained as being due to very slight differences in their 2 values. The spores grown in trypticase yeast extract are of considerably lower heat resistance. As shown in Tables 6 and 8 and Figures 11 to 13, their D250 values decrease with the recoVery medium in the following order: beef infusion, yeast extract, eugonbroth. The 2 values vary from 17. 00F for yeast extract to 16. 00F for beef infusion and to 15. 50F for eugonbroth. The results obtained in general are in agreement with those re- ported for P. A. 3679 in the literature: infusion type media generally yield a higher recovery than formulated media with the exception of yeast extract which in turn gives a better recovery than the dehydrated media. FRANK AND CAMPBELL (1955) reported contrary to the findings bf WYNNE, SCHMIEDING AND DAYE (1955) a higher recovery of spores of P. A. 3679 with yeast extract starch agar than with yeast extract starch bicarbonate agar. This discrepancy is believed to stem! from the fact that the former authors used the same media for both initial and survi- vor counts, while FRANK AND CAMPBELL derived their initial counts 75 only from one medium namely eugonagar, regardless of the type of medium used to count the survivors. As shown in Table 9, initial counts vary considerably with the type of medium used. It is therefore quite conceivable, that if FRANK AND CAMPBELL had used the same media for both the initial and survivor counts, their results would have been in agreement with those reported by WYNNE, SCHMIEDING AND DAYE. As far as the use of eugonagar is concerned, FRANK AND CAMPBELL reported lowest D values when compared to any of the other media tested. Even supplementation of eugonagar with soluble starch, malt extract, yeast extract or liver infusion resulted only in a very slight in- crease in the D value. WHEATON AND PRATT (1961) obtained similar results. By far the best recovery was obtained with pork pea infusion as the recovery medium. Beef infusion gave somewhat lower results, as did yeast ex- tract. Eugonagar again gave the lowest recovery. Yeast extract how— ever resulted in a higher number of survivors than liver infusion. With reference to the relative heat resistance of beef heart grown spores and trypticase yeast extract grown spores the results obtained are also in agreement with those of WHEATON AND PRATT (1961). As pointed out earlier, D values in some cases have been reported to be affected by the initial spore concentration. However, according to the reports cited earlier, ten or one hundred fold differences in initial spore concentration result in relatively small changes in D values. It is therefore concluded that the small differences in the initial spore 76 TABLE 9. Comparative initial counts in various recovery media Recovery Medium Beef Heart Spores Trypticase Spores Beef Infusion 4. 35 x 104/cup ll. 0 x 103/cup Liver infusion 3. 93 x 104/cup --- Pea Infusion 3. 94 x 104/cup --- Yeast Extract 4. 29 x 104/cup ll. 3 x 103/cup Trypticase 3.10 x 104/cup "— Eugonbroth 2. 98 x 104/cup 9. 46 x 103/cup Synthetic 2. 54 x 104/cup 77 concentrations between the two types of spores as shown in Table 7, are not large enough to account for the extreme difference in their relative heat resistance, and that therefore these results are to be considered factual rather than coincidental. The thermodynamical data reported should be viewed with caution. These values are meant to give information regarding the events that take place during heat inactivation of the spores. Therefore they are only applicable in the case where the subculture medium completely ful- fills the nutritional requirements of the surviving Spores. In the case of this investigation, beef infusion is believed to represent such a medium. Therefore, the energies of activation and the entr0py values calculated from the data obtained with this medium are to be considered the true values. Nevertheless, the thermodynamical data calculated from any of the other recovery media, provide useful indications in comparing moist and dry heat resistance of bacterial spores. Table 10 shows the results of an experiment in which the effect of the addition of vitamin B12, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) to the synthetic medium was studied with the purpose of find- ing out, whether the addition of certain compounds would result in a higher recovery. The results indicate that supplementing this medium with either vitamin B12 or RNA or a combination of any of two of these compounds or all three of these compounds resulted in an increased D value. However, experimentation was not extensive enough to allow firm 78 TABLE 10. The effect of the addition of RNA, DNA, and Vitamin B12 to the synthetic medium on the recovery of moist heat treated beef heart spores Compounds added No.1- of Nb. of D26O’ sec. replicates positive Control 20 0 f 8. 65 DNA (400mg/lt) 20 0 8. 65 RNA (800mg/lt) 20 2 8. 60 Vitamin B12 20 2 8. 60 DNA, RNA, B12 20 4 9.40 DNA, RNA 20 4 9. 40 RNA, B12 20 6 9. 70 DNA, B 20 4 9. 4O 12 79 conclusions. Rather, the results are to be taken as indicative to the direction in which further work might be fruitful. 3. Resistance of Spores to Dry Heat In accordance with other reportings, the spores of P.A. 3679 ex- hibit considerably higher resistance to dry heat than to saturated steam. Just as this was the case with moist heat treatments, both D and 2 values vary as shown in Tables 11 and 12 and Figures 4 to 10 are dependent on the type of subculture medium used. With the exception of beef infusion with a 2 value of 390F and the synthetic medium with a 2 value of 33°F, z values obtained with all other subculture media are of the magnitude of 36°F. At 2550F the D values obtained with the various subculture media are lowest when the heated spores were recovered in the synthetic medium. A slightly but distinctly higher D value was found when eugon- broth was used as the recovery medium. With all other subculture media substantially higher D values but of the same magnitude were obtained. At 320°F the use of beef infusion as the subculture medium gave by far the highest D values. D values obtained with yeast extract were slightly lower. Third highest D values were obtained with liver infusion, followed by the D values obtained with pea infusion and trypticase, which were of the same magnitude. Again, the use of eugonbroth resulted in second lowest, and that of the synthetic medium in the lowest D values. Results in Table 12 and Figures 4 to 9 show an increase in the D values during storage of the Spores. The first D values were obtained no longer than 90 days following Sporulation, whereas the last values, 80 “am A ran-w + flmom .N - HM on 8m .3m u 8333.. we 303cm u m J. - NH. 7:? as x Jam-w xmmom .~ u 5:233 mo 33:6 u m... I- -- I- m .3 ooo .3 o .8 3 .o ongofiim 3 03 .3 o .3 N .m o .3 CS .3 oxen N .m fiquomsm -- -- I- e .3 23.3 o .3. 3.3 333%; 5 cos .3 o .3 o .m o .3. 30.3 o .3 N .2 Hansen 38» I- -- I- o .3 03 .3 o .3. o .2 nosed: mom -- -- -- o .3 OS .3 o .3. o .3 5335 834 S. 25.3 o .3 To o .S 23.3 93 0:2 :33qu “mom to. am has £8. .83 *m *m hog 58 .83 89:52 >Ho>ooom monomm ommoflmrfh. monomm ”Edam moon E monfimfl a: :39? mm oucmumwmou ado: >33 new Gofiumcfiuomcw mo mummy: mdmvnommmnuoo mam. mafia-mgr N van oomQ .3 Mdméfifi @3.H-m~.avxmm.-o~.mv A3H.m-s.mv Amm.m-H3.mv Aao.m-ws.~v Awm.m-o.mv “03.3-om.mv 3m.~ em.~ ms.~ om.m om.~ o~.m vo.m ANH.N-ow.~. 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The trypticase yeast extract grown spores again exhibit considerably lower heat resistance than the beef heart grown spores, as shown in Tables 11 and 13 and Figures 11 to 13. The results reveal a decrease in D values with the recovery medium in the following order: beef in- fusion, yeast extract, eugonbroth. It is interesting to note that the E values which are all of the same magnitude, namely 40, 600 calories are approximately half of those obtained with the same type of spores with moist heat. This is not the case with the beef heart grown spores. The difference in recovery of the nonheated spores in the various subculturing media as shown in Table 9 follows more or less the same pattern as those reported for their corre8ponding D values, although to considerably smaller degree. The same holds true for the trypticase grown spores. 4. Statistical Methods of Analysis The results of a comparative study of four different methods of statistical analysis of the thermal resistance data are compiled in Table 14 and shown in Figures 14 and 15. With the moist heat resis- tance data, the D values obtained with the loglog or maximum likelihood method are consistently lower than those obtained with the Schmidt or the Spearman-Karber method. With the dry heat resistance data, an Oppo- site trend appears to hold. However the 2 values resulting from any of 83 TABLE 13. D values and their corresponding 95 percent confidence limits in minutes for dry heat resistance of trypticase yeast extract spores and various recovery media. ==== WW Temp. , oF Beef Infusion Yeast Extract Eugonbroth 255 101.9 100 56.5 (96.8 - 107.8) (72.4) (48.3-65.0) 130 96.0 57.0 (108-151) (84.2-107.7) (52.4-61.7) __ 105 __ (90.5-111) 109 _ " (103-115) ' 285 17.3 16.6 9.66 (16.7-17.9) (15.5-17.7) (9.1-10.5) 20.7 14.7 8.74 (19.2-22.2) (13.8-15.5) -- 18.5 14.0 7.85 (17.4-19.7) (12.3-15.7) (6.55-9.14) __ 16.3 __ (15.8-16.9) 300 6.33 6.82 4.01 (5.94-6372) (6.42-7.20) (3.51-4.41) -- 5.82 3.22 (5.44-6.19) -- 315 -- -- 1.05 (0.84-1.26) 320 1.55 1.56 1.Q3 (1.42-1.69) (1.39-1.72) (0.94-1.11) 1.91 1.53 1.23 (1.76-2.06) (1.37-1.68 (1.04-1.41) 1.80 (1.62-1.98) 1.89 - 1.68 -- (1.78-1.99) (1.56-1.80) 84 TABLE 14. D values and their corresponding 95 percent confidence limits utilizing different methods of calculation m —_======_—.=: D values and 95% CL* Heating Temp. , Medium 0F Modified Maximum ST UMBO Spearman- Schmidt likelihood ET AL. Karber # (LEWIS) hdoist 230 11.83 11.5 12.8 11.9 heat (10.50-13.08) -- -- (10.50-13.33) 250 1.09 1.00 1.08 1.12 (1.0-1.18) -- -- (1.07-1.16) 260 0.33 0.32 0.33 0.33 (0.31-0.37) -- -- (0.31-0.35) 280 0.033 0.031 0.033 0.033 (0.031-0.037) -- -- (0.032-0.035) lDry 255 181 192 192 191 heat (169-193) -- -- (187-195) 285 29.4 31.1 31.6 31.5 (27.9-30.8) -- -- (29.2-33.7) 300 11.1 10.9 11.0 11.0 (10.4-11.6) -- -- (10.7-11.4) 320 3.94 3.87 3.84 3.85 (3.8-4.10) -- -- (3.73-3.98) *Moist heat: Yeast extract recovery medium, D in seconds Beef infusion recovery medium, D in minutes Dry heat: 200 I00 8.0 6.0 4.0 3.0 2.0 10 0.8 0.6 'E 0.4 . Q 03:; —J 0.2 01 0.08 0.06 0.04 ' 0.02 f FIGURE 14. 85 = Schmidt, z=|95°F = Loglog, z=205°E = Stumbo, 2:195 °E = Spearman-Kr’irber, z=|9.5°F. 230 250 270 290 230 250 270 290 Temperature, °F. Thermal resistance curves obtained from D values calculated by four different methods from moist heat resistance data of beef-heart infusion spores sub- cultured in yeast extract Log 0, min. GOUD FIGURE 15. 250 86 1:39 =LogIog 2:39 °F :Stumbo 2:385 2 Spearmon K'drber, z=38.5°F 270 290 3|0 330 270 290 3I0 330 250 270 290 3|0 330 250 270 290 3 IO Temperature, °F. Thermal resistance curves obtained from D values calculated by four different methods from dry heat resistance data of beef-heart infusion spores sub- cultured in beef infusion 87 the four methods used were identical. Therefore, and because the dif- ferences in the D values obtained by the four methods were only small and for ease of calculation the Schmidt method was considered to be the most desirable one for the statistical treatment of all heat resistance data. 5. Rearrangement of Heat Resistance Data As shown earlier, the 2 values not only vary with the heating medium, but also in some cases with the type of recovery medium used. This means that the D values obtained with the various subcul- ture media are somewhat different in their temperature dependence. Thus the statement made by CURRAN AND EVANS (1937) that severe heat treatment causes bacterial spores to become more exacting in their nutritional requirements might show not only a time dependence regard- less of the temperature used, but it also might be temperature dependent. In order to demonstrate this more drastically, the data plotted in Fig- ures 4 to 10 were rearranged. Equation (6) was solved for N. 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