Quality and Stability of Canned Meats: A Symposium Sponsored by the Quartermaster Food and Container Institute for the Armed Forces, Quartermaster Research and Development Command, U.S. Army Quartermaster Corps, Palmer House, Chicago, March 31 - April 1, 1953 (1954)

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Second, when we find a better canned meat, will it have maximum shelf life with maximum acceptability? We know there are chemical changes that occur in other animal products that are not put in a can. This is an area which should be explored, but currently it is not active in the animal products program. II. Microbiological Factors Affecting Canned Meats CHAIRMAN ROBINSON The next section of our symposium concerns microbiological factors affecting canned meats. The first discussion on "Chemical and Physical Factors Affecting the Thermal Resistance of Bacterial Spores" is by Dr. Hiroshi Sugiyama of the University of Chicago. Chemical and Physical Factors Affecting the Thermal Resistance of Bacterial Spores HIROSHI SUGIYAMA The ability of the endospores of certain bacterial species to sur- vive drastic heat treatment is the important factor in the heat pres- ervation of foods. Numerous studies on the thermostability of bac- terial spores have resulted in the accumulation of a mass of data; sev- eral reviews of this literature are now available (5, 40, 43). Factors which determine the thermal tolerance of spores have been considered as intrinsic or extrinsic. Spores of certain species show heat resistance not much greater than that of vegetable cells, whereas others are probably among the most thermostable forms of life. Even within a given species, strain differences can be demon- strated (9, 13). It would seem that there are no practical methods which are foreseeable which can control what might be called the genetic behavior of the microorganisms. However, it is possible to take a given strain of sporulating bac- teria and significantly alter the thermostability of the spores; these may be considered the extrinsic factors.8 One point of empirical con- trol of the heat tolerance of spores is the environment in which spor- ulation occurs. Several workers have shown the importance of the temperatures of growth and sporulation as it relates to the heat sus- ceptibilities of the organisms that develop (24, 31, 42, 44, 48). Cer- tain types of peptones, carbohydrates (48), fatty acids, and cations (42, 48) in the sporulating medium enhance the thermostability of some spores. Probably to be placed in this category is the higher thermostability of spores of P.A. 3679, which develop in pasteurized or heat sterilized meats, as compared to those developing in raw • At this point it should be stressed that in most cases the organisms in- volved will not be named for the sake of brevity; however, the fact that spores of a certain species behave in a certain manner does not imply that spores of other species will react similarly when treated in the same way.

meats (45). The suitability of the culture medium for growth or for sporulation is not the factor determining the heat resistance of the spores (48, 50). Evidence has been adduced that the heat resistance of vegetative cells are not necessarily related to that of the spores; that is, the vege- tative cells may be of relatively high thermal tolerance while that of the homologous spores may be low (52). It would be interesting to determine whether the substrates which enhance spore thermosta- bility increase the resistance of the vegetative cells at the same time. Once the spores are formed, the age of the spores and the con- ditions of aging become of some importance. Some spores may not reach maximum resistance for 60 days under moderate temperature and moisture (27) ; rapid drying in vacua may result in a spore prep- aration whose heat resistance remains constant over a period of months (18). These factors, as well as the separation of a spore suspension into fractions of different heat resistances by centrifu- gation (54), are important in experimental pack studies. Factors in the control of spores. The immediate goal of the food technolo- gists is the control of spores produced in their natural habitat—spores which can be expected to contaminate the food product being subjected to heat processing. The conditions under which the spores are heated becomes of impor- tance under these circumstances. The pH of the menstruum in which the spores are present is of major concern, the resistances usually being highest near neu- trality (I3, 54), although the nature of the buffer or organic acid used plays a role (2, 18). In small amounts ascorbic acid increases while a vitamin K ana- logue decreases the thermotolerance of Clostridium botulinum spores (35.) Salt in different concentrations has a variable effect (2, 13). Sugars in high con- centrations generally increase resistance (2, 42). Oily material markedly in- creases the resistances of spores (26) and vegetable cells (53). Spores that cannot germinate and multiply can be considered as being non- viable. Any factor which will decrease the germination of the spores will have the apparent effect of making the spores more sensitive to heat. Although there is a difference of opinion, it would seem that the effect of meat-curing agents belongs in this category, rather than in increasing the lethality of a given heat process (20, 41, 55). A lower optimum temperature of incubation than that considered optimum for the growth of the vegetable cells has been found for the germination of heat-processed spores of Clostridium botulinum (51). Spores that survive severe heat treatment are more fastidious in their nutritional re- quirements (8) and become more sensitive to deleterious effects of rancid fatty acids (57) which normally suppress the germination of spores (14, 32, 33). The first report on the successful preservation of a variety of foods with a mild heat processing when subtilin was incorporated in the foods (1) seems to have been a temporary sporostatic instead of a sporocidal effect (3, 49). The appar- ently greater thermostability of suspensions of spores containing clumps may be attributed, at least partially, to a greater germination. Thus, the oxidation reduc- tion potential, the concentration of essential nutrients, etc., may be shifted more easily toward the optima in a localized area around a group of spores acting as a unit than when the same number of spores are separated. For example, the pH of Clostridium welchii which are centrifuged on a pad is much lower than that of the same cells dispersed in the medium (30). Of course, another contributing factor in the seemingly greater heat resistance of suspensions of clumped spores is the strictly numerical aspect—the greater the number of spores, the greater the heat tolerance (e.g. 40), the original suspension actually containing a greater number of organisms than colony counts would indicate. For the practical heat

processing of meats, the low degree contamination of raw meat products suggests that perhaps the present standards of heat processing based on large inocula of experimental packs may be excessive. Spores that survive heat or ultraviolet radiation lethal to part of a spore population appear more sensitive to further treatment of the same agent. In suitable combinations of heat and ultraviolet radiation, some spores are sensitized to the effect of heat by the action of light, although the converse does not seem to occur (12). A possible field of experimentation is the use of mild heat treatments of the spores. This type of heating results not only in an acceleration of spore germi- nation but also in an increase in the number of spores that germinate (7, 36) . This apparent increase in the viability of spores is important in the standardiza- tion of spore suspensions for experimental studies. Moreover, the stimulation of metabolic activities can be demonstrated subsequent to these heat shocking treat- ments. Thus in microrespirometer studies of aerobic spores, with checks made to show that actual germination of the spores does not occur, the Qo2 is meas- urably increased (4). In view of the fact that growing organisms are more susceptible to deleterious agents it would seem interesting to determine whether spores in the state of heat activation are altered as to heat resistance. Perhaps of some bearing on this point, is the observation that spores become more sensi- tive to the mutagenic effect of ultraviolet irradiation very early (5 minutes) in the period of spore germination (28). The basis of heat tolerance. What is the basis of the heat tolerance of bacterial spores? The most likely explanation would involve the change in the physio-chemical make-up of the vegetative cells in their transition into the spore state, resulting in a spore cytoplasm that becomes uniquely resistant to the ther- mal inactivation of its biological activities. The rather high temperature co- efficients shown in the heat destruction of spores suggest that protein denatura- tion is the basic mechanism. Thus, moderate hydrostatic pressure accelerates the killing of spores at ordinary temperatures and retards the destruction at high temperatures, an analogous behavior being shown by protein denaturation (22). Urethane, a protein denaturing agent, renders spores more sensitive to heat pro- cessing (21). Many of the observations already cited can be explained from this point of view (5, 42). That there is a difference in the chemical make-up of the spores as compared to the homologous vegetative cells is evident from the differences in the antigenic moities (e.g. 11), the calcium content (6), and the qualitatively different amino acid composition (10). Although the specific gravity of spores seems to be uni- formly greater than that of the vegetative cells (25) and the refractive index higher in spores than in vegetative cells (34), the total moisture in the spores is now thought to be of the same magnitude as that in the vegetative cells (19, 25) but a larger portion of the moisture in the spores is in the bound form (15). Since dry heat is much less efficient than moist heat as a sterilizing agent and since bound water does not participate in chemical reactions, this difference in the bound water content may be one of the basic reasons for the high thermostability of the spores. However, it should be pointed out that the method used in the measurement of the bound water is admittedly open to large experimental errors. Various studies would indicate a high content of nucleic acids in spores. It has been suggested that the relatively high content of the ribonucleic acid may contribute to the resistance of spores against ultraviolet rays and electrons (23). In addition, the fact that the desoxyribonucleates have a high degree of protec- tive action in increasing the thermal stability of egg albumen may be signifi- cant (16). This latter effect is a function of the weight ratios of the nucleate and protein and the presence or absence of salt. It has been postulated that the enzymes of spores may be combined in some way which render them inactive and also resistant to heat (46). Recently, however, an active enzyme has been found which exists in higher concentration in spores than in the homologous vegetative cells. This alanine racemase catalyses

the conversion of 2-alanine to d-alanine (39) and is more heat resistant in the spore than in the vegetative cells. The racemase activity of the spore has been partially resolved into 2 fractions, a heat-resistant particulate fraction and a heat-sensitive soluble fraction. This most significant observation would indicate that the spore heat resistance may reside in the manner in which the spore pro- teins are bound to particles (38). It is interesting that a rather similar theory has been made for the malic dehydrogenase and cytochrome systems extracted as a red fraction from a thermophile; this substance is stable at 56° C. This would indicate that the thermostability may not reside in the inherent structure of the protein molecule but rather in the fact that the red fraction behaves like a compact mass of protein which resembles an organization of molecules (29). Conclusion From the brief review presented it is evident that a considerable amount of work has already been done on the thermal resistance of bacterial spores. Nevertheless, it is also true that the heat preserva- tion of foods is still more empirical than deductive; processing factors must still be determined with experimental packs in most instances. It seems that only when the basic mechanism underlying the thermal resistance of spores is more fully understood can there be much hope of graduating from the present empirical trial and error methods to that of a sound scientific approach. Thus, much further work is needed; the use of newer techniques and approaches is particularly indicated—such as the recent work of Stewart and Halvorson on the alanine racemase of spores. Literature Cited 1. ANDERSEN, A. A., and MICHENER' H. D. Preservation of foods with anti- biotics. I. The complementary action of subtilin and mild heat. Food Technol., 4, 188-189 (1950). 2. ANDERSON, E. E., ESSELEN, W. B. JR., and FELLERS, C. R. Effect of acids, salt, sugar, and other food ingredients on thermal resistance of Bacillus thermoacidurans. Food Research, 14, 499-510 (1949). 3. CAMERON, E. J., and BOHRER, C. W. Food preservation with antibiotics: the problem of proof. Food Technol., 5, 340-342 (1951). 4. CROOK, P. G. The effect of heat and glucose on endogenous endospore respira- tion utilizing a modified Scholander microrespirometer. J. Bad., 63, 193-198, (1952). 5. CURRAN, H. R. Symposium on the biology of bacterial spores. Part V. Resist- ance in bacterial spores. Bact. Rev., 16, 111-124, (1952). 6. CURRAN, H. R., BRUNSTETTER, B. C., and MYERS, A. T. Spectrochemical analysis of vegetative cells and spores of bacteria. J. Bact., 45, 485-494 (1943). 7. CURRAN, H. R., and EVANS, F. R. Heat activation inducing germination in the spores of thermotolerant and thermophilic aerobic bacteria. J. Bact., 49, 335-346 (1945). 8. CURRAN, H. R., and EVANS, F. R. The importance of enrichments in the cultivation of bacterial spores previously exposed to lethal agencies. J. Bact., 34, 179-189 (1937). 9. DAVIS, F. L., JR., and WILLIAMS, 0. B. Studies on heat resistance. I. Increasing resistance to heat of bacterial spores by selection. /. Bact., 56, 555-559 (1948). 10. DAVIS, F. L., JR., and WILLIAMS, O. B. Chromatographic analysis of the amino acid composition of bacterial spores. J. Bact., 64, 766-767 (1952).

11. DOAK, B. M., and LAMANNA, C. On the antigenic structure of the bacterial spore. J. Bad., 55, 373-380 (1948). 12. DUGGAR, B. M., and HOLLAENDER, A. Irradiation of plant viruses and of microorganisms with monochromatic light. II. Resistance to ultraviolet radiation of a plant virus as contrasted with vegetative and spore stages of certain bacteria. J. Bact., 27, 241-256 (1934). 13. ESTY, J. R., and MEYER, K. F. The heat resistance of the spores of B. botulinus and allied anaerobes. XI. J. Infect. Diseases, 31, 650-663 (1922). 14. FOSTER, J. W., and WYNNE, E. S. Physiological studies on spore germination with special reference to Clostridium botulinum. IV. Inhibition of germination by unsaturated CIS fatty acids. J. Bact., 55, 495-501 (1948). 15. FRIEDMAN, C. A., and HENRY, B. S. Bound water content of vegetative and spore forms of bacteria. J. Bact., 36, 99-105 (1938). 16. GREENSTEIN, J. P. Enzymatic degradation of ribosenucleic and desoxyribo- nucleic acids with an addendum on the effect of nucleates on the heat stability of proteins. Fed. Proc., 6, 488-499 (1947). 17. HALVERSEN, W. V., and HAYS, G. L. The thermal death time of Clostridium botulinum spores at temperatures and pH values commonly encountered in home canning. J. Bact., 32, 466-467 (1936). 18. HEADLEE, M. R. Thermal death point. III. Spores of Clostridium welchii. J. Infect. Diseases, 48, 328-329 (1937). 19. HENRY, B. S., and FRIEDMAN, C. A. The water content of bacterial spores. J. Bact., 33, 323-331 (1937). 20. JENSEN, L. B. Microbiology of Meats. 2nd Ed., 1945, Garrard Press, Cham- paign, 111. 21. JOHNSON, F. H., and ZoBELL, C. E. The acceleration of spore disinfection by urethan and its retardation by hydrostatic pressure. J. Bact.. 57, 359-362, (1949). 22. JOHNSON, F. H., and ZoBELL, C. E. The retardation of thermal disinfection of Bacillus subtilis spores by hydrostatic pressure. J. Bact., 57. 353-358 (1949). 23. KNAYSI, G. The endospore of bacteria.'Bact. Rev., 12, 19-77 (1948). 24. LAMANNA, C. Relation of maximum growth temperature to resistance to heat. J. Bact., 44, 29-35 (1942). 25. LAMANNA, C. Symposium on the biology of bacterial spores. I. Biological role of spores. Bact. Rev., 16, 90-93 (1952). 26. LANG, O. W., and DEAN, S. J. Heat resistance of Clostridium botulinum in canned sea foods. J. Infect. Diseases, 55, 39-59 (1934). 27. MAGOON, C. A. Studies upon bacterial spores. I. Thermal resistance affected by age and environment. J. Bact., 11, 253-283 (1926). 28. MEFFERD, R. B., JR., and WYSS, 0. The mutability of Macillus antracis spores during germination. J. Bact., 61, 357-363 (1951). 29. MILITZER, W., SONDEREGGER, T. B., TuTTLE, L. C., and GEORGI, C. E. Thermal enzymes. II. Cytochromes. Arch. Biochem., 26, 299-306 (1950). 30. MITCHELL, P. Physical factors affecting growth and death. In Werkman, C. H., and Wilson, P. W. Bacterial Physiology. 1951, Academic Press, New York. 31. MUNDEL, O., and SCHMID, E. Uber Resistenzanderung von Erdsporen durch thermische Einflusse. Arch. Hyg., 119, 20-25 (1937). 32. MURRELL, W. G., OLSEN, A. M., and SCOTT, W. J. The enumeration of heated bacterial spores. II. Experiments with Bacillus species. Australian J. Sci. Research, Series B., 3, 234-244 (1950). 33. OLSEN, A. M., and SCOTT, W. J. The enumeration of heated bacterial spores. I. Experiments with Clostridium botulinum and other species of Clostridium. Australian J. Sci. Research, Series B., 3, 219-233 (1950). 8

34. POWELL, J. F. The speculation and germination of a strain of Baeillut megatherium. J. Gen. Microbiol, 5, 933-1000 (1951). 35. REYNOLDS, H., and LICHTENSTEIN, H. Effect of certain growth factors on the heat resistance of anaerobic spores. Soc. Amer. Bact. Proc., 28 (1950). 36. REYNOLDS, H., and LICHTENSTEIN, H. Germination of anaerobic spores in- duced by sublethal heating. 49th Gen. Meeting, Soc., Amer. Bacteriologists 9, (1949). 37. ROTH, N. G., and HALVOHSON, H. O. The effect of oxidative rancidity in unsaturated fatty acids on the germination of bacterial spores. J. Bact., 63,429-436 (1952). 38. STEWART, B. T., and HALVORSON, H. O. Heat resistant enzymes from bacterial spores. Abstract, Winter Meeting of Illinois Bact. Soc. (1953). 39. STEWART, B. T., and HALVORSON, H. 0. Studies on the spores of aerobic bac- teria. I. The occurrence of alanine racemase. J. Bact., 65,160-166 (1953). 40. STUMBO, C. R. Thermobacteriology as applied to food processing. Advances in Food Research, 2, 47-115 (1949). 41. STUMBO, C. R., GROSS, C. E., and VINTON, C. Bacteriological studies relating to thermal processing of canned meats. II. Influence of meat-curing agents upon thermal resistance of spores of a putrefactive anaerobic bacterium in meat. Food Research, 10, 283-292 (1945). 42. SUGIYAMA, H. Studies on factors affecting the heat resistance of spores of Clostridium botulinum. J. Bact., 62, 81-96 (1951). 43. SUGIYAMA, H., and DACK, G. M. Thermal resistance of bacterial spores. Proc. Second Conference on Research, American Meat Institute, 44-50 (1950). 44. THEOPHILUS, D. R., and HAMMER, B. W. Influence of growth temperature on the thermal resistance of some bacteria from evaporated milk. Agr. Exp. Sta. Iowa State College, Res. Bull. No. 244 (1938). 45. VINTON, C., MARTIN, S., JR., and GROSS, C. E. Bacteriological studies relating to thermal processing of canned meats. VII. Effect of substrate upon thermal resistance of spores. Food Research, 12, 173-183 (1947). 46. VIRTANEN, A. I. On the enzymes of bacteria and bacterial metabolism. J. Bact., 28, 447-460 (1934). 47. VIRTANEN, A. I., and PULKKI, L. Biochemische untersuchungen uber bakter- iensporen. Arch, fur Mikrobiologie, 4, 99-112 (1933). 48. WILLIAMS, O. B. The heat resistance of bacterial spores. /. Infect. Diseases, 44,421-465 (1929). 49. WILLIAMS, 0. B., and FLEMING, T. C. Subtilin and the spores of Clostridium botulinum. Antibiotics and Chemotheraphy, 2, 75-78 (1952). 50. WILLIAMS, O. B., and HARPER, 0. F., JR. Studies on heat resistance. IV. Sporu- lation of Bacillus cereus in some synthetic media and the heat resistance of the spores produced. J. Bact., 61, 551-556 (1951). 51. WILLIAMS, 0. B., and REED, J. M. The significance of the incubation tempera- ture of recovery cultures in determining spore resistance to heat. J. Infect. Diseases, 71, 225-227 (1942). 52. WILLIAMS, 0. B., and ZIMMERMAN, C. H. Studies on heat resistance. III. The resistance of vegetative cells and spores of the same organism. J. Bact., 61, 63-65 (1951). 53. YESAIR, J., BOHRER, C. W., and CAMERON, E. J. Effect of certain environmental factors on heat resistance of micrococci. Food Research, 11, 327-331 (1946). 54. YESAIR, J., and CAMERON, E. J. Centrifugal fractionation of heat resistance in a spore crop. J. Bact., 31, 2-3 (1936). 55. YESAIR, J., and CAMERON, E. J. Inhibitive effect of curing agents on anaerobic spores. Canner, 94, 89 (1942). 9

CHAIRMAN ROBINSON Thank you, Dr. Sugiyama. The next paper on the microbiologic subjects will be presented by Dr. Williams of the University of Texas. Factors Causing Sporulation and Vegetation of Spoilage Organisms in Canned Meats 0. B. WILLIAMS Applied and basic research have recently been differentiated by Newton (10) on the grounds that basic research is motivated by natural curiosity—the desire for knowledge, whereas applied research has as its incentive a desire for improvement—improvement of a product, a procedure, or an industry. The program of research on bacterial spores here discussed has both basic and applied aspects. Fundamentally, the research is an integral part of a continuing- effort to improve the quality of the preserved meat food items of the ration. Canned meat food products in general heat by conduction and require a long exposure to elevated temperature to destroy the spore contamination. There is a very general belief that physical and chemical changes or deterioration are functions of total time and temperature to which the food is subjected. Presumably, storage life would be extended if less heat could be applied during the pro- cessing. Certainly, if the severity of processing could be reduced, a better grade of raw product could be utilized in place of the tougher, less flavorful, tissue required to withstand the current schedules of processing. One approach to the objective of improved quality of the pre- served product is in the use of other methods of sterilization, or pres- ervation, than the conventional exposure of hermetically-sealed con- tainers to an atmosphere of steam under pressure. Exploratory ex- perimentation in this area is in progress, but it is not certain at the present time how soon, if ever, a substitute for steam sterilization will be both possible and practicable. Another approach lies in basic investigations of the biology, in the broadest sense of the word, of bacterial spores. These are the structures which make severe heat treatment necessary. The success of a canning operation depends upon the destruction, or inactivation, of resistant spores, yet com- paratively little is known about them. The present state of knowledge as to why some bacteria form spores, for example, is about as it was in 1932 when Cook (3) summarized it in the statement ". . . they form spores because they form spores." The situation as to spore germination and sporulation by spore-forming species is comparable. The need for greater knowledge has become more widely recognized in recent years, as is evidenced by the increased volume of published research, and it may be predicted confidently that the jig saw puzzle 10

of spore biology will begin soon to take definite shape. Process deter- mination was strictly an empirical operation for a hundred years following Appert's discovery. It is still empirical so far as a knowl- edge of spore biology is concerned, and it will remain empirical until we fill in many of the gaps and expand our understanding of these critical cellular structures. We need to know why some bacteria, but not others, form spores; what conditions favor and what hinder spore formation; what promotes and what interferes with spore germination and subsequent growth; what is concerned in spore resistance to various adverse environmental conditions; what is the distribution of significant spore formers; what may be expected in the way of numbers in food for processing; and many other items of varying degrees of importance. The development of this knowl- edge is basic research—basic research directed toward an objective of application. The section of this general field of research with which I have been most closely associated has been concerned chiefly with the for- mation and the germination of spores. Most of the work has been done with fabricated media because of the technical difficulty of fol- lowing changes quantitatively when working with a product. Fur- thermore, it is possible to control the composition of the substrate more accurately with a fabricated medium than with the natural product, and the data which have been developed by ourselves and others emphasize the importance of substrate composition for repro- ducible results. It is not possible to discuss in a short time, even in a cursory way, all of the work which has been accomplished. Some of the results have been published, and do not deserve more than men- tion ; other work is still in the preliminary or developmental stage and cannot be discussed extensively. Spore germination. When the current program of spore research was initiated little was known about the factors operative in spore formation, and practically nothing, beyond the morphological changes, about spore germination. The first undertaking was the development of a procedure for the study of spore germination; this was achieved by Wynne (20) for Clostridium botulinum. He showed for this very important organism that germination follows a course which is essentially logarithmic. Thus, the greater the initial number of spores present in a favorable environment, the longer will be the time necessary for germination to be complete. This observation needs to be extended to other spore-bearing organisms of significance to the food industry, and an effort made to explain the orderly nature of the process. Through the use of the standard procedure developed it was found that there are species differences in the requirements for germination. Clostridium botulinum requires carbon dioxide for germination in a chemically defined medium, or a by-pass for carbon dioxide in complex media, whereas several other species of anaerobes and aerobes did not show the carbon dioxide effect. A further species difference in spore germination was shown in the effect of trace amounts of C18 unsaturated fatty acids, which interfered sharply with Clostridium botulinum, but to a much lesser extent with the well-known strain P. A. 3679, and not for all aerobic types tested. This observation has been extended by Roth and 11

Halvorson (75) who showed that the inhibitory effect of fatty acids is associated with rancidity, not with the fatty acid per se. The concept of a toxic factor (s) in medium as inhibitory to spore formation originated in the work of Roberts and Baldwin (13) who, in 1942, recorded observations made some years earlier which showed that media prepared with certain peptones could be increased in spore productivity by treatment with adsorbing agents, especially charcoal. Starch has long been used to increase the value of media for the growth of certain non-spore-formers. Heated spores are generally thought to be somewhat more exacting in requirements for growth than unheated spores of the same lot. In 1946, Olsen and Scott (11) reported that the number of colonies developing from heated spores was significantly increased by incorporation of small amounts of starch in the recovery medium, suggesting that at least one factor concerned in the germination of heated spores was an adsorbable substance, more effective for heated than for unheated spores. We have confirmed many times the value of starch in recovery media for heated spores of both mesophilic and thermophilic aerobic forms. Again there seems to be a species difference since Hays (5) has noted beneficial results from charcoal, but not for starch, with strain P. A. 3679. The incorporation of starch in each of several media tested for spore produc- tivity with Clostridium botulinum did not increase the spore yield. Whether this means that these media did not contain inhibitory substances is uncertain. There may be a difference in sporulation response to starch among strains of the same organisms, as is suggested- by results with Bacillus stearothermophilus, strains of which responded unequally to the presence of starch in the medium. It is, however, certain that not only growth but spore formation and spore germination for some types is sharply affected by adsorbable substances in the medium. Hardwick, Guirard, and Foster (4) associated the antisporulation factor with certain saturated fatty acids. It may be that here also rancidity of the acid is the determining factor. This idea would appear to be supported by results which show the superiority of media prepared from fresh ingredients over dehydrated media, or possibly also extract media. We have recently noted a marked difference in productivity, i.e., germination and subsequent growth, from heated spores for brain-heart-infusion medium prepared from fresh tissue over the dehydrated product. Numerous workers have studied spore formation from vegetative cells and spore germination in the presence of one or more nutrient materials under con- ditions which would not support continued growth. An interesting observation regarding germination was made by Hills (7) in the demonstration of the significance of certain specific nutrients, especially L-alanine, for the process. We have been able to confirm this observation for several species, and to extend it by demonstrating that spores produced in a chemically defined medium in general show an accelerated germination when compared with those produced in a complex medium. This observation has not been fully explored so that the constituent (s) of the medium significant in the production of spores susceptible to specific stimulatory effects in germination could be defined. A comparison by the technique of Hills of spores of strains of the same species of unequal resistance to heat suggests that the more resistant spores have a reduced germination rate. A phenomenon associated with spore germination which has been very intriguing is that of heat activation. Numerous workers have observed that spore suspensions exposed to nonlethal temperature for a period of time gave appreciably higher counts than before heating. Recently, activation of bacterial spores by a chemical agent has been noted (9). A considerable portion of our efforts for the past year has been devoted to an attempt to find out something about this phenomenon. The results to date can be summarized very briefly. Spore suspensions have been prepared in the usual way, washed and freed of vegetative cells by exposure to high frequency sonic vibration. It has been 12

established that suspensions exposed to sound waves will gradually decrease to a constant count which is not altered by as much as 90 minutes exposure. Sonic vibration has not been observed to activate the spores. A considerable number of species, and strains of some species, have been tested under a variety of conditions. Many negative data have been obtained. Contrary to expectations, activation was irregular and inconstant. Appreciable activation was finally noted with a strain of Bacillus subtilis obtained from Dr. Harold Curran when it was exposed in commercial peptone solution, acid hydro- lyzed casein solution, and especially in skim milk. No activation was obtained with other suspending media. Experiments are now actively in progress to establish the characteristics of the phenomenon and to determine some of the factors concerned. Results now in hand indicate that a fivefold increase in count is obtained by 10 minutes exposure at from 80° to 95" C., and activation to some extent at a temperature as low as 65° C., within 10 minutes. Maximum effects are observed for both undiluted and 50% skim milk, with a gradual decline in effect as the concentration of milk is lowered, some effect still being apparent at the 5% level. We have a backlog of experience with negative results which is expediting the experiments that do yield positive results. Spore formation. One of the ideas held by many bacteriologists who are inexperienced in the field, I am sure, is that most of the vegetative cells of a spore-forming species enter the spore state. This idea will be quickly dispelled if one makes spore percentage counts over a period of time. Some species are extremely reluctant to form spores, and even some strains of a species are poor spore formers, whereas other strains, incubated in parallel, regularly produce good spore crops. Percentage counts after 12 days incubation of 5 complex media gave a spread of from 25% to 49% for the well-known strain 62A of Clostridium botulinum. Repeated testing with this strain indicates that a yield as high as 40% spores among the countable cells is exceptional. The studies are complicated by the autolysis of many of the vegetative cells so that counts after 5-7 days are inaccurate. A comparison of 50 strains of Bacillus stearothermophilus after B days incubation in a basamin-mineral salts medium gave a spread of from 1% to 95% spores. One explanation of unequal spore yield possibly is to be found in unequal response among species and strains to antisporulation substances in the medium, but this is certainly not the sole explanation. It would be very enlightening if the trigger mechanism which sets off the sporulation process could de defined. Recently Charney, Fisher, and Hegarty (2) have reported the stimulatory effect of manganese on sporulation by several species of the genus Bacillus. Upon examination of some of our old data it was found that a similar observation had been made in 1947 by Ward (16) for Bacillus coagulans, one of the poor sporulating species. Recently Laskin (8) has confirmed the observation for Clostridium nigrificans, another poor sporulat- ing type. How general this observation may be, and the significance of other trace minerals, has not yet been established. Synthetic media, with the close control, both qualitatively and quantitatively, over the constituents, offer an excellent approach to the study of the effect of a specific material on almost any cell activity. As early as 1926 (17) I attempted to produce spores of a strain of Bacillus snbtilis in synthetic media for com- parative heat resistance determinations, but without success. Good growth could be secured, but spores could not be detected in any of a number of chemically defined nutrient solutions. Roberts (12) was successful in developing a synthetic medium in which this same strain sporulated, and Heiligman (6) formulated a modification suitable for Bacillus ccreus. A synthetic medium for the cultivation of Clostridium botulinum was developed by Roessler and Brewer (14) but this medium is not productive of spores. Blair (1) assumed the task of investigating spore production by Clostridium botulinum in synthetic media, starting with the nutrient solution of Roessler and Brewer. An increase in spore yield was 13

obtained following the addition of any one of several substances, greatest with "-alanine, glutamic acid or ornithine, and to a lesser degree with glycine, sodium butyrate, sodium valerate, arginine or proline. No effect was noted for serine, norleucine, lysine, glutamine, choline, creatine or sodium glyceraldehyde phosphate. Glucose over the range of 0.4% to 1% gave a heavier growth but reduced spore yield. A reduction in spore yield was also recorded for phenylalanine and tyrosine. No correlation between structure and effect was apparent from these preliminary experiments. A more extensive study of the effect of various substances on sporulation in simple media by this organism is now in progress. Attempts to produce spores, both in complex media and in synthetic media, have not been successful with Clostridium thermosaccharolyticum. This organism has potential spoilage significance for meat food products especially those con- taining cereal. It can be cultivated in synthetic medium, and this fact considered with its meagre sporulation capacity, offers a very fertile field for a study of the effect of both nutritive and environmental factors on sporogenesis since any perceptible number of spores will represent an increase over what is customary. Extensive studies, however, have been consistently negative. No stimulation has been observed for any one of the 18 common acids, nor for any one of the growth factors—thiamine, nicotinamide, pantothenate, pyridoxin, biotin or paraamino- benzoic acid. The work with synthetic media emphasizes what has long been observed; namely, that good growth without sporulation may be obtained. Incidentally, it may be noted that the heat resistance of spores of Bacillus cereus produced in synthetic media was independent of the luxuriance of both growth and sporulation, and was not conditioned by any one of a number of amino acids, or growth factors. Valine was required for growth. Lack of paraaminobenzoic acid resulted in a sharp drop in the percentage of spores (20). Water and spore activity. One series of experiments which may be mentioned briefly is that concerned with the water content of the substrate and spore germination, growth, and spore formation. A report on this phase of the research recently appeared in Food Re- search (19). A great deal of unexpected trouble was encountered in these experiments. The procedure consisted of grinding liver tissue very fine, drying and powdering in a ball mill. The liver powder was sterilized with ethylene oxide, moisture determinations were made in the usual way on a sample, and the desired moisture content was ob- tained by adding the appropriate volume of Clostridium botulinum spore suspension. The mixture was filled into small tubes, a strip of detinned base plate added to rust out residual oxygen, and the tubes sealed in the blast lamp. Observations for spore germination, growth, and spore formation were made in the usual manner. The first ex- periment gave excellent results. Succeeding experiments were vari- able. Rarely, a satisfactory series of results would be recorded. More commonly there would be no growth, or good growth in one tube and no growth in a replicate, etc. Eventually it was found that some tubes of liver powder had a pH unfavorable to Clostridium botulinum. When the powder was rehydrated in future experiments, provision to adjust and control the pH eliminated the erratic results except in tubes near the limiting moisture concentrations. One additional point which deserves mention is the method of determining the moisture activity of the various moisture levels 14

tested. It is generally recognized that the governing factor is not the absolute amount of moisture present in a system, but the moisture activity. In our experiment, moisture activity was determined by taking advantage of the relative humidity of closed systems con- taining saturated solutions of various salts selected to give a range of values. Such a system is self-adjusting so long as the salt solution remains saturated, and is independent of the volume of gas, liquid or solid. Our data indicate that at moisture concentrations below 40%, growth and sporulation are unlikely, although spore germination seemingly can take place. Literature Cited 1. BLAIR, E. B. Bact. Proc., 62 (1950). 2. CHARNEY, JESSE, FISHER, W. P., and HEGARTY, C. P. J. Bact., 62, 145 (1951). 3. COOK, R. P. Biological Rev., 7, 1 (1932). 4. HARDWICKE, W. A., GUIRARD, BEVERLY, and FOSTER, J. W. J. Bact., 61, 145 (1951). 5. HAYS, G. L. (Personal communication). 6. HEILIGMAN, F. (Thesis for M.A. degree, Univ. of Texas, 1949). 7. HILLS, G. M. Biochem., 45, 363 (1949). 8. LASKIN, A. I. (Thesis for M.A. degree, Univ. of Texas, 1952). 9. MEFFERD, R. B., JR., and CAMPBELL, L. L., JR. J. Bact., 62, 130 (1951). 10. NEWTON, R. C. National Canners Association, Information Letter 1426, 40 (1953). 11. OLSEN, A. M., and SCOTT, W. J. Nature, 157, 337 (1946). 12. ROBERTS, J. L. Science, 79, 432 (1934). 13. ROBERTS, J. L., and BALDWIN, I. L. J. Bact., 44, 653 (1942). 14. ROESSLER, W. G., and BREWER, C. R. (Personal communication). 15. ROTH, N. G., and HALVORSON, H. 0. J. Bact., 63, 429 (1952). 16. WARD, B. Q. (Thesis for M.A. degree, Univ. of Texas, 1947). 17. WILLIAMS, O. B. J. Infect. Diseases, 44, 421 (1929). 18. WILLIAMS, 0. B., and HARPER, 0. F., JR. J. Bact., 61, 551 (1951). 19. WILLIAMS, O. B., and PURNELL, H. G. Food Research, 18, 35 (1953). 20. WYNNE, E. S. (Doctoral dissertation, Univ. of Texas, 1948). CHAIRMAN ROBINSON Thank you, Dr. Williams. We have 2 more papers this morning, and if we have a few min- utes, we will have a short discussion of some of the information given. Dr. Ayres of Iowa State College is our next speaker. Microorganisms Associated with the Spoilage of Thermal Processed Meats JOHN C. AYRES The microorganisms which may ultimately contaminate and even decompose canned meat are disseminated in and on these products by diverse pathways. For example, they may develop directly in the blood vessels, be transferred to the carcass from the water in the 15

scald tank or during the washing and rinsing operations, come from the air, or be contributed by the hands and tools of workmen during eviscerating and processing operations. The hide and hair of meat animals provide excellent lodging places for many types of microorganisms. For example, Jensen and Hess (19) indicated the presence of from 100,000 to 1,500,000,000 aerobes and from 10,000 to 2,000,000,000 aerobes on 2 square inches of neck skin of unwashed hogs at the place where the animal's jugular vein generally is stuck. Also, the intestinal tract contains billions of organisms, some of which may penetrate the intestinal wall and be carried to the various parts of the body by the blood. Fortunately, proper handling of the animal during slaughter and dressing mini- mizes chances for the flora from the hide or viscera of the slaughtered animal coming in contact with the dressed meat. It is difficult to achieve complete freedom from such contamination and, therefore, it is not strange that the organisms associated with the animal's en- vironment and those found in and on meats are the same—or closely related—species. Various explanations have been offered for the small bacterial load on the surface of the intact carcass. One opinion held among workers in the meat industry seems to be that some reduction in humidity facilitates preservation of all types of meat. However, there is evidence in the literature (33, 36) which indicates that the influence of relative humidity has little effect in delaying microbial growth on meat surfaces. Certainly, in a dry atmosphere the diffusion of water from the interior of meat helps to maintain a higher moisture content near the surface than is indicated by the relative humidity of the surrounding air. Haines (13) offered a possible explanation for the growth on uncut surfaces being limited, namely, that the carcass is covered by a layer of fat and connective tissue and has poor nutrient qualities for most organisms. Also, many of the organ- isms coming in contact with the meat are mesophiles which grow poorly at low temperatures and, when the animal is chilled, die before conditions in and on the meat are again favorable for their growth. On the other hand, the cut flesh is subject to marked increase in numbers even though stored at refrigeration temperatures. Juices released from the intact cell provide excellent nutrients for the growth of many types of microorganisms. When meats are comminuted, bacteria are afforded an excellent opportunity to develop because grinding not only releases juices but distributes the organisms and provides more aerobic conditions as well as presenting them with a larger area of surface from which to obtain nutrients. Numbers of organisms associated with fresh meats. Samplings made from the native surfaces of 37 matched sets of knuckle, inside round and outside round of cutter and canner grade beef (2) sent to the Iowa State College laboratory from a Chicago meat packing plant indicated that aerobic loads usually ranged from 10,000 to 1,000,000 bacteria per sq. cm. or from 100,000 to 10,000,000 per 16

gram. For areas that had been sliced at the packing plant just before the meat was shipped, microbial populations ordinarily varied from 10,000 to 1,000,000 per sq. cm. or per gram. It should be pointed out that the surface flora on the native areas was permitted to develop from the time the animal was skinned, while organisms on sliced portions may not have been introduced until after the quarter was dissected. The number of aerobes differed considerably among samplings. For example, 2 surface tests indicated the presence of less than 100 bacteria per sq. cm. while 2 others had 10 million. Since about as many organisms were recovered by swabbing the surface as by mincing a gram of meat with an equivalent amount of surface, the results might be said to confirm Moran's (28) statement that spoilage in stored, unfrozen beef is primarily a surface phen- omenon. Types of bacteria found in tissues of slaughtered animals. A large number of investigators (Ayres et al, 3; Brooks and Hansford, 6; Empey and Scott, 8; Empey and Vickery, 9; Haines and Scott, 14; Jensen, 17, 18; Jensen and Hess, 19; Kirsch et al. 22; Klein, 23; McBryde, 26; Niven, 29; Ogilvy and Ayres, 34; Yesair, 49) have studied the taxonomic distribution of microorganisms isolated from carcasses of apparently healthy animals. Members of the following genera have been identified: Bacteria: Pseudomonas, Xanthomonas, Azotobacter type, Micrococcus, Gaffkya, Sarcina, Neisseria, Diplococcus, Streptococcus, Leuconostoc, Lactobacillus, Eubacterium, Microbacterium, Alcaligenes, Achromobacter, Flavobacterium, Escherichia, Aerobacter, Paracolobactrum, Serratia, Proteus, Salmonella, Bacteroides, Ristella, Hemophilus, Bacterium, Bacil- lus, Clostridium, Streptomyces, Actinomyces; Molds: Zygorhynchus, Mucor, Thamnidium, Rhizopus, Penicillium, As- pergillus, Sporotrichum, Cladosporium, Alternaria, Geotrichum (Oidium, Oospora, Geotrichoides), Monascus; Yeast-like fungi and yeasts: Candida (Monilia, Blastodendrion, Myco- torula), Torulopsis (Cryptococcus, Torula), Rhodotorula, Debaryomyces (Wardomyces), and Sacchoromyces. Kinds of organisms associated with meat spoilage. Many of the organisms that have been isolated from inspected meats are quite fastidious in their tem- perature requirements. On the other hand, some of the bacteria, yeasts, and molds listed do not die if the meat remains unfrozen. Empey and Scott (8) found that less than 1% of the microbial population growing on the surfaces of beef at 20° C. were viable at —1° C. and, although bacteria represented 97% of the contamination acquired by beef surfaces at the higher temperature, yeast and molds made up a greater share of the population at — 1° C. The 4 principal genera of low temperature bacteria comprising the initial flora were said to be: Achromo- bacter, 90%; Micrococcus, 7%; Flavobacterium, 3%; and Pseudomonas, less than 1%. In an earlier study, Empey and Vickery (9) observed that 95% of the initial flora of beef capable of growth at — 1°C. consisted of members of the genus Achromobacter; the remainder were species of Pseudomonas and Micrococcus. During storage the relative numbers of Achromobacter and Pseudomonas increased while those of Micrococcus decreased. In the case of uncured animal tissues, a number of workers (Ayres, 1; Empey and Scott, 8; Empey and Vickery, 9; Haines and Scott, 14', Jensen, 17; Moran, 28; Scott, 36) have indicated that the formation of surface slime on meat is primarily due to organisms of the Achromobacter and Pseudomonas types. As a result of changes in classification made in the more recent editions of Bergey's Manual (5) a number of types previously isolated from slimy beef by Haines (13), Empey and Vickery (9), and Empey and Scott (8) no doubt would be considered as members of the genus Pseudomonas in the present schema since the organisms in question were reported to have monotrichous ftagella. Recently Kirsch et al. (22) advanced this same opinion. 17

In an examination of colonies isolated from frozen ground beef trimmings (1), 78% of the flora were found to be comprised of Achromobacter and Pseudo- monas organisms while they represented but 18% of the isolates from freshly ground beef. At the time of spoilage these 2 genera accounted for 98% of the flora from both meats. Sulzbacher and McLean (42) studied the distribution of bacteria found in fresh pork sausage and found that 75% of the organisms isolated were members of the genera Pseudomonas, Microbacterium, Alcaligenes, Achromobacter, Bac- terium, and Bacillus. They observed that species of Microbacterium made up a rather large proportion of the flora and, consequently, associated these organisms with the deterioration of sausage during storage. They considered the organisms similar to Microbacterium lacticum except that they were non-heat-resistant, surviving heating to 63 °C. for 3 minutes but not for 5 minutes. Also, Sulzbacher and McLean recovered 28 cultures of Bacillus and 4 of Xanthomonas; the spice was believed to contribute most of these. Members of the genus Lactobacillus were recently reported by Kirsch et al. (22) to be frequent contaminants in refrigerated hamburger. The joint fluid and bone marrow as well as the flesh of hams from dressed hog carcasses seldom were sterile as early as 45 minutes after slaughter. Boyer (4) isolated both aerobic and anaerobic bacteria; among the spore-forming anaerobes which he identified were Bacillus putrefaciens (Clostridium putre- faciens), Bacillus histolyticus (Clostridium histolyticum), Bacillus sporogenes, (Clostridium sporogenes), Bacillus tertius (Clostridium tertium), and an un- identified organism resembling Bacillus oedematicus (Clostridium novyi). The last-named organism is probably the same as that described by Haines and Scott (14) which they associated with bone taint of beef. Workers at Armour & Com- pany (15) used McClung and Toabe's egg-yolk suspension to classify tentatively a number of organisms that they had isolated from freshly ground pork trimmings. According to the reactions obtained, the organisms were considered to be: Clos- tridium perfringens, Clostridium bifermentans, Clostridium novyi A and B, Clostridium sporogenes, and Clostridium putrefaciens. Steinkraus and Ayres (57) used temperature and oxygen relationships and the ability to grow in the presence of 1-200,000 crystal violet as a screening test for 120 cultures of putre- factive spore-forming organisms found in pork. Of the 21 cultures selected as obligate anaerobes, species tentatively identified by biochemical reactions were considered to be most nearly related to: Clostridium tetanomorphum, Clostridium novyi, Clostridium carnis, Clostridium paraputrificum, Clostridium tetani, Clos- tridium histolyticum, Clostridium sporogenes, and the well-known spoilage organism P. A. 3679. The last-named organism was culturally and serologically similar to putrefactive anaerobes previously isolated by Gross, Vinton, and Stumbo (12) as the causative agents in spoilage of canned meats. Information concerning the microorganisms causing spoilage of cured meats has been advanced by many investigators. With these products not only the flora found in and on the meat but also that introduced with curing salts, sugar, and spices contribute to the total contamination. Sturges and Heideman (41) isolated 101 bacterial cultures from curing solution brines. They encountered difficulty when they attempted to classify these organisms according to Bergey's Manual (1st ed.) and concluded that classification utilizing the organism's salt relations was less confusing. Heideman (16) studied 5 organisms which produced ropiness in curing solutions. He considered the presence of carbohydrate and reaction of the medium of vital importance for the production of a ropy curing solution. There is general agreement among workers regarding the effect of sodium chloride in checking putrefactive anaerobic spoilage, but various investigators (Niven, 29; Niven et al., 3O; Norton and Roderick, 31; Ogilvy and Ayres, 34) have found micrococci and lactobacilli able to tolerate rather high salt concentra- tions. Differing opinions have been advanced regarding the preservative action of sodium nitrate and sodium nitrite (7, 20, 38, 39, 43, 44, 45, 46, 50). Most of the studies that have been made tested the effect of these agents in preventing 18

spoilage by putrefactive anaerobes. However, Tarr (47) tested sodium nitrite using a broader bacterial spectrum and found that the growth of the following genera at pH 5.7-6.0 was either inhibited or prevented by 0.02% NaNO2: Achro- mobacter, Flavobacterium, Pseudomonas, Micrococcus, Escherichia, Aerobacter and one species of Torula. Also, Tarr found that, in this pH range, sodium nitrite inhibited Clostridium botulinum, Clostridium sporogenes, Eberthella typhosa (Salmonella typhosa), and Staphylococcus aureus (Micrococcus pyogenes var. aureus). Discoloration, wherein oxidation pigments were formed, was associated by Greenwood et al. (10), with action of excess nitrite, oxygen and microorganisms upon cured meat pigments. According to Jensen (17) the typical gaseous swell of canned cured meat is generally due to the fermentation of sugar by species of the genus Bacillus. Earlier, Jensen et al. (21) found that only when nitrate, sugar, and cured meat were present together would the bacilli ferment the sugar. Bulman and Ayres (7) found that bacilli, forming spores and capable of growing aerobically, were responsible for gas production and discoloration of pork trim- mings containing nitrate levels as high as 4.7%. These organisms reduced nitrate to nitrite and were able to tolerate 10% NaNO3. Evidence which indicates that certain types of Lactobacillus and Leuconostoc species cause surface and internal discolorations of cured meats has been presented by Niven et al. (3O). Norton and Roderick (3I) believed the micrococci were the organisms re- sponsible for slime on sausage. In a study in this laboratory (34) not only micro- cocci but bacilli and sacrinae were numerous in cured meats and lactobacilli and gram-negative bacteria were also present in somewhat smaller numbers. Some- times sizable populations of yeasts as well were encountered. None of the types of micrococci could be identified definitely with any of the species of Micrococcus described in Sergey's Manual (5). The organisms differed from the micrococci characterized by Norton and Roderick in that they liquefied gelatin and many peptonized milk. Some 30% of the cultures were found to be similar in many respects to Micrococcus caseolyticus. The bacilli were frequently found on the surface and always in the interior of fresh frankfurters. The term lactobacillus, as used, was employed rather loosely to refer to organisms which may include several members of the family Lactobacteriaceae. It is possible that some of the organisms isolated may belong to the genus Leuconostoc, although this was not indicated by morphological characteristics. Some cultures grew fairly well aerobically and these, perhaps, would fall into the genus Microbacterium. Kraft (24) found some of the lactobacilli cultures to be catalase-positive. Ham souring, bone stink, or bone taint are terms used in the industry to in- dicate a putrefactive spoilage of particular importance in large thick pieces such as the hindquarters of pork and beef. In 1908, Klein reported an anaerobic bacil- lus from "miscured hams" which he called Bacillus foedans (Eubacterium foedans). Three years later, McBryde (26) reported Bacillus putrefaciens (Clostridium putrefaciens) to be the etiologic agent involved. Tucker implicated Clostridium putrificum (Clostridium lentoputrescens) and Moran (27) showed that Clostridium sporogenes could cause ham souring. Jensen and Hess (19) catalogued various types of ham sours and asserted that salt-tolerant bacteria which grow at 0° to 3.3°C. in bone marrow can cause any kind of sour. They named these bacteria: Achromobacter, Bacillus, Pseudomonas, Proteus group, Serratia, Clostridium, micrococci, streptobacilli, and a miscellaneous group. Thermal resistance characteristics of typical spoilage organisms. Although many different groups of bacteria have been found to grow in abundance in meat, their presence in products that are to be heat processed is of less significance if they are not thermoduric. For ex- ample, bacteria belonging to the genera Achromobacter and Pseudo- monas, although they have been found to grow to enormous numbers 19

on chilled meats, failed to survive when incubated at temperatures exceeding 45°C. Jensen (18) writes: "We have never isolated the Coliform group of bacteria, Serratin, Achromobacter, Pseudomonas, Flavobacteria, Chromobacteria, Lactobacilli, Proteus, and the mis- cellany of nonsporing rods from canned cured meats which have received a light process." On the other hand, Niven (29) isolated some lactobacilli from cores of sausage which were moderately resistant to heat, surviving temperatures of 65.5°C. for 2 hours and 71°C. for 8 minutes. In the study of flora in and on beef round referred to earlier in this paper, a surprisingly large number of bacteria survived a 20- minute heating period at 80 °C. and still were capable of reproducing when grown under atmospheric conditions. The number of heat- resistant cells or spores growing aerobically differed widely among samples. In some cases there were less than 10 organisms per gram while in others there were more than 100,000. It would not be advisable to consider that all of the surviving organisms were spores. It is quite possible that heat-tolerant micrococci, Microbacterium, Lactobacilli, actinomycetes, and' other vegetative cells can withstand 80°C. for 20 minutes in a meat substrate. Ruyle and Tanner (35) observed that cocci have often been found in canned meats which were given processes considered to be adequate. Verbal reports from rep- resentatives of at least 2 of the commercial meat packing plants (11) and unpublished findings of work conducted at the Iowa State College laboratories indicate the presence of heat-tolerant aerobes and, in particular, spore-forming bacteria—both in fresh pork trimmings and in products receiving mild processes. In the past, most spoilage of canned meat has been considered to be caused by the putrefactive anaerobic bacteria. Also, processing schedules advocated for sterilizing the canned product are calculated to destroy large number of Clostridium botulinum, spores as well as to prevent spoilage by huge populations of extremely heat-resistant putrefactive spores. It is interesting, then, that none of the labora- tories which have sampled the raw meat products have encountered any of the Clostridium botulinum organisms. Jensen (18) states: "During 2 decades of bacteriological control of canned meats, neither we nor any of our colleagues in this work have ever isolated a spon- taneously occurring strain of Clostridium botulinum from canned meats." Insofar as the other mesophilic anaerobic spores are concerned, only limited numbers of these organisms have been encountered. In an unpublished survey, one group of workers took samples of freshly ground trimmings from 99 production days over a period of 9 months and found the maximum number of spores to be 42 spores per gram while the average was 1.5. In this laboratory 80 separate samplings of fresh pork trimmings were obtained from 4 packing plants in Iowa. Less than 3 putrefactive 20

TABLE 1 INCIDENCE OF TOTAL VIABLE ORGANISMS AND OF SPORES GROWING AEROBICALLY OR ANAEROBICALLY IN PACKAGED RAW BEEF Kind of Growth Unit Measured Surface Examined Number of Organisms x 100 Smallest Usual Range Largest Total Aerobic no./sq. cm. uncut cut 1 <1 100 - 10,000 100 - 10,000 200,000 33,000 no./g. uncut cut 50 1,000 - 10,000 100 - 10,000 4,000,000 680,000 75 Total Anaerobic no./sq. cm. uncut cut 1 <1 10 - 1,000 10 - 1,000 40,000 1,000 no./g. uncut cut 1 4 1,000 - 10,000 1,000 - 10,000 1,600,000 700,000 no./sq. cm. uncut cut <1 10 - 1,000 10 - 1,000 24,000 1,700 Spores Growing Aerobic <1 no./g. uncut cut <1 <1 10 - 1,000 10 - 1,000 140,000 19,000 Spores Growing Anaerobic no./l00 g. <0.6 0.7 - 6.0 140 anaerobic spores per gram were recovered from 22% of the samples. Only 3 lots of trimmings had more than 8 spores per gram; the max- imum spore count in any sample tested was 51. Ten samples of fresh beef from one packing plant contained an average of 6.5 spores per gram after the meat had been comminuted. Also, of 111 samplings from the 37 matched sets of knuckle, inside round, and out- side round of cutter and canner grade beef, none of the putrefactive anaerobic spore counts exceeded 1.4 spores per gram (see Table 1). TABLE 2 RETORT TEMPERATURE—PROCESSING TIME SCHEDULE Retort Temperature (°F.) Time in minutes at: Row 225° 243° 261° 279° 297° 315" 1 10 8 7 5 3 2 2 20 17 14 10 7 4 3 30 25 21 16 11 6 4 40 34 27 21 14 8 5 50 42 34 26 18 10 6 60 50 41 31 21 12 7 70 59 48 36 25 14 8 80 67 54 42 29 16 9 90 76 61 47 32 18 10 100 84 68 52 36 20 11 110 93 75 57 40 22 12 120 101 82 62 43 24 21

Co W 01 Z 4 O oo o Co K o o CO B: £ b U U oa t. o M Z o O DH U O n <; « S s o o O O O ffl S tn Z o PS O BS £ S H j g as CQ « o w tO CM 1C H CO <J i-H '* CM PQ 3 CO CO t- 05 IM .< '* Co t- rH rH PQ tO to IM S to o OS rH rH t- IM * CO "^ 0 0 rH rH PQ CO (M 3 r- 1 to CSI « rH rH z a « o la ^1" CO IM « § CO N M § g 0 0 rH CO kg « 0 0 O O ** IM CJ ° o "£ ^ lo ^ ^ ^ 5 o o is o ^ is i— ( w S '§ jj * « S ^ S 2 il II rH ** Hg* _j !» <! M 11 w i n HH 1—1 ^ 1 « 1 i a No spores survived processing times longer than: 1 o o g g g S ~£ T3 "tjj^ V ^ -2 rS .S S '3 c M C '£ W bo M « t* h * 0 M Si H r-1 O ffl I "a QJ " II 22

Rectangular chunks of meat were forced into 300 x 308 tin cans and, after subjecting the canned product to the several retort tem- peratures shown in Table 2, bacteriological tests were made of core and peripheral portions of the processed meat. The thermal pro- cesses which were required to destroy all spores from 100 g. of beef or to reduce total counts of organisms growing aerobically or anaero- bically to less than one organism in 10 g. of meat are shown in Table 3. No spores were recovered either from aerobic or anaerobic cultures from beef that had received processes exceeding those recorded in the 4th or 5th row of retort time designations shown in Table 2. Also, cultures which survived the 20-minute heating period at 80°C. and subsequently grew under anaerobic conditions were not subcultured from meat that had been processed for times exceeding those shown in the 3rd row of process-time designations. On the other hand, total aerobic growth persisted in many cans of processed beef from which anaerobic spores could no longer be isolated. It is probable that these cans contained no heat-resistant spores. In view of the low numbers of putrefactive anaerobic spores recovered in raw beef, these findings are in agreement. It is not known whether this same condition prevails in the industry but, if it does, it would appear to indicate that it is possible to successfully utilize a thermal process known to be inadequate to destroy spores of some of the more heat- resistant anaerobes. It is significant that viable organisms were found in cans from which no anaerobic spores were recovered. Work in this laboratory and elsewhere indicates that members of the genus Bacillus are com- monly found in cured perishable canned meat products. Their role in canned beef is not known and has received insufficient study. Literature Cited 1. AYRES, J. C. Some bacteriological aspects of spoilage of self-service meats. Iowa State Coll., J. Sci., 26, 31-48 (1951). 2. AYRES, J. C., and ADAMS, A. T. Occurrence and nature of bacteria in canned beef. Food Technol., 7, 318-323 (1953). 3. AYRES, J. C., OGILVY, W. S., and STEWART, G. F. Post-mortem changes in stored meat. I. Microorganisms associated with development of slime on eviscerated cut-up poultry. Food Technol., 4, 199-205 (1950). 4. BOYER, E. S. A contribution to the bacteriological study of ham souring. J. Agr. Res., 33, 761-768 (1926). 5. BREED, R. S., MURRAY, E. G. D., KITCHENS, A. P., et al. Bergey's Manual of Determinative Bacteriology. 6th ed., (1948). The William and Wilkins Com- pany, Baltimore, Md. 6. BROOKS, F. T., and HANSFORD, C. G. Mould growths upon cold-store meat. Trans. Brit. Mycol. Soc., (London) ser. B. 107, 248-69 (1923). 7. BULMAN, C., and AYRES, J. C. Preservative effect of various concentrations of curing salts in comminuted pork. Food Technol., 6, 255-259 (1952). 8. EMPEY, W. A., and SCOTT, W. J. Investigation on chilled beef. Part I. Microbial contamination acquired in the meatworks. Australian Coun. Sci. Ind. Res. Bui. 126 (1939). 23

9. EMPEY, W. A., and VICKERY, J. R. The use of carbon dioxide in the storage of chilled beef. Australian J. Coun. Sci. Ind. Res., 6, 233-43 (1933). 10. GREENWOOD, D. A., URBAIN, W. M., JENSEN, L. B., and LEWIS, W. L. The heme pigments of cured meats. IV. Role of sugars in color of cured meats. Food Research, 5, 625-35 (1940). 11. GROSS, C. E., and OGILVY, W. S. Study of incidence of spoilage organisms in canned meat products manufactured under commercial conditions. Sym- posium on Canned Meats, Chicago, March, 1953 (Unpublished). 12. GROSS, C. E., VINTON, C., and STUMBO, C. R. Bacteriological studies relating to thermal processing of canned meats. V. Characteristics of putre- factive anaerobe used in thermal resistance studies. Food Research, 11, 405- 410 (1946). 13. HAINES, R. B. The bacterial flora developing on stored lean meat, especially with regard to "slimy" meat. J. Hyg. (Gr. Brit.) 33, 175-82 (1933). 14. HAINES, R. B., and SCOTT, W. J. Anaerobic organism associated with "bone taint" in beef. J. Hyg. (Gr. Brit.) 40, 154-161 (1940). 15. HARRIMAN, L. A., DELGIUDICE, V. J., SHINN, B. M., and HANSEN, R. The incidence of putrefactive anaerobes in pork. Paper delivered to the Society of Illinois Bacteriologists' fall meeting, Peoria, Illinois, Oct. 15, 1948. (Ab- stract of paper with meeting notice). 16. HEIDEMAN, A. G. 42. Abnormalities of meat-curing solutions. Viscid Brine: so-called "Ropy Pickle." Preliminary Report. Abs. Bact., 8, 14 (1924). 17. JENSEN, L. B. Microbiological problems in the preservation of meats. Bact. Revs., 8, 161-188 (1944). 18. JENSEN, L. B. Microbiology of Meats. 2nd ed., 1945, Garrard Press, Cham- paign, Illinois. 19. JENSEN, L. B., and HESS, W. R. A study of ham souring. Food Research, 6, 273-326 (1941). 20. JENSEN, L. B., and HESS, W. R. A study of the effects of sodium nitrate on bacteria in meat. Canner, 92 (12), 82 (1941). 21. JENSEN, L. B., WOOD, I. H., and JANSEN, C. E. Swelling in canned chopped hams. Ind. Eng. Chem., 26, 1118-1120 (1934). 22. KIRSCH, R. H., BERRY, F. E., BALDWIN, C. L., and FOSTER, E. M. The bac- teriology of refrigerated ground beef. Food Research, 17, 495-503 (1952). 23. KLEIN, E. On the nature and causes of taints in miscured hams. The Lancet, 174, 1832-4 (1908). 24. KRAFT, A. A. Information on prepackaged meats. (Private communication), Ames, Iowa (1951). 25. MALLMANN, W. L., ZAIKOWSKI, L., and RUSTER, M. The effect of carbon dioxide on bacteria with particular reference to food poisoning organisms. Mich. Agr. Exp. Sta. J., 489, 25-40 (1940). 26. McBRYDE, C. N. A bacteriological study of ham souring. U.S.D.A., B.A.I., Bui., 132 (1911). 27. MORAN, J. A. The metabolism of certain anaerobic bacteria concerned with food spoilage. Univ. of Chicago, Science Series VI, 355-360 (1929). 28. MORAN, T. Post-mortem and refrigeration changes in meat. J. Soc. Chem. Ind., 54, Pt. 2, trans. 149T (1935). 29. NIVEN, C. F. Sausage discoloration of bacterial origin. Am. Meat Institute Foundation Bui. 13 (1951). 30. NIVEN, C. F., CASTELLANI, A. B., and ALLANSON, V. A study of the lactic acid bacteria that cause surface discolorations of sausages. J. Bact., 58, 633-41 (1949). 31. NORTON, J. F., and RODERICK, L. M. Color control and conservation of sausage and cured meats. Inst. Am. Meat Packers. Unnumbered Bui. pp. 15-16 (1936). 32. OGILVY, W. S., and AYRES, J. C. Post-mortem changes in stored meats. II. The effect of atmospheres containing carbon dioxide in prolonging the storage life of cut-up chicken. Food Technol, S, 97-102 (1951). 24

33. OGILVY, W. S., and AYRES, J. C. Post-mortem changes in stored meats. III. The effect of atmospheres containing carbon dioxide in prolonging the storage life of frankfurters. Food Technol., S, 300-303 (1951). 34. OGILVY, W. S., and AYRES, J. C. Post-mortem changes in stored meats. V. Microbiology of frankfurters stored in atmospheres containing carbon dioxide. Food Research, 18 (1953). 35. RUYLE, E. H., and TANNER, F. W. The microbiology of certain canned meat products. Zentbl. f. Bakt., Abt. 11, Bd. 2, 436-449 (1935). 36. SCOTT, W. J. The growth of microorganisms on ox muscle. The influence of water content of substrate on rate of growth at — 1°C. Australian J. Coun. Sci. Ind. Res., 9, 177-190 (1936). 37. STEINKRAUS, K. H., and AYRES, J. C. Biochemical and serological relation- ships of putrefactive anaerobes isolated from meat. In preparation. 38. STUMBO, C. R., GROSS, C. E., and VINTON, C. Bacteriological studies relating to thermal processing of canned meats. Food Research, 10, 260-72 (1945). 39. STUMBO, C. R., GROSS, C. E., and VINTON, C. Bacteriological studies relating to thermal processing of canned meats. II. Influence of meat-curing agents upon thermal resistance of spores of a putrefactive anaerobic bacterium in meat. Food Research, 10, 283-292 (1945). 40. STUMBO, C. R., GROSS, C. E., and VINTON, C. Bacteriological studies relating to thermal processing of canned meats. III. Influence of meat-curing agents upon growth of a putrefactive anaerobic bacterium in heat-processed meat. Food Research, 10, 293-302 (1945). 41. STURGES, W. S., and HEIDEMAN, A. G. 43. Studies of halophilic microorgan- isms. II. The flora of meat-curing solutions. Abs. Bact., 8, 14-15 (1924). 42. SULZBACHER, W. L., and MCLEAN, R. A. The bacterial flora of fresh pork sausage. Food Technol., 5, 7-8 (1951). 43. TANNER, F. W. Food-Borne Infections and Intoxications. 1933, Twin City Printing Co., Champaign, Illinois. 44. TANNER, F. W., and EVANS, F. L. Effect of meat-curing solutions on anaerobic bacteria. I. Sodium chloride. Zentbl. f. Bakt. Abt. 11, 88, 44-54 (1933). 45. TANNER, F. W., and EVANS, F. L. Effect of meat-curing solutions on anaerobic bacteria. II. Sodium nitrate. Zentbl. f. Bakt. Abt. 11, 89, 48-54 (1933). 46. TANNER, F. W., and EVANS, F. L. Effect of meat-curing solutions on anaerobic bacteria. III. Sodium nitrite. Zentbl. f. Bakt. Abt. 11, 91, 1-14 (1934). 47. TARR, H. L. A. The bacteriostatic action of nitrites. Nature, 147, 417-8 (1941). 48. TUCKER, W. H. Studies on Clostridium putrificum and Clostridium putre- faciens. Inst. Am. Meat Packers, Chicago, 111. (1929). 49. YESAIR, J. Color control and conservation of sausage and cured meats. Inst. Am. Meat Packers. Unnumbered Bui. pp. 33-38 (1936). 50. YESAIR, J., and CAMERON, E. J. Inhibitive effect of curing agents on anaerobic spores. Canner, 94, 89-92 (1942). 51. YESAIR, J., CAMERON, E. J., and BOHRER, C. W. Comparative resistance of desiccated and wet micrococci heated under moist and dry conditions. J. Bact., 47, 473, A3 (abstr.) (1944). 25

CHAIRMAN ROBINSON Thank you, Dr. Ayres. Now we shall hear from Dr. Gross. Study of the Incidence of Spoilage Organisms in Canned Meat Products Manufactured under Commercial Conditions " C. E. GROSS This discussion will be limited to the so-called "commercially sterile" or shelf-type canned meat items. They will be considered in contrast to those receiving pasteurization processes, in the trade termed "perishable," which must be held under refrigeration. Fur- ther, only those canned meats made solely from meats and meat by- products as defined by the Regulations Governing the Meat Inspec- tion of the USD A (12) with approved curing agents added, will be considered. The more complex mixtures of meats and cereals and/or vegetables introduce more complex bacteriological problems and should receive separate study. The group of canned meats specifically cov- ered in this discussion are the pork luncheon meats. Even in this limited field there are complex bacteriological prob- lems not fully understood. Problems involving technique are likewise difficult. Only the exploratory phases of the indicated research needed have been conducted and published. The major considerations appear to be to make safe products from both the public health and spoilage loss standpoints and in so doing preserve the utmost possible desir- able qualities of flavor, texture, and appearance. These considerations are much like those of the commercial canning industry generally. However, the problems are more acute in low-acid products heated by conduction, under which classification are the canned meats. The bacteriological problem is to destroy or cause to remain dor- mant any viable organisms that may be present as well as to inacti- vate enzyme systems. Essentially that is what is meant by a "com- mercially sterile" process. In this discussion an attempt has been made to point out the limits of the over-all problem with respect to aerobic and anaerobic spore-forming organisms. The purposes have been to evaluate the lethal effect of a range of thermal processes and to determine whether there has been effective inhibition of growth of viable spores when present. The technique used might be termed fractionation by thermal methods. Data are derived from samples of product handled in plant-scale operations as well as from competi- tors' products purchased on the open market. • This paper is in the nature of a review of data presented earlier; added to it are the findings of the year 1952. It closely follows the form and outline of a paper presented at the Fourth Research Conference at the University of Chicago last year. 26

Experimental Methods Although the technique used has been covered in the literature and in con- siderable detail by Stumbo, Gross, and Vinton (I3), a short review and com- ments on deviations or other special handling may be appropriate. Unprocessed cans of product were obtained from the production line by chance selection. Product shipped from another plant was selected the same way and immediately packed with dry ice sufficient to deliver to destination with a small surplus. The cans of frozen product were defrosted by flowing tap water over them for a sufficient period to defrost, but keeping the temperature under 40°F. Two hours have been found sufficient for properly defrosting a 6-pound can. The cans were opened in the usual manner with good aseptic bacteriological technique. After removing the top portion, a sterile Alemite gun was filled from the center of the can. The desired number of clean, sterile 10x75 mm. chemical test tubes were then filled with approximately one g. of meat. The tubes were sealed in a blast lamp. A 5-minute preliminary process in an oil bath at 175°F. was used to ensure that all tubes were the same initial temperature when proc- essing began. Processing was done in an oil bath controlled to plus or minus 0.1°C. and upon removal immediately transferring to cooling water at 70°F. Part of the tubes used at each heating level were subcultured into glucose brain broth, and the remainder were to be held under 25° to 30°C. incubation conditions for a minimum of 5 years. The number of tubes used varied at different periods in the investigation but was never less than a total of 6. The results of sub- culture were judged by the usual criteria including examination of microscopic slides. The tubes under incubation were checked by visual inspection at gradually increasing time intervals. Any tubes not appearing normal were classed "suspect" and opened. By organoleptic tests, microscopic examination, and subculture a decision was made as to whether there had been any bacterial activity or, in rare cases, leakage. At the end of 5 or more years under incubation conditions all tubes were to be opened and rated for condition by organoleptic tests and micro- scopic examination. In any doubtful cases more extensive tests were to be made. The processing values used were expressed in terms of F0 units. b Another method of approach to the problem was to fill 3 %-ounce cans (208x109) with the product being sampled for the standard thermal fractionation procedure and to process them to reach a center temperature of 156°F. by heating 48% minutes in a water bath controlled at 160°F. This pasteurization process is comparable to those given to perishable products which must be held tmder refrigeration. The number of viable organisms surviving this pasteurization process gives an estimate of the number of viable organisms, probably in spore form, which the normal "shelf-type" processes would have to inactivate. Gross and Schaub (4) proposed a system of nomenclature for pasteurization processes similar to that of Ball (1) for the highest temperature processes. The above process would be designated as a process value of F'=34. In Table 1 are shown the lethal effect of various processes. The number of samples containing viable bacteria varies indirectly with level of processing. During the 1944-47 period very high processes were required to reach any high percentage of sterility. It has since been established that the main reason for this was that a large number of the samples contained spores of P.A. 3679 which are known to have a high thermal resistance. The incidence of P.A. 3679 decreased from approximately 40% in the 1944-47 period, to 5% in 1949, to 0.9% in 1950-51, and to 0% in 1952. As the incidence of P.A. 3679 dropped, the number of samples positive at given processing values dropped also. In the 1949 through 1952 period a process of F0=0.6 sterilized from 89% to 95% of the product with an apparent tendency for the percentage sterility to increase from year to year. The point b A thorough discussion of this method of expressing thermal processing results can be found in the Canned Food Reference Manual (14). 27

TABLE 1 EFFECT OF THERMAL PROCESSING Per Cent Samples Positive for Viable Bacteria c No. of Samples Processing Level in F0 Values Period 0.05 0.2 0.6 1.0 2.0 1944-47 1949 1950-51 1952 380 337 218 138 98 79 78 87 43 43 36 67 11 9 5 40 0 1.4 0 19 0 77 Incidence of P.A. 3679: 1944-47—approx. 40%; 1949—5%; 1950-51—0.9%; 1952—0%. for 100% sterilization during the 1949 through 1952 period apparently lay some- where between F0=0.6 and 1.0. The 3 samples not sterilized at F0=l in the 1950-51 period contained more resistant organisms, two of which were P.A. 3679 and the third Clostridium sporogenes. During the 1952 work no P.A. 3679 was found. Even though spores of P.A. 3679 are still present—and may be isolated from scalding tub water—it now appears that the numbers now present are much lower. The extra tubes not subcultured immediately after processing were to be held under 25° to 30°C. incubation conditions for a minimum of 5 years. The actual holding period on the product reported in Table 1 varied from one month to over 7 years. The group of samples listed as 1944-47 were held a minimum of 51 months and a maximum of 87 months. With the exception of a very few selected samples which are to be held still longer, all tubes for the 1944-47 period were opened. Samples representing each month of the 1944-47 period were examined for viability of the organisms which were known to be present by sub- culture immediately after processing. The duplicate tubes from each processing level on each sample of the 1944-47 series were examined at intervals. Any tubes that were discolored or for any other reason appeared to have changed appearance were examined by organoleptic tests and examination of microscopic slides. These tubes were then subcultured into glucose brain broth to check viability. These checks were made in March and August 1946, January 1947, March 1948, and February 1952. All samples examined microscopically and by organoleptic tests were found to be unaltered at all inspection times, and there was no microscopic evidence of bacterial growth on any. It is interesting that at lower processing levels the contents of the tubes when opened showed no evidence of microbial action by organoleptic tests; the aroma was pleasant and similar to that of product opened a few days after processing. Negative results in themselves are some- times considered inconclusive, but additional evidence may be cited indicating that the methods are satisfactory. In other such processing and incubation tests on more complex canned meat products such as a liver loaf, spoilage was observed on the lower processing levels after prolonged incubation. The spoilage was detectable by changes in appearance and by a vile, putrefactive odor when the tube was opened. Viable organisms were present when subcultured. Also in this series there was one period when a high percentage of all samples changed to gray and green. This was conclusively traced to microscopic leaks due to off quality of the glass in one lot of test tubes. 28

TABLE 2 VIABILITY OF SPORES AFTER INCUBATION Viable Organisms Present Process F. Months Incubation Immed. after Process After Incubation Yes No Yes No 0.01 5-13 19 0 1 18 19-30 10 1 5 6 73-78 36 0 0 36 0.2 6-18 16 2 2 16 20-37 12 1« 4d 9 50-70 18 0 2 16 71-87 29 3 0 32 0.6 13-21 6 5« 4« 7 26-39 3 1 2 2 40-60 14 4 0 18 70-87 35 5 0 40 1.0 13-24 2 6 2 6 28-36 6 8 2 12 40-60 27 2 1 28 60-80 55 4 0 59 81-86 21 2 0 23 2.0 6-12 1 3 0 4 20-33 1 1 1 1 50-60 19 1 0 20 60-71 22 6 0 28 d Sample was negative immediately after processing and positive after incubation —total of 2 samples. Table 2 shows the results of the subculture of the incubated tubes compared 'with the results of subculture immediately after processing. In 2 instances viable organisms were found in tubes in a series which were negative immediately after processing. The reverse is also true; this is to be expected since very small numbers of organisms are involved. In spite of these variations there appears to be a trend of loss of viability after prolonged incubation. This is especially marked after about 3 years of incubation. Samples were selected from the 1944-47 series which had shown viable organisms on subculturing immediately after processing. These samples were subcultured with results as shown in Table 3. No viable organisms were found. Appropriate controls and checks had been used on the media and technique so that l-hese negative results are thought to be significant. The data show that ap- parently all viable organisms at all processing levels had lost their viability during the 51- to 87-month incubation period. These results were checked on the 1950-51 series of tests and checks made at more frequent time intervals in order to attempt to check the time when viability is apparently lost. Present data would seem to indicate that time to be between 26 and 36 months although 2 samples showed viable organisms after 52 and 61 months, respectively. 29

TABLE 3 VIABILITY OF ORGANISMS AFTER PROLONGED INCUBATION Viable Organisms Present Process Months No. Immed. after Subcultured F. Incubation Samples e Process Feb. 1952 Yes No 0.05 51-87 34 34 0 0.2 51-87 37 37 0 0.6 51-87 52 52 0 1.0 51-87 95 95 0 2.0 51-87 40 40 0 e Samples selected that were positive after processing. Table 4 shows data on the effect of thermal processing on meats for canning covering plants in 7 midwestern states. These data were reported at the Fourth Annual Meeting of the Associates, Food and Container Institute (11). An industry cooperative group, using a technique similar to that used in the Morrell Laboratory, made the survey. Only one sample of the 177 examined was presumed to be P.A. 3679 or approximately 0.6%. Data in Table 4 correlate well with those in Table 1 indicating that Morrell Laboratory experience is not snecific or unusual. In at least an important segment of the total industry the level of thermal processing for a given percentage of samples to be sterile is approximately the same for different plants located in 7 midwestern states. After an incubation period of 30 to 36 months there has been only one tube showing visual evidence of spoilage. One tube processed at F0=:0.05 showed definite digestion of the meat in March 1953. When opened, typical putrefactive odors were present. Microscopic examination and subculturing demonstrated that a TABLE 4 EFFECT OF THERMAL PROCESSING LEVELS f Samples Positive for Viable Bacteria * No. of Samples Processing Level in F0 Values Plant 0.05 0.2 0.6 1.0 1 57 45 17 0 0 2 29 20 9 3 0 3 25 23 12 1 1 4 21 18 13 1 0 5 18 16 6 0 0 6 16 12 6 0 0 7 11 10 5 0 0 Total 177 144 68 5 1 Total as per cent 81.4 38.4 2.8 0.6 ' From Associates' survey of 1950 covering 7 states. " One sample was presumed to be P.A. 3679 or 0.56%. 30

TABLE 5 SURVEY OF 12-ouNCE LUNCHEON MEAT 1949-52 No. of Subculture P.A. 3679 present h Company Samples Positive Negative Samples Per Cent A 35 6 29 4 11.4 B 19 6 13 0 0 C 17 10 7 2 11.8 D 13 5 8 0 0 E 17 3 14 1 5.9 P 17 2 15 1 5.9 X1 38 10 28 2 5.3 Total 156 42 114 10 Total per cent 27 73 6.4 h Checked by serological methods. 1 Represents several samples from each of 6 different companies. viable organism was present. In the original examination this organism was shown to be Clostridium sporogenes. On this sample all the tubes from the various processing levels were then opened and subcultured. No evidence of any off condition was noted when they were opened after the 31-month incubation period. However, on subculture 50% of those positive after processing were now found to be negative. The results of bacteriological examination of cans of 12-ounce luncheon meat available on the retail market throughout the country are shown in Table 5. The data include samples from the production of 12 companies although those of 6 companies were listed together because of the limited number of samples for each. The over-all average shows that these data are comparable to those in Table 1 for the 1949, 1950-51, and 1952 periods, and in Table 4. The exact thermal processes used on the market product are of course not available but are presumed to lie in the range 0.2 to 0.6 F0. Three samples from a total of 130 competitors' samples examined during 1952 contained P.A. 3679 or 2.3 %. Among Morrell products one such sample was found in 166 samples examined or.0.6%. Thus, it would appear that the 1952 period shows a decline in the percentage of samples containing P.A. 3679. TABLE 6 TOTAL SPORE LOAD IN TRIMMINGS Range of Viable Spores after Pasteurization 0 to 10 11 to 100 over 100 Plant Samples Samples Per Cent Samples Per Cent Samples Per Cent 1951 A B 65 32 24 11 37 34 23 14 35 18 7 28 22 44 1952 A B 130 33 33 9 25 67 21 52 64 30 3 23 27 9 Total 260 77 30 125 48 58 22 31

Some work was done on determining the total number of spores present in normal well-handled meats such as are used to make canned luncheon meat. The Armour (6) and Iowa State College (2) groups have found very small numbers of putrefactive anaerobes normally present. The total load of spores would be of importance in thermal processing and so in Table 6 are data from the survey; routine bacteriological methods were used and the counts were made after pasteurization to destroy vegetative cells. Most counts were made on samples later used to show the effect of thermal processing (Table 1). These data con- firm the general observations in the literature that the total numbers of spore- forming bacteria may be quite high in normal, well-handled trimmings such as are used to make canned luncheon meats. On the other hand these numbers are quite low compared to the levels normally used in inoculated pack studies. Discussion The purpose of the conventional thermal processing of canned meats is to destroy microbial life so that the product may be kept at ordinary storage temperatures without deterioration due to microbial action. There is considerable evidence to show that complete destruc- tion of microbial life is not always essential. In many instances actual viable organisms are present that remain dormant or are inhibited from germination by some factor (s). Thus, a so-called safe commer- cial process does not always require the complete destruction of life but in some instances only a complete and proved inhibition of future microbial growth during the desired maximum shelf life of the product. Usually the thermal processing required by conventional proce- dures for absolute sterility is so high that the desirable organoleptic characteristics are adversely affected. In attempting to reduce the excessive thermal processing required, attention has been directed to ways and means of producing satisfactory products as determined by test pack procedures and actual production experience rather than by theoretical bacteriological considerations and arbitrarily ruling that each and every can of every product must be completely sterile. Among the theoretical considerations is usually the fact that 3 putrefactive anaerobes have great significance because of the high thermal resistance of the spores and the possible public health sig- nificance of one of them. The 3 usually considered in meat canning technology are Clostridium sporogenes, Clostridivm botulinum, and P.A. 3679. Directly affecting this consideration is the fact that num- bers of organisms present affect the thermal processes required to destroy them. Data on the normal numbers of spores of putrefactive anaerobes present would be of great value. The work of the Armour (6) and Iowa State College (2) groups has been of great value in indicating that the numbers are normally very low. Data from papers by Esty and Meyer (3) and Houston (?) indicate that the thermal death-time of small numbers of spores of Clostridium botulinum is very low. Unpublished data from this laboratory indicate that the thermal death-time of small numbers of spores of Clostridium sporo- 32

genes in meat products is relatively low, in the range F0—0.2 to 0.5. On the other hand, Gross and co-workers (5) have reported that very high thermal processes are required to destroy very small num- bers of spores of P.A. 3679. Normal processes given canned meat products do not destroy small numbers of spores of P.A. 3679. Data in the literature (10) indicate that spores of other common putre- factive anaerobes have a lower thermal resistance than Clostridium sporogenes. From these considerations it would seem that the re- quired thermal processes could be drastically reduced if spores of P.A. 3679 could be eliminated and the number of spores of Clostridium sporogenes and other putrefactive anaerobes were very low. The fact is that in most normal meats the spore load of putrefactive anae- robes is very low. If P.A. 3679 were absent, then the required ther- mal process should be very low. From the data given in this report such is not the case. Other investigators, especially Jansen and Aschehoug (8), have concluded that in many cases aerobic spore formers of the genus bacilli are important factors in spoilage of canned meats. Jensen (9) reported that such organisms were important in the spoilage of the perishable class of canned meat items. Various sources report that much greater numbers of such organisms are nor- mally present in meat. The Morrell Laboratory has found under normal conditions of operation and handling that total spore loads in the hundreds are not uncommon as shown in Table 6. Unpublished work has shown that the resistance of such numbers of spores of some aerobic organisms may be as high as 0.2 F0. This resistance is con- siderably higher than is reported in the literature but it has been found that in a meat substrate the thermal resistances may fre- quently be much higher. One problem that may not have been explored sufficiently is the point of temperature or total thermal process where the organoleptic properties deteriorate seriously. If this information were available, a reasonable estimate of the possibilities of processing to commercial sterility conditions with conventional thermal processing procedures could be made. The small amount of data available would seem to indicate that the possibilities of improvement of organoleptic qualities through thermal process reduction appear to be very limited by the conventional procedures. If this can be confirmed, it should only serve to still further stimulate the existing research efforts on other possible procedures such as electronic heating and high-energy radiation. It is interesting that even though as much as 98 % of the product processed at F0=0.05 was not sterile, there was no evidence of germi- nation of spores, subsequent growth, and spoilage, until recently. Perhaps this one exception is in itself significant. One tube out of 4 from the 0.05 processing level of plant 4 in Table 4 showed typical digestion and reddening at some time between 18 and 30 months under 33

incubation at 30°C. This sample was positive immediately after process through F0 0.6 and a putrefactive anaerobe was isolated which was identified by serological methods as Clostridium sporogenes. To this date no other samples have ever germinated and grown from any of the extensive series of experiments on normal production-line pork luncheon meat products. Many of these were known to contain viable spores of both P.A. 3679 and Clostridium sporogenes. Appar- ently the standard curing agents used are effective inhibiting agents, or perhaps, more accurately, bacteriostatic agents. This is especially noteworthy when the incubation time of up to 87 months at 30° C. is considered. It was known and demonstrated that in many of these samples viable spores of P.A. 3679 were present as well as Clostridium sporo- genes and aerobic spore formers. The effectiveness of bacteriostatic action with respect to viable spores of anaerobes is no longer definite since the one case of germination and growth of Clostridium sporo- genes has been found. Another bacteriostatic factor in addition to cur- ing agents is also present and may seriously exert an effect in re- stricting the germination of some organisms. This factor is a lowered oxygen tension. The oxidation-reduction potential of cured meats no doubt has some effect, either favorable or adverse, depending on the class of organism. One fact becomes all the more important: namely, the factors affecting spore formation, germination, and inhibition which must be studied and understood. The reasons as to why there was an almost completely effective inhibition of germination need to be known before the safety and reliability of depending on such inhibition can be judged. Another interesting observation was the demonstrated viability of spores in canned meat products over long periods of time under conditions of 30° C. incubation but with an eventual loss of viability at still longer times. Apparently from 26 to 31 months under such conditions is the normal period of loss of viability although 2 excep- tions surviving a processing level of 0.2 were viable after 52 and 61 months. Summary and Conclusions The level of thermal processes required to reach sterility in cured canned luncheon meat products has been lowered in the past few years. The incidence of occurrence of P.A. 3679 has decreased from that of great importance at 40% to relative unimportance of from 0.6% to 2.3% on Morrell and competitors' products, respectively, in 1952. During one extensive survey in 1952 covering 2 plants no P.A. 3679 was found in pork luncheon meat products. The percentage of product sterile at a processing level of F0 0.6 increased from 33% in the 1944-47 period to 95% in 1952. Similar figures for a processing level of F0—0.02 would be 13% to 63%. On the other hand, at the lowest level of F0=0.05 the increase was only 2% to 27%. 34

Processes to assure sterility of a very high percentage of the product would have to exceed F0=0.6. This is supported by the find- ings on a 12-ounce market product presumed to be processed in the range F0=0.2 to 0.6. There was only one instance of spoilage due to germination and growth of viable spores surviving thermal processes during the 9 years of this survey work. This was due to one tube out of 4 pro- cessed at F0=0.05 spoiling sometime between 18 and 30 months incu- bation at 30° C. The. spoilage was due to germination and growth of spores of Clostridium sporogenes. None of the other tubes at that processing level or at the 0.2 or 0.6 levels have shown evidence of growth after 30 months incubation at 30° C. The tubes from all other samples were incubated as long as 87 months and spores were shown to be viable as long as 61 months. The loss of viability of spores surviving thermal processes apparently usually took place dur- ing 26 to 31 months of incubation at 30° C., although 2 samples showed survival periods of 52 and 61 months. There appears to be evidence that aerobic spore formers are an important factor and perhaps a limiting factor in attempting to lower thermal processes. The point to which processes may be lowered is in doubt as is also whether or not the final point would be at a level yielding worth-while improvement in organoleptic properties. Further fundamental work is indicated as necessary in order to understand and control the apparent inhibitory or bacteriostatic ac- tion of curing agents on viable spores present in a rather substantial percentage of commercial cured canned luncheon meats. Work is also needed to determine whether or not improvement in organoleptic properties can be realized by lowering thermal processes at all, using the normal thermal processing procedures. Conversely, increased at- tention should be given to unorthodox procedures such as electronic heating, high-short processes, and high-energy radiation. Literature Cited 1. Ball, C. O. Mathematical solution of problems on thermal processing of canned foods. Univ. of California Publications in Public Health, 1, 15-245 (1928). 2. Burke, Martin V., Steinkraus, Keith H., and Ayres, John C. Methods for deter- mining the incidence of putrefactive anaerobic spores in meat products. Food Technol., 4 (1), 21-25 (1950). 3. Esty, J. R., and Meyer, K. F. The heat resistance of the spores of B. botuli- nus and allied anaerobes. /. Infect. Diseases, 31, 650-63 (1922). 4. Gross, C. E., and Schaub, D. J. Evaluation of lethality of pasteurization pro- cesses used in the meat packing industry. Proc. Inst. Food Technologists, 139- 145 (1945). 5. Gross, C. E., Vinton, C., and Stumbo, C. R. Bacteriological studies relating to thermal processing of canned meats. VI. Thermal death-time curve for spores of test putrefactive anaerobe in meat. Food Research, 11 (5), 411- 418 (1946). 6. Harriman, L. A., DelGuidice, V. J., and Shinn, B. M. Spore formers in pork. Annual Meeting, 111. Soc. Amer. Bacteriologists (1948). 35

7. Houston, C. W. Heat resistance studies on Clostridium botulinum in meat. Ph. D. Thesis, Univ. of Illinois (1947). 8. Jansen, Erling, and Aschehoug, Valborg. Bacillus as spoilage organisms in canned foods. Food Research, 16 (6), 457-61 (1951). 9. Jensen, L. B., and Hess, W. R. Fermentation in meat products by the genus Bacillus. Food Research, 6, 75-83 (1941). 10. McCoy, Elizabeth, and McClung, L. S. The Anaerobic Bacteria. A Short Bibliography. Vols. 1 and 2 (1939) and Supplement 1938-39 (1941). Uni- versity of California Press, Berkeley. 11. Proceedings of the Fourth Annual Meeting, Research and Development Asso- ciates, Food and Container Institute, Inc., 1951, Chicago, 111. 12. Regulations Governing the Meat Inspection of the U.S.D.A. 1947, U.S. Gov- ernment Printing Office. 13. Stumbo, C. R., Gross, C. E., and Vinton, C. Bacteriological studies relating to thermal processing of canned meats. I. Laboratory methods employed for studying thermal processes required to prevent bacterial spoilage of canned meats. Food Research, 10 (3), 260-72 (1945). 14. The Canned Food Reference Manual. 3rd ed., 1947, American Can Company, New York. 15. Vinton, C., Martin, Sterling, Jr., and Gross, C. E. Bacteriological problems in thermal processing of canned meats. Proceedings of the Fourth Research Conference. Sponsored by the Council on Research, American Meat Insti- tute at the University of Chicago (1952). 36

III. Mode and Rate of Heat Transfer in Canned Meats TUESDAY AFTERNOON SESSION March 31, 1953 The meeting reconvened at 1:15 o'clock with Dr. Robinson pre- siding. CHAIRMAN ROBINSON This afternoon while the subject is still fresh in your minds we would like to call on Dr. Halvorson of the University of Illinois to comment on his impression of the morning session. Dr. Halvorson. H. O. HALVORSON The purpose of this meeting, I take it, is to review our knowledge to see where we are at this time and to see whether we can do anything else to approach the goal—how to obtain sterile products that have not been overcooked. Such meetings as this are very useful for that purpose. And now my observations on the papers: Dr. Gross made an impressive point when he showed that even now a fairly large percentage—from 40% to 60 %—of the so-called pasteurized meat products that have been cooked at a relatively low temperature are sterile. I am sure if he dared, he could well go to his management and say, "Now, look and see what we have done. By improving our housekeeping, we have eliminated these bad organisms, and therefore improved our products." Perhaps that is the reason— housekeeping—but I am afraid there may be more subtle reasons that we don't know or understand as yet. Some people, probably sonie in management, would say, "If you can do this to 44^ of the cans, why can't you do it to all of them?" Perhaps we can, but at present we lack some essential information. What this industry needs, I think, is the appropriation of funds for more fundamental work, and I am very happy to see from the papers given this morning that more fundamental work is being done. I spent some 20 years serving as a consultant to this industry, making use largely of the information that was available at the beginning of that time. During those years I did no fundamental research. As a result, I didn't contribute any- thing. If the Armed Forces want to solve this problem, I think it is abso- lutely essential that they appropriate some of their money to support fundamental research. When we get enough fundamental data, we can then apply it, and perhaps solve the problem. Also, I think the industry or the Armed Forces, whoever has the funds, should appropriate money so that you can bring together people who are doing fundamental research on spores—12 or 15 people, not 37