Time-Temperature Effects on Salmonellae and Staphylococci in Foods

Transcription

1 R. ANGELOTTI, M. J. FOTER, AND K. H. LEWIS 308 [VOL. 9 SHERMAN, J. M The streptococci. Bacteriol. Rev. 1:3-97. Subcommittee on Methods for the Microbiological Examination of Foods Recommended methods for the microbiological examination of foods, p American Public Health Assoc., New York. WINTER, C. E., AND L. A. SANDHOLZER Studies of the fecal streptococci. 1. The isolation of enterococci from natural sources. Fishery Leaflet 201. U. S. Fish and Wildlife Service. ZABOROWSKI, H., D. A. HUBER, AND M. M. RAYMAN Evaluation of microbiological methods used for the examination of precooked frozen foods. Appl. Microbiol. 6: Time-Temperature Effects on Salmonellae and Staphylococci in Foods III. Thermal Death Time Studies ROBERT ANGELOTTI, MILTON J. FOTER, AND KEITH H. LEWIS Milk and Food Research Program, U. S. Department of Health, Education, and Welfare, Public Health Service, Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio Received for publication October 11, 1960 ABSTRACT ANGELOTTI, ROBERT (U. S. Department of Health, Education, and Welfare, Cincinnati, Ohio), MILTON J. FOTER, AND KEITH H. LEWIS. Time-temperature effects on salmonellae and staphylococci in foods. III. Thermal death time studies. Appl. Microbiol. 9: Thermal death time studies were conducted at 5 F intervals from 130 to 150 F with strains of salmonellae and enterotoxigenic staphylococci. Heat-resistant Salmonella senftenberg strain 775W, Staphylococcus aureus strains 196E and Ms149, and non-heat-resistant Salmonella manhattan were studied in custard, chicken a la king, and ham salad. The F140 values (minutes of exposure at 140 F required to effect 100% destruction) were as follows: S. senftenberg 775W in custard 78, and chicken a la king 81.5; S. manhattan in custard 19, and chicken A la king 3.1; S. aureus 196E in custard 59, and chicken a la king 47; S. aureus Ms149 in custard 53, and chicken a la king 40. The end points of survival-kill at all the test temperatures for both salmonellae and staphylococci in ham salad were considerably less than for the other foods studied. D140 values (minutes of exposure at 140 F required to effect a 90 % reduction in numbers) were also calculated from the data and presented. Values for ZF and ZD (slope of the thermal-death-time and decimal-reduction-time curves) are also presented and discussed in relation to type of food, organism, and temperature. These data indicate that heating perishable foods of the type studied to 150 F and holding every particle of food at this temperature for at least 12 min reduces 10 million or less salmonellae or staphylococci per gram to nondetectable levels. The same degree of destruction is achieved in similarly contaminated foods when held at 140 F for 78 to 83 min. On the basis of the calculation procedures employed, it is estimated that 45-min exposure at 140 F would be necessary to reduce 1,000 organisms per gram to nondetectable levels. Because of the continued occurrence of staphylococcal food poisoning and foodborne salmonellosis in the United States, a study has been undertaken on the conditions necessary for thermal destruction of the causative agents in foods. Of the many factors which affect the response of microorganisms to their environment, temperature and time can be controlled most readily to prevent food-poisoning episodes caused by these bacteria. Though the effects of time-temperature relationships on the activities of microorganisms have been under investigation for many years and are presently recognized as having a profound effect upon microbial life, application of this basic knowledge to the control of bacterial growth or toxin production in perishable foods has not been as extensive as in the canning or dairy industries. Instead, temperature control, as an environmental factor important in food sanitation, is applied largely on the basis of practical experience. The popularity of precooked ready-to-serve foods, the rapid development of catering services, the increased

2 19611 THERMAL DEATH OF SALMONELLAE AND STAPHYLOCOCCI IN FOOD use of vending machines to dispense perishable meals, and the growth of the restaurant industry have greatly complicated the problem of insuring adequate timetemperature control over perishable products. For this reason, a series of studies has been undertaken to develop experimentally an organized body of technical data on the critical time-temperature relationships applicable to the safe handling of these foods during processing, storage, transportation, and preparation for serving. Our previous reports (Angelotti et al., 1959; 1961a, b) demonstrated that the practice of storing perishable foods at a temperature of 50 F for more than 1 day may result in increases of the microbial population; but, because of the slow microbial growth at this temperature, it is possible to store foods safely for short periods, provided the foods are not grossly contaminated with salmonellae or staphylococci. It was also shown that growth of salmonellae, staphylococci, and Clostridium botulinum types A, B, C, D is prevented in perishable foods when the internal temperature is at or below 42 F, and salmonellae and staphylococci do not multiply in foods heated to temperatures of 116 F and above. The purpose of this present study is to report upon the thermal resistance of food-poisoning salmonellae and staphylococci in selected foods as determined from thermal death time values obtained for each group in the temperature range of 130 to 150 F at five-degree intervals. MATERIALS AND METHODS Preparation of Cultures Previous research, conducted in our laboratory, on the heat tolerance of a number of Salmonella species representative of those associated with foodborne salmonellosis and on several enterotoxigenic strains of Staphylococcus aureus, permitted us to select the following organisms as those displaying the limits of heat tolerance typical of salmonellae and staphylococci (Angelotti et al., 1959): high heat resistance, Salmonella senftenberg 775W and Staphylococcus aureus Ms149; low heat resistance, Salmonella manhattan and S. aureus 196E. Initially, all the organisms were grown on nutrient agar slants incubated at 37 C for 24 hr. They were harvested in sterile phosphate buffered dilution water (APHA, 1953), inoculated into sterile skim milk, and lyophilized. The lyophilized cultures were maintained as stocks at 5 C. To prepare a primary culture, a lyophilized stock was opened, transferred to a tube of Bactol brain heart infusion broth, and incubated 18 hr at 37 C. A transfer I Bacto products manufactured by Difco Laboratories, Inc., Detroit, Mich. Note: Mention of commercial products throughout is not to be construed as endorsement by the Public Health Service. 309 was made from the broth to a Bacto nutrient agar slant (primary culture) which was incubated for an additional 24 hi at 37 C. This primary culture was stored at 20 C and served as a source of inoculum for secondary cultures for 5 consecutive days. At the end of 5 days, the primary culture was discarded, a fresh lyophilized stock opened, and the cycle repeated to obtain a new primary culture. Secondary cultures were prepared by inoculating a Bacto nutrient agar Roux bottle with cells from the primary culture and incubating at 37 C for 24 hr. After incubation, the cells were washed from the surface with sterile phosphate-buffered dilution water, shaken thoroughly with beads to break up clumps, and adjusted to the desired concentration with a Coleman Junior spectrophotometer at 620 m,u. In every experiment, the cells employed to inoculate the test foods were obtained from a 24-hr-old secondary culture which had undergone only one transfer beyond the primary culture. This technique was used to avoid variation and inadvertent selection of types whose heat resistance differed from that of the parent strain. Preparation of Sterile Foods Custard, ham salad, and chicken A la king were prepared as described previously, except that the water was omitted from the ham salad and chicken a la king (Angelotti et al., 1959). The foods were weighed out in 250-g aliquots and placed in I-lb, screw-cap jars with rubber seals. The jars of ham salad and chicken 'a la king were autoclaved at 121 C for 45 min and the custard at 115 C for 45 min. Each batch of food prepared throughout the study was tested for sterility by removing the contents of a few jars and plating in Bacto plate count agar and inoculating into Bacto fluid thioglycolate medium. The results of these tests indicated that in every instance a sterile product was obtained. The sterile jars of food were stored at 5 C. For each experiment, a single jar of food was employed. One milliliter of the appropriately adjusted culture, described above, was inoculated into the cold jar of food and resulted in a cellular concentration of approximately 1 X 107 organisms per gram as determined by plate count. (See last paragraph of the following section.) The inoculum was thoroughly mixed into the food with a sterile tongue depressor. Immediately after inoculation, the jar of food was placed in a 2.8 C water bath to prevent multiplication of the organisms during the interval required for setting up the thermal death time (TDT) tube filling apparatus. This interval was never more than 10 min. Procedure for Filling, Holding, and Heat Treating TDT Tubes In view of the success reported by Stumbo, Gross, and Vinton (1945), in filling TDT tubes with solid

3 310 R. AN-GELOTTI, M. J. FOTER, AND K. H. LEWIS [VOL. 9 foods, an Allstate2 spring-fed grease gun was used to fill the TDT tubes. The grease fitting was replaced with a length of borosilicate glass tubing (7 by 120 mm) held in place with a short section of rubber tubing. This attachment permitted the injection of the food intothe bottom of the TDT tube in a continuous column free of entrapped air bubbles. Each tube received that amount of food delivered by one stroke of the gun. The average variation in weight delivered upon replicate strokes of the gun was found to be plus or minus 0.1 g for all three foods. The mean weight delivered for each food differed slightly as follows: chicken a la king 1.1 g; custard 1.0 g; and ham salad 1.1 g. All parts of the grease gun were thoroughly coated with a light covering of nontoxic, high melting point, technical grade, white grease3 to prevent rusting during autoclaving. The head and barrel assemblies were wrapped separately in craft paper and autoclaved at 121 C for 30 min. After autoclaving, the assemblies were oven dried at approximately 90 C and refrigerated to 5 C. To fill the TDT tubes, the sterile, cold gun was reassembled, filled aseptically with the cold inoculated food, and clamped to a ring stand. Soft glass (100 by 10 mm), TDT tubes were employed. For each thermal resistance test performed, 72 sterile, cotton-plugged tubes were distributed into 6 copper serological test tube racks containing 12 tubes per rack. As each rack of tubes was filled, the rack was placed in a 2.8 C (±0.1 C) water bath to prevent multiplication of the test organisms. After filling all 72 tubes, one tube at a time was removed from the 2.8 C bath, sealed in an oxy-gas flame, and returned to the bath. After sealing, the ends were permitted to cool sufficiently to prevent cracking and the TDT tubes were completely submerged in the 2.8 C bath to equilibrate for 10 min. The complete filling, sealing, and cooling operation required approximately 60 to 90 min. Multiplication of the test organisms was prevented during this time by keeping the temperature of the food continuously below 7 C. Following equilibration to 2.8 C, all 6 racks of tubes were removed from the cold bath and plunged simultaneously into a water bath heated to the desired test temperature. The hot bath maintained the test temperature within ±0.1 C. Timing was begun at the instant of submersion and at selected intervals individual racks were withdrawn from the bath, plunged instantaneously into the 2.8 C bath, and cooled for 10 min. For each test exposure, 10 TDT tubes were examined for survivors. The heat-treated TDT tubes were examined for survivors by two methods. In the case of 2 Allstate, steel, spring-fed grease gun manufactured by K-P Manufacturing Company, Minneapolis, Minn., and purchased from Sears Roebuck and Company. 3Petrol-Gel (white), McGlaughlin Oil Company, 3750 E. Livingston Ave., Columbus 13, Ohio. chicken a la king and custard, the cooled tubes were incubated for 5 days at 37 C, after which they were scored with a motor-driven glass tubing cutter, snapped open, and a loopful of the contents transferred to a tube of Bacto phenol red broth containing 1 % dextrose. The inoculated broth tubes were incubated at 37 C for 24 hr and examined for turbidity and acid production. TDT tubes containing ham salad were handled similarly to the others, except that they were opened and the contents transferred immediately after cooling 10 min. These two methods, out of several variations tried, resulted in the largest number of tubes showing survivors. In ham salad, a 5-day incubation resulted in fewer survivors than the method finally adopted. It was also found necessary to employ a broth with an acid-base indicator, because the loopful of food added to the broth resulted initially in some turbidity. All tubes showing survivors were examined by Gram stain. The salmonellae survivors were streaked onto Bacto brilliant green agar plates and incubated 48 hr at 37 C. Following incubation, colonies were picked and tested against Salmonella polyvalent serum. Surviving staphylococci were streaked onto Bacto staphylococcus no. 110 medium and incubated 48 hr at 37C. After incubation, colonies were picked and tested for coagulase production. To determine the number of organisms per gram in the inoculated foods, four additional TDT tubes were filled, sealed, and handled in a manner identical to the TDT tubes of the test series. Following the 10-min cooling period, the control tubes were opened, their contents combined, and two 1.0-g aliquots weighed out. Appropriate 10-fold dilutions of the aliquots were prepared in sterile, phosphate-buffered, dilution water, and duplicate plate counts of each were made in Bacto plate count agar. The plates were incubated at 37 C for 48 hr. In all cases the food was found to contain between 9 X 101 to 11 X 106 cells per gram. Determination of Rate of Heat Penetration Five TDT tubes were filled with one of the test foods as described above. An iron-constantan thermocouple (B & S 32 gauge) was placed in one tube at a time with the hot junction at the geometric center of the food column. The thermocouple was centered in the tube by means of three plastic rods which held it firmly, and the tube was sealed with a water-tight rubber cap, through which the leads extended. The tube was submerged in the 2.8 C water bath, and the temperature was recorded every 7.2 sec by means of a Leeds and Northrup Speedomax type G temperature recorder. When the internal temperature reached 2.8 C, the tube was transferred to the hot water test bath and the interval required to raise the internal temperature to that of the test bath, as registered by a control thermocouple, was recorded. On attaining this temperature, the tube was

4 19611 THERMAL DEATH OF SALMONELLAE AND STAPHYLOCOCCI IN FOOD 311 returned to the 2.8 C bath and the interval required to lower the internal temperature to 113 F was recorded. This procedure was repeated with five tubes of each food at 5-degree intervals from 120 to 150 F. The average temperature per time interval was calculated for each bath temperature employed. The tempeature of 113 F was selected as an end point, because previous work in our laboratory revealed that thermal inactivation of the test organisms does not occur at this temperature or below (Angelotti et al., 1959.) Using the average values, heat penetration curves were drawn for the three foods for each bath temperature (Ball, 1943; Schultz and Olson, 1940; Anellis, Lubas, and M1orton, 1954). 'Correction for "thermal lag" (time lag required for internal food temperature to reach bath temperature when TDT tubes are plunged into the bath) and "lethality during lag" (thermal destruction of bacteria in the food during the interval required for the food to reach the bath temperature) were made employing the procedure described by Anellis et al. (1954), and resulted in a mean correction factor for each of the foods as follows: custard, 1.6; chicken a la king, 1.5; and ham salad, 1.4. These values correspond very closely to those reported by others for various foods in glass TDT tubes (Ball et al., 1937; Sognefest and Benjamin, 1944; Anellis et al., 1954; Osborne, Straka, and Lineweaver, 1954). These values, subtracted from the total exposure time in the bath, gave endpoints of "survival-kill" corrected for thermal lag and lethality due to lag. Employing these points, corrected thermaldeath-time curves were constructed and defined in terms of their F140 (minutes of exposure at 140 F necessary to reduce the inoculum to a level which is not detectable) and ZF values (number of degrees F necessary for the thermal-death-time curve to traverse one log cycle). These corrections are insignificant when the times are of relatively short duration. For this reason, a greater degree of certaintv is associated with the survival-kill points of the corrected TDT curves at the longer exposure times than at those for exposures of less than 10 minutes. D values (minutes of exposure at 140 F necessary to effect a 90% reduction in original inoculum) and ZD values (number of degrees F necessary for the decimal reduction time curve to traverse one log cycle) were calculated by the probability procedure of Schmidt (1957) and decimal reduction time curves were constructed. RESULTS The results obtained in custard and chicken a la king with the test salmonellae and staphylococci are summarized in Table 1. The F140, ZF, D140, and zd values obtained in duplicate experiments along with average values are shown in Tables 2 and 3. These data reveal several points of interest in respect to the thermal resistance of the two groups of organisms in chicken a la king and custard. As would be expected, rather large differences in the heat resistance of the salmonellae and staphylococci were observed. Excluding, momentarily, the results obtained with the exceptionally heat-resistant S. senftenberg 775W (Solowey, Sutton, and Calesmick, 1948; Anellis et al., 1954; Osborne et al., 1954; Angelotti et al., 1959), the staphylococci displayed greater heat resistance than the remaining salmonella species. This is in keeping with the findings for broth cultures reported earlier (Angelotti et al., 1959). Also, in keeping with these findings, very little difference in heat resistance was observed among the staphylococci; however, S. aureus 196E gave greater F140 values than Ms149. This was unexpected, in view of the earlier work which indicated that broth cultures of Ms149 were more heat resistant than 196E. S. senftenberg 775W displayed the greatest heat resistance of any of the organisms studied. The F140 values in custard (78.0) and chicken a la king (81.5) were quite similar. It is important to note that this organism was originally isolated from egg products; therefore, it is reasonable to assume it may, on occasion, contaminate prepared foods containing eggs. It is equally important to note, that the extreme heat tolerance of S. senftenberg 775W is not a common property of this serological type, as was shown by Anellis et al. (1954), with strains 523, 2623, and 3252, which were also isolated from egg products. S. manhattan was the least heat-resistant organism studied and yielded F140 values more in keeping with those generally recognized for salmonellae (Solowey et al., 1948; Anellis et al., 1954; Osborne et al., 1954). Custard afforded greater protection against heat destruction than chicken a la king, and the slope of the TDT curve in custard was not as steep. This was the only instance observed in which the food menstruum apparently affected the slope of the TDT curve. Kaplan, TABLE 1. Thermal resistance of salmonellae and staphylococci in custard and chicken d la king Minutes required to kill 1 X 107 organisms per gram* Exposure Staphylococcus S. aureus 196E Salmonella Salmonella Temp aureus Ms149 senftenberg 775W manhattan Cus- Chicken Cus- Chicken Cus- Chicken Cus- Chicken tard a la king tard a la king tard a la king tard a la king t t * Average value from duplicate experiments. t Extrapolated.

5 312 R. ANGELOTTI, M. J. FOTER, AND K. H. LEWIS [VOL. 9 Reynolds, and Lichtenstein (1954), have shown that z values are independent of the menstruum in which the organism is heated, and that the observed variation in individual z values for different foods fall within statistical limits of experimental error. The variation for z observed with S. manhattan in custard and chicken a la king may represent such experimental error in that both are low acid foods and neither contains any substances in great enough concentration to inhibit survivors upon product incubation at 95 F (Angelotti et al., 1959). In custard and chicken a la king, both strains of S. aureus were found to possess an intermediate degree of heat resistance between the high of S. senftenberg 775W and the low of S. manhattan (Tables 2 and 3). For comparison with the F140 values, D140 values are also presented in Tables 2 and 3. The D140 data emphasize the relatively short period of time, from that of the whole heat treatment, required to kill 90% of the bacterial population. Because D is independent of initial bacterial c oncentration (Youland and Stumbo, 1953; Pflug and E sselen, 1954; Schmidt, 1957), although some evidence exists to the contrary (Reed, Bohrer, and Cameron, 1951; El-Bisi and Ordal, 1956a, b), a greater Organism TABLE 2. flexibility is available for interpreting heat resistance data than is possible by thermal-death-time determinations alone. From the time of Bigelow and Esty (1920), it has been recognized that the heat resistance of an organism is dependent upon initial concentration. Because of this, F values for different suspensions are not comparable, unless the initial inocula were identical. By employing D, it is possible to determine the heat resistance of an organism and make direct comparisons between data in which various sized inocula were employed. In freeing the investigator from dependence upon inoculum size, a method is available that enables him to determine thermal resistance in a given substrate under various conditions of bacterial load. This fact is particularly important in a study of the type presented here. From a practical standpoint, it is impossible to measure the heat resistance of an organism in prepared dishes under all levels of bacterial concentrations. But in calculating D values, a measure of thermal resistance is derived, based on a limited number of experiments, which is applicable through the many predictable bacterial concentrations attainable in a given food. Under practical conditions, ham salad would not be Heat resistance of salmonellae* in custard and chicken d la king Custard Chicken a la king Fl40t ZFt Di4o ZDT Fl40 ZF D140 zd Salmonella senftenberg W S. senftenberg 775W Avg S. manhattan S. manhattan Avg * Approximately 10,000,000 organisms per tube. t Minutes of exposure at 140 F necessary to reduce the inoculum to a level which is not detectable. t Number of degrees F necessary for the thermal-death-time curve to traverse one log cycle. Minutes of exposure at 140 F necessary to effect a 90% reduction in original inoculum. Number of degrees F necessary for the decimal reduction time curve to traverse one log cycle. Organism TABLE 3. Heat resistance of staphylococci* in custard and chicken d la king Custard Chicken a la king Fijot ZF DI40 ZD F140 ZF D140 ZD Staphylococcus aureus E S. aureus 196E Avg S. aureus Ms S. aureus Ms Avg * Approximately 10,000,000 organisms per tube. t See footnotes to Table 2 for explanation of symbols.

6 19611 THERMAL DEATH OF SALMONELLAE AND STAPHYLOCOCCI IN FOOD 313 served as a hot food; however, a few trials were made with this food for comparative purposes. These experiments revealed that both the salmonellae and staphylococci gave lower end points of survival than in custard or chicken a la king. Because holding temperatures for hot foods are best based on the survival of the most heat-resistant organism likely to be encountered in a food affording it maximum heat protection, little merit was associated with determining TDT and D values for the test organisms in ham salad. However, Table 4 shows the "survival-kill" points attained with S. sen tenberg 775W and S. aureus Ms149 in this food. DISCUSSION Judging from the values presented for D140, 90 % destruction of the population of the most heat-resistant organism studied (S. senftenberg 775W) occurred in approximately 10 to 11 min in both custard and chicken a la king, and the same degree of destruction for the staphylococci occurred in approximately 5 to 8 min. When the number of salmonellae remaining in a food after a 90 % reduction is not equivalent to an infective dose for the susceptible group under study and provided subsequent contamination or growth of the remaining organisms is prevented, little danger is associated with the ingestion of such a food. According to McCullough and Eisele (1951), the adult oral infective dose for several strains of S. melagridis and S. anatum ranged between 587 thousand to 67.2 million. However, in those instances where a 90 % reduction still results in numbers approximating an infective dose for the susceptible group under study, danger does exist. With S. senftenberg 775W in chicken a la king, all the cells were killed after 81.5 min of exposure at 140 F and 90% reduction occurred after 9.61 min. From the slope of thermaldeath-time curve (ZF 11.45) and the decimal-reductiontime curve (ZD 11.83), we find that a temperature of 152 F would yield the same results, but with better than a tenfold reduction in time. However, even this information does not tell the investigator whether an TABLE 4. Heat resistance of Salmonella senftenberg 7751W and Staphylococcus aureus Msl49 in ham salad* Time of exposure Organism Exposure Last interval First interval at which some at which all tubes were tubes were positive negative F min min S. senftenberg 775W S. auireus Ms * Approximately 10,000,000 organisms per tube. infective dose exists after such a heat treatment, unless he knows the initial concentration of cells; a situation rarely encountered in practice. But, by employing D alone, any arbitrary degree of destruction can be achieved irrespective of the initial concentration. For example, if D = 90% reduction, then a process of 2D = 99 % reduction, etc. Assuming an exaggerated situation in which 1 X 107 salmonellae per gram of food are present, a process equivalent to 6D would yield a % reduction in numbers, or 10 salmonellae per gram. From the data presented, it is seen then that a process of 6D would have reduced ten million S. senftenberg 775W per gram of custard to an innocuous level of 10 salmonellae per gram, after min of exposure at 140 F. At the same time, this exposure results in complete destruction in either food of S. manhattan and the staphylococci. However, an extended exposure of this type may not be practical. From observing the decimal reduction time curve (see Fig. 1), it is seen that 90% reduction of S. senftenberg 775W in custard occurs after 1.68 min at 150 F. A process of 6Di5o (10.08 min at 150 F) results in the same degree of destruction of S. senftenberg 775W in custard, as cited in the above example and simultaneously gives complete destruction of this organism in chicken A la king, as well as complete destruction of the remaining organisms in all the foods. Because z has been shown to be independent of the menstruum in which vegetative organisms are heated, and because D provides a measurement of heat resistance independent of initial concentration of cells, a method is available of evaluating holding times and temperatures necessary to eliminate salmonellae and staphylococci from foods. The extreme heat resistance of S. senftenberg 775W and its occurrence in egg products demands that any heat treatments proposed to eliminate salmonellae from foods be based on the requirements necessary to destroy this organism. Fortunately, the F140 value for other strains of salmonellae, as shown here and reported by others (Winter et al., 1946; Solowey et al., 1948; Goresline et al., 1951; Osborne et al., 1954; and Anellis et al., 1954) is of the order of 3 to 4. These values are considerably less than required for S. senftenberg 775W. Heat resistant, enterotoxigenic staphylococci also require shorter heating times (F140 of 40 to 59). Therefore, a process capable of destroying S. senftenberg 775W would necessarily destroy other salmonellae and staphylococci with some margin of safety. Because the extended time necessary to kill at 140 F may be impractical under actual conditions of food service, higher holding temperatures may be advantageous. The results reported here indicate that heating perishable foods of the type employed in this study to 150 F and holding every particle of food at this temperatuire for no less than 12 min reduces 10 million or fewer salmonellae or staphylococci per gram to levels not detectable by our methods. In practice, exposure

7 314 s fi_ ~ _ I-m B.. -A~~+F ~~~~~Y 0 ~ ~ m3a5-iw9ns:.kt R. ANGELOTTI, M. J. FOTER, AND K. H. LEWIS w 10 e 1-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 0~~~~~~~~~~~ =F _ F3= W~~~~~~~~~~~~~T M ERTRE 0 EXPOSUR TEMPERATURE OF EXPOSURE_ ; _. FIG. 1. Heat resistance of Salmonella senftenberg 775W in custard; approximately l X 107 cells per tube [VOL. 9

8 1961] THERMAL DEATH OF SALMIONELLAE AND STAPHYLOCOCCI IN FOOD 315 times of longer durationi at 150 F or of equivalent duration at temperatures above 150 F would provide a safety factor. Because destruction time increases with a decrease in temperature, prolonged holding at 140 F is necessary to achieve results comparable to those cited above. However, if the level of contamination were approximately 1,000 organisms per gram of the test food, it is estimated that a process equivalent to 4D140 or 45 min at 140 F would, in most instances, eliminate detectable survivors. LITERATURE CITED American Public Health Association Standard methods for the examination of dairy products. 10th ed., New York. p. 95. ANELLIS, A., J. LUBAS, AND M. R. MORTON Heat resistance in liquid eggs of some strains of the genus Salmonella. Food Research 19: ANGELOTTI, R., E. WILSON, M. J. FOTER, AND K. H. LEWIS Time-temperature effects on salmonellae and staphylococci in foods. I. Behavior in broth cultures and refrigerated foods. The Robert A. Taft Sanitary Engineering Center Technical Report F59-2. ANGELOTTI, R., M. J. FOTER, AND K. H. LEWIS. 1961a. Time-temperature effects on salmonellae and staphylococci in foods. I. Behavior in refrigerated foods. Am. J. Public Health 61: ANGELOTTI, R., M. J. FOTER, AND K. H. LEWIS. 1961b. Time-temperature effects on salmonellae and staphylococci in foods. II. Behavior at warm holding temperatures. Am. J. Public Health 61: BALL, C Short-time pasteurization of milk. Ind. Eng. Chem. 36: BALL, C. 0., C. N. MERRIL, C. 0. WILLIAMS, AND D. J. WESSEL Unpublished data presented by P. Sognefest and H. A. Benjamin Heating lag in thermal-deathtime cans and tubes. Food Research 9: BIGELOW, W. D., AND J. R. ESTY Thermal death point in relation to time of typical thermophilic organisms. J. Infectious Diseases, 27: EL-BISI, H. M., AND Z. S. ORDAL. 1956a. The effect of certain sporulation conditions on the death rate of Bacillus coagulans var. thermoacidurans. J. Bacteriol. 71:1-9. EL-BIsI, H. M., AND Z. S. ORDAL. 1956b. The effect of sporulation temperature on the thermal resistance of Bacillus coagulans var. thermoacidurans. J. Bacteriol. 71: GORESLINE, H. E., M. H. KIRBY, R. E. MOSER, M. A. HOWE, JR., AND E. E. DREWNIAK Pasteurization of liquid whole egg under commercial conditions to eliminate Salmonella. U. S. Dept. Agr. Circular No U. S. Govt. Printing Office, Washington. KAPLAN, A. M., H. REYNOLDS, AND H. LICHTENSTEIN Significance of variations in observed slopes of thermaldeath-time curves for putrefactive anaerobes. Food Research 19: MCCULLOUGH, N. B., AND C. W. EISELE Experimental human salmonellosis. I. Pathogenicity of strains of Salmonella melagridis and Salmonella anatum obtained from spray-dried whole egg. J. Infectious Diseases 88: OSBORNE, W. W., R. P. STRAKA, AND H. LINEWEAVER Heat resistant strains of salmonella in liquid whole egg, egg yolk, and egg white. Food Research 19: PFLUG, I. J., AND W. B. ESSELEN Observations on the thermal resistance of putrefactive anaerobe no spores in the temperature range 250 to 300 F. Food Research 19: REED, J. M., C. W. BOHRER, AND E. J. CAMERON Spore destruction rate studies on organisms of significance in the processing of canned foods. Food Research 16: SCHMIDT, C. F Thermal resistance of microorganisms. In G. F. Reddish [ed.], Antiseptics, disinfectants, fungicides, and physical and chemical sterilization. ch. 32, 2nd ed. Lea and Febiger, Philadelphia. 975 p. SCHULTZ, 0. T., AND F. C. W. OLSON Thermal processing of canned foods in tin containers. III. Recent improvements in the general method of thermal process calculations. A special coordinate paper and methods of converting initial and retort temperatures. Food Research 6: SOGNEFEST, P., AND H. A. BENJAMIN Heating lag in thermal-death-time cans and tubes. Food Research 9: SOLOWEY, M., R. R. SUTTON, AND E. J. CALESMICK Heat resistance of Salmonella organisms isolated from spray-dried whole-egg powder. Food Technol. 2:9. STUMBO, C. R., C. E. GROSS, AND C. VINTON Bacteriological studies relating to thermal processing of canned meats. Food Research 10: WINTER, A. R., G. F. STEWART, V. H. MCFARLANE, AND M. SOLOWEY Pasteurization of liquid egg products. III. Destruction of Salmonella in liquid whole egg. Am. J. Public Health 36: YOULAND, G. C., AND C. R. STUMBO Resistance values reflecting the order of death of spores of Bacillus coagulans subjected to moist heat. Food Technol. 7: