ENGINEERING RESEARCH INSTITUTE THE UNIVERSITY OF MICHIGAN ANN ARBOR Progress Report No. 2 COMBINED USE...OF HEAT AND RADIATION TREATMENT FO.STERILIZ-AT IOT OF. FOODS Period: 7 August 1955 - 7 October 1955 Lloyd' L Kenmpe Official Inves ti-gatoi J. T. Graikoski Nancy J. Williams Peter F. Bonventre Collaborators Project 2391 QUARTERMASTER RESEARCH AND DEVELOPMENT COMMAND NATICK, MASSACHUSETTS CONTRACT NO. DA-19-129-qm-388, PROJECT NO. 7-84-01-002 October 1955

TABLE OF CONTENTS Page INTRODUCTION 1 EVALUATION OF CONSECUTIVE IRRADIATION AND HEAT TREATMENT FOR STERILIZING CANNED MEAT 2 PROCEDURE 2 EFFECTS OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES 8 EFFECTS OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES 16 SUMMARY 16 METHODS AND MATERIALS 16 Organisms 16 Chemicals 17 Control 17 Irradiation Procedures 17 Results 17 PRELIMINARY COMPARISON OF THE Z VALUE OF IRRADIATED AND UNIRRADIATED SPORES OF PA 3679 21 MATERIALS AND METHODS 21 RESULTS 22 CONCLUSION 22 ii

CONTRACT RESEARCH PROJECT REPORT QUARTERMASTER FOOD AND CONTAINER INSTITUTE FOR THE ARMED FORCES, CHICAGO Hq, QM Research and Development Command, QM Research and Development Center, Natick, Mass. The University of Michigan Engineering Research Institute Ann Arbor, Michigan Official Investigator: Lloyd L. Kempe Collaborators: J. T. Graikoski Nancy J. Williams Peter F. Bonventre Title of Contract: Combined 1 Treatment Project No. 7-84-01-002 Contract DA-19-129-qm-388 File No. S-510 Report No. 2 (Progress) Period: 7 August 1955 to 7 October 1955 Initiation Date: 7 JCnTe 1955 Use of Heat and Radiation for Sterilization of Foods INTRODUCTION The work accomplished during this second reporting interval will be presented under four headings, viz.: (1) Evalution of consecutive irradiation and heat treatment for sterlizing canned meat. (2) Effects of temperature during irradiation on the survival of bacterial spores. (3) Effects of chemical composition of the medium during irradiation on the survival of bacterial spores. (4) Determination of the Z value of irradiated C. botulinum spores. 1

EVALUATION OF CONSECUTIVE IRRADIATION AND HEAT TREATMENT FOR STERILIZING CANNED MEAT Previous work has shown that anaerobic bacterial spores are sensitized by preliminary irradiation so that they are killed much more rapidly by subsequent heating than are unirradiated spores. The present study is designed to determine whether the sensitization phenomenon can be successfully applied for reducing the amount of heat required to sterilize canned meat. During the present reporting interval the following has been accomplished. (1) The autoclave and accessory equipment necessary for following the temperature inside No. 1 picnic tin cans of ground beef has been installed. For this purpose 0. F. Eckland thermocouples and a Leeds and Northrup Portable Precision Potentrometer are in use. (2) A standard operating procedure has been developed for packing, inoculating, irradiating, and heat-processing ground beef in No. 1 picnic cans. (3) The procedure described in (2) has been used to determine the Fo required to sterilize unirradiated ground beef in No. 1 picnic cans, which is the necessary starting point for this study. PROCEDURE a. Packing. —Lean ground beef is purchased locally from The University of Michigan food stores. The meat is placed in shallow pans and autoclaved at 15 psig steam pressure for one-half hour. Excess liquid is poured off and the hot meat is packed into 28 No. 1 picnic cans, four of which have been fitted previously with 0. F, Ecklund thermocouples. Covers are set loosely on the cans, which are then placed in an autoclave where they are sterilized at 17 psig steam pressure for one hour. Next, each can is removed individually from the hot autoclave, inoculated with 2 ml of a spore suspension, sealed in a commercial-type closing machine, immersed in cold tap water for about 20 minutes, and finally placed in ice water for an hour. Experimental cans are then either irradiated or temporarily stored in a refrigerator as required. The 8 controls are placed in a 370~C incubator immediately, while the experimental cans are incubated after processing. Processed cans are, of course, quickly cooled to 370~ by immersion in cold water before incubation. 2

b. Irradiation.-The University of Michigan cobalt-60 source will accommodate 8 No. 1 picnic tin cans at once in the "center well," where the dosage rate is,now somewhat less than 200,000 rep per hour. In the warm weather seasons it is necessary to refrigerate the cans during irradiation. This is satisfactorily accomplished with dry ice, but it is more convenient to do this work from late October to April, when the "cave" temperature is near 40C. Irradiation can also be accomplished outside the source. c. Heat Processing.-Several false starts were made before a heat-processing technique was developed that yielded reproducible data. The process is now carried out as follows: 1) A 3-gallon pail is positioned in the upper part of a steamheated autoclave and the pail is half-filled with water at. 180~F. 2) Six cans of meat, two of which contain thermocouples, are removed from the refrigerator, thermocouple leads are attached to the two control cans, and then all six cans are placed in the 180~F water. Temperature measurements of the water in the pail and in the center of each can containing thermocouples are begun immediately. 3) The autoclave cover is clamped shut, and steam is introduced at such a rate that the water surrounding the cans is maintained at 180~F until the two thermocouples in the cans of meat show identical temperatures of 170~F or more. A temperature of 180aF was selected because (1) it represents "hot filling" temperatures of industrial practice, (2) it is not sufficient to cause appreciable killing of spores anywhere in the can during temperature equilibration, and (3) it is high enough to make "come-up" rates to the processing temperature essentially uniform in all the cans. 4) When the cans have equilibrated at some temperature between 170~F and 180~F, the water-bath temperature is quickly brought to 230~F by introducing steam into the autoclave. Processing time to attain the desired Fo value, less the cooling increment, is now provided. 5) At the proper time, the autoclave is quickly opened and the cans are plunged into ice water. Temperature measurements are continued until the temperature at the center of the cans reaches 180oF. 6) The four experimental cans of meat are incubated at 37~C. The two cans containing thermocouples are again refrigerated; they are used 3

a second time only. 7) Four sets of cans are autoclaved for each run, using arbitrarily selected processing times designed to provide suitable Fo increments. After the runs are completed, the actual Fo accomplished for each set of four cans is computed. This, together with the incubation results, constitutes the basic data acquired. 8) Fo values are calculated according to Halvorson's technique (see Kempe, Applied Microbiology, November, 1955, in press), which is a modification of Bigelow's General Method, apparently similar to that described by Ball in 1928. In addition, the calculations are checked by 0, T. Schultz's graphical modification of C. 0. Ball's General Method. In these calculations the Z value of irradiated spores is assumed to be 18. However, this is 6nly an assumption, since it is quite possible that irradiation may alter the Z value. Experimental data are being collected to establish the Z value of irradiated spores. It is concluded from the data shown in Table 1 that ground beef packed in No. 1 picnic tin cans containing approximately 40,000 C. botulinum 213B spores per gram will require an Fo of 1.0 for sterilization. Under otherwise similar conditions, except that No. 2 cans were used, it has been reported previously that 3,500,000 rep of gamma radiation from cobalt-60 were required for the sterilization of ground beef canned in a similar manner. (Kempe et al., Applied Microbiology 2, 330-332,1954.) These data set the limits for the next phase of this work, which will involve determination of the Fo required to sterilize canned beef after it has been irradiated with less than 3,500,000 rep of gamma radiation from cobalt-60. 4

TABLE 1 37~C Incubation Results for No. 1 Picnic Cans of Ground Beef That Have Been Inoculated with C. Botulinum 213B Spores, Irradiated with Gamma Rays from Cobalt-60, and Heat Processed with Increasingly Severe Heat Treatments. Run C-1 Can size - No. 1 Picnic (2-11/16" x 4") Product - Ground Beef Spores - 10,700,000 C. botulinum 213B spores per can Irradiation - None.Can No. Gas Days to Gas Fo Can No. Formation Formation Inoculated 1 + 2 Control 2 + 2 3 + 2 4 + 2 Uninoculated 1 0 Control 2 0 3 0 4 0 F 1 + 4.5 Can 1 0.26 2 + 5 Can 2 0.31 3 + 5 Avg 0.29 4 + 5 Fo 5 + 6.5 Can 1 1.00 6 + 7 Can 2 1.00 7 + 6 Avg 1.00 8 +.11 Fo 9 + 6.,5 Can 1 0.80 10 + 7.5 Can 2 1.25 11 + 7.5 Avg, 1.02 12 + 6.5 Fo, 13 + 6 5 Can 1 0.53 14 + 5~5 Can 2 0.57 15 + 7*5 Avg 0.56 16 + 5.5 Result: Fo required is more than, but very close to, 1.0, Remarks: Used preheat water-bath temperature at 210~F and bath at 230~F. 5 sterilizing

TABLE 1 (Cont.) Run C-2 Can size Product Spores Irradiation No. 1 Picnic (2-11/16" x 4") - Ground Beef - 7,200,000 C. botulinum 213B per can - None Can No. Gas Days to Gas F, Can No. ______ u ________ _____Format ion Format ion Inoculated 1 + 1.5 Control 2 + 1 5 3 + 1.5 4 + 1.5 Uninoculated 1 0 Control 2 0 3 0 4 0 F 1. + 3.5 Can 1 0.28 2 + 3.5 Can 2 0.39 5 + 3.5 Avg 0.534 4 + 35.5 Fo 5 0 Can 1 0.70 6 0 Can 2 0.73 7 0 - Avg 0072 8 + 3..5 Fo 9 + 3.5 Can 1 0.75 10 + 45 Can 2 1.01 11 + 4.5 Avg 0.88 12 0F 13 0 Can 1 1.24 14 0 Can 2 1.29 15 0 Avg 1.27 16 0 Result: Fo required is between 0.8 and 1.3. Remarks: Used preheat water-bath temperature bath at 230~F. at 210~F and sterilizing 6

TABLE 1 (Concl.) Run C-3 Can size Product Spores Irradiation - No. 1 Picnic (2-11/16" x 4") - Ground Beef - 10,700,000 C. botulinum 213B spores per can - None Gas Days to Gas Fo Can No. Formation Formation Inoculated 1 + 1.5 Control 2 + 1.5 3 + 1.5 4 + 1.5 Uninoculated 1 0 Control 2 0 3 0 4 0 Fo 1 + 6 Can 1 00 6 2 + 6 Can 2 0.49 3 + 6 Avg 0.43 4 + 6 Fo 5 0 Can 1 0.9 6 0 Can 2 0.93 7 0 Avg 0.93 8 0 Fo 9 0 Can 1 1.18 10 0 Can 2 1.53 11 0 Avg 1.36 12 0 Fo 13 0 Can 1 1.59 14 0 Can 2 1.82 15 0 Avg 1.71 16 0 Result: Fo required is between 0.4 and 0.9. Remarks: Used preheat water-bath temperature bath at 230~F. at 190~F and sterilizing 7

EFFECTS OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES During the past few months, three lines of investigation have been followed to evaluate some of the combined effects of temperature and irradiation on anaerobic bacterial spores suspended in M/15 phosphate buffer at pH 7.0. First, we have investigated the effect of temperature during irradiation on the survival of these spores; second, we have been interested to learn whether irradiation at a high temperature affected the sensitization of spores to the subsequent lethal action of heat; and third, we have tested the effect of different holding temperatures following irradiation of the spores. Data in Table 2 and Fig. 1 show that C. botulinum 213B spores are least sensitive to the lethal action of gamma rays in the room temperature range of 15 to 30~C. PA 3679 spores exhibit a similarly narrow temperature range in which they are least sensitive to gamma radiation, but the range is higher, lying between 70 and 100~C. Table 3 and Fig. 2 present data to show that spores of C. botulinum 213B are sensitized to the subsequent lethal action of heat at 100~C whether they are preirradiated at 10 or 90~C. The slight difference in sensitivity indicated between spores irradiated at these temperatures may be more apparent than real, Table 4 and Figs. 3 and 4 present data showing the effect of heating previously irradiated spores. Figure 3 shows that irradiated C, botulinum spores must be heated to a minimum, critical temperature of 800C before killing occurs. This temperature is also the critical lethal temperature for unirradiated spores. So, although preirradiation causes C. botulinum spores to be much more rapidly killed by subsequent heating, it does not alter the fact that the spores must be heated to the high temperature of 800C before the lethal effect of temperature is manifest. Figure 4 points out the increased importance of the irradiation sensitization of C. botulinum 213B spores as progressively higher killing temperatures above 80'C are utilized. 8

TABLE 2 Effedt of Temperature During Irradiation with Gamma Rays From Cobalt-60 on the Survival of Anaerobic Bacterial Spores Suspended in M/15 Phosphate Buffer at pH 7,0 Percent Survivors Temp, oC Dosage Megarep 0_.550 1 0.647 1 0.740 PA 3679 5 1.75 1.05 30 4.65 0.697 0.500 56 8.75 0.675 58 12.3 0.229 80 12.7 3.62 85 58.5 20.0 17.2 95 47.5 11.7 C. botulinum 213B -70 8.7 2.8 0.8 -7 2.0 0.3 5 23. 0.06 27 11.6 10.5 30 14.o 4.4 0.88 56 10.0 0.3 58 2.9 0.4 80 2.9 o.14 95 o.16 9

0 | I2 -J Ii 8 -I00 -80 -40 0 40 80 -100 -80 -40 0 40 80 100 TEMPERATURE ~C Fig. 1i Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of anaerobic bacterial spores suspended in M/15 phosphate buffer at pH 7.0. 10

TABLE 3 Effect of Temperature During Irradiation on the Survival of C. Botulinum 213B Spores When They are Subsequently Heated at 100~C, Both Irradiation and Heating Being Carried Out in M/15 Phosphate Buffer at pH 7.0 Control Irradiated at 10~C Irradiat Minutes at Spores Spores Spores 100~C per ml % Log % per ml %Log % per ml Actual Effectivel Survivors Survivorsl Survivors Survivors Sury 0 0 2,200,000 100.0 2.00 230,000 100.0 2.000 600,000 100. 10 8.54 1,100,000 50.0 1.70 160,000 69.5 1.842 61,000 10. 20 18.54 460,000 20.9 1.32 9,700 4.21 0.624 3,200 0. 30 28.54 85,000 3.86 0.587 1,600 0.695 -0.158 750 0. 40 38.54 22,000 1.00 0,000 36 0.156 -0.807 160 o. 50 48.54 4,400 0.200 -0.699 30 0.0130 -1.886 13 0,,0 2.000 2 1.009 533 -0.273.125 -0.903.0267 -1.574.00216 -2.666

2.0C I.OC O.OC Cf) ar -I.OC V) 0 o -2.0C -3.OC Key X - Unirradiated Control o-Irradiated at 10 ~C \A-Irradiated at 90 ~C 1-^-,. X-I j x 0 20 40 60 EFFECTIVE HEATING TIME AT 100 ~C - MINUTES 80 Fig. 2. Effect of temperature during irradiation on the survival of C. botulinum 213B spores when they are subsequently heated at 1000C, both irradiation and heating being carried out in M/15 phosphate buffer at pH 7.0. 12

TABLE 4 Survival of C. Botulinum 213B Spores Suspended in M/15 Phosphate Buffer at pH 7.0, Which Have Been Irradiated at 5~C with Gamma Rays From Cobalt-60 and Then Held for One Hour at the Indicated Temperature H \4 Control 100,000 rep 200,000 rep _ 400,000 rep Temp Spores Log % Spores Log % Spores Log % Spores Log ~C per ml Survivors per ml Survivors per ml Survivors per ml Survivors 5 1,050,000 2.000 820,000 2.000 730,000 2.000 370,000 2.000 50 1,050,000 2.000 1,000,000 2.083 630,000 1.940 320,000 1.937 60 930,000 1.947 670,000 1.912 970,000 2.124 240,000 1.813 70 960,000 1.961 620,000 1.879 550,000 1.877 450,000 2.083 80 590,000 1.749 620,000 1.879 720,000 1.984 330,000 1.950 90 630,000 1.778 520,000 1.803 240,000 1.513 106,000 1.457 95 310,000 1.450 11,000 1.335 50,000 0.914 2,800 0.013 100 1,700 -0.791 170 -1.894 40 -2.261 0

I t (I y C OQ rl. () 03 (1) D H c-1 d n.-' 0 Pi O > n > ( > ~ ~ o _'o (D /) j 0 CD ) 3 lao iS V 0 " 0 (D ct (D c+n ff Ha ( *'4' O S O O (D {1 C- C) ru 0l Ul F CD 2.00 o, -, I _ 2,4 %2 I:2 -I- iQ I __________________________________________________.I I 1.00 0.00 -1.00 -2.00 -3.00 Key 0- Control Held for I hour at \ I- Irradiated for 100,000 rep each temperature 2- Irradiated for 200,000 rep 10l spores/ml 4- Irradiated for 400,000 rep I. I iI 0 50 60 70 TEMPERATURE ~C 80 90 100

2.00 1.00 ME m A; - t -'~~~omw*.,Q li1 II 7 C) 0 > 0) o 0 -J i 0.00 II 1 I P M I.uu -2.00 -3.00 Key X- 80~C o -- 900C A - 95 ~C,.._oI 0 D-100 OC 0 0.1 0.2 0.3 0.4 Rep x 106 Fig 4. Survival of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0, which have been irradiated at 5~C with gamma rays from cobalt-60 and then heated for one hour at the indicated temperature.

EFFECTS OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES SUMMARY It has been previously reported that certain chemicals present in the suspending medium during irradiation of C. botulinum 62A spores, reduce the lethality of gamma radiation from cobalt-60 for these spores. This work has been extended, using C. botulinum 213B and some additional chemical agents. It has been found that the chemicals that reduce the lethality of gamma radiation for spores of C. botulinum 62A also reduce the lethality of such radiation for spores of C. botulinum 213B. Sodium hydrosulfite and glutathione have been tested with both strains of C. botulinum spores and have been observed to be quite effective in reducing the lethality of gamma radiation, sodium hydrosulfite being the most active protective agent tested so far. This established fact, that many chemicals reduce the lethality of gamma radiation for anaerobic bacterial spores, emphasizes the conclusion that caution must be exercised in stating the lethal dose of irradiation to be used for a specific food or in any particular application. METHODS AND MATERIALS Organisms C. botulinum 213B spores were grown for three weeks in a casitone medium at 300C, washed in sterile water to remove the mother liquor and vegetative cells, and then collected by centrifugation. The spores were then heated at 850C for 15 minutes to kill any remaining vegetative cells. Immediately before use, the spores were shaken with sterile glass beads for 3 minutes to disperse clumps. These stock suspensions contained approximately 4 x 108 spores per ml. For use, appropriate dilutions were made to provide more than one million spores per ml. PA 3679 spores were grown for three weeks in pork extract broth at 30~C. They were then harvested and pretreated in an identical manner to that previously described for C. botulinum 213B spores. 16

Chemicals All solutions were prepared in 0o02-M concentration. Appropriate amounts of the chemicals were weighed out aseptically into sterile weighing bottles, mixed with the required solution, and used in experiments without sterilization. Glutathione.-0o312 gm of glutathione was added to 50 ml of sterile M/15 phosphate buffer of pH 7.0. Sodium Hydrosulfite..-0.174 gm of sodium hydrosulfite was added to 50 ml of sterile M/15 buffer of pH 7.0, Methionine, -0.298 gm of methionine was added to 100 ml of sterile phosphate buffer of pH 7.00 Control In every case, sterile M/15 phosphate buffer of pH 7.0 was used as the control solution. Irradiation Procedures All samples were irradiated in flame-sealed glass vials that contained approximately 4 ml of solution. These were placed in an es-pecially designed container which provided temperature control at 40C. The containers and vials were then irradiated for proper intervals in the center well of the large cobalt-60 gamma radiation source. Results The data taken during this reporting interval with C. botulinum 213B spores substantiate; previous data taken with C. botulinum 62A spores, by showing that several chemicals reduce the lethality of gamma radiation for C. botulinum spores. Methionine, glutathione,and sodium hydrosulfite all have marked effects in this respect as is shown in Tables 5 and 6 and in the companion figures 5 and 6. 17

TABLE 5 Effect of Methionine in M/15 Phosphate Buffer of pH 7~0 on the Lethality of Gamma Radiation from Cobalt-60 for the Spores of C. Botulinum 213B Control Methionine Irradiation Spores Log % Spores Log % Dose Rep per ml Survivors per ml Survivors 0 8,700,000 2.000 7,800,000 2.000 360,000 3,150,000 1.559 5,350,000 1.836 540,000 2,260,000 1.462 720,000 83,00oo -0.020 750,000 0.983 900,000 4,500 -1.287 190,000 0.387 1,080,000 128 -2.831 27,100 -0.459 TABLE 6 Effects of Glutathione and Sodium M/15 Phosphate Buffer at pH 7.0 on Hydrosulfite Added to the Lethality of Gamma Radiation from Cobalt-60 for the Spores of C, Botulinum 213B..Control Glutathione Sodium Hydrosulfite Irradiation Spores Log % Spores Log % Spores Log % Dose Rep per ml Survivors per ml Survivors per ml Survivors 0 7,770,000 2.000 7,500,000 2.000 7,300,000 2.000 360,000 3,600,000 1.670 5,200,000 1.841 7,100,000 1.988 540,000 645,000 0.923 3,700,000 1.693 5,700,000 1.893 720,000 57,000 -0.131 2,370,000 1.500 3,850,000 1.722 900,000 54,000 -1.154 1,100,000 1.166 2,480,000 1.531 1,080,000 49 -3.192 410,000 0.738 1,119,500 1.214 1,260,000 6 -4.108 112,500 0.176 615,000 0.926 1,440,000 0 49,500 -0.180 241,000 0.519 18

+2 +1 C() 0 _r 0 0 -J 0 X% I —\ —x! Key(213-B) Key(213-B) - Control X-Methionine iI -I -2 -3 0 4.8 12 MEGA- REP Fig. 5. Effect of methionine in M/15 phosphate buffer of pH 7.0 on the lethality of gamma radiation from cobalt-60 for the spores of Co. botulinum 213B. 19

+2 \.\ Cr) O -I -3 -3 O \ Key: 213 -B 0 - Control X - Glutathione -Na2S2O4 -5 0.8 1.2 1.6 MEGA - REP Fig. 6. Effects of glutathione and sodium hydrosulfite added to M/15 phosphate buffer at plI 7.0 on the lethality of gamma radiation from cobalt-60 for the spores of C. botulinum 213B.,,,, 20

PRELIMINARY COMPARISON OF. THE Z VALUE OF IRRADIATED AND UNIRRADIATED SPORES OF PA 3679 An investigation designed to determine whether a difference exists in the Z value of irradiated and unirradiated spores was deemed necessary since (1) all calculations made thus far in studies pertaining to inactivation of spores which had been exposed to irradiation were made on the assumption that the Z value was 18, (2) any appreciable change in the Z values caused by irradiation would markedly affect the rationale of the methods now employed in calculating the Fo values when combined heat and irradiation techniques are usedo Consequently, preliminary investigations are being conducted to determine the Z value of irradiated and unirradiated spores. MATERIALS AND METHODS Spores of PA 3679 were obtained by growth in pork-extract broth for 3 weeks at 30~C. Cells were harvested at 4~C and washed 10 times in sterile distilled water. They were resuspended, and vegetative cells were inactivated by heat shocking at 990C for 5 minutes. The concentrated spore suspension was diluted in neutral phosphate buffer until it contained approximately 106 spores/ml. Four ml of such a spore suspension were introduced aseptically into 5-ml neutral glass vials which were then sealed in an oxygen flame One group of vials was exposed to gamma irradiation so that the spores received a dosage of 250,000 rep while another group was not irradiated. Both series of vials were subsequently heat treated at temperatures between 1000C and 1200C for varying lengths of time. This was accomplished by immersing the vials in a constant-temperature oil bath and removing samples at the desired time intervals. A correction was made for the time required for the contents of the vials to reach the temperature of the bath. The entire contents of a vial was inoculated into a Prickett tube containing pork-extract agar, layered with agar and paraffin, and allowed to incubate for 3 weeks. Prolonged incubation was found necessary since the severe heat shocking delayed the germination of the viable spores. Survival points were then obtained as indicated by growth or no growth in the Prickett tubes. 21

RESULTS Tables 7 and 8 and Figs. 7 and 8 show the composite data of two independent runs. Z values were obtained by plotting the logarithm of the time required to inactivate all the spores against the temperature in degrees centigrade. Thermal inactivation curves were drawn in such a way that no points indicating growth lie above the line. The Z values obtained are essentially the same as those reported by Reed et al.* The value of 23 found for irradiated spores was approximately 2 units higher than the value of 21.3 found for nonirradiated spores. Investigations will be continued to determine whether or not this difference is statistically significant. CONCLUSION Preliminary investigations indicate a slight difference for Z values of irradiated and unirradiated spores. Experiments will be continued until valid statistical analysis can be made. Future investigations will also include determinations of the Z value of spores exposed to various levels of gamma irradiation. *Reed, J. M., C. W. Bohrer, and E. S. Cameron, Food Research, 16, 383-408 (1951). 22

TABLE 7 Z Value Determination for Irradiated PA 3679 Spores Suspended in M/15 Phosphate Buffer at pH x 106 Spores Per Tube 7.0 Using 1,8 Heating Growth on Heating Growth on Heating Growth on Time Incubation Time Incubation Time Incubation (min) at 30~C (min) at 30~C (min) at 30~C 125~C 1200C 11"5~ 3 + 4 + 12 + 4 + 6 + 14 + 5 + 8 + 16 + 6 - 10 + 18 + 7 - 12.5 - 20 + 22 + 24 + 26 + 28 30 110~C 105~C 20 25 30 35 4o 45 50 55 60 65 70 75 80 85 + + + + + + + + + + ~.. 90 100 110 130 14o 160 170 180 190 200 210 + + + + + + + 23

TABLE 8 Z Value Determination for Nonirradiated Spores of PA 3679 Suspended in M/15 Phosphate Spores Buffer at pH Per Tube 7.0 Using 4 x 106 Heating Growth on Heating Growth on Heating Growth on Time Incubation Time Incubation Time Incubation (min) at 300C* (min) at 30~C* (min) at 30~C* 125~C 120~C 115~C 5 + 10 + 10 + 6 11 + 15 + 7 12 + 19 + 8 13 - 20 + 9 14 - 25 + 10 15 - 30 + 11- 16 - 35 + 12 17 - 40 45 105~C 40 45 50 55 60 65+ 70 80 90 100 105 + + + + + + + 100 150 175 225 250 240 250 260 270 280 500 + + + + + * + growth - no growth 24

3001 2001 A I I k Irradiated (250,000 rep) Key Growth - * No growth- z 2 F%00J 00 80 60 ---- 4 K 40 - PI —------- 30 —d —-- 20: OI. I x 8 6 -.- Z 23.0 2 4 —--------------------------------------— n~~~~~~~~~~~~~~~ 3 —-------------------------------------- ** --— ~~~~~~~~~~~? i ------------------------ I 105 110 115 TEMPERATURE (~C) 120 125 Fig. 7. Thermal death time curve for irradiated PA 3679 spores suspended in M/15 phosphate buffer at pH 7.0. 25

J 200 100o I 41 K r B!!! OF 0 0 k < Non - irradiated Key Growth- * No growth-0 z 2 l - F 80 60 40; 30, 20 1I0 8 —-— <-Z 21.3 6'' 2 4 —----------------------- 105 110 115 120 TEMPERATURE (~C) Fig. 8. Thermal death time curve for nonirradiated PA 3679 spores suspended in M/15 phosphate buffer at pH 7.0. 125 26