ENGINEERING RESEARCH INSTITUTE THE UNIVERSITY OF MICHIGAN ANN ARBOR Final Report COMBINED EFFECTS OF HEAT AND RADIATION IN FOOD STERILIZATION 7 Jite 1954 to 7 October 1955 Lloyd Lo Kempe Collaborators J. T. Graikoski Nancy J. Williams Project 2272 QUARTERMASTER ACTIVITIES DEPARTMENT OF THE ARMY CONTRACT NOo DA44-109-qm-1826 PROJECT NO. 7-84-01-002 October 1955

TABLE OF CONTENTS Page LIST OF TABLES ivLIST OF FIGURES vi OBJECT viii ABSTRACT.ix SUMMARY 1 I. CONSECUTIVE TREATMENT OF BACTERIAL SPORES WITH HEAT AND GAMMA RADIATION 4 A. SUMMARY 4 B. COMBINED EFFECTS OF HEAT AND GAMMA RADIATION IN FOOD STERILIZATION 4 II. SPECIAL EXPERIMENTS 26 A. EFFECT OF A STORAGE INTERVAL BETWEEN THE TIME OF IRRADIATION AND THE TIME OF PERFORMANCE OF THERMALDEATH TIME STUDIES ON THE HEAT RESISTANCE OF IRRADIATED BACTERIAL SPORES 26 B. EFFECT OF USING UNHEATED BACTERIAL SPORES IN PLACE OF SPORES THAT HAD BEEN HEATED FOR 15 MINUTES AT 85~C WHEN STUDYING THE PROBLEM OF RADIATION SENSITIZATION OF BACTERIAL SPORES TO HEAT 26 III. EFFECT OF TEMPERATURE ON THE SURVIVAL OF BACTERIAL SPORES 31 A. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES 31 B. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SENSITIZATION OF BACTERIAL SPORES TO THE SUBSEQUENT LETHAL ACTION OF HEAT 36 ii

TABLE OF CONTENTS (Concluded) Page IV' EFFECT OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES 42 A. MATERIALS AND METHODS 42 B. IRRADIATION PROCEDURE 43 C. DISCUSSION 43 V. BIBLIOGRAPHY 48 iii

LIST OF TABLES Page I. Effect of preliminary irradiation by gamma rays from cobalt-60 on the heat resistance of C, botulinum 62A spores 7 II. Effect of preliminary irradiation by gamma rays from cobalt-60 on the heat resistance of C. botulinum 213B spores 14 III. Effect of preliminary heat treatment on irradiation resistance of C, botulinum 62A spores to gamma rays from cobalt-60 21 IV. Effect of preliminary heat treatment on irradiation resistance of C. botulinum 213B spores to gamma rays from cobalt-60 24 V, Effect of storage at 4~C between time of irradiation and heating on the sensitivity to heat induced by preirradiation on C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0 28 VI. Effect of preirradiation with subsequent heating at 99~C on unheated C. botulinum 215B spores and on similar spores that had been heated at 85~C for 15 minutes to kill vegetative cells 29 VII. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of C. botulinum 213B when suspended in M/15 phosphate buffer at pH 7.0. 32 VIII. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of PA 3679 when suspended in M/15 phosphate buffer at pH 7-0 54 iv

LIST OF TABLES (Concluded) Page IX. Effect of a combined treatment consisting of irradiation with gamma rays from cobalt-60 followed by heating for 1 hour at 99~C on the survival of PA 3679 spores suspended in M/15 phosphate buffer at pH 7.0 37 X. Effect of temperature during irradiation on the subsequent resistance of C, botulinum 62A spores to heating at 1000C 40 XI. Effect of various chemicals on the lethality of gamma radiation from cobalt-60 on the spores of C. botulinum 62A suspended in M/15 phosphate buffer at pH 7.0. 44 v

LIST OF FIGURES Page 1. Effect of preliminary irradiation with gamma rays from cobalt-60 on the heat resistance of C. botulinum 62A spores suspended in M/15 phosphate buffer at pH 7.0 11 2. Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance of C. botulinum 62A spores suspended in nutrient broth at pH 6.6 12 3. Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance of C. botulinum 62A spores suspended in 10% gelatin at pH 7,0 13 4. Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance'of C. botulinum 213B spores suspended in nutrient broth at pH 6.7 19 5. Effect of preliminary irradiation with gamma rays from cobalt-60 on the heat resistance of C. botulinum 213B spores suspended in 10% gelatin at pH 7.0 20 6. Effect of preliminary heating at 99~C on the subsequent radiation resistance of C. botulinum 213B spores suspended in 10% gelatin at pH 7.0 23 7. Effect of 3 monthts storage at 4~C between the times of irradiation and heating on the sensitivity to heat induced by preirradiation of C. botulinum 213B spores suspended inM/15 phosphate buffer at pH 770 27 8. Effect of preirradiation with 250,000 rep of gamma radiation followed by heating at 99~C on unheated C. botulinum 213B spores and on similar spores that had been heated at 85~C for 15 minutes to kill vegetative cells 30 vi

LIST OF FIGURES (Concluded) Page 9. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of C. botulinum 213B suspended in M/15 phosphate buffer at pH 7.0 33 10. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of PA 3679 suspended in M/15 phosphate buffer at pH 7.0 35 11. Effect of irradiation at 5~C followed by heating for 1 hour at 99~C on the survival of PA 3679 spores in phosphate buffer at pH 7.0 38 12. Effect of irradiation at 95~C followed by heating for 1 hour at 99~C on the survival of PA 3679 spores in phosphate buffer at pH 7.0 39 13. Effect of temperature during irradiation on the subsequent resistance of C. botulinum 62A spores to heating at 100 C while suspended in M/15 phosphate buffer at pH 7.0 41 14. Effect of various chemicals incorporated into M/15 phosphate buffer at pH 7.0 on the lethality of gamma radiation from cobalt-60 on the spores of C. botulinum 62A suspended in the solutions so obtained 46 vii

OBJECT The object of this project is to develop an improved food sterilizing process. This work was carried out to investigate the value of a food sterilization technique involving a combined heat and gamma radiation treatment. If the two energy forms were found to be synergistic in their lethal action on spores, it is possible that some combined heat and irradiation process might be equivalent insofar as the effect on microorganisms is concerned but, at the same time, reduce the damage to the food resulting when either form of energy is used alone for sterilization purposes. Specifically then, this project was concerned with determining whether the combined, lethal effect of heat and gamma radiation on bacterial spores is independent or synergistic in nature. viii V1 11

ABSTRACT Spores of Clostridium botulinum strains 62A and 213B as well as those of PA 3679 have been found to be more rapidly killed by heat after irradiation with gamma rays from cobalt-60 than are unirradiated spores. However, heated spores were not sensitized to the subsequent lethal action of radiation. Hence, if a combined heat and radiation treatment is to be used for food sterilization, irradiation should be carried out first. It was found that storage between the time of irradiation and heating did not reduce the sensitization of the spores to heat caused by the irradiation treatment. When bacterial spores were irradiated at temperatures below those where heat damage occurred it was found that C. botulinum 215B spores were more rapidly killed by gamma radiation as the temperature was increased from -70 to 95~C but PA 3679 spores reacted oppositely, being killed more rapidly at temperatures below 58~C than above 80~C. Studies of the effect of certain chemicals in the medium on the lethality of gamma radiation for bacterial spores showed that many chemicals reduce the lethality of these rays. In particular, reducing agents and sulfur-containing compounds were found to be active in reducing the lethality of gamma radiation for bacterial spores. ix

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 Project No. 7-84-01-002 Contract DA44-109-qm-1826 File No. S-510 Report No. 7 (Final) Period: 7 June 19547 October 1955 Initiation Date: 7 June 1954 Title of Contract: Combined Effects of Heat and Radiation in Food Sterilization SUMMARY As set forth in the contractual agreement, studies have been carried out during the past year to determine the following: 1. Whether the combined killing effect of heat and radiation on food spoilage bacteria present in buffer and in foods is independent, additive, detrimental, or synergistic in nature. It has been found that when C, botulinum spores, suspended in phosphate buffer, nutrient broth, or gelatin, are irradiated., they are subsequently more rapidly killed by heating at 99~C than are the unirradiated spores. A similar effect was found for PA 3679 spores in buffer. This indicates that irradiation and heat are synergistic in their lethal action on spores when heating follows irradiation. On the other hand, when the spores were irradiated after heating no difference in the rate of killing by irradiation was observed. This indicates that the two agents are inde 1

pendent in their lethal action on spores when irradiation follows heating. 2. Whether bacterial spores react differently to the lethal actions of gamma radiation when irradiated at different temperatures. This study is not yet complete but at present it can be said that the effect of temperature during irradiation on the lethality of gamma radiation for bacterial spores suggests that C. botulinum and PA 3679 spores are oppositely affected. The former spores are killed more rapidly by gamma radiation as the temperature of irradiation increases but the latter are less rapidly killed at higher temperatures, 3. What effect any process found in (1) above will have on similar spores in meat. Equipment has been purchased and is being placed in operation to investigate this problem rigorously, since it is the logical application of the positive results reported in (1) above. 4. Whether preliminary irradiation will endow a food with any antiseptic qualities that will increase the effectiveness of a subsequent thermal process. Studies were carried out in nutrient broth, which is essentially a bouillion soup; in gelatin, which is a food; and in guar gum, which is used as a sausage component. The studies indicate that these food materials do not develop detectable antiseptic qualities when irradiated. In fact, the first two protected the spores to a slight degree during irradiation. 5. Whether chemicals or treatments that affect development of flavor in foods by irradiation alter the lethal effects of irradiation. This study is necessarily a continuing one. Up to the present, certain amino acidst sulfur containing compounds, and chemicals that are classed as reducing agents, have been shown to protect spores in varying degrees against the lethal action of radiation. 6. Whether or not a correlation exists between heat and radiation resistance of spores, 2

Work reported by the contractor in Applied Microbiology, Vol. 2, No. 6, p. 530-332, 1954 (but supported by the Michigan Memorial-Phoenix Project) showed that Clostridium botulinum spores are more resistant to gamma radiation than are PA 3679 spores. It is common knowledge, on the other hand, that PA 3679 spores are more resistant to heat than C. botulinum spores (Food Research, Vol. 19, No. 2, p. 173-181, 1954). So at least for these two spores an inverse correlation exists between heat and radiation when used as sporocidal agents. 7. Whether a biological standard for calibrating the effectiveness of radiation sources can be developed based on a bacterial spore of known radiation sensitivity, Observations of variations in heat and radiation sensitivity of bacterial spores used in this work demonstrated the need of extensive controls. This indicates that, while a biological standard could possibly be developed based on the killing rate of a known spore preparation, the reliability and general utility of such a test would likely be inferior to other calibration methods now available. This work was therefore abandoned. 8. Some extra experiments were conducted to answer specific questions that arose during the project Two of these were as follows: a. Does a time interval between irradiation and heat processing affect the sensitization of spores toward heat developed by irradiation? Preliminary experiments indicate that there is either no difference or a slightly increased sensitivity of irradiated spores to heat after 3 montht storage in a refrigeratoro This should permit irradiation of food at one geographical location and shipment to another for heat processing with no decrease in the advantage offered by preirradiation. b. We used spores that had been preheated at 85~C for 15 minutes to free the suspensions of vegetative cells. Would unheated spores have given the same results? Results of a test experiment show that no differences exist between spores preheated for 15 minutes at 85~C and unheated spores insofar as the purpose of our studies are concerned. 3

I. CONSECUTIVE TREATMENT OF BACTERIAL SPORES WITH HEAT AND GAMMA RADIATION A. SUMMARY Clostridium botulinum spores, strains 62A and 213B, were tested for development of radiation sensitivity subsequent to heating and for heat sensitivity following irradiation. Both strains were tested while suspended in M/15 phosphate buffer at pH 7.0, in nutrient broth at pH 6.7, and in 10% gelatin at pH 7.0* Data presented in tables and graphs show that when these spores were first irradiated with gamma rays from cobalt-60 they were killed much more rapidly by subsequent heat treatment than were similar spores that had not been irradiated. For example, preliminary treatment with 900,000 rep of gamma radiation reduced the subsequent heat resistance of C. botulinum spores approximately fourfold when the spores were suspended in the gelatin medium. With regard to heat shocking, the data indicate that preliminary heating did not change the rate at which the spores were inactivated by subsequent irradiation. The nature of the medium in which the spores were suspended during irradiation was found to affect the rate at which the spores were killedo Both strains of C. botulinum were killed more slowly in the nutrient broth and gelatin than in the M/15 phosphate buffer. B. COMBINED EFFECTS OF HEAT AND GAMMA RADIATION IN FOOD STERILIZATION Ionizing radiations are not presently used for sterilizing foods because undesirable flavor changes and other adverse effects often result at the required dosage levels. Morgan and Reed1 have shown that preliminary exposure to ionizing radiations lowers the resistance of some bacterial spores to the lethal effects of subsequent heat treatment. However, no work has been presented to indicate the effects of combined heat aid radiation treatments on the spores of Clostridium botulinum, which are of critical importance in food sterilization. Therefore, this study has been directed toward determining the effect of preirradiation on the heat resistance of C. botulinum spores, as well as the subsequent irradiation resistance of these spores after heat shocking 4

The two strains of C. botulinum that were used in this work were obtained from the Hooper Foundation for Medical Research at the University of California. The spore suspensions were prepared according to the procedures of Reed, Bohrer, and Cameron2 except that Difco bacto-casitone was substituted for casein digest in the growth of medium specified by these workers. Stock spore suspensions were suspended in sterile distilled water and stored at 4~C. The types of toxins produced in the growth media were verified by neutralization tests. Antitoxin used for this purpose was supplied by the New York State Department of Health and the tests were carried out in mice. Before titering, the stock spore suspensions were heated at 85~C for 15 minutes to kill the vegetative cells. The number of viable spores were then determined by dilution counts, using the Prickett tube technique and pork infusion agar containing 0.1% soluble starch. For irradiation tests, the spore suspensions were appropriately diluted in M/15 phosphate buffer, nutrient broth, or 10% gelatin. The buffer and gelatin were adjusted to pH 7.0, but the nutrient broth had a pH of 6.7. The solid gelatin kept the spores suspended during irradiation, but both the buffer and broth allowed the spores to settle. Two series of tests were carried out with both strains of C. botulinum in each of the three media. For the first series, aliquot portions of a spore suspension were placed in each of three 18- x 170-mm pyrex glass test tubes. Two of the tubes were immersed in boiling water; the first was removed after 8 minutes and the second after 25 minutes; the third was an unheated control. Following heat treatment, all three of these tubes containing the spore suspensions were irradiated with gamma rays from cobalt-60 at the rate of 170,000 rep per hour. The temperature of irradiation was not controlled and varied from 5 to 17~C as the temperature of the irradiation room changed with the season. At the end of every hour, the irradiation was interrupted for about 10 minutes while samples were pipetted from each tube for spore counting. When the spores were suspended in "tsolid" gelatin, it was necessary to melt the gelatin by immersing the tube in a 50~C water bath before a sample could be removed. The gelatin was resolidified by plunging the tube into ice water. For tests of the second series, the spore suspensions were placed in test tubes as before. The suspensions were then irradiated in the same way except that no samples were withdrawn. When the irradiation was complete, 5

thermal-death time tests were conducted by placing the tubes in water boiling at 99~C. Samples for counting were pipetted from the tubes and transferred to cold dilution water at the proper time intervals. The number of viable spores were then counted as before. Since the temperatures of the spore suspensions did not rise instantly to 99~C when the test tubes were placed in boiling water, it was necessary to compute the effective-heating time at 990C before a thermal-death time curve could be plotted. A modification of Halvorson's3 procedure was used for this purpose. Data presented in Table I and Figs. 1 through 3 show that preirradiation of C. botulinum 62A spores with gamma rays from cobalt-60 sensitized these spores to subsequent heat inactivation. In every case, whether the spores were suspended in phosphate buffer, nutrient broth, or gelatin, they were more rapidly killed by subsequent heating than were the unirradiated spores. Table II and Figs. 4 and 5 show similar data for the spores of C. botulinum 213B. Table III and Fig. 6 show that initial heat shocking of C. botulinum 215B spores suspended in gelatin does not affect the lethal action of subsequent irradiation with gamma rays from cobalt-60. Similar data were obtained for C. botulinum 62A spores in all three suspending media as shown in Table IV. For purposes of comparison, the Fo value of the spores was assumed to be that amount of time in minutes at 250~F required to cause the thermal-death time curve to pass through eight logarithmic cycles, and the Z value was taken as 18. On this basis, when C. botulinum 213B spores were suspended in gelatin and then were- irradiated with 900,000 rep.of gamma rays, their Fo value was reduced from 1o48 to 0.46. If the lethal effect of the preliminary irradiation with 900,000 rep is included in the overall effect, the Fo value could be considered to. have been still further reduced to 0.35. This represents approximately a fourfold reduction in the Fo value. In conclusion, it can be said that preirradiation with gamma rays from cobalt-60 sensitizes the spores of C. botulinum to the subsequent lethal action of heat, but that preliminary heat shocking at 99~C does not affect the lethal action of subsequent gamma irradiation. It was also found that increased amounts of gamma radiation caused increased degrees of sensitization to the subsequent lethal action of heat. This was evidenced by the increasing steepness of the thermal-death time curves developed with those spores that received increasingly higher dosages of gamma radiation. It was also shown that the Fo value of C. botulinum 6

TABLE I EFFECT OF PRELIMINARY IRRADIATION BY GAMMA RAYS FROM COBALT-60 ON THE HEAT RESISTANCE OF C. BOTULINUM 62A SPORES ~ 1_CControl radiated.E-', - e H e u 9 V, 0 0 0 V *eH n 1o o0 O 0 I o l ~ H O *H bD Hr-1' Q),'H b - A) Suspended in M/15 phosphate buffer at pH 7.05 Run 2 (170,000 rep) O 0 1,980,Q00 5 3.3 965, 00o 10 8.3 800,000 20 18.3 320,000 30 28.3 230,000 in 16100.0 48.7 40.4 16.2 11.6 x 150-mm-OD 2.00 1 688 1.606 1.210 1.065 pyrex test 1,300,000 670,000 270,000 101,000 46,000 tubes 100.0 51e.5 20.8 7.8 3.54 2.000 1o712 1.318 0.892 0.549 Run 4 (340,000 rep) 0 0 1,020,000 10 8.3 290,000 20 18.3 190,000 40 38.3 59,000 60 58.3 13,900 80 78.3 3,950 in 16- x 100 28.4 18.6 5.5 1.36 0.387 150 -mm-OD 2.000 1.454 1.270 0.741 0.134 -0.412 pyrex test tubes 440,000 100 48,000 10.9 9,500 2016 255 o0058 4 0.00091 2 o0ooo45 2.000 1.380 0.335 -1.236 -3.041 -3.346 Run 3 (575,000 rep) 0 0 810,000 10 8.3 310,000 20 18.3 250,000 40'38.3 49,000 6o 58.3 11,000 80 78.3 4,500 in 16- x 150-mm-OD 100.0 2.000 38.3 1.584 30.9 1.491 6.1 0.786 1.36 0.134 0.55 -0.259 pyrex test 95,000 12,300 1,100 10 1 0 tubes 100 1300 1..16 0.010 0.001 2.000 1.114 o.o65 -1o979 -2.979 Run 5 (340,000 rep) 0 0 1,280,000 10 6.1 20 15.23 218,000 30 24.53 40 33.83 60,600 50 43.20 60 52.40 22,750 70 61.70 80 71.03 7,000 in 22- x 175-mm-OD 100 2.00 17.01 4.74 1.231 0.676 pyrex test tubes 440,000 100 92,500 21.0 25,570 5.85 7,050 1.60 2,850 0o648 448 0.102 16o 0.354 56 0.0127 14 o0.o318 2.000 1.323 0.768 0.204 -0.198 -0o991 -1.451 -1.896 -2.497 1.775 0.252 0. 546 -0.262 7

TABLE I (continued) A'....Control Irradiated H a \ o a) N a o - > r l- 0r-l P C a 0 bO c 0F-it O) bDH P)F? *H ct3. CO C CQ COC 0C CO B) Suspended in nutrient broth at pH.6.68 Run 18 (340,000 rep) 0 0 910,000 15 10.8 220,000 30 24.6 112,000 45 37.5 54,500 60o 52.4 19,000 75 66.3 12,600 Run 19 (500,000 rep) 0 0 910,000 15 10.8 220,000 30 24.6 112,000 45 37.5 54,500 60 52.4 19,000 75 66.3 12,600 in 22- x 175-mm-dD 100.00 2.0000 24.20 1.3838 12.31 1.0903 6.00 0.7782 2.09 0.3202 1.385 o.1415 in 22- x 175-mm-OD 100.00 2.0000 24.20 1.3838 12.31 1.0903 6.00 0.7782 2.09 0.3202 1.385 0.1415 pyrex test tubes 640,000 100.00 227,000 35.5 43,ooo 6.72 9,700 1.515 4,850 0.758 695 0.185 2.0000 1.550 0.8274. 18o4 -0.1203 -0.7328 2.0000 1.2788 0.1650 -0.7235 -1.1518 -2.1355 pyrex test tubes 410,000ooo 100.0 78,000 19.0 6,ooo 1.462 775 0.189 290 0.0705 30 0.00732 C) Suspended in 10% gelatin at pH 7.0 Run 31 0 0 15 10.8 30 24.6 45 38.5 6o 52.4 75 66.3 90 8o.o (300,000 r 6,870,C 5,050,C 63o50,c 630,C 104,C 14,C 2,2.ep) )00 100.0 )00 44.4 )00 9.18 )00 1 515 )00 0,204 -50 0.03275'00 0.01020 rep) (Same control as Run 31) 2.00 1.647 0.963 0.181 -0.690 -1.485 -1.991 4,930,OC 680,oc 40,oc 3,0~ 5c 12 3 )0 100.0 )0 13.77 )o 0.810 )0 0.0618 )0 0.0101'o 0.00243 4 0. ooo68 )0 100.0 o 26.2 )0 1.1155 i0 0.0515 >5 o.oo485.0 0.000625 5 0.000156 Run 31A (500,000 O 0 15 10.8 30 24.6 45 38.5 60 52.4 75 66.3 90 80.0 2.00 1.139 -0.091 -1.209 -1.995 -2.614 -3.167 2.00 1.418 o 048 -1.288 -2.314 -3.204 -3.807 3,200,OC 840,oc 37,00 1,6 8

TABLE I (continued)'... Control Irradiated *H US V V O ) E1 ( o o 0 0 k 0 ON aa) k 9 > > Q) t t> > E- 7)o o'o ~W _______ g > uD o9 / u > eO O F-4U) n F bfl' OR...., ~, _,, -........... - +' eciid CO;j CO LO 01 c o COC CO U) rW Xr _ _ _ _ _ _ Run 31B 0 0 15 10.8 30 24.6 45 38.5 60 52.4 75 66.3 90 8o.o (900,000 rep) (Same control as Run 31) 550,000 9,300 15 8 0.5 out out 100.0 1.69 0.00272 0.00146 2.00 0.228 -2.567 -2.855 Run 33 0 0 15 10.8 30 24.6 45 38.5 60 52.4 75 66.3 90 8o.o (340,000 rep) 1,660,000 1( 310,000 57,000 4,000 350 135 45 30 18.7 3.43 0.241 0.0211 0.00812 0.00271 2.00 1.272 0.556 -0.618 -1.676 -2.090 -2.567 1,100,000 197,000 12,000 375 45 1.5 0.5 100.0 17.8 1.145 0.0341 0.0041 0.000136 2.00 1.251 0.059 -1.467 -2.587 -3.866 Run 33A (500,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 33) 75 66.3 90 8o.0 850,000 93,500 2,200 15 out out out 100.0 11.0 0.259 0.00176 2.00 1.042 -0.587 -2.754 Run 35 0 0 15 10.8 30 24.6 45 38.5 60 52.4 75 66.5 90 80o0 (300,000 1 505,( 175, 12C, lc rep) )00 100.0 )oo 54.65 500 2.48 300 o.158 )st 20 0.00396 7 0.001585 2.00 1.540 0.395 -0.801 -2.402 -2.858 390,000 23,000 900 40 5.5 out out 100.0 5.90 0.2305 0.0105 0.000896 2.00 0.771 -0o637 -1.979 -5.047 9

TABLE I (concluded).. Control Irradiated *H.4a)o 15 10.8 1 00 6 O - o886 60 24 as Run 35) 1 000512 -3290 4-75 6 3 out g CO CO CO Run 35A (500,000 rep) 0 0 8195000 100.0 2 00 15 10.8 771000 O690 0, 30 24.6 250 0.128 -2o.892 45 8.5 (Same control 15 0.00768 2.114 60 52.4 as Run 35) 1 0.000512 -5.290 75 66.53 out 90 8.o out.,:.. i..,.,, Run 55B (900,000 rep) 0 85,oo000 100.0 2.00 15 o10.8 770 0.905 -0.045 50 24.6 1 0,00178 -2.749 45 58.5 (Same control 60 52.4 as Run 55) 75 66.5 90 80.0o 1-0

0 5 Ct) > 0 a: 5|\ 170,000 REP Bs' -0.00 0 5 -2.00 340,000 REF 575,000 REP \ -3.00 1 i 3 -4.00 0 20 40 60 80 ( EFFECTIVE TIME AT 990C- MINUTES Figo 1. Effect of preliminary irradiation with gamma rays from cobalt-60 on the heat resistance of C. botulinum 62A spores suspended in M/15 phosphate buffer at pH 7.0. 30 11

" 1.00 0 o -I.00 -2.00 -3.00 0 20 40 60 80 EFFECTIVE TIME AT 990C -MINUTES Fig. 2. Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance of C. botulinum 62A spores suspended in nutrient broth at pH 6.6. 100 12

2.00 1.00 0 U() 0 > 0 -o So -1.00 -2.00 -3.00 -4.00 -500 0 20 40 EFFECTIVE TIME 60 MINUTES 80 Fig. 30 Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance of C. botulinum 62A spores suspended in 10% gelatin at pH 7.0.

TABLE II EFFECT OF PRELIMINARY IRRADIATION BY GAMMA RAYS FROM COBALT-60 ON THE HEAT RESISTANCE OF C. BOTULINUM 213B SPORES " ____ Control __ Irradiated E +)d~Hzz:Hc f' E- r4 ~1Q 0 0 CD FEI 0 0 ~0 (^0\ q ^> Uq 0 > > OpH 0 0 OP4 Q? P _4- C.,.._O CO.CO A) Suspende A) Suspended in M/15 phosphate buffer at pH 7.0 Run 12 (200,000 rep) 0 0 1,490,000 100 lo 6,O1 20 15.23 295,000 19.8 30 24.-53 40 33.83 190,000 12.75 50 43.2 60 52.4 68,000 4.56 70 61.7 80 71.0 44,500 2.99 2.00 990,000 375,000 1.2706 99, 500 43,000 1.1055 27,000 10,800 0.6590 4,950 2,450 0.4757 1,570 100 37.8 10.05 4.34 2.725 1.091 0.500 0.2480 0.1589 2.00 1.5775 1.0022 0.6375 0.4354 0.0378 -0.3010 -0.6055 -0.8089 Run 10 (340,000 rep) 0 0 2,610,000 10 6.10 20 15.23 30 24.53 4o 33.83 50 45.2 60 52.4 70 61.7 8o 71.03 705,000 390,000 213,000 188,000 92,000 70,500 48,000 55,000 100 27 14.9 8.16 7.21 3.52 2.70 1.84 2.02 2.000 1.422 1.174 0.912 o.858 0.547 0.432 0.265 0.306 1,310,000 228,000 57,000 14,300 4,400 1,700 615 134 125 100 17.4 4.35 1.092 0.336 0.130 0.0469 0.01025 0.00953 2.000 1.241 0.639 0.039 -0.473 -0.886 -1.329 -1.989 -2.021 Run 14 (500,000 rep) 0 0 369,000 15 10.8 106,000 30 24.6 60,000 45 37.5 25,900 60 52.4 11,900 75 66.3 5,100 100 00 28.70 16.25 7.03 3.23 1.382 2.000 1.4579 1.2109 0.8470 0.5092 o.1405 35,000 1,400 240 20 2 0 100.0 4-.00 o.686 0.0572 0.00572 2.0000 0.6021 -0.1637 -1.2426 -2.2426 m 14

TABLE II (continued)'~ _.. Control I___ Irradiated H 0 | D o 0 > O> ~*HH P -P bO CO C0 b. C0 CO E > S.> O >. > 4-);> w w C CO CO CO C k,. U. k o. o. ~ 0 f,_,, o 3 0 o3 o o. q E,_ 00 _,..__..._____ B) Suspended in nutrient broth* at pH 6.68 Run 13 (200,000 rep) 0 0 1,580,000 15 10.8 225,000 30 24.6 67,500 45 37.5 12,600 6o 52.4 9,900 75-66.3 2,490 100 14a25 4.28.798.627.1575 2.000 1.1538 0.6314 -.0980 -.2027 -.8027 1,065,000 100,000 58,ooo 2,400 255 47 100 9.40 5.45 0.2255 0.02210 0.00442 2.000.9731.7364 -.6468 -1.6556 -2.3546 Run 11 (340,000 rep) 0 0 1,45o,oo000 10 6.10 20 15.23 30 24.53 40 33.83 50 43.2 6o 52.4 70 61.7 80 71.03 Run 15 (: 0 0 15 1008 30 24.6 45 37.5 6o 52.4 75 66.3 510,000 230,000 129,000 50,000 19,000 9,800 4,900 2,900' 100 35.2 15.86 8,88 3.45 1.31 0.675 0.348 0.200 100 18.8 7.55 2.42 o 835 0.224 2.0000 1.5465 1.2000 0.9484 0.5378 0.1173 -0.1807 -0o 4584 -0.6990 2.00 1.2742 0.8780 0.3838 -0.0783 -0.6497 980,000 206,000 64,000 20,100 5,300 1,020 103 18 6 129,000 6,600 1,040 40 2 0 100 21.0 6.54 2.05 0.541 o. 14 0.0105 o.oio5 0.001835 0 000613 100.0 5.12.8o8.031.0015 2.0000 1.5222 0.8156 0.3118 -0.2668 -0.9830 -1.9788 -2.7345 -3.2125 2.00 0.7093 -0.0926 -1.5086 -2.8259 500,000 rep) 345,oo000 65,000 26,000 8,350 2,880 775 *Nutrient broth containing 3 tone, 5 g NaCl plus 1000 ml g Difco beef extract, 10 g Difco pepdistilled water. 15

TABLE II (continued) _...Control Irradiated El~~~0 0 0 01 |r Eh i g h E-M o o a)o o (o,>.> V l > C D 0 O > L' O 1 > ObD -1 > o ~ oP 0 ~P | o|. c' CC I -....c C. C) Suspended in 10* gelatin at ph 7.0. Run 21A 0 0 15 10.8 30 24.6 45 38.5 60 52.4 75 66.3 (340,000 rep) 1,530,000 100.00 290,000 18.95 45,ooo 2.94 17,500 1.143 4,700 0.3075 960 0.0627 2.00 1.278 0.469 0.059 -0.512 -1.202 1,030,000 132,000 36,500 8,000 1,940 380 100.0 12.81 5.542 0.776 0.1883 0.0369 2.00 1.108 0.538 -0.110 -0.7245 -1.433 Run 22A (500,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 21A) 75 66.3 750,000 65,500 10,100 1,900 340 30 Run 27 0 0 15 10.8 30 24.6 45 58.5 6o 52.4 75 66.3 90 80.o (340,000 rep) 720,000 217,000 79,000 18,200 6,000 2,650 1,090 100 00 30.20 10.97 2055 0.834 0.368 0.115 2.000 1. 48o 1.o40 0.403 -0.079 -o 434 -0.939 285,000 79,500 24,400 8,250 2,230 1,000 73 100.0 8.74 1.347 0.254 0.0454 o.oo40 100o 27.9 8.56 2.89 0.783 0,351 0.0256 100.0 18.55 3.285 0.855 0.1865 0.0210 0.0057 2.00 0.842 0.129 -0.595 -1.343 -2.396 2.000 1.246 0.933 0.461 -0,106 -0.454 -1.592 2.00 1.269 0.517 -0.o68 -0.729 -1.678 -2,244 Run 27A (500,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 27) 75 66.3 90 8o.o 295,000 63,000 9,700 2,520 550 62 17

TABLE II (continued)' I ". Control _ ___ Irradiated *H U F1I > 0 0 0 0 *H * H~ 4 94 a a.o o lP:' I ti |., 1. |'....., 4- p C U U U u U C 3 0% CCQ ci 0r ^0 ~~ I _ 0_ U1 _____ Run 28 (500,000 rep) 0 0 1,140,000 15 10.8 30 24.6 45 38.5 60 52.4 75 66.3 90 8o.o I 455,000 168,000 74,500 22,100 9,550 2,450 100.0 39.9 14.75 6.54 1.94 o.838 0.215 2.00 1.601 1.169 o.815 0.288 -0.076 -o.667 643,000 124,000 24,400 5,400 1,400 160 19 100.0 19.28 3.79 0.839 0.218 0.0249 0.00285 2.00 1.285 0.579 -0.076 -0.661 -1.603 -2.545 Run 28A (900,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 28) 75 66.3 90 8o.o 124,000 4,200 360 7 0.5 out out 100.0 3.39 0.291 0.00565 0.o00040 2.00 0.530 -0.536 -2.248 -53598 Run 50 (500,000 rep) 0 0 5,300,000 15 10o8 3,100,000 30 24.6 795,000 45 38.5 470,000 60 52.4 159,000 75 66.3 76,000 90 80.0 18,300 100.0 58.5 15.0 8.87 35.00 1.432 0.345 2.000 1.767 1.176 0.948 0.478 0.157 -0.462 4,300,000 980,000 370,000 120,000 44,500 6,700 1,540 100.0 22.8 8.6o 2.79 1.033 0.1556 0.o358 2.00 1.358 0.935 0.446 0.015 -o 8o8 -1,446 Run 30A 0 0 15 10.8 30 24,6 45 58.5 60 52.4 75 66.3 90 8o.o (900,000 rep) (Same control as Run 30) 58o,ooo 104,000 11, 500 2,280 2355 24 2.5 100.0 17.95 1.982 0.393 0.0405.0oo414 0.000432 2.00 1.254 0.298 -0.445 -1.392 -2.383 -3.364 17

TABLE II (concluded)...... Control Irradiated H.H +~ *naio o (D. O C> Y > > hD > ERun ( 00,000 rep 0 0 10,300,000 100.0 2.00 5,030,000 100.0 2.00 b cO\ 10. 3100 6 25 0 124 1.0 F6 0 O5 P4 0 4 -0.6 18 - 75 66.- 25,500 0.2285 -.640 260 0P4 00517 -2.286 - &.0 C O0 CO CO CO CO58 -3 Run 34 (500,000 rep) 0 0 10,00,000 100.0 2.00 00 100.0 2.00 15 10.8 5,100,000 50.1 1.479 25,000 12.4 1.094 30 24. 48o 46 0.669 15,2000 2.64 0.422 45 38.5 145,000 1.41 o.149 18,560 0.570 -o42.7 60 52.4 75,500 0.714 - o.146 u1,90 0.0578 -1.422 75 66.5 25,500 0.2285 -.64o 26 0.00517 -2.286 908 4,0 o.o48 -1 8 18 0.000558 -o.446 Run 54A (700,000 rep) 0 0 910,000 100.0 2.00 15 10.8 47,000 5.17 0.724 50 24.6 1,260 0.159 -0.857 45 58.5 (Same control 15 0.00165 -2.782 60 52.4 as Run 54) out 75 66.3 out 90 8o.o out

I, J-2.0011 -.00_______ < K _ E -4.00 \ 500,000 RE: > \ A P O 0 20 40 60 80 1 EFFECTIVE TIME AT 990C -MINUTES cobalt-60 on the subsequent heat resistance-of C..botulinum ) -1.00 -- - -2.00........ 200,000 REP x^ (340,000 REP -3.00 X 500,000 REP -4.00 ___________________________ 0 20 40 60 80 100 EFFECTIVE TIME AT 99~ C - MINUTES Fig. 4. Effect of preliminary irradiation with gamma rays from cobalt-60 on the subsequent heat resistance of C. botulinum 213B spores suspended in nutrient broth at pH 6.7. 19

2.00 2.00 RUN 28 0 CONTROL 0\\~ iID 500,000 REP 1 \.2 0s X 900,000 REP 1.00 Q\,-\ —CONTROL 0 o 0, > -1.00 o x 0, \X 900p00oo kREP 1\ -3.00 -4.00 ________________ 0 20 40 60 80 EFFECTIVE TIME AT 99~C Fig. 5. Effect of preliminary irradiation with gamma rays from cobalt-60 on the heat resistance of C. botulinum 213B spores suspended in 10% gelatin at pH 7.0. 20

TABLE III EFFECT OF PRELIMINARY HEAT TREATMENT ON IRRADIATION RESISTANCE OF C. BOTULINUM 62A SPORES TO GAMMA RAYS FROM COBALT-60 0 aI I d C o lb S - 0 HO 4H o rd 4-) f JVkH0 A)I~~~ S A Ssd in M1 p hoo b e at o pye ts N) H Run 6 0 170, 000 340,000 510,000 680 9000 1,620,000 1,480,000 810 000 460,000 114,000 100 91,2 50 0 28,4 7.03 2.000 1.961 1.699 1.454 0.847 9 minl 630,000 560,o000oo 46o,ooo 25460,000 230 000 70,000 8 min3 20,000 15,000 14,000 2,500 350 100 89.0 73.0 36.5 11ol 2.000 1.950 1,864 1.563 1.046 20 min2 430,00oo 450,000 384,000 258,000 197,000 72,000 25 min4 4,500 6,500 8oo 800 330 81 100 89.3 6o.o 45.8 16,7 2o000 1.951 1.778 1,661 1.223 152 min equivalent time at 99~C 215o2 lent min equivatime at 99~C Run 7 0 170,000 540,000 510,000 680,000 1335000 64,500 33,000 7,500 550 100 48.5 24,8 5.64 0.414 2.00 1.686 1.395 0.751 -0.383 100 75 70 12.5 1075 2.00 1.875 1.846 1.097 0,244 100 144 17.5 7.5 1.8 2.00 2.158 1.243 0.864 0.256 34.4 min equivalent time at 99~C 420 min equivalent time at 99~C

TABLE III (concluded) o0 Ca Co Co CO IT!. II 4-) a0)4-' 0 rd-P0 0 0 rdP 0 Cd30 o0 0k 00 0o2 00 (U rd W (L) 0 bDrHHJ bO~ HO P C m ^i -jq - pq ^ f-q:J pq ^i-^^ ___0 _ _ I c CO COCco _____-A____________CO ______ ____________________ ___ ________________ Run 8 0 170,000 340, 000 510,000 680,000 850,000 1,020,000 430,000 490,000 1935,000 41, 000 8,100 900 45 100 114 44.8 9.55 1.88 0.209 0.0109 2 000 2.057 1.652 0o 980 0 274 -o.68o -1.962 8 min3 74,000 54,500 435,000 15,600 35600 537 18 100 73.6 58.1 18.4 4086 0.725 0,245 2.00 1.868 1.765 10265 o0.688 -0o14 -1.614 25 min4 7,000 5,400 4,300 5,000 515 40 3 100 77.2 61.5 42.8 7.36 0,572 0.0428 2.000 1.888 1.789 1.632 0.878 -0.242 -1.368 15.2 n lent ann equivatime at 99~C ro <215.2 min equivalent time at 99~C 34o4 min equivalent time at 990C 420 min equivalent time at 99~C B) Suspended in nutrient broth at pH 6068 Run 20 0 170,000 340,000 510,000 680,oo000 850,000 1,020,000 910,000 910, 000 770,000 350,000 105,000 235,700 5,200 100.0 100.0 84.6 38.5 11.54 2. 60 0.572 2.0000 2.0000 1.928 1.586 1.063 o.416 -0.242 8 min1 415,9000 364,000 310,000 141,000 91,500 25,200 8,250 100.0 87.8 74.7 34.2 22.05 6.07 1.985 25 min2 2.00 620,000 1.944 475,000 1.874 315,000 1.534 2035,000 1.344 57,000 0.784 16,5300 0.298 5,900 100.0 76.6 50.8 32.7 9.19 2.63 0.629 2.000 1.885 1.706 1.515 o0.963 0. 420 -0.202 C) Suspended in 10% gelatin at pH 7.0 Run 32 0 340,000 680,000 850,000 1,020,000 1,230,000 1,5360,000 720,000 590,000 169,000 55,000 13,100 2,900 1,200 100 82.0 25.5 7.64 1.82 0.403 0.1667 2.000 1.914 1.571 0.885 0.260 -03.594 -0.778 8 min 570,000 385,000ooo 160,oo000 101, 000 26,000 4,000 1, 100 100 67.6 28.1 17.7 4.56 0.702 0.193 2.00 1.83 1.449 1.248 o0.660 -0.153 -0.714 25 min 183 5,000 154,000 735,000 27,500 7,250 1,020 315 100.0 84.0 39.9 15.02 3.96 0.556 0.172 2.00 1.924 1. 6o1 1.177 0.598 -0.254 -0.764

) I.00 (I) o 0 0 4 8 12 16 REP x 105 Fig. 6. Effect of preliminary heating at 99~C on the subsequent radiation resistance of-C. botulinum 213B spores suspended in 10% gelatin at pH 7.0 o -1.00 —... -2.00 ——. -3.001 0 4 8 12 16 REP x 1O5 Fig. 6. Effect of preliminary heating at 99~0C on the subsequent radiation resistance of C. botulinum 215B spores suspended in 10% gelatin at pH 7.0. 25

TABLE IV EFFECT OF PRELIMINARY HEAT TREATMENT ON IRRADIATION RESISTANCE OF C. BOTULINUM 213B SPORES TO GAMMA RAYS FROM COBALT-60 p4CQ 0 P 0?. o Cm rdG ( a 0 Ho o 0> o > o C^&!0 l V & 1 0 0 V k a I o vi i ( O I I 11 m CO A) Suspended in phosphate buffer at pH 7.0 Run 16 0 1,290,000 170,000 1,100 000 5- 0340,000 690,000 510,000 333,000 68,000o 70,000 850,000 10,100 1,020,000 930 100 853. 53.5 25.8 5.43 0 783 0.0721 2.000 1.9310 1.7284 1.4116 0.7348 -0.1062 -1.1421 8 min1 750,000 520,000 354,O00 162,000 46,o00 9,500 1,140 100.0 69.3 47.2 21.6 6.13 1.266 0.152 2.000 1.8407 1.6739 1.3345 0.7875 0,1024 -0.8182 25 min2 267,000 249,000 168,500 98,500 21,000 3,590 520 100.0 93.3 63.1 36.85 7.87 1.345.1945 2.000 1.9699 i.800o 1.5664 0.8960 0.1287 -0.7111 144. min equivalent time at 99~C 220 min equivalent time at 99~C B) Suspended in nutrient broth at pH 6.68 Run 17 0 170 000 340,000 510,000 680,000 850,000 1,020,000 1,580,000 1,460,000 995,000 820,000 285,000 98,500 17,600 100.0 97.5 63.0 51.9 18.03 6.25 1.113 2.0000 1.9890 1.7993 1.7152 1.2560 0.7959 0.0465 8355,000 905,000 545,ooo 385,ooo 177,000 84,500 21,600 100.0 o18.3 65.3 46.2 21.2 10.12 2.59 2.0000 2.0346 1.8149 1.66461.3263 1.0052 0.4133 310,000 275,000 180,000 165,000 83,000 48,000 16,100 100.0 88.7 58.1 53.25 26.8 15.5 5.2 2.0000 1.9479 1.7642 1.7263 1.4281 1.1903 0.7160

TABLE IV (concluded) o a) - 1.HQ C Q $ o 1 > 1 CO II U C!) cd S0 0 I rd - 0 0 rd ) 0 0 | | d (D o a) >o 0 I> _ _>.0 120 I_> _ a~~L,~~0 co0 k0 H- I ^CQCQCOO rC P^ CO C) Suspended in 10% gelatin. Run 23 8 min 25 min 0 1,580,000 100.0 2.00 o 5,000 10 2.00 1,230,000 1000 0 2.00 170,000 1,310,000 82.9 1.919 520,000 92.9 1.968 104,000 84~5 1.927 340,000 1,240,000 78.5 1o895 280,000 50.0 1.699 74,000 6o01 1.780 510,000 720,000 45.6 1.659 290,000 51.7 1.714 74,000 60ol 1.780 68o,00 3550,000 22o15 1.345 150,000 26.8 1.428 40,000 32.5 1.512 850,000 230,00 14.56 1.1163 73,000 13.02 1.115 22,000 17.9 1.253 Run 29 8 min 25 min 0 1,100,000 100,0 2.000 480000 100.0 2.00 184,000 100.0 2.00 340,000 635,000 57.7 1.762 255,000 53.1 1.726 200,000 108.8 2.037 680,000 210,000 19.1 1.281 186,000 38.75 1.588 77,000 41.8 1.612 850,ooo 81,000 7.36 0.868 54,500 11.34 1.055 33,500 18.2 1.260 1,020,000 14,700 1.55336 0.1126 16,700 3.48 0.542 10,500 5.59 0.747 1,190,000 3,600 0.3275 -0.485 3,000 0.625 -0.204 2,o60 1.1118 o0.0o48 1,360,ooo 450 0,0409 -1.388 550 0.1144 -0.941 155 0.0842 -1.o85 iu)

spores could be reduced approximately fourfold by preirradiation. The amount of this effect was found to be less in gelatin and nutrient broth than in phosphate buffer. II. SPECIAL EXPERIMENTS A. EFFECT OF A STORAGE INTERVAL BETWEEN THE TIME OF IRRADIATION AND THE TIME OF PERFORMANCE OF THERMAL-DEATH TIME STUDIES ON THE HEAT RESISTANCE OF IRRADIATED BACTERIAL SPORES When planning our experiments soon to be carried out in meat and also in response to a letter from Captain Reuben Pomerantz, it was necessary to answer the following question with experimental data: Is the increased heat sensitivity induced in bacterial spores by irradiation affected by storage? Three lines of attack on the problem were used, lo Runs 35, 35A, and 35B were conducted by allowing the irradiated Co botulinum 62A spores to stand in the refrigerator overnight before the thermal-death time studies were conducted. The data obtained were normal for C. botulinum 62A spores suspended in 10% gelatin at pH 7.O.and: irradiated before heating (Table 1C)o 2. Runs 36 and 36A were designed to test the effect of a 3month storage interval at 4~C after irradiation.' C. botulinum 213B spores were used in M/15 phosphate buffer for this purpose. The data are presented in Table V and Fig.7. A comparison with Run 14 (Table IIA) indicates that storage did not reduce the sensitization effect in this instance, but rather appeared to have accentuated it. 3. A long-time experiment has been set up and will be reported on at a later date. B. EFFECT OF USING JUNfEATED BACTERIAL SPORES IN PLACE OF SPORES THAT HAD BEEN HEATED FOR 15 MMIUTES AT 850C WHEN STUDYING THE PROBLEM OF RADIATION SENSITIZATION OF BACTERIAL SPORES TO HEAT This problem arose as a result of a question in the discussion period following presentation of QMC Paper 544 at the Society of American Bacteriologists Annual Meeting in New York in May. 26

2.00 0 - CONTROL A - 450,000 rep BEFORE STORAGE V) WI 1.00 o \ () 0 - -2.00 -3.00 RUN 36 -4.00 __ 0 20 40 60 80 EFFECTIVE TIME AT 99~C Fig. 7. Effect of 3 month's storage at 40C between the times of irradiation and heating on the sensitivity to heat induced by preirradiation of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0. 27

TABLE V EFFECT OF STORAGE AT 4~C BETWEEN TIME OF IRRADIATION AND HEATING ON THE SENSITIVITY TO HEAT INDUCED BY PREIRRADIATION ON C. BOTULINUM 213B SPORES SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 Control ____ Irradiated ___ *H H -P -p 0 ". - 0 -i ~ 0 5 3 3 60 00 65 C 70 0 - OH OH O0 or1 6 *Hl a u o ~ o uo o o'h % k 0,. 0 0'. O. EH ( > o ( 1) 0!! ~O> 0 P4 F OP4'H - H-Pp-' P4 P4 -I C CO COCO CO C C Run 36 (450,000 rep) and storage from Jan. 31, 1955, to May 1, 1955, at 4~C 0 0 1,050,000 100 2.00 130,000 100.0 2.00 5 3.3 690,000 65.7 1.818 110,000 84.5 1.927 10 8.3 460,000 43.8 1.642 27,000 20.8 1.318 15 13.3 290,000 27.6 1.441 8,700 6.69 0.825 20 18.3 550,000 33.3 1.523 2,700 2.08 0.318 50 28-5 150,000 14.3 1.156 250 0.192 -0.716 40 38.3 30 0.023 -1.638 Run 356A (540,000 rep) and storage from Jan. 31, 1955, to May 1, 1955, at 40~C 0 0 64,000 100.0 2.00 5 3.3 26,000 40.6 1.610 10 8.3 3,500 5.46 0.738 15 13.3 (Same control as 1,900 2.97 0.473 20 18.3 Run 356) 320 0.50 -0.301 30 28.3 8 0.125 -1.903 40 38.3 out While it is realized that heating for 15 minutes at 85~C is a very mild treatment and is standard procedure among investigators working with anaerobic bacterial spores, it was considered desirable to check briefly this criticism. Run 37 was conducted for this purptose.In this experiment, previously preheated C. botulinum 213B spores were grown and harvested. They were diluted into M/15 phosphate buffer at pH 7.0 and then four portions were withdrawn. Two of these portions were heated at 85~C for 15 minutes. Then, one portion of the heated 28

and one portion of the unheated samples were set aside as controls and the other two were irradiated at 250,000 rep in the cobalt-60 gamma-ray field, Thermal-death time studies were then made of all four samples. The results are presented in Table VI and Fig. 8. It will be observed that there is no essential difference, for our purposes, between unheated spores and similar spores that have received a preliminary heat treatment at 85~C for 15 minuteso TABLE VI EFFECT OF PREIRRADIATION WITH SUBSEQUENT HEATING AT 99~C ON UNHEATED C. BOTULINUM 213B SPORES AND ON SIMILAR SPORES THAT HAD BEEN HEATED AT 85~C FOR 15 MINUTES TO KILL VEGETATIVE CELLS....Control.. Irradiated _ *H.) - H ri H -i -p U)3 CQ Co R II CI H. c a)?'H tH. S H Ok H _ I >I o oi 11 HI to I o Cd Di tC Run 39 Heated, but not irradiated Heated and irradiated o. 2...... ( 250,000 rep) O 0 1,260,000 100o0 2o00 1,730,000 100.0 2.00 15 10.8 725,000 57.5 1.76 178,000 10.3 -1.013 30 24.6 140,500 11016 1o48 3,750 0.2165 -0.664 45 38o5 30,500 2,42 0o384 150 0.00oo865 -2.063 60 52.4 3,200 0o254 00.595 25 0.001445 -2.837 75 66.3 900 0.0715 -10346 4 0.000231 -3.636 Not heated, not irradiated Not heated, but irradiated (250,000 rep) O 0 *12 100,000 100.0 2o00 8,300,000 100.0 2.00 5 - 8,550,000 -- 5,350,000 64.5 1.81 10 4,450,0ooo - 1,350,000 16.3 1.212 15 10.8 2,380,000 57.5 1,760 530,000 6.39 0.807 30 24.6 425,000 10.27.o012 6,o50 0.0729 -1.137 45 38.5 83,000 2o00 0.301 1,000 0.0121 -1.917 60 52.4 9,900 0.239 -0o.62 175 0.00211 -2.676 75 66.3 2,150 00052 -1.284 50 000oo603 -3.219 Contains vegetative cells; a calculation was made indicating 4,140,000 spores were present in this sample at 0 minutes. 29

2.00 1.0( C I 2 r, f) 0 D oU 0 0 -J -.0( - 2.0C * HEATED-NOT IRRAD. \O HEATED-IRRAD. (250,000 rep) * NOT HEATED-NOT IRRAD. 0 NOT HEATED-IRRAD. (250,000 rep) 0\~ NOTE: USED CALCULATED VALUE FOR INITIAL SPORE COUNT FOR NOT HEATED NOT IRRADIATED VALUES ) — a; —A: 0 -- -\____________________'% ______ -3.0C -4.0( 6) RUN 39 0 20 40 60 EFFECTIVE TIME AT 99~C 80 Figo 8. Effect of preirradiation with 250,000 rep of gamma radiation followed by heating at 99~C on unheated C. botulinum 213B spores and on similar spores that had been heated at 85~C for 15 minutes to kill vegetative cells. 30

IIl. EFFECT OF TEMPERATURE ON THE SURVIVAL OF BACTERIAL SPORES A. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES This study involves the irradiation of spore suspensions contained in small, heat-sealed glass vials. Irradiation is carried out principally in the center well of the cobalt-60 gamma radiation source. During irradiation the vials are fixed in an especially designed container which permits immersion of the vials in a fluid bath whose temperature can be controlled. After irradiation, the surviving spores are counted and the numbers found are compared with those surviving in suitable controls. Counting is carried out according to standard techniques similar to those described by Reed, Bohrer and Cameron.2 Table VII and Fig. 9 show that when C. botulinum 213B spores were suspended in M/15 phosphate buffer at pH 7,0, they were progressively more rapidly killed as the temperature of irradiation was increased from -70 to 95~Co Table VIII and Fig. 10 indicate that the spores of Putrefactive Anaerobe No. 3679 reacted in the opposite manner, since whenn these spores were:suspended in M/15 phosphate:bu'ffer at pH 7.0 they were killed more rapidly at irradiation temperatures of 58~C or lower than at temperatures of 80~C and above. This phenomenon is being studied for verification and explanation. If it should be' established that the lethality of gamma radiation for C. botulinum spores varies directly with the temperature of irradiation and that PA 3679 spores react oppositely, such information might be of considerable significance in food sterilization. This arises from the fact that Co botulinum spores are among the most resistant to gamma radiation; so, from the microbiological standpoint, it might be advantageous to irradiate foods at high temperatureso This would tend to make C. botulinum and PA 3679 spores more.nearly equal in radiation sensitivity which would cause the spoilage of food to be the first sign of inadequate irradiation rather than toxin production. Also, the protection afforded Co botulinum spores by low temperature should be considered when evaluating suggestions for the prevention of flavor development by irradiation in the frozen state. 31

TABLE VII EFFECT OF TEMPERATURE DURING IRRADIATION WITH GAMMA RAYS FROM COBALT-60 ON THE SURVIVAL OF SPORES OF C. BOTULINUM 213B WHEN SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 SO"C IO.30C.5 0C Dose,* Number of og Numbe o Lg Number ofNumber ofLogNumber megarep Spores Survivor s Spores Survivors Spores Survivors 0 6,200,000 2.000 2,500,000 2.000 3,500,000 2.000 o0.185 2,000,000 1508 --------- 1,300,000 1.570 0.370 5,700,000 0o964 900,000 1.560 5,200,000 1.170 0.550 14o,000 0.354 350,000 1,146 100,000 0.456 0.647 --------- ----- 110,000 0.644 -------- 0.740 41,000 2.820 22,000 -0.055 15,000 -0.369 0.832 ---------.. —-... 1,000 -1o398 ---- ---- 0.925 3,900 -2.201 150 -2.222 1,500 -13569 1.017 150 -35617 85 -3.469 ---- ----- 1.110 3 -4.315 ----- -----. —-. --- 8e0C S95~.C Heat Control Dose,* Number of Log % Number of Log Hr. 800C 850C megarep Spores Survivors' Spores Survivors 0 5,500,000 2.000 2,700,000 2.000 0 5,500,000 2,700,000 0.185 35,200,000 1o 765 ------ ---- 2- 35,000,000 0.370 1,400,000 1.407 2,300,000 1.930 3 --- --- 2,500,000 0.550 16o0,000ooo o.465 14,000 -0.215 4 —---- 1,500,000 0.740 7,800 -o.848 45 -1o777 5 3,000,000 1,400,000 0o832 3,500 -1o197 0 --—. -—. —. — 0.877 1,4oo00 -lo595 0 ----- -70 ~c -70C I 270C Dose,** Number of Log o Number of Log To Number of Log o megarep Spores Survivors Spores Survivors Spores Survivors 0 950,000 2o000 520,000 2.000 670,000 2.000 0.227 720,000 1.903 520,000 2.000 530,000 1.898 0.454 350,000 o1522 79,000 1.225 260,000 1.589 0.680 75,000 0.940 9,700 0.314 78,000 1.o65 0.794 25,000 o.44o —. -.. —..... —. — -- 0.907 12,000 -o0.o060 1,800 -0.523 71,000 1.025 1.020 5,000 -0.246 200 -1.366 ------- 1.134 1,500 -0o7753 -- ----- 790 -0.928 Frozen control 9'0 x 105o *Dosage rate = 0.185 megarep per hour. **Dosage rate = 0.227 megarep per hour (vials not immersed in liquid). 52

cr C -1.00 0.,J -70 U 80 %95 -2.00 -3.00 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 MEGAREP Fig. 9. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of C. botulinum 213B suspended in M/15 phosphate buffer at pH 7.0.... 33

TABLE VIII EFFECT OF TEMPERATURE DURING IRRADIATION WITH GAMMA RAYS FROM COBALT-60 ON THE SURVIVAL OF SPORES OF PA 3679 WHEN SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7*0 I -~ 5~C _ 5c300C 560 C Dose,* Number of Log f Log % Number Log erof Log megarep Spores Survivors Spores Survivors Spores Survivors 0 850,000 2.000 280,000 2.000 400,000 2.000 0.185 700,000 1.911 ------ --- 300,000 1.875 0.370 220,000 1.409 54,000 1.286 180,000 1.653 0.550 15,000 0.243 13,000 0.668 35,000 0.942 0.647 ------- ---- 1,900 -0.157 - 0.740 900 -0.979 1,400 -0.301 2,700 -0.171 0.852 ------- ----- 220 -1.105 ----- o.832 o 220 -1 o 103 0.925 16o -1.731 20 -2.146 -----. 1.017 35 -2.392 4 -2.845 ---- -. 1.110 1 -3.935 --- _ ----- *Dosage rate = 0.185 megarep per-hour ".; 58~0C 80~C 85~ - Dose,* Numberr of Log Number of Number of Log megarep Spores Survivors Spores Survivors. Spores Survivors 0 480,000 2.000 1,100,000 2.000 700,000 2.000 0.185 480,000 2.000 950,000 1.937 ------ - 0.370 210,000 1.640 530,000 1.684 470,000 1.826 0.550 59,000 1.089 140,000 1.104 410,000 1.767 0.647 ----- -- ---.- ---- 140,000 1.301 0.740 1,100 -0.641 40,000 0.558 120,000 1.235 0.832 ------- 10,000 -0.041 55,000 0.895 0.925 120 -1.602 4,000 -0.439 13,400 0.267 1.017 ---- ------ ----- - - -- 1.110 -..9'5~0.C Heat Control Dose, Number of Log ~PA 5679 mpgarep Spores Survivors Hr 85C 950C 0 1,200,000 2.000 - -- ---- 0.185 ----- - - 0 700,000 1,200,000 0.370 900,000 1.875 2 950,000 1,400,000 0.550 570,000 1.676 3 ----- 1,600,000 0.647 -- -.- --- 4 810,000 1,400,000 0.740 140,000 1.068 5 ---- 1,200,000 0.832 51,000 0.628 5.5 790,000 -- 0.925 30,000 0.398 - ----- ------ 1.017 10,000 -0.061 - - 1.110 3,100 -0.558 ---- --

Is~. 0 0 084 I 0 > -1.00':) 0 0 -2.00 -3.00 -4.007 0.2 0.4 0.6 0.8 1.0 1.2 MEGAREP Fig. 10. Effect of temperature during irradiation with gamma rays from cobalt-60 on the survival of spores of PA 3679 suspended in M/15 phosphate buffer at pH 7.0. 355

B. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SENSITIZATION OF BACTERIAL SPORES TO THE SUBSEQUENT LETHAL ACTION OF HEAT Other workers have suggested that irradiation of food at low temperatures may reduce off-flavor development due to the irradiation treatment. The question naturally arises then as to whether irradiation at low temperatures will still sensitize bacterial spores to the subsequent lethal action of heat. Also, how will high temperatures during irradiation influence this phenomenon? The following experiments have been carried out to answer these questions: 1o PA 3679.-PA 3679 spores were suspended in M/15 phosphate buffer at pH 7.0 and then placed in several glass ampoules which were sealed. The spores were then irradiated at 5 and 95~C, ampoules being withdrawn from the irradiation chamber at specified intervals. Following irradiation, all the vials containing spores were heated at 99~C for 1 hour. An unirradiated control was also included. Data in Table IX and Figs. 11 and 12 indicate that irradiation sensitizes PA 35679 spores in some manner that causes a portion of them to be killed by 1 hour of heating at 99~C. Unirradiated PA 3679 spores show little if any decrease in numbers when heated for 1 hour at 99~C. This finding will be investigated further since it could be significant if a process of irradiation of food followed by heat treatment should be applied in food preservation. 2, C. botulinum 62A.-C. botulinum 62A spores were suspended in M/15 phosphate buffer at pH 7.0, distributed into vials, and then irradiated with 500,000 rep of gamma radiation from cobalt60 while held at 5 or -70~C. Thermal-death time tests were then carried out on the irradiated spores by holding them at 99~C for the periods of time indicated. The data shown in Table X and Fig. 13 show that C. botulinum spores are killed at essentially the same rate by subsequent heat treatment at 99"C in both instances. This tentatively indicates that irradiation at -70~C does not alter the phenomenon of sensitization to heat developed by preirradiation. It would thus appear that food could be irradiated in the frozen condition and still be processed by subsequent heat treatment in such a way as to take advantage of the decreased heat resistance of bacterial spores following irradiation. 36

TABLE IX EFFECT OF A COMBINED TREATMENT CONSISTING OF IRRADIATION WITH GAMMA RAYS FROM COBALT-60 FOLLOWED BY HEATING FOR 1 HOUR AT 99~C ON THE SURVIVAL OF PA 3679 SPORES SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 Dosage, Spores per,. Log % rep ml Survivors Survivors A) Irradiated at 5~C and heated for 1 hour at 99~C 0 2,300,000 370,000 450,000 550,000 14,000 832,000 700 1,000,000 2 92.0 19.5 0.61 0.0304 oooo00008 1.964 1.29 -0.2147 -2.5171 -4.0605 B) Irradiated at 95~C and heated 1 hour at 99~C 0 370,000 550,000 740,000 883,000 925,000 1,100,000 1,100,000 1,100,000 1600,000 120,000 8j700 1,200 260 36 8 100.0 54.5 10.9 0.791 0.109 0.0236 0.00328 0.000726 2.00 1.7364 1.0374 -0.1018 -0.9626 -1.6271 -2.4840 -301391 Note: See Table VIII for control data of radiation alone 57

2.00 B -i I no \ I.UU 0 U) cr 0 > > {, 0 0 o -1.00 -2.00 O-HEATED AT 990C I hr AFTER IRRADIATION \ D-IRRADIATED (ONLY) AT 50 C. \{ I_ -- I I i I -3.00 -4.00 0 0.4 0.8 MEGAREP 1.2 1.4 Fig. 11. Effect of irradiation at 5~C followed by heating for 1 hour at 99~C on the survival of PA 3679 spores in phosphate buffer at pH 7.0. 38

2.0C I.OC 0 m~. (C, Cr 0 > D 0 0 _J -I.OC -2.0C 0 0 O-HEATED at 99~C I hr AFTER IRRADIATION I-IRRADIATED (only) AT TEMP OF 950C (SEE TABLE VIII) )1 -------------------------— _ _______ -3.0C -4.0C 0 0.4 0.8 MEGAREP 1.2 1.4 Fig. 12. Effect of irradiation at 95~C followed by heating for 1 hour at 99~C on the survival of PA 3679 spores in phosphate buffer at pH 7.0. 39

TABLE X EFFECT OF TEMPERATURE DURING IRRADIATION ON SUBSEQUENT RESISTANCE OF C'. BOTULINUM 62A SPORES TO HEATING AT 100~C.... i-w...l...'. i! i i i. 1 i. L i. i ~'- I:roh - trol.I.. i'H'H..H m 4m. O OH E'-o o o-'. o:.o0 o o' cB 0 C O:> O>.,.. b.; -I I k 11 R& I I Om 0 P4 o P4 A) (500,000 rep at -70~C) 0 0 370,000 100.0: 2,00 90):000 100.0 2.00 5 2.6 58,000 64.5 1.4416 10 7.6 220,000 59.5 1.774 16,000 17.7 1.150 15 12.6 150,000 4o.5 1.608 7,700 8.55 0.7161 20 17.6 110,000 29.8 1.474 290 0.322 -0.4959 30 27.6 26,000 7.03 0.847 40 37.6 9,900 2.68 0.428 50 47.6 3,000 0.811 -0.091 60 57.6 1,100 0.298 -0.526 B) (500,000 rep at +5~C) o 86,0oo 100.0 2.00 5 71,000 82.5 1.9165 10 Same. control as (A.) 4,300 5.0 0.699 15 1,000 1.16 o..06 20 55 0.04 -1.398.......;.......~................. 4o

0 A 0'ob 0 r \ -1.00 0- CONTROL x - 500,000 rep at -70 C X -500,000 rep at +5 C -2.00 0 20 40 60 80 -EFFECTIVE TIME (MINUTES) Fig. 13. Effect of temperature during irradiation on the subsequent resistance of C. botulinum 62A spores to heating at 100~C while suspended in M/15 phosphate buffer at pH 7.0. 41

IV. EFFECT OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES Morgan and Reed.- have.reported. that certain media components affect the rate at which bacterial spores are killed by gamma radiation. Also, work in this laboratory on the present QMC contract has shown that gelatin and nutrient broth reduce the lethality of..gamm radiation for the spores of C. botulinum, It is evident that information regarding, the effect of such media components on the- lethality of gamma radiation for bacterial spores is needed to interpret the effectiveness of ionizing radiations for food sterilization.. With this in mind, several chemicals have been investigated in a preliminary fashion to determine their effect on the lethality of gamma radiation for Co botulinum 62A spores. A. MATERIALS AND METHODS A 1:5 dilution of a 108-per-ml spore suspension was prepared in M/15, pH 7.02, phosphate buffer. One ml of the suspension was added to each 20 ml of the solution to be used for irradiation. Organism: C. botulinum 62A Control: Spore suspension in M/15, pH 7.02, phosphate buffer Chemicals 0.5% lecithin 0.5% ferrous sulfate 0.5% lead (plumbous) chloride 0.5% nicotinic acid 0.5% riboflavin 0.5% biotin 0.5% glutathione 0.5% vitamin A* 0.5% vitamin K* For the fat-soluble vitamins the phosphate buffer spore suspen*For the fat-s.oluble vitamins, the phosphate buffer spore suspensions were mixed with the oil solution of the vitamin and allowed to stand from 4:00 p.m. on one day until 9:00 a.m. the following morning. The suspensions were placed in a dark cabinet at room temperature. The oil and aqueous emulsion was then separated by centrifugation and the spores (in the aqueous portion) were pipetted off into phosphate buffer for irradiation purposes. 42

0.5; gelatin 0. 5% I-lysine monohydrochloride 0-5% dl-methionine — All-except lead chloride were prepared in M/15, pH 7.02, phosphate buffer (lead chloride in sterile water). B. IRRADIATION PROCEDURE Each of the chemicals was weighed out aseptically into sterile -weighing bo.ottles and then added to 20 ml of sterile M/15 phosphate buffer at pH 7-0 to provide a 005% concentration. Then 1 ml of 1.:5 dilution of C. botulinum 62A spores was added to each flask. Samples of -4 ml of the suspension were placed in sterile glass vials and heat:-sealed. They were then irradiated by the cobalt-60 gamma radiation source following which the number of sur" viving spores were counted in pork infussion agar in Prickett tubes. C. DISCUSSION.The — data-shiwn in Table XI and Figs-o 14a through 14e indicate that many chemicals reduce the lethality of gamma radiation for spores of C. botulinum 62Ao Among the more important of these are reducing substances and chemieals containing sulfur. This work will be continued next year in an attempt to establish the mechanisms of spore protection involved.

TABLE XI EFFECT OF VARIOUS CHEMICALS ON THE LETHALITY OF GAMMA RADIATION FROM COBALT-60 ON THE SPORES OF C. BOTULINUM 62A SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7o0 Control (M/15 Phosphate Chemical in M/15 Phosphate Buffer Buffer atP 70) at H 70 c Ufr a3 p ssH.7. a t'H.7 0H CuQ hD O H > D >0O^ > > 0I0)D O( ^ 0^P^<U?I 0^ CO O O P n hI> 0 k 1 Si P t A P ho 3 0 A) P4, P0 i~ U PU a) 0 P,,,._ _. _,,....., * Vitamin A O 520,000 100 2.00 6,000o 100 2.00 0.342 143,000 27.50 1.439 198,000 29.12 1.464 0.672 7,200 1.38 0.140 14,300 2.10 0.322 Vitamin K 0.0 480,000 100 2.00 0.342'", 200,000 41.67 1.620 0.672 8,400 1.75 0.243 Lecithin 0.0 620,000 100 0 2.00 0.342 " 240,800 40.0 1.602 0 672 20,000 3.23 0o509 Ferrous Sulfate 0 580,000 100.0 2.000 0.342 "2 " 208,000 35.86 1.555 0.672 60,000 10 34 1.015 Plumbous Chloride O 650,000 100.0 2.00 0.342 " " " 165,000 25.38 1.405 0.672 3,000 0.46 -0o335 Biotin 0 450,000 100.0 2.00 0.342 " 175,000 38089 1.59 0.672 14,400 3,20 0.505 Riboflavin 0 650,000 100.0 2.00 0.342 " " 250,000 38.31 1.583 0.672 26,ooo 4.00 0.602

TABLE XI (concluded) Control (M/1-5 Phosphate'. Chemical in M/15 Phosphate Buffer Buffer at pH 7-,0):__ at pH 7.0 ~,.] P']''OC] bP 4 a o ~) o -) 0o. oE to P,' > Q > P > >,.., 0|. __ __ ii _ QbD O1 rHM 0 0.342 0.672 0 0.169 0.338 0.676 0.845 1.014 0 o.169 0.338 0.676 0.845 1.014 520,000 143,000 7,200 1,030,000 765,000 500,000 53,000 8,050 1,075 610,000 453,100 315,000 56,000 8,050 1,075 100 " 27.50 1.38 100.0 74.27 48.54 5.146 0.782 0.104 100 74.28 51.64 9.18 1.32 0.176 2 00 1.439. 140 2.00 1.871 1.686 0.711 -0.107 -0.983 2.00 1.871 1.713 0.963 0.121 -o0754 Nicotinic Acid 420, 000 203,000 17,100 Glutathione 95,00oo 965,ooo 775,000 264,500 104,000 24,500 Gelatin 970,000 745,-00 510,000 156,000 64,000 25,000 100.0 48.33 4.07 100.0': 98.47 79.08 27.00 10.61 2(&50 100.0 76.8 52.58 16.o8 6.598 2.577 2.00 1.684 0.610 2.00 1 995 1.898 1.431 1.026 0.598 2. 00 1.885 1.721 1.206 0.819 0,411 0 0.. 169 0.338 0.676 0.845 1.014 l-Ly~Sine 530,00 400,000 84,000 18,250 3,95P dl -Methionine 540,000 365,000 179,500 93,000 35,000 100.0 2.00 75.47 15-85 3.44 0.745 100.0 67.60 33.24 17.22 6..48 1.878 1.200 0.537 2.00 1.850 1.522 1.2356 0.812 0 0,169.3358 0.676 0.845 1.014 It

2.0 2. ^\ nI o0 > 1. cn o 0 (9 -I. -J -1.'I,> O-CONTROL X-LECITHIN D-NICOTINIC ACD' It U C). (n cr o a: 0 0:: 0 0 0 -J.. O -CONTROL X- IRON (FERROUS). O-LEAD (PLUMBOUS) f% I ^. 0 0 0.4 0.8 MEGAREP Fig. 14a 1.2 I.u o -1.0 -2.0 - 0 0.4 0.8 1.2 MEGAREP Fig. 14b 2.0 iO - CONTROL X- VITAMIN A 0- VITAMIN K...~-_-.... —---—.... 2.:) cro -J 0-2. -2.:) Q5 0 (9 o 0 (01 -I. -1.01'4 -2.0L 0 0 0.4 0.8 MEGAREP Fig. 14c 1.2 0.4 0.8 MEGAREP Fig. 14d Effect of various chemicals incorporated into M/15 phosphate buffer at'pH 7.0 on the lethality of gamma radiation from cobalt-60 on the spores of C. botulinum 62A suspended in the solutions so obtained.

2.00 1.00 0) 0 n" C2 D (C) 0 O - CONTROL X X - LYSINE A - METHIONINE -GELATIN\ X - LYSIN \ \ o0 -2.00 0 0.4 0.8 MEGAREP 1.2 Fig. 14e Effect of various chemicals incorporated into M/15 phosphate buffer at pH 7.0 on the lethality of gamma radiation from cobalt-60 on the spores of C. botulinum 62A suspended in the solutions so obtained.

V. BIBLIOGRAPHY 1. Morgan, Bruce H., and Reed, James M., Resistance of Bacterial Spores to Gamma Radiation", Food Research,. 19, 357-359 (1954). 2. Reed, J. M., Bohrer, C. W., and Cameron, E. J., "Spore Destruction Rate Studies on Organisms of Significance in the Processing of Canned Foods", Food Research, 16, 383-408 (1951). 3. Halvorson, H. Orin, Personal Communication, 1946.