ENGINEERING RESEARCH INSTITUTE THE UNIVERSITY OF MICHIGAN ANN ARBOR Final Report COMBINED USE OF HEAT AND RADIATION TREATMENT FOR STERILIZATION OF FOODS Period 7 June 1955 to 31 August 1956 Lloyd L, Kempe Official Investigator J. To Graikoski Nancy J. Williams Peter Fo Bonventre Collaborators QUARTERMASTER RESEARCH AND DEVELOPMENT COMMAND NATICK, MASSACHUSETTS CONTRACT NO, DA-19-129-qm-388, PROJECT NO. 7-84-01-002 September 1956

TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS iii GENERAL SUMMARY 1 PHASE I - EFFECT OF PREIRRADIATION OF INOCULATED PACKS OF CANNED GROUND BEEF ON THE Fo SUBSEQUENTLY REQUIRED FOR STERILIZATION 4 SUMMARY 4 INTRODUCTION 4 MATERIALS AND METHODS 5 RESULTS 6 DISCUSSION 36 BIBLIOGRAPHY 36 PHASE II - EFFECT OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES 37 SUMMARY 37 INTRODUCTION 37 MATERIALS AND METHODS 38 RESULTS 39 BIBLIOGRAPHY 51 PHASE III.- EFFECTS OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF THE SPORES OF ANAEROBIC BACTERIA 55 SUMMARY 55 INTRODUCTION 55 MATERIALS AND METHODS 56 RESULTS 56 DISCUSSION 70 BIBLIOGRAPHY 70

LIST OF ILLUSTRATIONS Table Page I-P Fo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with Approximately 5,000,000 C. botulinum 213B Spores Per Can 7 II-P Gamma-Radiation Dosage Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with Approximately 5,000,000 C. botulinum 213B Spores Per Can 10 III-P Fo Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 5,000,000 Co botulinum 213B Spores Per Can, and Then Irradiated with Gamma Rays from Cobalt-60 Before Heat Processing 12 IV-P Fo Value Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 300 C. botulinum 213B Spores, and Then Heat Processed 22 V-P Gamma-Radiation Dosage Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with Approximately 300 C. botulinum 213B Spores Per Can 26 VI-P F Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 300 C. botulinum 213B Spores Per Can, and Then Irradiated with Gamma Rays from Cobalt-60 Before Heat Processing 29 I-C Effect of Methionine in M/15 Phosphate Buffer at pH 700 on the Lethality of Gamma Radiation from Cobalt60 for the Spores of C. botulinum 213B 39 II-C Effects of Glutathione and Sodium Hydrosulfite Added to M/15 Phosphate Buffer at pH 7.0 on the Lethality of Gamma Radiation from Cobalt-60 for the Spores of C. botulinum 213B 40 III-C Effect of Protective Chemicals Added to the M/15 Phosphate Buffer Used as a Suspending Medium for PA 3679 Spores During Irradiation with Gamma Rays from Cobalt-60 43 IV-C Effect of the pH of the Medium in Which C. botulinum 213B Spores Were Suspended During Irradiation on the Lethality of Gamma Rays from Cobalt-60 for These Spores 45 V-C The Effect of Different Molar Concentrations of pChloromercuribenzoate on the Lethality of Gamma Rays from Cobalt-60 on the Spores of C. botulinum 213B 47 iii

List of Illustrations (Cont.) Table Page VI-C Effect of Oxygen Tension on the Protection Afforded C. botulinum 213B Spores by Sodium Hydrosulfite and Glutathione Against the Lethal Effects of Gamma Rays of Cobalt-60 49 I-T 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 57 II-T 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 59 III-T Survival of Co 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 Temperatures 61 IV-T Effect of Temperature During Irradiation with Gamma Rays from Cobalt-60 on the Survival of C. botulinum 213B Spores Suspended in M/15 Phosphate Buffer at pH 7o0 64 V-T Effect of Temperatures During Irradiation with Gamma Rays from Cobalt-60 on the Survival of C. parabotulinum 457A Spores Suspended in M/15 Phosphate Buffer at pH 7.0 66 VI-T Effect of Temperatures During Irradiation with Gamma Rays from Cobalt-60 on the Survival of PA 3679 Spores Suspended in M/15 Phosphate Buffer at pH 7.0 67 VII-T Effect of Postirradiation Heating for One Hour at Various Temperatures on Previously Irradiated PA 3679 Spores 68 Figure 1-P F0 required to sterilize ground beef packed in No. 1 picnic tin cans, inoculated with approximately 5,000,000 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 34 iv

List of Illustrations (Cont.) Figure Page 2-P Fo required to sterilize ground beef packed in No. 1 picnic tin cans inoculated with approximately 300 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 35 1-C Effect of methionine in M/15 phosphate buffer of pH 7.0 on the lethality of gamma radiation from cobalt60 for the spores of C. botulinum 213B. 41 2-C Effects of glutathione and sodium hydrosulfite added to M/15 phosphate buffer at pH 7.0 on the lethality of gamma radiation from cobalt-60 for the spores of C. botulinum 213B. 42 3-C Effect of protective chemicals at 0.02 M concentration when present in the M/15 phosphate buffer (pH 7.0) used as a suspending medium for PA 3679 spores during irradiation with gamma rays from cobalt-60. 44 4-C The effect of different molar concentrations of pchloromercuribenzoate on the lethality of gamma rays from cobalt-60 on the spores of C. botulinum 213B. 48 5-C The effect of oxygen tension on the lethality of gamma rays from cobalt-60 on the spores of C. botulinum 213B suspended in M/15 phosphate buffer at pH:7.0. 52 6-C The effect of oxygen tension on the protection afforded C. botulinum 213B spores by glutathione against the lethal effects of gamma rays from cobalt-60o 53 7-C The effect of oxygen tension on the protection afforded C. botulinum 213B spores by sodium hydrosulfite against the lethal effects of gamma rays from cobalt-60. 54 1-T 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. 58 2-T 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. 60 v

List of Illustrations (Conc.) Figure Page 3-T Survival curves for C: botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0, which have been first irradiated with gamma rays from cobalt-60 and then held for one hour at the indicated temperatures. 62 4-T 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. 65 5-T Effect of temperature during irradiation on the survival of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7~.0 65 6-T Effect of postirradiation heating for one hour at various temperatures on previously irradiated PA 5679 spores. 69

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 Project No. 7-84-01-002 Contract No. DA-19-129-qm-388 File No. S-510 Report 8 (Final) Period 7 June 1955 to 31 August 1956 Initiation Date: 7 June 1955 Title of Contract: Combined Use of Heat and Radiation Treatment for Sterilization of Foods GENERAL SUMMARY In order that irradiation processing of foods can be intelligently considered, basic factual information is needed. Such information includes the effects of temperature on the lethality of ionizing radiations for anaerobic bacterial spores that are important in food sterilization, the effects of medium components, as well as the combined effects of temperature and such radiations. Where it may be planned to use irradiation alone for processing foods, it is necessary to know whether or not such a treatment is.effective at refrigerating or freezing temperatures which protect the food before irradiation. Further, do chemical additives used in the preparation of the food affect the sterilizing effectiveness of ionizing radiation? These and other questions have provided the necessity for and objectives of this workO The work has so far established the following: 1o An induction dosage of approximately one megarep of gamma radiation is required before the combined irradiation-heat processing of

food shows advantage. For example, using 300 Co botulinum 213B spores in a No, 1 picnic can of ground beef, sterility was attained with an Fo of 0015 following 1.0 megarep of radiation. But, following 0.5 megarep or less, an Fo of 0.4 was needed to produce sterile cans of meat. 2. Reduced sulfhydryl groups present in anaerobic bacterial spores protect these spores against the lethal activities of gamma radiation, but oxidized sulfhydryl groups are ineffective. 3. Different anaerobic bacterial spores have different temperature ranges in which they are less sensitive to the lethal effects of gamma radiation. However, when such radiations are to be used alone for food sterilization, refrigeration or freezing temperatures are best suited for the process since both C. botulinum and PA 3679 spores are more sensitive to gamma radiation at low temperatures than they are at temperatures between 5~C and 95~C. This is compatible with processing schedules designed to protect foods by refrigeration or freezing before and during irradiation. 4. Combined irradiation —heat processing of foods will involve heating the foods to at least 95~C, either subsequent to or during irradiation. This follows from the discovery that a critical temperature exists at about 95~C, below which irradiated anaerobic bacterial spores are no more sensitive to heat than are nonirradiated spores. However, above 950C, previously irradiated spores are much more easily killed than are nonirradiated spores of C. botulinum or PA 5679. It is expected that future work will establish whether preirradiation of canned raw meat produces similar reductions in the amount of heat required to sterilize previously irradiated foods. Also, the Z value of irradiated spores of C. botulinum has been assumed to be the same as that of nonirradiated spores. This must be factually established before combined irradiation-heat processing schedules can be developed or even properly evaluated. Since the present study of the combined irradiation-heat processing of foods has been confined to ground beef, and C. botulinum spores, this work should be extended to include other food products and other anaerobic bacterial spores. Several papers are in preparation as a result of this work. They will include the following: 1. Combined irradiation-heat sterilization of canned ground beef: by Lloyd L. Kempe, J. T. Graikoski, and P. F. Bonventre. 2

2. The effect of chemicals on the lethality of gamma radiation for anaerobic bacterial spores: by Nancy J. Williams and Lloyd L. Kempe. 3. The effect of temperature during irradiation on the lethality of gamma radiations for anaerobic bacterial spores: by J. T. Graikoski and Lloyd L. Kempe. 4. Sensitization of anaerobic bacterial spores to temperatures above 85~C by gamma radiation: by J. T. Graikoski and Lloyd L. Kempe. 3

PHASE I EFFECT OF PREIRRADIATION OF INOCULATED PACKS OF CANNED GROUND BEEF ON THE FO SUBSEQUENTLY REQUIRED FOR STERILIZATION SUMMARY Canned ground beef, packed in Noo 1 picnic tin cans and inoculated with Clostridium botulinum 2135B spores, was sterilized using preirradiation and heat processing, Two spore concentrations were tested. ao When 5,000,000 C. botulinum 2133B spores were used per can of meat, heat processing alone required an Fo of approximately 1.0 for sterilizationo Similarly, irradiation alone required between 3.4 and 3.9 megarep to produce sterility. But when preirradiation with 1.2 megarep of gamma radiation was used, sterility was attained with an Fo of approximately 0.2. b. Using 300 C. botulinum 213B spores per can of meat, sterilization was attained with an Fo of approximately 0.4, while with irradiation alone, approximately 1.7 megarep of gamma radiation from cobalt-60 were required. With preirradiation using 1.0 megarep, sterility was attained with an F0 of approximately 0.15. INTRODUCTION Combined radiation and heat processing of canned foods to produce commercially sterile products has been considered possibly more desirable than sterilization with either energy form alone.122,3 This phase of the present project is designed to test such an idea. It has been previously demonstrated that canned meat can be sterilized by gamma radiation alone,l and, it is common industrial practice to sterilize foods with heato When a study of the combined processing was considered, it was found desirable first to irradiate the inoculated packs of canned ground meat and follow this by heat processing. The sequence of irradiation followed by heat processing is based on the previously observed finding2Y3 that preirradiation sensitized the anaerobic bacterial spores to the lethal properties of heat and that the sensitization persists during subsequent storage until heat processing is effected. This work was designed to establish combined irradiation-heat processing treatments that would sterilize canned ground meat. From these treatments, it would be possible to select the most desirable combined 4

process for further investigation. The study has been concerned with sterilizing inoculated packs prepared from previously heat-sterilized ground beef packed in No. 1 picnic tin cans before inoculation. Data have been accumulated to establish possible combined sterilization treatments under these conditions at two levels of inoculation with Clostridium botulinum 213B spores. MATERIALS AND METHODS 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 tin cans, four of which have previously been fitted with 0. F. Ecklund thermocouples. Covers are set loosely on the cans of meat 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 and the meat is inoculated with 2 ml of a spore suspension. The cans are then sealed in a commercial-type closing machine, immersed in cold tap water for about 20 minutes, and finally placed in ice water for an houro Experimental cans are then either irradiated or temporarily stored in a refrigerator, as required. The 8 controls are placed in an incubator immediately, while the experimental cans are incubated after processing. Processed cans are quickly cooled by immersion in cold water before incubation. Incubation is carried out at 37~C or 30~C, as indicated. b. Irradiation.-The canned meat is irradiated in the "center well" of the large cobalt-60 source here at The University of Michigan. The- temperature of the meat is kept below 4~C during irradiation. c. Heat Processing.-Several false starts were made before a heat-processing technique was developed that yielded reproducible data. As finally developed, 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 1800F. 2. Six cans of meat, two of which contain thermocouples, are removed from the refrigerator, thermocouples 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. 5

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 180~F was selected because (a) it represents "hotfilling" temperatures of industrial practice, (b) it is not sufficient to cause appreciable killing of spores anywhere in the can during temperature equilibration, and (c) it is high enough to make "come-up" rates to the processing temperature essentially uniform in all the cans. 4o When the cans have equilibrated at some temperature between 170~ and 1800F, the water-bath temperature is quickly brought to 250~F by introducing steam into the autoclaveo Processing time to attain the desired Fo value, less the cooling increment, is now provided. 5o At the proper time, the autoclave is quickly opened and the cans are plunged into ice watero Temperature measurements are continued until the temperature at the center of the cans reaches 180~F. 60 The four experimental cans of meat are incubated. The two cans containing thermocouples are again refrigerated; they are used a second time onlyo Incubation was carried out at 37~C for the first series of runs, i.e., for those packs inoculated with approximately 5,000,000 C. botulinum 213B spores per can. The second series, or those cans inoculated with approximately 300 spores per can, were incubated at 29~Co 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 acquiredo 8. Fo values are calculated by Oo T. Schultz's graphical modification of C. 0. Ball's General Method. In these calculations the Z value of both irradiated and nonirradiated C. botulinum 213B spores is assumed to be 18. d. Spores.-The spores of anaerobic bacteria used in these studies are prepared and used according to techniques described in previously published work from this laboratoryoRESULTS Canned ground beef packed in No. 1 picnic tin cans was sterilized 6

by combined irradiation-heat processing technique. a. As shown in Figs. 1-P and 2-P and Tables I-P through IV-P, an induction level of preirradiation with gamma rays was required before the sensitization of spores became significantly important. This level was found to be approximately 1 megarep for C. botulinum 213B spores. b. The data in Figo 1-P show that 5,000,000 C. botulinum 213B spores were used per can of meat, heat processing alone required an Fo of approximately 1.0 for sterilization. Similarly, irradiation alone required between 354 and 359 megarep to produce sterility. But when preirradiation with 102 megarep of gamma radiation, was used, sterility was attained with an. Fo of approximately 0.2. c. From Fig. 2-P it will be observed that when using 300 C. botulinum 213B spores per can of meat, sterilization was attained with heat alone using an F0 of approximately 0.4, while with irradiation alone approximately 1.7 megarep of gamma radiation from cobalt-60 were required. On the other hand, when irradiation and heat processing were combined, sterility was attained with an Fo of approximately 0.15 following 1.0 megarep of radiation. TABLE I-P Fo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with Approximately 5,000,000 C. botulinum 213B Spores Per Can Pun No. - C-1 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Spores - 10,700,000 Co botulinum 213B spores per can Incubation Temperature - 37~ Fo Can No Days to Gas Formation Inoculated control 1 2 2 2 3 2 4 2 7

Fo__ Can No, Days to Gas Formation Noninoculated control Can 1, 0.26 Can 2, 0.31 Can 1, 1.00 Can 2, 1.00 Can 1, 0.80 Can 2, 1.25 Can 1, 0.53 Can 2, 0.57 1 2 3 4 1 2 3 4.5 5 5 5 5 6 7 8 6.5 7 6 11 9 10 11 12 6.5 7.5 -.5 6.5 13 14 15 16 6.5 5.5 7.5 5.5 Conclusion: The Fo required to sterilize ground beef packed in No. 1 picnic tin cans and inoculated with 10,700,000 C. botulinum 2135B spores is more than, but close to, 1.00. Remarks: Used equilibration bath temperature of 210~F and sterilizing bath at 230~F. Run No.o - C-2 Can Size - No 1 Picnic (211 x 400) Product - Ground Beef Spores - 7,200,000 C. botulinum 213B spores per can Incubation Temperature - 37~C _F0.Can No. Days to Gas Formation Inoculated control 1 3 2 4 8 1.5 1.5 1.5 1.5

_0 Can No. Days to Gas Formation Noninoculated control Can 1, 0.28 Can 2, 0.39 Can 1, 0.70 Can 2, 0.73 Can 1, 0.75 Can 2, 1.01 Can 1, 1.24 Can 2, 1.29 1 2 3 1 2 4 3.5 3.5 3.5 5.5 5 6 7 8 3.5 9 10 11 12 3.5 4.5 4.5 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans and inoculated with 7,200,000 C. botulinum 213B spores per can was sterilized in the Fo range of 0.75 to 1.29. Remarks: A preheat water-bath temperature of 210~F and sterilizing bath temperature of 230~F were used. Run No. - C-3 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Spores - 10,700,000 C. botulinum 2133B spores per can Incubation Temperature - 37~C F0_ r Can No. Days to Gas Formation Inoculated control 1 2 3 4 1.5 1.5 1.5 1.5 9

Fo Can No.. Days to Gas Formation Noninoculated control Can 1, 0.36 Can 2, 0O49 Can 1, 0.93 Can 2, 0.93 Can 1, 1.18 Can 2, 1.53 Can 1, 1.59 Can 2, 1.82 1 2 3 4 1 2 3 4 6 6 6 6 5 6 7 8 9 10 11 12 13 14 15 16 Conclusion: Ground beef packed in No. 1 with 10,700,000 C. botulinum 213B spores range of 0.36 to 0.935 picnic tin cans and inoculated per can was sterilized in the Fo Remarks: A preheat water-bath temperature of 190~F and a sterilizing bath temperature of 2300F were usedo TABLE II-P Gamma-Radiation Dosage Required to Sterilize Ground Beef in Noo 1 Picnic Tin Cans Previously Inoculated with Approximately 5,000,000 C. botulinum 213B Spores Per Can Run No. - CB-15 Can Size Product Inoculum Incubation - No. 1 Picnic (211 x 400) - Ground Beef - 5,000,000 C. botulinum 213B spores per can Temperature - 37~C 10

Radiation Dosage d(megarep) D eCan Noo Days to Gas Formation (miegarep) Noninoculated control - no irradiation Inoculated control - no irradiation 3.42 3.96 1 2 3 21 22 23 24 1 2 3 2 2 2 2 4 4 4 5 6 7 8 2.52 9 10 11 12 4 3 3 3 2.16 13 14 15 16 2 3 3 3 4 3 5 4 4 2.88 17 18 19 20 Conclusion: Ground beef packed in No. 1 picnic tin cans and inoculated with 5,000,000 C. botulinum 213B spores per can was sterilized with 3.42 to 3.96 megarep of gamma radiation from cobalt-60. 11

TABLE III-P Fo Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 5,000,000 C. botulinum 213B Spores Per Can, and Then Irradiated with Gamma Rays from Cobalt-60 Before Heat Processing Run No. - CB-3 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 2,300,000 Co botulinum 213B spores per can Preirradiation - 1.35 megarep Incubation Temperature - 37~C Fo Can No. _Days to Gas Formation Inoculated control Noninoculated control 0.36 1.03 0.23 0.63 1 2 3 4 2 2 2 2 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 - ---- 12

Run No. CB-4 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 5,200,000 C. botulinum 213B spores per can Preirradiation - 1,350,000 rep Incubation Temperature - 37~C F0o Can No. Days to Gas Formation Inoculated control Noninoculated control Irradiation control Without heat processing but using 1,550,000 rep Can 1, 0.11 Can 2, 0.11 Can 1, 0.21 Can 2, 0.21 Can 1, 0.42 Can 2, 0.42 Can 1, 0.62 Can 2, 0.62 1 2 3 1 1 1 1 2 3 4 17 18 19 20 1.5 1.5 1.5 1.5 4 m. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 5,200,000 C. botulinum 213B spores per can, and then irradiated with 1.35 megarep of gamma rays from cobalt-60, subsequently required an Fo between 0.11 and 0.21 for sterilization. 13

Run No. CB-5 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 6,300,000 C. botulinum 213B spores per can Preirradiation - 675,000 rep Incubation Temperature - 37~C F0 Can No. Days to Gas Formation __ _ _ __~~~~~~~~ Inoculated control Noninoculated control Can 1, 0.12 Can 2, 0.12 1 2 3 4 2 2 2 2 1 2 3 4 1 2 3 4 3 3 3 3 5 Can 1, Can 2, Can 1, Can 2, 0.36 0.36 0.47 0.47 5 6 7 8 5 4 4 4 9 10 11 12 8 Can 1, 0.58 Can 2, 0.58 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 6,300,000 C. botulinum 213B spores per can, then irradiated with 0.675 megarep of gamma rays from cobalt-60, subsequently required an Fo greater than 0.58 for sterilization. *Toxin present by animal inoculation test.

Run No. CB-6 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 6,000,000 C. botulinum 213B spores per can Preirradiation - 1,000,000 rep Incubation Temperature - 57~C Fo Can No. Days to Gas Formation Inoculated control Noninoculated control Can 1, 0.24 Can 2, 0.39 Can 1, 0.41 Can 2, 0.59 Can 1, 0.80 Can 2, 0.80 Can 1, 0.04 Can 2, 0.09 1 2 3 4 2 2 2 2 1 2 3 4 1 2 3 4 5 6 6 7 5 6 7 8 7 7 9 10 12 13 11 14 15 16 5 4 5 4 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 6,000,000 C. botulinum 213B spores per can, and then irradiated with 1.00 megarep of gamma radiation from cobalt-60, subsequently required an F between 0.41 and 0.80 for sterilization. 15

Run No, CB-7 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 3,800,000 C. botulinum 213B spores per can Preirradiation - 1,200,000 rep Incubation Temperature - 37~C Fo Can No. Days to Gas Formation Inoculated control Noninoculated control Can 1, 0.66 Can 2, 0.77 Can 1, 0.55 Can 2, 0.55 Can 1, 0.25 Can 2, 0.29 Can 1, 0.09 Can 2, 0 11 1 2 2 2 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 15 14 15 16 6 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 3,800,000 C. botulinum 213B spores per can, and then irradiated with 1.200 megarep of gamma radiation from cobalt-60, subsequently required an F between 0.09 and 0.29 for sterilization. 16

Run No. CB-8 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 5,000,000 C. botulinum 213B Preirradiation - 500,000 rep Incubation Temperature - 37~C Fo Can No. Days to Gas Formation Noninoculated control Inoculated control Can 1, 1.40 Can 2, 0o85 Can 1, 0.745 Can 2, 0.639 Can 1, 1.05 Can 2, 0.73 Can 1, 0.881 Can 2, 0.540 Nonirradiated controls Can 1, 0.86 Can 1, lo18 1 2 3 4 2 1 2 3 4 2 2 2 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 17

Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 5,000,000 C. botulinum 213B spores per can, and then irradiated with 0.500 megarep of gamma radiation from cobalt-60, required an F0 greater than 0.85 for sterilization. Run No. CB-9 Can Size - No. 1 Picnic Product - Ground Beef Inoculum - 5,000,000 C. botulinum 213B spores per can Preirradiation - 1,500,000 rep Incubation Temperature - 37~C Fo Can No. Days to Gas Formation Noninoculated controls Inoculated controls Can 1, 0.029 Can 1, 0.10 Can 2, O.ll Can 1, 0.015 Can 2, 0.019 Can 1, 0.062 Can 2, 0.081 1 2 5 1 2 3 4 2 2 2 2 1 2 3 4 2 2 2 2 5 6 7 8 3 3 3 5 9 10 11 12 2 2 3 3 13 14 15 16 5 4 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 5,000,000 C. botulinum 213B spores per can, and then irradiated with 1.500 megarep of gamma radiation from cobalt-60, required an Fo greater than 0.11 for sterilization.

Run Noo CB-10 Can Size - No. 1 Picnic Product - Ground Beef Inoculum - 6,000,000 C. botulinum 213B spores per can Preirradiation - 500,000 rep Incubation Temperature - 37~C Fo Can No. Days to Gas Formation L Noninoculated controls Inoculated controls Can 1, 0.43 Can 2, 0.26 Can 1, 0.56 Can 2, 0.51 1 2 3 4 17 18 19 20 42 2.5 2.5 2.5 2.5 1 2 4 4 5 6 5 6 5 6 7 8 6 6 6 Can 1, 1.o6 9 10 11 12 Can 1, 0.84 Can 2, 0.77 13 14 15 16 9 7 Nonirradiated controls Can 1, 0.81 Can 2, 0.68 20 21 22 23 7 7 6 7 Can 1, 0.98 24 25 26 27 8 19

Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 6,000,000 C.-botulinum 213B spores per can, and then irradiated with 0.500 megarep of gamma radiation from cobalt-60, required an Fo between 0.77 and 1,06 for sterilization. Run Noo CB-11 Can Size - No. 1 Picnic Product - Ground Beef Inoculum - 12,000,000 C. botulinum 213B spores per can Preirradiation - 1,500,000 rep Incubation Temperature - 37~C F0 Can No. Days to Gas Formation Noninoculated controls 1 2 3 4 5* Inoculated controls 17 18 19 20 2 2 2 2 Can 1, 0.329 Can 2, 0.206 1 2 4 4 5 Can 1, 0.125 Can 2, 0.072 5 6 7 8 3 3 Can 1, 0.047 Can 2, 0.033 9 10 11 12 3 3 3 3 Can 1, 0.147 Can 2, 0.087 13 14 15 16 3 6 4 Conclusion: None *Toxin present by mouse inoculation test. 20

Run No CB-12 Can Size - No. 1 Picnic Product - Ground Beef Inoculation - 5,000,000 C. botulinum 213B spores per can Preirradiation - 1,500,000 rep Incubation Temperature - 37~C Fo Can No. Days to Gas Formation Noninoculated controls 1 2 3 4 Inoculated controls Can 1, 0.15 Can 2, 0.04 Can 1, 0.27 Can 2, 0.26 Can 1, 0oo63 Can 1, 0.39 Can 2, 0.25 17 18 19 20 2 2 2 2 1 2 3 4 3 3 3 3 5 6 7 8 9 10 11 12 2 3 2 5 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 5,000,000 C. botulinum 213B spores per can, and then irradiated with 1.500 megarep of gamma radiation from cobalt-60, subsequently required an Fo between 0.063 and 0.27 for sterilization. 21

TABLE IV-P Fo Value Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 300 C. botulinum 213B Spores, and Then Heat Processed Run No. CB-17 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 250 C, botulinum 213B spores per can Incubation Temperature - 37~C Fo Can No. Days to Gas Formation Noninoculated control Inoculated control 0.21 1 2 3 4 17 18 19 20 3 3 5 5 1 2 3 4 0.51 5 6 7 8 0.28 9 10 11 12 0.37 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 250 C. botulinum 213B spores per can, and then heat processed, requires an Fo of at least 0.21 for sterilization when incubated at 37~C. 22

Run Noo CB-18 Can Size - No 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 250 Co botulinum 213B spores per can Incubation Temperature - 29~C Fo Can No Days to Gas Formation Remarks _ -_ _...i..... Noninoculated control Inoculated control Can 1, 0.31 Can 2, 0.26 1 2 3 4 4 14 no toxin no toxin 17 18 19 20 1 2 3 4 3 3 3 3 toxin 7 no toxin Can 1, 0.14 5 6 7 8 4 4 4 4 Can 1, 0.079 Can 2, 0~058 9 10 11 12 3 3 3 3 3 3 3 3 5 toxin no toxin toxin toxin toxin cans, inoculated with processed, requires an 0.035 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin 250 C. botulinum 213B spores per can, and then heat FO between 0.14 and 0.31 for sterilization. I- --- - - - - ----- -—, --- — ~- - - - _- -cl - - - _ _ __ _ c_ 23

Run No. CB-19 Can Size - No. 1 Picnic (211 x 400) Product - Canned Beef Inoculum - 400 C. botulinum 213B spores per can Incubation Temperature - 29~C Fo Can No. Days to Gas Formation Remarks Inoculated control Noninoculated control Can 1, 0003 Can 2, 0o045 Fo = 0.084 Fo = 0.15 Can 1, 0.26 0.45 19 20 21 22 4 1 4 4 toxin 1 2 17 no toxin 1 2 3 4 6 6 4 6 5 6 7 8 5 4 4 5 9 10 11 12 8 toxin toxin toxin no toxin 13 14 15 16 4 Remark: An additional experiment on autoclaving carried out as part of this run established the fact that the cans were not being adequately processed by our preliminary autoclaving treatment. This explained the positive controls obtained in this and, occasionally, in previous runs. After considerable effort, it was found that the automatic exhausting system was almost completely plugged with meat particles. So the cans were being processed in an incompletely exhausted autoclave. This was immediately corrected. Conclusion: Based on toxin tests in mice in addition to the observation of gas production, this run shows that ground beef packed in No. 1 picnic tin cans, inoculated with 400 C. botulinum 213B spores per can, requires an Fo between 0.15 and 0.45 for sterilization. 24

Run No. CB-24 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Heat Processed Only Incubation Temperature - 29~C Fo Can No. Days to Gas Formation Noninoculated controls Inoculated control Can 1, 0e26 Can 2, 0.26 Can 1, O.36 Can 2, 0.54 Can 1, 0.33 Can 2, 0.33 Can 1, 0.47 1 2 5 17 18 1 2 3 4 5 6 8 6 6 5 5 7 7 7 9 10 11 12 7 13 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 300 C. botulinum 213B spores per can, was sterilized by heat processing to an Fo value between 0.33 and 0.47. 25

TABLE V-P Gamma-Radiation Dosage Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with Approximately 300 C. botulinum 213B Spores Per Can Run No. CB-14 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 250 C. botulinum 213B spores per can Irradiation Only Incubation Temperature - 37~C Radiation Dosage Can No. Days to Gas Formation (megarep) Noninoculated control 1 2 3 4 33 41 t* Inoculated controls 1.117 2.300 2.900 3.400 17 18 19 20 2.5 2.5 2.5 2.5 33 34 35 36 37 38 39 40 3 4 3 3 4 3 4 29 30 31 32 25 26 27 28 17 18 19 20 26

Radiation Dosage Radiatn Dage Can No. Days to Gas Formation (megarep) 4.000 21 22 23 24 Remarks: 1. The indication of few bacterial spores not having been killed by the presterilization treatment does not invalidate this run. 2. The incubation temperature was 37~C instead of the 29~C used on all other runs with low spore concentration. Conclusion: Ground beef packed in No. 1 picnic tin cans and inoculated with 250 C. botulinum 213B spores per can was sterilized with between 1.117 and 2.300 megarep of gamma radiation from cobalt-60. Run No. CB-21 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Irradiation Only Incubation Temperature - 29~C Radiation Dosage Radiatin D e Can No. Days to Gas Formation (megarep) Noninoculated control - no irradiation 1.02 1.36 1.80 1 2 3 1 2 3 4 6 6 6 6 5 6 7 8 7 6 7 6 8 9 11 12 27

Radiation Dosage Radiation Ds Can No. Days to Gas Formation (megarep) 2.10 13 14 Conclusion: Ground beef packed in No. 1 picnic tin cans and inoculated with 300 Co botulinum 213B spores per can was sterilized with 1.36 to 1.80 megarep of gamma radiation from cobalt-600 Run No CB-27 Can Size - Noo 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 Co botulinum 213B spores per can Incubation Temperature - 29~C Radiation Dosage Radiatin D e Can No. Days to Gas Formation (megarep) Noninoculated control - no irradiation Inoculated control - no irradiation 1.10 1 2 3 17 18 19 20 6 6 6 5 7 14 16 6 6 6 6 11 1 12 3 8 6 m 1o92 4 6 13 15 Conclusion: Ground beef packed in Noo 1 picnic tin cans and inoculated with 300 C. botulinum 213B spores per can was sterilized with 1.64 to 1092 megarep of gamma radiation from cobalt-60. 28

TABLE VI-P Fo Required to Sterilize Ground Beef Packed in No. 1 Picnic Tin Cans, Inoculated with Approximately 300 C. botulinum 213B Spores Per Can, and Then Irradiated with Gamma Rays From Cobalt-60 Before Heat Processing Run No. CB-20 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Preirradiation - 0.500 megarep Incubation Temperature - 29~C F.0 Can No. Days to Gas Formation Remarks Noninoculated controls Inoculated controls 1 2 3 4 17 18 19 20 1 2 3 4 5 5 5 5 toxin Can 1, Can 2, Can 3, Can 1, Can 2, Can 3, 0.12 0,17 0.15 0.095 0.085 0.063 7 7 7 8 7 7 7 5 6 7 8 Can Can Can 1, 0.022 2, 0.025 3, 0.028 9 10 11 12 6 10 6 Can 1, Can. 2, Can 3, 0.32 0.40 0.38 13 14 15 16 5 10 toxin 29

Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 300 C. botulinum 213B spores per can, and then irradiated with 0.500 megarep of gamma radiation from cobalt-60, subsequently required an Fo greater than 0.40 for sterilization. Run No. CB-22 Can Size - No 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Preirradiation - 0.500 megarep Incubation Temperature - 29~C Fo Can No6 Days to Gas Formation Noninoculated controls Inoculated controls 0o48 0.41 0.29 Can 1, 0.15 Can 2, 0.095 Conclusion: None. 1 2 3 4 17 18 19 20 5 5 5 6 1 2 3 4 8 12 10 5 6 7 8 11 m 9 10 11 12 13 14 15 16 10 10 - -- -— ~ I'' I - --- ---- I -- - ---- - L- - 30

Run No. CB-25 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Preirradiation - 500,000 rep Incubation Temperature - 29~C Fo Can No. Days to Gas Formation Noninoculated controls Inoculated controls Can 1, 0.31 Can 2, 0.31 1 2 3 4 1 2 3 3 6 1 2 5 4 Can Can Can 1, 0.326 2, 0.481 3, 0.510 5 6 7 8 Can 1, 0.60 Can 1, 0.46 Can 2, 1o28 9 10 11 12 153 14 15 16 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 300 C. botulinum 213B spores per can, and then irradiated with 0..500 megarep of gamma radiation from cobalt-60, subsequently required an Fo between 0.31 and 0.51 for sterilization. 31

Run No. CB-26 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Preirradiation - 0.750 megarep Incubation Temperature - 29~C Fo Can No. Days to Gas Formation Noninoculated controls Inoculated controls Can 1, 0.34 Can 2, 0.34 Can 1, 0.22 Can 2, 0.22 Can 1, 0.12 Can 2, 0.12 Can 1, 0.057 Can 2, 0.065 1 2 5 3 4 17 18 19 4 4 4 1 2 3 4 5 6 7 8 8 9 10 11 12 8 13 14 15 16 6 Conclusion: Ground beef packed in No. 1 picnic tin cans, inoculated with 300 C. botulinum 213B spores per can, and then irradiated with 0.750 megarep of gamma radiation from cobalt-60, subsequently required an Fo between 0.22 and 0.34 for sterilization. 32

Run No. CB-23 Can Size - No. 1 Picnic (211 x 400) Product - Ground Beef Inoculum - 300 C. botulinum 213B spores per can Preirradiation - 1.000 megarep Incubation Temperature - 29~C Fo Can No. Days to Gas Formation Noninoculated control Inoculated control Can 1, 0o18 Can 2, 0.16 Can 1, 0.25 Can 2, 0.16 1 2 3 4 17 18 19 20 5 5 5 6 1 2 3 4 5 6 7 8 Can 1, 0.35 Can 2, 0.30 9 10 11 12 Can 1, 0.086 13 14 15 16 8 Conclusion: Ground beef packed in No, 1 picnic tin cans, inoculated with 300 C. botulinum 213B spores per can, and then irradiated with 1.000 megarep of gamma radiation from cobalt-60, was subsequently sterilized by heat processing with an Fo between 0.086 and 0.18. 33

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 I I z Li Io3 I2. 0.1.09.08.07.06.05 IS I - -\~ — I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Irradiation alone 3.4 —3.9 MR i I l.04.03.02.0 1 0 1.0 2.0 MEGAREP 3.0 Fig. 1-P. Fo required to sterilize ground beef packed in No. 1 picnic tin cans, inoculated with approximately 5,000,000 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 34

w I -.0.9 ----- z.08.07 I.06 io.05.04.03.02 Irradiation alone 1.64 — 1.80 MR..01 _ 0 1.0 2.0 3.0 MEGAREP Fig. 2-Po Fo required to sterilize ground beef packed in No. 1 picnic tin cans inoculated with approximately 300 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 35

DISCUSSION The spores of C. botulinum were chosen for the initial portion of this work because of their importance in food poisoning. Also, the toxin developed by their growth permitted testing of the processed cans to insure that spoilage was caused by growth of the injected spores and not by contaminants. It was necessary to develop techniques for the heat-processing phase of this work that would produce consistent results. The method described under "Materials and Methods" provided reasonable certainty in the reported results, but ultimately the process should be tested with a larger number of canso Data presented in Section III of this report show that irradiation sensitization of spores to heat was only useful if temperatures above 85~Cwere used. For this reason it was possible to preheat the cans to 180~F in the autoclave before heat processing without affecting the results. The preheating permitted the development of much more uniform Fo values among the cans being processed than when they were processed with lower starting temperatures. It will be noted from the results that the spore concentration difference between 300 and 5,000,000 spores per can did not cause a great deal of difference in the combined processing treatment required. On the other hand, where either of these treatments was used alone, spore concentration was an extremely significant factor.l14 BIBLIOGRAPHY 1. Kempe, L. L., Graikoski, J. T., and Gillies, R. A., "Gamma Ray Sterilization of Canned Meat Previously Inoculated with Anaerobic Bacterial Spores," Appl. Microbiol., 2, 330-332 (1954). 2. Kempe, L. L., "Combined Effect of Heat and Radiation in Food Sterilization," Appl. Microbiol., 3, 346-352 (1955). 35 Morgan, B. Ho,, and Reed, J. M., "Resistance of Bacterial Spores to Gamma Radiation," Food Research, 19,357-366 (1954). 4. Stumbo, C. R., "Thermobacteriology as Applied to Food Processing," Advances in Food Research, Vol. 2, Academic Press, 1949, page 73 et seqo 36

PHASE II EFFECT OF CHEMICAL COMPOSITION OF THE MEDIUM DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES SUMMARY The presence of reducing agents in the chemical environment of anaerobic bacterial spores during irradiation lowers the lethal effectiveness of ionizing radiations from cobalt-600 Conversely. the presence of oxygen enhances the lethal effects of such radiations. This acquires importance when evaluating chemical additives suggested for reducing adverse flavor production by ionizing radiations since most of the chemical additives so far proposed are chemical reducing agentso For example, in the presence of sodium hydrosulfite there was only one log cycle reduction in the number of C. botulinum 213B spores produced by one megarep of gamma radiation, whereas in phosphate buffer alone a reduction of approximately six log cycles was found with a similar amount of radiation. Experiments with mercury compounds indicate that the probable sites of the lethal actions of ionizing radiations within anaerobic bacterial spores are associated with the presence of sulfhydryl groups at such sites. This could be the basic reason why chemical reducing agents protect these bacterial spores and oxygen acts in the opposite manner when such spores are irradiated. INTRODUCTION It has been shown that the most important biological effects of ionizing radiations result from their ability to generate oxidizing substances such as free radicals and hydrogen peroxide, which are assumed to be effective over several molecular diameters. It has also been shown that such reactions are strongly enhanced by the presence of oxygen and that the presence of easily oxidizable materials during irradiation counteracts the oxidative effectso Our previous work1 has shown that these observations can be extended to aid in an understanding of the lethal actions of gamma radiations on the spores of anaerobic bacteria that are important in food preservation. The present studies have been limited to phosphate buffer or similar solutions prepared from materials of known composition as suspending media for C. botulinum or PA 35679 spores during irradiation. The work has been 37

directed toward learning whether the chemical additives suggested for minimizing the adverse organoleptic properties of ionizing radiations also protect these spores during irradiation. The possibility that such might be the case is obvious since both effects are considered to be at least partly due to oxidative reactions. The results of this work will be presented under these headings, viz:. a. Effect of reducing agents. b. Effect of a mercury compound. c. Effect of oxygen. MATERIALS AND METHODS ao Organisms.-C o botulinum 213B spores were grown for three weeks in a casitone medium at 30~C, 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 cellso 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 mlo PA 3679 spores were grown for three weeks in pork extract broth at 30~Co They were then harvested and pretreated in a manner identical to that previously described for C. botulinum 213B spores. b. Chemicals. —All solutions were prepared in 0.02-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. The following are described as examples: Glutathione —0.312 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 7.0. 38

c. Control. —In every case, sterile M/15 phosphate buffer of pH 7.0 was used as the control solution. ds Irradiation Procedures. —All samples were irradiated in flame-sealed glass vials that contained approximately 4 ml of solution. These were placed in an especially designed container which provided temperature control at 4~Co The containers and vials were then irradiated for proper intervals in the center well of the large cobalt-60 gamma radiation sourceo RESULTS a. Effect of Reducing Agents —The data taken with Co botulinum 213B spores substantiate previous datal taken with Co 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 I-C and II-C and in the companion Figs. 1-C and 2-Co TABLE I-C Effect of Methionine in M/15 Phosphate Buffer at pH 7.0 on the Lethality of Gamma Radiation from Cobalt-60 for the Spores of Co botulinum 213B Control Methionine Radiation ConoRadiation Spores Log % Spores Log %o -Dose Rp per ml Survivors per ml. Survivors 0 8,700,000 2.000 7,800,000 2.000 36o,00o 3,150,000 1.559 5,350,000 1.836 540,000 2,260,000 1.462 720,000 83,000 -0.020 750,000 0.983 900,000 4,500 -1o287 190,000 0.387 1,080,000 128 -2o831 27,100 -o 459 39

TA BLE IS-C Effects of Glutathione and Sodium Hydrosulfite Added to M/15 Phosphate Buffer at pH 7.0 on the Lethality of Gamma Radiation from Cobalt-60 for the Spores of Co botulinum 213B Radiation Control Glutathione Sodium Hydrosulfite Radiation,-... I Dose R 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 3,6oo,ooo 1 670 5,200,00o0 1i841 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 3,850,00 1.722 900,000 54,000 -1.154 1,100,000 1.166 2,480,000 1.551 1,080,000 49 -3.192 410,000 0.758 1,119,500 1.214 1,260,000 6 -4.1o8 112,500 0.176 615,000 0.926 1,440,000 0 49,500 -0o180 241,000 0.519 Data presented in Table II-C and Fig. 3-C show that glutathione and sodium hydrosulfite protect PA 35679 spores against the lethal action of gamma radiation to essentially the same degree as was shown for C. botulinum 213B spores. The effect of pH of the suspending medium during irradiation on the lethality of gamma radiation for Co botulinum 215B spores was also studied over the range of 3523 to 8.4. 0o1iN acetate buffer was used in the pH range of 3253 to 500, while an M/15 phosphate buffer was used from 6.1 to 8.4. No significant difference was noted in the percent of C. botulinum 2153 spores surviving irradiation at any pH in the range of 3523 to 804, This is shown in Table TV-C. There appears to be some effect on the number of spores, since there were roughly 100 times as many survivors following O.9-megarep irradiation in the first series of experiments as were found in the second series. However, since each series of runs agrees within itself, the basic conclusion is not affected by this disparity. b. Effect of a Mercury Compound -it is well known that organic compounds can be protected during chemical manipulation by the preparatory formation of compounds whose nature permits regeneration of the original compound at a later time by suitable chemical or physical operations. Since the sulfhydryl groups are suspected of being involved in the sensitivity of anaerobic bacterial spores to the lethal action of gamma radiation, and since there is microbiological precedent for reversing the 4o

+2 +I o 0 >( _J 0 0 -j 0 -I -2Key(213B) Key(213-B) o - Control X - Methionine -3 0 4.8 1.2 MEGAREP Fig. 1-Co Effect of methionine in M/15 phosphate buffer of pH 7.0 on the lethality of gamma radiation from cobalt60 for the spores of C. botulinum 213B, 41

+2 C/) 0-r0 -I C/)., -3 Key: 213 -B 0 - Control X - Glutathione A -Nao2SO04 -5 0 4.8 1.2 1.6 MEGAREP Fig. 2-C. Effects of glutathione and sodium hydrosulfite added to M/15 phosphate buffer at pH 7.0 on the lethality of gamma radiation from cobalt-60 for the spores of C. botulinum 213B. 42

TABLE III-C Effect of Protective Chemicals Added to Buffer Used as a Suspending Medium for PA Irradiation with Gamma Rays from the M/15 Phosphate 3679 Spores During Cobalt-60 Rep Spores per ml Percent Survivors Log Percent Survivors a) Control - M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,440,000 3,130,000 1,120,000 16,200 1,210 129.5 1.5 0.5 100 35-78 0.5176 0.0587 0.00414 0.0000479 o.oooo000016 2.000 1.554 -0.286 -1.412 -2.383 -4.320 -4.796 b) 0.02 M Glutathione Solution in M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,440,000 3,600,000 1,925,000 720,000 260,000 128,500 36,500 6,250 100 53.47 20.00 7.22 3.57 1.014 0.1756 2,000 1.728 1.501 0.859 0.553 o.oo6 -0.760 c) 0.02 M Sodium Hydrosulfite Solution in M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,400,000 3,570,000 2,700,000 1,700,000 750,000 440,000 195,000 88,000 100 75.63 47.62 21.01 12.33 5.462 2.465 2.000 1.879 1.678 1.322 1.091 0.737 0.392 43

2.004 1.00 0 to 0.00 co -I2.00 -3.00 - 4.00 -5.00 MEGAREP Fig. 3-C. Effect of protective chemicals at 0.02 M concentration when present in the M/15 phosphate buffer (pH 7.0) used as a suspending medium for PA 5679 spores during irradiation with gamma rays from cobalt -60.

TABLE IV-C Effect of the pH of the Medium in Which C. botulinum 213B Spores Were Suspended During Irradiation on the Lethality of Gamma Rays from Cobalt-60 for These Spores Rep _.Spores per ml Percent Survivors Log Percent Survivors pH 3.4 0 10,900,000 100 2.000 360,000 8,400,000 77.06 1.887 720,000 1,910,000 17.52 1.244 900,000 725,000 6.651 0.823 1,o80,000ooo 195,500 1.794 0.254 pH 6.75 0 13,000,000 100 2.000 360,000ooo 8,650,000 66.54 1.823 720,000 5,300,000 40.77 1.610 900,000 2,050,000 15.77 1.198 1,080,000 945,ooo 7.27 0.862 pH 7.01 0 10,800,000 100 2.000 36o,ooo 8,200,000 75.93 1.88o 720,000 3,o65,ooo 28 38 1.453 900,000 1,940,000 17 96 1.254 1,080,000 665,000 6.157 0.789 pH 8,4o 0 360,000 540,000 720,000 900,000 6,000o,000 2,230,000 330,000 42,000,10oo 100 37.17 5.50 0.70 0.0517 2.000 1.570 0.740 -0.155 -1.287 pH 7.17 0 360,000 540,000 720,000 900,000 5,800,000 2,155,000 240, 000 69,500 5,650 100 37.16 4.138 1.200 0.0974 2.000 1.570 0.617 0.079 -1.011 45

TABLE IV-C (Concl.) Rep Spores per ml Percent Survivors Log Percent Survivors pH 6 10 0 60,o000 540,000 720,000 900,000 pH 5. 00 0 360,000 540,000 720,000 900,000 4,870,000 1,910,000 255,000 56,000 3,740 4,730,000 1,180,000 174,000 20,400 1,625 100 39.32 5.236 1.150 0,0768 100 24.95 3.679 0.4313 0.0344 2.000 1.594 0.719 0. o61 -1.115 2.000 1.597 o. 66 -0.365 -1.463 pH 4o00 0 36o,000 540,000 720,000 900,000 pH 3.23 0 360,000 56o,ooo 540,000 720,000 900,000 3,900,000 845,ooo 139,500 8,650 176 1,553,000 420,000 75,000 5,500 850 100 21.67 3.577 0.2218 0.04513 100 27.04 4.829 0.354 0.05473 2.000 1.336 0.554 -0.654 -1.346 2.000 1.432 0.684 -0.451 -1.262 "bactericidal" effects of mercury compounds, it appeared reasonable that such compounds might be useful for learning more about the mechanism of radiation damage to such spores. For this purpose C. botulinum 213B spores, prepared as previously described, were suspended in solutions of p-chloromercuribenzoate. Subsequent to irradiation, the spores were counted in the usual pork extract media to which sufficient sodium thioglycollate had been added to insure reversal of mercury combinations with the spores. Incubation was carried out at 30~C. Table V-C and Fig. 4-C present initial evidence indicating that some protection is offered to C. botulinum spores by p-chloromercuribenzoate

and that this protection varies directly with the concentration of this compound in the suspending mediumo TABLE V-C The Effect of Different Molar Concentrations of p-Chloromercuribenzoate on the Lethality of Gamma Rays from Cobalt-60 on the Spores of C. botulinum 213B Concentration of Number of Log % Megarep Hg cpd Present egep Spores Survivors 0.02 M 0.000 4,930,000 2.000 0.775 1,950 -1.403 1.085 15 -3.517 1.240 0.5 -4.994 0.05 M 0.00 6,700,000 2.000 0.775 12,000 -0.747 1.085 93 -2.858 1,240 4 -4.282 0,08 M 0.000 7,600,000 2.000 0.775 18,550 -0.612 1.085 170 -2.652 1.240 7 -4.o36 0.10 M 0o000 6,800,000 2.000 0O775 24,950 -0.435 1.085 305 -2.348 1o240 23 -3.480 c~ Effect of Oxygen. -Since oxygen is known to enhance the damaging effects of ionizing radiation, s the influence of oxygen on the effects of sodium hydrosulfite and of glutathione present in the suspending media was studied. For this purpose C. botulinum 213B spores were suspended in (1) phosphate buffer, (2) phosphate buffer containing 0.02 M glutathione, and (3) phosphate buffer containing 0.02 M sodium hydrosulfite. The phosphate buffer was M/15 and had a pH of 7.0. Each batch of tubes containing spore suspension was treated in one of the following ways: e1 The tubes were sealed in an atmosphere of air. 2. Pure nitrogen was bubbled through this liquid, and then the tubes were sealed in an atmosphere of nitrogen.

-I Cf) (. 0 0.2 0.4 0.6 0.8 1.0 MOLAR CONC. OF CHLOROMERCURI BENZOATE _ —-4 -5 0 0.2 0.4 0.6 0.8 1.0 MOLAR CONC.OF p-CHLOROMERCURIBENZOATE Figo 4-C. The effect of different molar concentrations of p-chloromercuribenzoate on the lethality of gamma rays from cobalt-60 on the spores of C. botulinum 213B. 48

3. Pure oxygen was bubbled through the medium, and the tubes were then sealed in an atmosphere of oxygen. The data in Table VI-C and Figs. 5-C through 7-C show the effects of varying oxygen tension during irradiation on the lethal action of gamma rays from cobalt-60 for the spores of C. botulinum 213B when different suspending media were used. Figure 5-C shows such data for a suspending medium composed of M/15 phosphate buffer at pH 7.0, Fig. 6-C for glutathione, and Fig. 7-C for sodium hydrosulfite. It will be noted that the protective effect of sodium hydrosulfite is reduced in an atmosphere of pure oxygen but that there is little difference between an air and a nitrogen atmosphere in this respect. On the other hand, glutathione shows considerably reduced protective ability in an air atmosphere as compared with a nitrogen atmosphere, and there is even further reduction of the protective effect of glutathione when pure oxygen is substituted for air. From all of these experiments, we conclude that reduced sulfhydryl groups present in the spores protect the spores against the lethal action of gamma rays from cobalt-60o On the other hand oxidized sulfhydryl groups do not provide such protection. TABLE VI-C Effect of Oxygen Tension on the Protection Afforded C. botulinum 213B Spores by Sodium Hydrosulfite and Glutathione Against the Lethal Effects of Gamma Rays of Cobalt-60 Gaseous eg pNumber of Log % Atmosphere garp Organisms Survivors Phosphate Buffer Air 0.000 5,730,000 2.000 0.465 13,10,000 1.359 0.775 31,500 -0.260 0.930 675 -1.929 1.085 190 -2.479 Oxygen 0.000 6,530,000 2.000 0.465 485,000oo 0.871 0.775 1,500 -1.639 0.930 27 -3.384 i.o85 1 -4.815 49

TABLE VI-C (Cont.) Gaseous Number of Log % Megarep Or Atmosphere Organisms ___ _Survivors Nitrogen 0.000 5,270,000 2.000 0.465 1,810,000 1.536 0 775 144,000 0.437 0.930 20,700 -o.406 1.o85 3,000 -1.245 Glutathione Air 0.000 6,630,000 2.000 0.482 1,350,000 1.309 0.775 420,000 0.802 0.930 208,500 0.498 1.085 37,500 -0.247 Oxygen 0.000 7,750,000 2.000 0.482 140,000 0.267 0.775 2,650 -1456 0.930 255 -2.473 1.085 21 -3-557 Nitrogen 0.000 6,o100000 2.000 0.465 2,350,000 1.586 0.775 985,000 1.208 0.930 710,000 1.066 1.085 250,000 o.613 Sodium Hydrosulfite Air 0.000 5,400,000 2.000 o 465 5,300,000 1.992 0.775 2,465,000 1.659 0.930 1,510,000 1.447 o 085 780,000 1.160 Oxygen 0.000 3,830,000 2.000 o.465 3,150,000 1.915 0.775 1,140,000 1.474 0.930 131,000 0.534 1.085 90,000 0.371 50

CQ -P CQ CQ 0 Q) ^ 0) (^ ^. 0 ^ > 0 OJ 00 00 1>- ^ ~ 0 bD -H 0 OJ OJ 0 \s0 H bO S 0 >Ot"-^DlT\OJ?= j "I ^ **<~** r^ PL ^_> ^OJr^r-lr-Ji-1 <D 0 CQ fl ^ 0) *H -P ^ rQ?-1 0 S o ^ 0 ft PH ___ u <u p^ ^ VD *N 1-1 OJ * c6 oo *-) a r-1 -H I * FH S CHCQO OO O O SO1 osooooo ^ i CQ o oo o o a'0^ ^-(*H *s~s~\~\~\ S ^iO ^ <ua o oo o o ojOr^ J3CO O LT\O ir\Lr\ *H^H l r-^ S hO ^OOr-1K^K\ r-l-P-:CJ?!?-< *\~S~K~S~\ r-l(^^S O I^-K^r^OJr-^ -H N< 0 IS *H 0 0 r^ N^-^ r^j ~r-| 43 0?-I 0 0 OS 0) C0 ( ------ -P?-1 M ^ CQ -P > * P! &-1 ^ 0 w 00 tZ * o p: h-o h U *- /^'^^

2. 1.0 - 1.0 0 > o -2.0 -3.0 C -4.0 Key A-P04 Buffer+ N2 0 - PO4 Buffer-+ Air 5.0 - P04 Buffer + 02 -5.0 0 0.2 0.4 0.6 0.8 1.0 1.2 MEGAREP Fig. 5-C. The effect of oxygen tension on the lethality of gamma rays from cobalt-60 on the spores of C. botulinum 213B suspended in M/15 phosphate buffer at pH 7.0. 52

o -I.0 I) 0 -2.0 0 -J 0 -3.0 Key: A - Glutathione + Nitrogen o -Glutathione + Air D -Glutathione + Oxygen -4.0, 0 0.2 0.4 0.6 0.8 1.0 1.2 MEGAREP Fig. 6-C. The effect of oxygen tension on the protection afforded C. botulinum 213B spores by glutathione against the lethal effects of gamma rays from cobalt-60. 53

2 I 1 (/) n 0 o 9? D U) 0e (D 0 -I ^.0 I ~\.0.0.0.0.0 -Key A - Na2S2 04 + N2 o-N02S204 + Air O - Na2S2 04 + 02.0 -2 -3 -4 0 0.2 0.4 0.6 0.8 MEGAREP 1.0 1.2 Fig. 7-C. The effect of oxygen tension on the protectiOn afforded C. botulinum 213B spores by sodium hydrosulfite against the lethal effects of gamma rays from cobalt-60. 54

PHASE III EFFECTS OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF THE SPORES OF ANAEROBIC BACTERIA SUMMARY When anaerobic bacterial spores were irradiated at different temperatures, a temperature zone was found within which the spores were less susceptible to the lethal action of such rays. The temperature zone was different for different sporeso However, for the most effective sterilizing results, temperatures of 5~C or below were most desirable for all the spores tested, except where advantage was to be taken of the combined irradiation-heat process. When the combined irradiation-heating process was used as a sequence of operations, it was found necessary to heat previously irradiated anaerobic bacterial spores above a critical temperature of approximately 95~C before advantage of the combined process was evident. Also, at temperatures above 95~C, the combined effect of irradiation and heat developed during the irradiation treatment. INTRODUCTION The effect of temperature during irradiation on the lethality of gamma radiations for anaerobic bacterial spores becomes important when the irradiation sterilization of food is considered. Since processes involving irradiation at temperatures all the way from -60~C to above 100~C have been proposed, it is necessary to know whether or not these temperature differences are important. Also, does the previously established sensitivity of irradiated spores to subsequent heating apply when radiation and heat are applied simultaneously? If so, this may affect the design of processes for the application of this combined process to food sterilization. Therefore, our studies in this area of the work have followed three lines. First, the effect of different temperatures during irradiation was observed; second, the effect of irradiation at high temperatures was studied to observe the sensitization effect; and finally, the effect of different holding temperatures following irradiation was noted. 55

MATERIALS AND METHODS a. Spores of anaerobic bacteria used in these studies were prepared and used according to techniques described in previously published articles from this laboratory.1'2 b. For irradiation the spores were suspended in M/15 phosphate buffer at pH 7.0 and then placed in glass ampoules which accommodated approximately 4 ml of solution. Then the ampoules were heat sealed and placed in an especially designed carrier. The carrier fitted into an apparatus which controlled the temperature ~ 1~C during irradiation. Irradiation was carried out in the center well of the large cobalt60 gamma ray source at the Fission Products Laboratory of The University of Michigan. RESULTS Data in Table I-T and Fig. 1-T 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 II-T and Fig. 2-T 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. Table III-T and Figs. 3-T and 4-T present data showing the effect of heating previously irradiated spores. Figure 3-T shows that irradiated C, botulinum spores must be heated to a minimum, critical temperature of 80~C before killing occurs. This temperature is also the critical lethal temperature for nonirradiated 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 80~C before the lethal effect of temperature is manifest. Figure 4-T points out the increased importance of the irradiation sensitization of C. botulinum 213B spores as progressively higher killing temperatures above 80~C are utilized. Table IV-T and Fig. 5-T show that C. botulinum 213B spores, suspended in M/15 phosphate buffer at pH 7.0, are killed somewhat more rapidly at 4~C than at -70~C; similar results for C. parabotulinum 457 and for PA 3679 spores are shown in Tables V-T and VI-T, respectively. This confirms similar data previously presented for these and other anaerobic bacterial spores. 56

TABLE I-T 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 Percent Survivors Temp, C Dosage Megarep _0.550 0.647 J 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 2,3 o.o6 27 11.6 10.5 30 14.0 4.4 o088 56 10.0 0.3 58 2.9 0.4 80 2.9 0.14 95 0o16 57

Key PA 3679 --- C. botulinum 213 B Radiation dose = 0.740 megarep I I e-I I (n) 5r >1 or:D (f) 0 0 _J b. a 2 I0 I' 8 T II I 4 I O~~ -100 -80 -40 0 TEMPERATURE C 40 80 100 Fig. l-T. 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. 58

TABLE II-T Effect of Temperature 213B Spores When They and Heating Being During Irradiation on the Survival of C. botulinum are Subsequently Heated at 100~C, Both Irradiation Carried Out in M/15 Phosphate Buffer at pH 7.0 Minutes at 100~C I \O Actual Effective Control Spores per ml % SLog % Survivors Survivors Irradiated at 10~C Irradiated at 90~C Spores Spores per ml %Log % per ml % Log % Survivonr Srvi vn'r.,ql'r'Tiv nr P,rs'T'-r'. ~~~~~~~~~~~~~~~~~~~~~~~~~~~.,.....I_.__.....................':........,I......_'''.- -. I I..L V L.J I V -L,-L 0 0 2,200,000 100.0 2.00 230,000 100.0 2.000 600,000 100.0 2.000 10 8.54 1,100,000 50.0 1.70 160,000 69.5 1.842 61,000 10.2 1.009 20 18.54 460,000 20.9 1.32 9,700 4.21 0.624 3,200 0.533 -0.273 30 28.54 85,000 3.86 0.587 1,600 0.695 -0.158 750 0.125 -0.903 40 38.54 22,000 1.00 0.000 360 0.156 -0.807 160 0.0267 -1.574 50 48.54 4,400 0.200 -0.699 30 0.01L30 -1.886 13 0.00216 -2.666

2.00 I.OC O.OC 0.00 () | -I.OC 0 c) o0 (3 0 -2.0C -3.OC ) Key X - Nonirradiated Control - Irradiated at 10 ~C A A- Irradiated at 90 ~C L * \\\O I )x.,'o^I A \ I\ *~- ii ii^!-\-~~~ 0 20 40 60 EFFECTIVE HEATING TIME AT 100~C- MINUTES 80 Fig. 2-T. 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. 6o

TABLE III-T 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 Temperatures 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 1,0000000 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 1.803 240,00 1.513 106,000 1.457 95 310,000 1.450 11,0000 1335 5 00 0.914 2,800 0.013 100 1>700 -0.791 170 -1.894 40 -2.261 0

I r, O. lr% - - - - - - I j-_.l I. - 2,4 1,2 1,)2 - 2,4~~~ 4\~ I 1.00 U) 0 o ro 0 -J 0.00 -1.00 Key 0- Control- Held for I hour at 0- Contro I I- Irradiated for 100,000 rep each temperature 2- Irradiated for 200,000 rep 106 spores/ml 4- Irradiated for 400,000 rep \ i iI ----— T~~~~~~~~~~~~l ________________i I 1_iI -2.00 -3.00 0 50 60 70 80 90 100 TEMPERATURE ~C Fig. 3-T. Survival curves for C. buffer at pH 7.0, which have been and then held for one hour at the botulinum 213B spores suspended in M/15 phosphate first irradiated with gamma rays from cobalt-60 indicated temperatures.

2.00 1.00 I=) X F. so 7 i n (I) 0 or Cl 0 0 -J 1 0.00 -1.00 k N \'4 -2.00 -3.00`9 Key X- 800C o -90~C A - 95 ~C o-1 00 ~C K \ 0 0.1 0.2 0.3 Rep x 106 0.4 Fig. 4-T. 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. 63

TABLE IV-T Effect of Temperature During Irradiation with Gamma Rays from Cobalt-60 on the Survival of C. botulinum 213B Spores Suspended in M/15 Phosphate Buffer at pH 7,0 Dose Rep Spores per ml Percent Survivors Log Percent Survivors a) Irradiated at 50C 0 630,000 100 2.000 340,000 172,000 27.3 1.438 510,000 72,000 11.4 1.057 595,000 11,500 1.85 0.263 680,000 6,100 0.968 -o0.014 765,000 760 0.121 -0.917 850,000 370 0.0587 -1.231 b) Irradiated at -70~C 0 7,500,000 100 2.000 Frozen 9,700,000 129.0 2.076 340,000 5,600,000 57.7 1.761 510,000 1250000 10 12.9 1.110 595,000 710,000 7.33 o.865 680,000 270,000 2.78 0.440 765,000 186,000 1.92 0.283 850,00 3,035 00 0.361 -0.443 64

2. ) 1.00 L (r 0 w \ x - 7 Q O C 2. 00 -1.00 - 0 Irrad at 5~C 00* Irrad at-70~C a -2 00 0.2 0.4 0.6 0.8 1.0 1.2 REP x 106 Fig. 5-T. Effect of temperature during irradiation on the survival of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0 65

TABLE V-T Effect of Temperatures During Irradiation with Gamma Rays from Cobalt-60 on the Survival of C. parabotulinum 457A Spores Suspended in M/15 Phosphate Buffer at pH 7.0 Dose Rep Spores per ml Percent Survivors Log Percent Survivors a) Irradiated at 5~C 0 490,000 100 2.000 340,000 145,000 36.6 1.593 510,000 29,000 5.92 0.772 595,000 9,250 1.89 0.277 680,000 2,550 0.52 -0.284 765,000 950 0.193 -0.714 850,000 145 0.0296 -1-535 b) Irradiated at -70~C 0 1,900,000 100 2.000 Frozen 2,300,000 121.0 2.083 34o,000 1,030,000 44.8 1.651 510,000 385,ooo 16.7 1.223 595,000 123,000 5.4 0.732 68o,ooo 58,ooo00 2.52 0.4 765,000 17,100 0.774 -0.111 850,000 7,700 0.334 -0.476 66

TABLE VI-T Effect of Temperatures During Irradiation with Gamma Rays from Cobalt-60 on the Survival of PA 3679 Spores Suspended in M/15 Phosphate Buffer at pH 7.0 Dose Rep Spores per ml Percent Survivors Log Percent Survivors a) Irradiated at 5~C 0 700,000 100 2.000 340,000 230,000 32.8 1.516 510,000 30,000 4.28 0,631 595,000 9,850 1.41 0.149 68o,ooo 2,950 0.237 -0.625 765,000 890 0.127 -0.896 850,000 175 0.025 -1.602 b) Irradiated at -70~C 0 590,000 100 2.000 Frozen 650,000 110 2.014 340,000 270,000 41.5 1.618 510,000 46,000 7.07 0.849 595,000 46,000 7.07 0.849 680,000 8,600 1.32 0.121 765,000 3,300 0.508 -0.294 85o,000 1,200 0.185 -0.733 67

Data in Table VII-T and Fig. 6-T show that PA 3679 spores in M/15 phosphate buffer at pH 7.0 are not killed in any significant number by heat until a temperature of 1050C is reached. Although such spores that have previously been irradiated with 400,000 or 800,000 rep die in significant numbers when heated to 100~C, it is apparent that a critical temperature of about 95~C is still necessary before irradiated PA 3679 spores become susceptible to killing with heat, The data in Table VII-T also indicate that although the heat resistance of irradiated PA 3679 spores is reduced considerably above 95~C, until this critical temperature range is reached there is no significant difference between the heat resistances of irradiated and nonirradiated spores. TABLE VII-T Effect of Postirradiation Heating for One Hour at Various Temperatures on Previously Irradiated PA 3679 Spores Temperature, ~ Spores per ml Percent Survivors Log Percent Survivors a) Nonirradiated Control 70 80 90 95 100 105 110 860,000 650,000 660,000 640,000 500,000 580,000 310,000 0 100 75.5 76.7 74.5 58.2 67.5 34.9 0 2.000 1.878 1.885 1.872 1.765 1.829 1.543 b) Irradiated with 400,000 rep Control 70 80 90 95 100 105 110 150,000 170,000 160,000 162,000 175,000 134,000 3,600 0 100 113 106 108 116 89.5 2.40 0 2.000 2.053 2,025 2.033 2.065 1.952 0.380 c) Irradiated with 800,000 rep Control 70 90 95 100 105 110 1,340 200 305 56o 65 0 0 100 14.9 22,8 41.7 4.85 0 0 2.000 1.1732 1.358 1.620 0,686 g 68

O.,, 0-.'Oft I I A- _A__ _ A 2 "`\ Mi \ I 01 0 --- -H114 1 1 I 1 0n 0 o >u CO 0 -.00 3 -1.00 -9 nn 0 Control A 400,000 Rep, a 800,000 Rep I I I I I I I I I I I i 70 80 90 100 11 TEMPERATURE CENTIGRADE I 0 Fig. 6-T. Effect of postirradiation heating for one hour at various temperatures on previously irradiated PA 3679 spores. 69

DISCUSSION These data indicate that gamma radiations are most lethal for C. botulinum spores at temperatures below 5~C or above 40~C. In the range of 5~ to 40~C, an unexplained protective effect exists. Similarly, PA 3679 spores exhibit a temperature range in which they are less sensitive to gamma radiation; but, for PA 3679 spores this protective temperature range lies between 50~ and 95~C. Consequently, it appears to be a fortunate, circumstance that the most advantageous irradiation temperature would appear to exist at or below the usual refrigeration temperatures. When advantage is to be taken of the combined process, temperatures above 950C appear to be desirable. Data are being collected at present on the effect of temperatures above 80~C on these spores, but there is only the limited amount shown available at this time. Even these limited data indicate that irradiated spores die very rapidly at temperatures of 100~C and above. It is important to note that any combined heat and irradiation sterilization treatment must be designed only for food that can be heated to at least 95~C since below this critical temperature even irradiated spores are unaffected by heatingo BIBLIOGRAPHY 1. Kempe, L. Lo, Graikoski, J. T,, and Gillies, R. A., "Gamma Ray Sterilization of Canned Meat Previously Inoculated with Anaerobic Bacterial Spores," Appl. Microbiol., 2, 330-332 (1954). 2. Kempe, L. Lo, "Combined Effect of Heat and Radiation in Food Sterilization, " Appl. Microbiol. 3, 3 46-352 (1955). 70