THE UNIVERSITY OF MICHIGAN INDUSTRY PROGRAM OF THE COLLEGE OF ENGINEERING THE SIMULTANEOUS LETHAL EFFECT OF TEMPERATURE AND GAMMA RADIATION ON BACTERIAL SPORES Johre To Graikoski. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the University of Michigan 1961 February 1961 IP-495

ACKNOWLEDGMENTS I wish to express my gratitude to Dro Lo Lo Kempe for his guidance throughout this workO My sincere appreciation is extended to Dro Wo Jo Nungester, DrO Wo So Preston, Dro Lo Eo Brownell and Dro Ho Co Eckstein, the other members of my doctoral committee, for their time and cooperationo Many thanks to Dro P, Co Rajam for his willingness to join my doctoral committee at a moments noticeo Acknowledgment is extended to Dro Bo Church for the spores of B_ subtilis, to Dro Ro MacDonald for help in the experiment with B0 cereus and to Dro Jo Vo Nehemias for helpful advice on radiation techniques My appreciation is also extended to the Quartermaster Food and Container Institute for the Armed Forces for their support of part of this research and to the staff of the Industry Program of the College of Engineering for the final preparation of the manuscripto My thanks to my wife for her encouragment and help in the preparation of the final stages of this thesiso Affectionately, this thesis is dedicated to my mother and to the memory of my fathero ~ii

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS............................................ ii LIST OF TABLES................................................. iv LIST OF FIGURES................................................. vi INTRODUCTION................................. 1 HISTORICAL............................................ 5 Introduction............................. 5 Effect of Temperature During Irradiation on Bacterial Spores....................9..... 9 Photosensitization of Proteins to Heat............... Photosensitization of Protoplasm to Heat.............. 13 MATERIALS AND METHODS Organisms....................., 21 Maintenance of Stock Cultures......,.......... 21 Production of Spore Suspensions....................... 22 Preparation of Spore Suspensions for Irradiation...... 26 Counting Technique and Recovery Media................. 28 Irradiation Facility................................ 29 Irradiation Techniques......................... 30 Measurement of Dose Rates................... 32 EXPERIMENTAL RESULTS....................... 32 The Effect of Temperature During Irradiation on the Survival of Cl. botulinum and Putrefactive Anaerobe spores........................................... 38 The Effect of Temperature During Irradiation on the Survival of Bacillus subtillis var. niger Spores.... 52 The Effect of Post-Irradiation Heating on the Survival of Bacterial Spores...................... 63 The Effect of Heating Cl. botulinum and putrefactive anaerobe Spores Prior to Irradiation.............. 64 The Effect of Irradiation on the Subsequent Germination of Bacterial Spores................1......... 71 The Effect of Temperature During Irradiation on the Subsequent Heat Resistance of Bacterial Spores...... 78 DISCUSSION..,.,....................................,.......... 98 SUMMARY....................................... 117 BIBLIOGRAPHY.....,....................................... 120 iii

LIST OF TABLES Table Page I Dose Rate at Various Positions in the Irradiation Chamber, August, 1954 o........................... 0 35 II Effect of Temperature During Irradiation with Gamma Rays from Cobalt-60 on the Survival of Spores of Cl. botulinum 213-B When Suspended in M/15 Phosphate Buffer at pH 7~0,o o o o,.. o O a..... o. o o, 41 III Effect of Temperature During Irradiation With Gammi Rays from Cobalt-60 on the Survival of Anaerobic Bacterial Spore Suspended in M/15 Phosphate Buffer at pH 7~0,O 43 IV 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....lo,..<.o<o<..o.oo.o.o....0,...000.0000 45 Effect of Temperature During Irradiation on the Survival of Cl1 botulinum 62-A Spores Suspended in Phosphate Buffer at pH 7.0, o o. O. o o o, 48 VI Effect of Temperature During Irradiation on Survival of Clo botulinum 213-B Spores.,,,.....oo...oo,.o 54 VII Effect of Irradiation at 10~C and 90~C on Survival of putrefactive anaerobe NCA 3679 o..0.00...... Q aOaao*aOO 57 VIII Effect of Temperature During Irradiation on Survival of Clo parabotulinum 457-A Spores.,..ooooo<.o..ooo..o 59 IX Effect of Temperature During Irradiation on Survival of Bo subtilis var. niger Spores..oo..oooo.,,...O3 O..o 62 X Survival of Clo botulinum 213B Spores Suspended in M/15 Phosphate Buffer at pH 7.0, Which Have Been Irradiated at 5~C With Gamma Rays from Cobalt-60 and Then Held for One Hour at the Indicated Temperature.oo0000000000o 65 XI Effect of Holding Irradiated Spores of Cl. botulinum 213 B for One Hour After Irradiation..,, o.. 0ooO 66 XII Effect of Postirradiation Heating for One Hour at Various Temperatures on Previously Irradiated PA 3679 SporesO..00oo0000000.o.00..o oOoo,.o.o. 68 iv

LIST TABLES (CONT D) Table Page XEI? Effect of Pre-Heating P Ao 3679 Spores at 900C for 7 Hours Prior to Irradiation........ o.... 75 XV Effect of Pre-Irradiation Treatment on Germination of B o cereus varo terminalis Spores............. 77 -XVI 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 0 00 0 00 00 00 0 0 a 0 0 0 0 0 O 0 O 82 7XVII Effect of Post-Irradiation Heating at 990C for Four Minutes on Survival of Bo subtilis varo niger Spores Irradiated at 5~ and 65~C 0 O.........0....... 85 XVTIIT Effect of Pre-Irradiation at 5 and "70~C With Subsequent Heating at 100~C on Survival of Clostridium botulinum 213-B..................... 87 XIX Effect of Pre-Irradiation at 5' and -72~ C With Subsequent Heating on Survival of Clo botulinum 213-B Spores........... o.......... 89 XZXZ Effect of Irradiation at 10~C and 900C With Post" Irradiation Heating at 100.0~C on Survival of Cl. botulinum 213-B Spores........... 92.XI Effect of Temperature During Irradiation on the Subsequent Resistance of Cl. botulinum 62A Spores to Heating at 100 C -94 XXII Effect of Temperature on the Subsequent Heat Resistance of PoAo 3679 Spores to Heating at 100'C.......... 96 V

LIST OF FIGURES Figure Page 1 Diagram of Facility for Irradiation of Spore Suspensions with Cobalt~60....o.......oOooooo......... 31 2o Dose Rate in Rep Per Hour x 103 at Various Positions in the Irradiation Chamber o...............o o o o o o.. 37 3o Effect of Temperature During Irradiation with Gamma Rays from Cobalt-60 on the Survival of Spores of Clo botulinum 2 3-B Suspended in M/15 Phosphate Buffer at p11 700 0 a 0 a 0 0. 0 0 0...oo 0 0 o 0 o..o.....40 4o Effect of Temperature During Irradiation with Gamma Rays from Cobalt 60 on the Survival of Anaerobic Bacterial Spores S.uspended in M/15 Phosphate Buffer at pH 7.0...,.,,,...................... o 42 5. Effect of Temperature During Irradiation with Gamma Radiation from Cobalt-60 on the Survival of Spores of PA 3679 Suspended in M/15 Phosphate Buffer at pH 7 o0 a.oooVo.oooo.....oooooo...ooD.ooooooooaooo 44 60 Effect of Temperaature During Irradiation on the Survival of C, hbo-ulinum 62-A Spores Suspended in M/15 Phosphate Buffe1re at pH 70 o o o a 0 0 0 0 0 0 0 0 47 7. Effect of Temperature During Irradiation on the Lethality of Gamma Radiation from Cobalt-60 for the Spores of Cl. botulinum 62A When They Are Suspended in M/15 Phosphate Bu.ffer at pH 7,0 and are Irradiated at the Rate of 0,218 Megarep pe Hor Houmr.....o...........o......o.ooo.. 51 80 Effect of Temperature During Irradiation on Survival of Cl. botul)inLm 213-B Spores Suspended in M/15 Phosphate Buffer at pH 7 0 o o o o o o o o o. o o o o.. o o 53 90 Effect of Temperatu.re During Irradiation on the Survival of putrefactive anaerobe Spores NCA 3679 Suspended in M/15 Phosphate Buffer at pH 7o0 oooo......... O.....O 56 10o Effect of Temperature During Irradiation on the Survival of Co bot.lin.um 457-A Spores Suspended in M/15 Phosphate Bu er~ at pH 7o0 o 0 o 0........o 0 o o 0 0 o o o o 58 711 Effect of Temperature During IrradiatJ on on Survival of Bacillus subtilis varo niger Spores, o o o o o o o. 61 vi

LIST OF FIGURES (CONTD) Figure Page 12, Survival Curves for Cl. botulinum 21.3-B 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..oooooooo.oo.... oooo...,...0. @, 0 @...@ 67 1.3 o Effect of Post-Irradiation Heating for One Hour at Various Temperatures on Previously Irradiated Putrefaetive anaerobe NCA 3679 Spores oo.o.o.o.o.o..,.o 69 14. Effect of Pre-Heating Cl. botulinum 213-B Spores at 65~C for 14 Hours Prior to Irradiation. oo...oo,....o 72 15, Effect of Pre-Heating putrefactive anaerobe Spores at 90~C for Seven Hours Prior to Irradiation....... 74 166 Effect of a Combined Treatment Consisting of Irradiation with Gamma Radiation from Cobalt-60 Followed by Heating for One Hour at 99 C on Sur-rival of putrefactive anaerobe Spores Suspended in M/15 Phosphate Buffer at pH 7o0 oO....................00.000.0000000o00000000 80 17 Effect of Irradiation at 5~ and 95 >C on the SuJ.:rvi~vai of putrefactive anaerobe NCA 3679 Spores,,,..,ooo... 81 18o Effect of Post-Irradiation Heating on. t:he Survival of Bacillus sub.liis var. niger Spores Irradiated at 5~C alid 65'C,,,,,,,0 0,,, 84 5 C ard 65 GC ooooooooooooooooooooooo oooooooeooooooo 84.19 Effect of Pre-Irradiation at 4~C and 70~C with Subsequent Heating at 100~' oon Survival o Cl, botulinum 213 B Spores, o OOo0ooo0oo ooooo OOoooooo 86 20, Effect of Pre-Irradiation at 5~C and =70~C with Subsequent Heating on Surfvival of Cl. botulin:am 2]3.B Spores o0oooooooooooooooooooooo00000ooooooooo 0000000 88 21o Effect of Irradiation at 10~C and 90~C with PostIrradiation Heating on 100~C on Surviv-al of C. o botui inum 22. Effect of Temperature During Irradiation on the Subsequent Heat Resistance of CI. botulinum 62-A Spores to to Heating at 100 Co C...o.ooOTD0...o..o.o.oooo.o.. 93 23o Effect of Temperature During Irradiation on the Subsequ.ent Heat Resistance of putrefactive anaerobe Spores to Heating at LOO ooo o ooooo ooooooooooooooooooo 95 vii

INTRODUCTI ON The lethal action of ionizing radiations on bacterial spores has been assumed. to be independent of the ambient temperature during irradiationo This conclusion was made by Lead Haines and Coulson (1936 9 1937) after studying the lethal effects of alpha and beta particles on Bacillus mesentericus spores which were exposed at various temperatureso This apparent lack of a temperature coefficient for the lethal effects of ionizing radiations on, bacterial spores was used. as strong evidence against a chemical or an indirect mechanism by which, radiations exert their lethal effects (Lea,1.947) Until the present decade these experiments represented the only detailed investigation in which bacterial spores were used. as a test system in, studies with, high energy radiationso Ionizing radiations have been suggested for sterilization purposeso Since the destruction of bacterial spores is usually the limiting factor in sterilization processes, the relative resistance of these spores to the various types of radiations and. the conditions prevalent d.uring irradiation become important in the determination of th.e sterilization lose o However9 when food sterilization is considered a further limiting factor is imposed upon the use of radiation^ namely the production, of off-flavors and odors in a variety of food stuffs The extent of offflavor and. odor production is dependent upon, the total. dosage Therefore any method which, would. help to lower the sterility dose would be of im.portance in the use of radiation sterilization for food. processingo - 1 -

-2In this respect the simultaneous action of heat and radiation for killing bacterial spores may become importanto Aside from the combined but independent effect of these two forms of energy) the synergistic action of radiation and heat may be of significance. The observation that the coagulation temperature of certain proteins could be lowered significantly by prior exposure to ultra-violet radiation -was early recognized in radiation studies (Bovie 19153; Clark, 1922- Stednan and Mendel, 1926). However, the mechanism of the phenomenon, referred to as photosensitization, has not been. elucidatedo. That cellul.ar protoplasm could also be sensitized by ultra-violet radiation was first demonstrated in Paramecium (Bovie and, Klein) 1919, Forbes and Dalandt, 9253 Bovie and Daland, 1923). Following the development of better techniques in the use of ultra-violet radiation., photosensitization of cellular protoplasm to heat was established in several species of bacterial spores by Curran and Evans (1938), in yeast cells by Duggar and Anderson (19399 1941) and reconfirmed by Giese and, Crossman (194R) in Parameciumn Giese and Heat (1948) were the first to demonstrate that X-rays were capable of inducing sensitization in Paramecium. Although not well established. these investigations suggested. that the reverse procedure of a prior heat treatment did not increase the sensitivity of cellular protoplasm to subsequent radiation treatmento Morgan and Reed (1954) made a preliminary study of the photosensitization phenomenon by cobalt-60 gamma radiation using spores from certain food spoilage organismso They found that spores from a thermophilic anaerobe were more sensi.tive to heat treatment after exposure to gamma radiationo The reverse treatment did not alter the

-3radiosensitivity of spores from Bacillus coagulans varo thermoacidurans or Bacillus stearothermophilus In more extensive experiments, and with spores from strains of Clostridum botulinum, it was demonstrated the Fo value (heating time in minutes at 250 F) could be lowered as much. as four fold if the spores were irradiated with, a total dose of 900,000 rep from cobalt-60 (Kempe 1955)o The practical significance of this method of sterilization was indicated when it was demonstrated, that spores from strains of Clostridium botulinum and, a putrefactive anaerobe could be adequately inactivated by lower heat treatments in canned peas, as well as in raw and cooked meat after preirradiation treatment (Kempe et alo, 1957, 19582 1959)o Kan et alo (1957) also demonstrated that putrefactive anaerobe spores, as well as aerobic spores of Bacillus cereus could be rendered more heat sensitive by cobalt-60 radiationso At present, little information is available concerning the effect of irradiation temperature upon bacterial spores, especially those of importance in food spoilageo Those temperature studies which have been undertaken have been mostly concerned with the relative resistance of bacterial spores in frozen and non-forzen stateso The results of these investigations are conflicting and, no adequate explanation for the diverse results has been advanced. In other radiobiological studies, where the role of temperature during irradiation has been studied., the significance of photosensitization of proteins has not been adequately considered. Therefore this study was initiated to explore the lethal effects of gamma radiations on bacterial spores using a wide range of sub-lethal

-4temperatures during irradiationo Spores produced. by food spoilage organisms were selected because of the practical considcerations involved and also because of their greater resistance to heat as compared, to those spores of the mesophilic9 aerobic9 spore-forming bacteria~ Aside from any theoretical implications involvedi the information obtained should. be useful, in evaluating the simultareous effects of heat and radiation as a method of steri.lizationo

HISTORICAL Introduction The interaction of radiation with living cells results in a complex series of reactions before death ensueso For clarity, it is convenient to separate into various stages the sequence of events which occur following radiation exposure Generally, three stages are recognized~ The first^ or primary action5 is the absorption of the incident radiation in, or near the cell; the second an. intermediate stage consists of a sequence of biochemical and biological reactions which lead to the third or final stage, death of the cello The interaction of radiant energy with, molecules of matter is well understoodo Also, the lethal effects following radiation exposure has been well documentel for many types of cells But, the mode of energy transfer to the site of the primary lesion within the cel.l, and the events originating from. this lesion which result in lethality are still incompletely understood in. radiobiologyo As working mode'ls two theories have been advanced to explain the effects of radiation on living cells. These theories are based upon the method by which the incident energy, is absorbed within the cel. l The target theory postulates thbat the primary absorption of energy occurs within or very near the key molecules in the cello The indirect action theory assumes that the primary absorption of energy occurs in. other molecular species, in or near the cell, with resultant production. of lethal chemicals that then react at the sensitive sites within the cello -5

-6The effect of temperature during irradiation assumes an important role in evaluating the two models, Since one model assumes a physical reaction for radiobiolI.ogical effects and the other assumes a chemical reaction, a temperature coefficient coul.d help to.etermine which model is operative Temperate perature epenency uring irradiation was one of the first parameters tested in radiobiological experiments. No satisfactory explanation was advanced, for the diverse temperature effects observed, with a wide variety of test systems (Mul.er,1954). To date9 no satisfactory explanation exists for the various ways by which temperature can modify radiobiological reactions (Pollard et al7 1.955> The early experimental basis and arguments for the target theory have been well. documented by Lea (!947) and. extended recently by Pollard (19535, 1955) The absorption of radiant energy in matter which results in excitations and ionization) is not considered to be influenced by temperatu re. According to this m.od.el the temperature coefficient for radiobiological reactions should, be orne or lesSo Any observerd temperature depen.:lency during irradLiaationr wouAd needk. to be attribuTted to other operative factors, for examples the effect of temperature on the reactions which lead to the recovery of the cell from the original.lesion produced by the radaiati on. Howevers recent investigations have questioned the importance of!the direct action model as the modu>s operanri'by which radiations exert their lethal effects.'The evid.ence for this negation is that the alteration of the environmental conditions during exposure of the cells is capa, ble of significantly altering the radiosensitivity of the ce.lls A notable

-7factor influencing the radiation sensitivity of the cell is the quantity of oxygen present during irradiationo Subsequent investigations have shown that other chemicals are also capable of altering the sensitivity of the cello Therefore, a number of radiobiological studies have been directed towards determination of the influence of oxygen on the lethality of ionizing radiations. Since viable cells contain approximately 80 per cent water and are intimately associated with an aqueous environment, a suggested approach for the mechanism by which oxygen modifies the radio-resistance of cells is based upon the proposed mechanism for the radiolysis of pure watero Early studies with X-rays demonstrated that hydrogen peroxide is formed in oxygenated solutions (Fricke, 1934 and Risse- 1929). A reaction mechanism, which incorporates the transient existence of intermediate free radicals with strong oxidative and reductive powers, has been postulated for production of this compoundo These entities may be responsible for the observed radiobiological lesions in the cell (Weiss 1944, 1947) Since the amount of peroxide formation is dependent upon the amount of oxygen present in the system, the oxygen effect has been attributed to the lack or non availability of this compound in the cello Historically, the activated solvent hypothesis or the indirect action model was proposed by Risse (1930) and Fricke (1934) for the biological effects of radiationso The quantitation and formulation of the indirect action model has been outlined by Zerkle and Tobias (1953). This model, referred to as the diffusion model. was designed to supplement the direct and indirect hypothesis

-8In order to explain the results in which a temperature dependency was noted during irradiation, several attempts have been made to ascertain the role of temperature during irradiation on the rate of free radical and peroxide formation by ionizing radiations in. pure watero Bonet-Maury and LeFort (1948) have demonstrated that the amount of hydrogen peroxide:formed in pu-re water duriTng {Irradiation is a function of temperature, twice as much peroxide being formed, at 20~ as at -4 ~C A definite discontinuity in peroxide formation occurs in the transition from the liquid to the solid phase. These investigators were not able to detect peroxide formation below -ll60C foll.owing a dose of 2 x 106 ro Gromley and Steward. (1956) were able to detect peroxide formation in ice at a temperature of -196OCo However, the amount detected was independent of the amount of oxygen present in. the systemo Their results showed more peroxide was formed, at -5~ than at -78~ or -196~C; the amount produced at the former two temperatures depended on the oxygen concentration in the systemo Hochandel (1952) studied peroxide formation in pure water irradiated in the temperature range of 25~ to 65~Co He found that less peroxide was formed, in pure water as the temperature increased in this range when cobalt-60 was used as the source of gamma radiationso This could indicate that the rates for thee back reactions increased as the temperature of the system was elevated,, thus resulting in lower effective peroxide concentrations Another means by which temperature could. exert its influence during irradiation involves a change in the rates of c.diffu.sion of free radical.s

-9to sensitive sites within the cell. The diffusion processes would be of considerable importance when irradiation is carried out in the frozen as contrasted with non-frozen stateso This concept for temperature dependence during irradiation can be adequately explained with the model proposed by Zirkle and Tobias (1953)o Because of their high thermal resistance. bacterial, spores offer an excellent test system for studying the effect of temperature during irradiation. Such spores can be subjected to heat treatments over a wide temperature range without significant loss of viabilityo Furthermore) because of their low metabolic activity) the influence of temperature oxygen and other chemical substances on enzyme reactions which might affect the viability of the spores is also minimizedo However spores can be considered abnormal cells in that they possess a greater resistance to deleterious influences than the vegetative cells from which they originateo This objection may be outweighed, in part) by a corollary study of spore resistance to two physical agentso The mechanism of spore resistance has not been elucidated, and the phenomenon presents an intriguing problem in microbiology. As a matter of practical significance, factors affecting spore resistance are of importance in the evaluation of sterilization techniques and processes Effect of Temperature During Irradiation on Bacterial Spores The early work by Lea et alo (1936) on the independence of temperature during irradiation on the survival of B mesentericus spores was used to support the target theory (Lea, 1947). The results of this investi. gation indicated no difference in the surviving fraction of Bo mesentericus

-10spores contained in dry gelatin and, exposed'to alpha-particles from polonium at temperatures of 500, 20, 2 and -200~C Similar effects -were observed with spores of the same organism when they were exposed to beta-particles from radium at temperatures of 41, 20 and -20~Co Edwards et al. (1954) using three mev electrons produced by a Van der Graaf accelerator, observed, no significant differences when Bacillus subtil.s spores were irradiated at temperatures of 0, 44 and -60~C with 34,000 repo However, when spores were exposed to 120,000 rep statistically significant differences were noted at these temperatures, for example,, survivors to this dosage were 5~46 per cent at 0~C, 3592 at -6o0~C and 1.5o71 at 44~Co They also observed that the increase in number of survivors at the higher dosage.level J.epended on the concentration of organismso These investigators did not advance an explanation for the results they observed. Also using high energy cathode rays produced by a three mev Van der Graaf accelerator and B subtilis spores, Proctor et alo (1955) observed that the spores were more sensitive when irradi.ateda at t78CC than at 4o4~Co These results showed. an opposite trend, which might be expected from a consideration of the indirect versus direct action theorieso No explanation for this anomaly was presented except that freezing might alter the sensitivity of the spores to irradiationo These results are in opposition to those obtained, by Edwards et aLo (1954) using both the same strain of organism and the same type of radiationo Houtermans (1956) studied, the lethal effect of alpha particles, as well as soft (50Kv) and. hard X-rays on B subtili s spores in the temperature range of -184 ~ to 20~Co The spores were exposed. to each type of

radiation in the presence and absence of moistureo The degree of hydration did not alter the sensitivity of the spores to hard and soft X-rays at -184~Co However, at room temperature a marked difference in the sensitivity of the spores was noted to X-rayso The dry spores were more sensitive to both wavelengths of X-radiation than the moist sporeso However, the slope of the dose inactiviation curve using soft X-rays indicated that this radiation was more efficient for inducing lethality than the shorter wavelengthso In a series of experiments in which the spores were exposed to a constant dose (60 and 90 second exposures) of hard X-rays and two conditions of hydration, a gradual decrease in sensitivity occurred from -184 to -40~C. At the latter temperature a marked change in sensitivit-y occurred, as evidenced by a break in. the survivor curveo No further change in sensitivity of the spores,, due to the radiation temperature was noted from 4G03 to -j+20~Co Although, the break in the sensitivity curve at -40~C occurred under both conditions of hydration and dosage levels, it was most pronounced'with dry spores which had been. exposed for 90 secondso With alpha particles from thoruim-B3 the least number of survi-, vors occurred with dry spores irradiated at -l84~Co The same degree of inactiviation occurred at 20 C with wet and, dry spores as with moist spores at -184 ~C With hydrated spores, irradiated at two constant dosage levels, no difference in the number of survivors was noted in the temperature range of -184 to 20~Co However with. dehydrated spores a marked change in the sensitivity of the spores to alpha particles occurred between -60 and -40~C. Proctor et al. (1958) utilized spores of Bo thermoaciduranss an organism of significance in food spoilage to study the effect of various environmental cond.itions during irradiation on. lethal effects of beta and

-12gamma radiationo Very little difference was noted. in the number of surviving spores when they were suspended in saline and irradiated in frozen (-72~) and non-frozen conditions Comparative results were presented for both gamma-rays from cobalt-60 and 3 mevo electrons, when the spores were irradiated under various physical states and gaseous conditions while suspended in 0o85 per cent sodium chlorideo The following cond.tions were employed; dry ice and room temperature9 and in the presence of pure oxygen9 nitrogen or air. No appreciable differences in the number of spores surviving irradiation treatment were observed under the various conditions present at the time of irradiation when the spores were suspended in saTline; however when the suspending medium was nutrient broth rather than saline9 slight differences were observedo The spores were found to be more resistant to irradiation in the frozen condition in this mediumo Also9 oxygen increased the radiJosensitivity of the spores to irradiationo The over all differences in resistance of spores suspended in broth andi saline solutions during irradiation was not appreciabl.e Webb et al. (1.958) studied the sensitivity of dry spores of Bacillus megatherium, to 50Kv X-rays at temperatures between 36~ and -268 ~Co The technique used by these investigators was to dry the spores on Millipore filters and. expose them to 50Kv X-rays at various temperatures in an atmosphere of heliumo No difference in sensitivity of the spores to radiat;ion was noted in the temperature range of -268~ to =12L~Co At the latter temperature a sharp break in radio-sensitivity occurredo In the temperature range of -121~ to 36 C it was found. that the log of the slope of the inactiviation curve was proportional to the reciporical of the absolute temperatureo No conclusions based on the various methods by which temperatlures

-13can alter the radio-sensitivity of spores was advanced, by these investigators for their results9 other than a discussion of the discontinunity in hydrogen peroxide formation at -1.68 C as was previously shown'by Ghromey and Stewart (1956) Comparison of these results obtained with Bo megatherium. spores with those of B subtilis (Houterman) 1956) showed a striking similarity in that a sharp change in sensitivity occurred at freezing temperatures O The temperature at which this break occurred varied slightly in the two investigationso Some difference was noted in that Bo subtilis became more radio-resistant as the temperature decreased to -176~C and. no change in sensitivity occurred in the 0~ to 500C temperature range Photosensitization of Proteins to Heat With the development of uLltra violet) X-ray and, radium sources at the turn of the cent'ury, one of the first effects observed was that visible coagulation of protein solutions occurred. when such solutions were exposed to these emanationso Coagulation of proteins was assumed to be the mechanism by which radiations affect cellso At first, i.t was the contenti.on of the earlier workers that coagul~at'ions produced by irradiation and heat were identical. Further studies showed that di.,fferences d:o exist,' although the data is meager (McLaren, 1949) The eariy work has been reviewed by Arnow (1936) It is of interest to note that Effont, in 19i53, suggested. that, the effects of radiations on protein soluAti.ons mi ght be -due to formation of hydrogen peroxide in water (Arnow, 1936)o An interesting observation with ultra-violet light showed that prior exposure of albumen to ultra-violet radiation resulted in lowering

-14the coagulation temperature of the protein (Bovie, 1913)o Stedman and Mendel (1926) irradiated 12 purified proteins from a wide variety of sources and found that the coagulation temperature was markedly lowered by pre-irradiation with ultra-violet lighto Clark (1935) made a systematic study of the sensitization of egg albumin by ultra-violet in order to elucidate the mechanism involved, She separated the reaction into three stepso 1) A slight denaturation of protein wherein a large amount of energy is absorbed, This reaction is independent of temperature, and can occur in the absence of water and over a wide pH range in presence of watero 2) The second step involves the reaction of altered molecules with water (hydrolysis). This step was considered, to be chemical in nature and occurs with a high temperature coefficient (temperature coefficient of 10)o 3) The third step, a physical reaction, manifests itself by the appearance of the visible coagulumo Although, this investigator offered the above mechanism to explain photo-sensitization, the manner in which radiation alters the protein molecule is not apparento Actually this proposed scheme is a modification of the classical theory of protein coagulation as caused by heat and suggested by Chick and Martin (1910, 1911). Photosensitization of Protoplasm to Heat The first observation that electromagnetic radiations rendered protoplasm of living organisms sensitive to heat was observed in Paramecium by Bovie (1919) with ultra-violet rays transmitted through fluorite (the radiations transmitted through fluorite are considered to be in the Schumann region of the electromagnetic spectrum between 1250 2000A)o In a series

-15of controlled and detailed experiments (Bovie and. Dalandl 19235 Forbes and Dal.and, 1925) with Paramecium caudatum. it was observed,, for exarmpl e that 88 per cent of the cells formed. vesicles when. heated at 34506C for five minutes after exposure to ultra-violet light whereas, when treated in-, dependently9 50 per cent formed. vesicles following heating and 25 per cent subsequent to irradiation. Sensitization of cells by ultra-violet light was also observed at temperatures of 27 o 50 and 530 5 C temnperatures wh-ich were not considered to be lethal for the animalso Preheating the animals did not alter their radiation sensitivity' Although the experiments of the above mentioned investigators were detaiied thre e was some question as to whether the sensitization was produced directly by the radiation or indirectly as a result of ozone produced by the 1utra-violet light sourceso With the advento of improved radiaa t;i.on, sources and. techniques for energy measurements, Curra:n and. Evans (.198) restudied the problem of photo-sensiti Ezationi using bacterial spores as a test systemo In their studies three strains of aerobic organilsms were used, Baci@tllus albolactis and Bacillus co'haerens and an u.nk.nown stra in belonging to the Bo mesenteri-cus group which they designated, as CC0 TI^To sources of u.itra-violet rad.iation were sed9 these were a mercur..amp which emit;ed 95 per cent of the radiation at a wavelength of 253 7A and a hydrogen ddi scharge tube with a fluorite filter The emission of the latter, lamp was in the 550 O 1600A regiono Using consecutive light exposures, heat treatnments at 98~C, and time intervals up to nine minutes In lengt:h3 th;:ey observed that an initial exposure to ultra-violet light made the spores of the three organisms more susceptible to subsequent heat treatmernt Alsog

-16in order for the heat sensitization phenomenon to manifest itself, it was necessary for the exposure to ultra-violet radiation to be of sufficient duration to be sporicidal for a large number of the organisms. Shorter wavelengths of ultra-violet radiation, in the range 350 - 1.600A, were found to be more efficient for inducing heat sensitizationo The most heat resistant organism tested, B. cohaerens, was the most susceptible to sensitizationo Duggar and Anderson (1939) observed that prior exposure of cells of Saccharomyces cerevisiae to ultra-violet radiation, (2650A) followed by heat treatment of 50~C, was two to five times more lethal than. the reverse procedure. They also observed that pre-irradiation increased the ability of the cells to take up methylene blue after heating, but that the respiration rate was not affectedo Respiration of the yeast cells was not affected by ultra-violet light even at exposures which prevented the cells from forming colonieso A conclusion reached by these investigators was that respiration and the ability to form colonies are governed by separate mechanisms It was suggested that nucleoprotein may be involved in sensitization because nucleoprotein strongly absorbs radiation of this wavelengtho A more extensive investigation by these investigators confirmed their original observation (Anderson and Duggar, 1941) Using the same criteria, namely, inhibition of colony formation, uptake of methylene blue and alteration of the respiration rate, they compared the effectiveness of irradiation at 2200A with that at 2650A in conjunction with heat treatments at 50~CO They observed that irradiation. at 2200A was as effective as at 2650A with the added observation that the respiration rate was depressed

-17when irradiation was carried out at 2200Ao They also observed. that heat was able to sensitize the cells to radiation at 2650A'but that the reversed. treatment, pre-irradiat-ion then heat treatment, was more effectiveo Using better radiation sources and more refined intensity measurements, Giese and Crossman (1945) re-investigated the photo-sensitization of Parmaci.um to heat by ultra-violet radiation The spectrum., at various wavelengths, between 2383A and, 3660A was studied. Maximum sensitization of the animals to heat treatment at 41lo5~C occurred. at 2383A' this was the shortest wavelength triedo There was a rapid decline in the sensiltizing effect towards the longer wavelengths with no observable effect apparent at 3669Ao The spectrum for photo-sensitization of pseuiogl.obiu`lin was observed to be similar to that for sensitization of the animals. They observed. that recovery was possible after exposure at all wavelengths of radiation.. as well as following heat trea.tment, bnut the recovery was sl.-owo The slowest recovery of all occurred after the cells were expose t at a wavelength of 2650Ao Since nucleoproteins have the maximum absorption at this wavelength and recovery was siowest+ indicat. rng greater damage2 it was con.cluded that nucleoprotein was.inrvolve f inr the photosensitizat-,ion phenomenon. Alt.hough only one temperatuire (4l cCC was usei. in their st;udi.es these investigators state. that, the sensi. tzation phenomenon manifest:ed i.tself onl-y if lethal temperatures were used. after irradiation. The first, observation that ionizing radia; tons are capable of inducing sensiti.zati~on in Paramecium was observed by Giese an. Heath (1918) with X-rays. An X-ray i.ose of 14-0,000 rep and heat. trea+tffent for 1,5 mi. nutes

-18at 42~C killed the animals. Either treatment alone had no effecte The opposite treatment, that is heating the cells at 42~C for 1l5 minutes followed by exposure to X-rays, also had no apparent effecto A direct correlation of heat lethality with radiation dosage was observed. Although the phenomenon of sensitization to heat by ultra-violet radiation has long been recognized, this study by Giese and Heath with ionizing radiation appears to stand aloneo Recently, because of implications of radiation sterilization, the combined effect of heat and irradiation has been studied on spores of importance in food processingo Morgan and Reed (1954) demonstrated that spores of thermophilic anaerobe NCA strain 3814 were more susceptible to heat treatment at 240oF after receiving a total dose of 250,000, 600^000, and. 1,000,000 rep from cobalt-60o They also observed that Bacillus coagulans varo thermacidurans and Bacillus stearothermophilis were not rendered more sensitive to gamma radiation by prior heat treatmento In a much more extensive investigation with Clo botulinum spores from strains 62-A and. 213-B it was observed that the Fo value in phosphate buffer coulld be reduced as much as four-fold following a pre-irradiation treatment of 900,00 rep (Kempe, 1956) Preheating the spores for periods as long as 24 minutes at 990 C did not alter their sensitivity to subsequent irradiationo The extent of the sensitization phenomena produced in the spores was dependent on the total radiation dosage received. The practical significance of this method of sterilization was demonstrated with canned beef and peas Depending upon the amount of gamma radiation used both the spores of Clo botulinum and putrefactive anaerobe were rendered

significantly more sensitive to subsequent heat treatment in these products significantl.y more sensitive to subsequent heat treatment in these products, (Kempe et al,, 19579 1958, 1959) Kan et alo (1957) also observed, that pre-irradiation treatment with gamma radiation from cobalt-60 rendered the spores more sensitive to subsequent heat treatmento Further experiments with spores from Bacillus cereus showed the phenomenon to be operative in this specieso The sensitization phenomenon has been observed, adventitiously in studies directed towards other objectiveso For example Wood (1955) in his study on the influence of temperature and phase state prevalent during radiation on the survival of yeast cells exposed, to X-radiation, observed that the sum of radiation and heat treatment was greater than if they had been applied independentlyo In a series of additive experiments in which the cells were exposed to various combinations of heat at 52o5~C and radi.ation treatmentsh his results indicated that the greatest nulmer of cell survivors occurred when the heat treatment -was appliel prior to irradiationo It is interesting to note that, this is exactly opposite to that observed in other investigations With bacteriophage strain T —., the simultaneous application of heat and radiation resulted, in greater destruction of the viruses than if either treatment was applied- independently (Adams an.d Pollard, 1.952) The phenomenon of sensitization of protoplasm to heat by electromagnetic radiations seems to be universal in, that it occurs in a wile variety of microorganismso However the relative importance of this phenomenon in terms of lethality of radiations must yet be ascertained Also the mechanism by which sensitization develops in the cells needs to be elucidatedo With bacterial spores^ an explanation of the sensitization phenomenor

-20~ assumes added importance; here such knowledge may help to produce an understanding of the basis upon which some bacterial spores exhibit unusually high resistance to damage by heato

MATERIALS AND METHODS Organisms The organisms used in the present study were originally obtained from the following sources. Clostridium parabotulinum 62-A Dro Fo Ko Meyer Clostridium botulinum 213-B Hooper Foundation for Medical Research San Francisco? California Clostridium parabotulinum4 457-A Dro Lo So McClung University of Indiana putrefactive anaerobe NCA Dro E Jo Cameron strain 3679 National Canners Assoco Washington, Do C. Maintenance of Stock Cultures Stock cultures of Clostridium strains were maointairne in' cooked meat phytone medium (a product of the Baltimore Biologi cal Laboratories;' This medium. consisting of dehydrated meat particles and. enzymaticallry digested soy beans? was rehydrated and sterilizedl for 30 minutes at 121."C It has been reported that cultures could be storej in t-.his medil.1umL for extended periods of time without losing their desireJ cultu;aral characteristics (Vera, 1.944). These cultures were transferred approximately every six. monthso For this purpose 01o ml of the stock cultures was transferred into cooked meat phytone media which was prepared in 1l6 x 120 mm screw cap tubes and, from which the di.ssolved oxygen had been removed by heating in boiling water The cultures were then incubated at 37 C until the meat was'backened; this usually require thhree or four days. The cultures were then -21

-22examined for purity and evidence of sporulation. If spores were present, the tubes were heated in a boiling water bath for 15 minutes in order to kill vegetative cells and to heat-shock the spores. After transferring into fresh media, and incubating at 37 ~C the cultures were stored at 4~Co The purity of the strains was verified by combining filtrates from cultures grown in 10% casitone broth with type specific anti-toxin obtained from. the New York State Health Departmento Stock cultures, produced as outlined above. have maintained sporogensis and toxicity for several years Production of Spore Suspensions The following medium was used for production of spore crops of Clostridium botulinum 213-Bo Bacto-Casitone 100. grams/Q Bacto-Beef Extract 0.5 grams/e Di-Sodium Phosphate 0.5 grams/i pH 6.8 - 7.0 This is essentially the same medium employed by Reed et alo (1951) in their studies on the heat resistance of anaerobic bacterial spores except that Bacto-casitone was substituted, for the freshly prepared pancreatin digest of casein recommended in their procedureo The inoculum used for seeding the sporulating media was prepared by transferring 0ol ml of a stock culture into 10 ml of this casitone medium after it had previously been exhausted of dissolved oxygen. The cultures were incubated at 30~C until growth occurredo Further transfers were made at daily intervals until the organisms became adapted, to the new medium wh ich was evident by early and vigorous growth in the culture tubes. A 24 hour culture, for

-23use as an inoculum, was then prepared and used to seed. 800 mil of media contained in 1000 ml Erlenmeyer flaskso Usually a five per cent inoculum was used, The flasks were sealed. with paraffin. paper and allowed to incubate at 30 C for three weeks After this, the flasks were removed from the incubator and then were placed in a refrigerator for seven to ten days This permitted the spores to settle, and the lysis of any remaining vegetative cells. For harvesting, the supernatant was aseptically aspirated from the settled spores in the culture flask and the spores were washed five times in 200 ml centrifuge tubes with cold, sterile^ distilled water. The initial, washings were done by sedimentation in an International. centrifuge located in the refrigeratoro Each centrifugation was carrie.d out at a rotational speed of 1500 rpm for 30 minutes. Additional washings were conducted at room temperature until a spore suspension was obtained that was practically free from vegetative cells and extraneous material as evidenced by microscopic examinationo This usually required eleven additional washingso As a final step, the spores were concentrated in a few ml of sterile distilled water and stored over glass beads at 4~Co Yields of approximately 106 spores per ml, based on the original culture broths were obtained by the above procedureo A fresh liver broth medium was used for the growth of spores of Clo botulinum 62-A. The preparation of the liver broth medium was as fol. lows: Chopped beef liver (fat free) was mixed with water in the proportion of 500 grams to 1000 ml This mixture was boiled slowly for one hour3 aljusted to approximately pH 7m2 with 10 sodium hydroxie and. boil.led for an an additional 10 minuteso This mixture was filtered through cheese cloth

-24and then made up to the original volumeo To this extract were added 10 grams of Bacto-peptone and one gram di-potassium phosphate per litero The medium was dispensed into tubes and five liter Erlenmeyer flasks, a few iron wires were added, and sterilization carried out for 45 minutes at 15 psi. The pH after sterilization was approximately 7~0~ A 24 hour culture, grown at 37 ~C was used to inoculate the five liter flasks. The large flasks were allowed to incubate for seven days at 37~Co Sporulation was observed to be complete after this timeo The cultures were cooled in a refrigerator after which the cells were harvestedo Preparation of the spore suspensions of this organism was essentially the same as described with strain 213-Bo Spore suspensions of putrefactive anaerobe NCA strain 3679 were prepared by the method, outlined by the National Canners Association (79) which is described in the next sectiono Fresh pork hams were trimmed of fat and the lean pork was ground and, mixed with distilled water using one pound of pork to one liter of watero This was boiled slowly for one houro The pH was adjusted to 7~4 with 10 per cent sodium hyroxide; approximately 110 ml of alkali were needed for 20 liters of extracto The meat was separated from the hot mixture by filn tration through several layers of cheese cloth. A portion of the resulting meat-cake was reserved and dried at 100~Co The pork extract was placed in the refrigerator overnight in order to facilitate removal of the solidified, fato After removal of the fat, the extract was made up to its original volume and the following ingredients were added per liter of extract, Bacto-peptone 5o00 grams Bacto-tryptone 1l50 grams Dextrose lo00 grams K2HPO4 Lo25 grams

-25After dissolving the above ingredients, the pH was readjusted to 7o4K The above medium, with the addition of lo5 per cent agar, was used as a recovery medium. However, during sterilization a troublesome precipitate always developedo This was removed by the following procedure five liters of medium contained in a Erlenmeyer flask were atuoclaved for 40 minutes at 15 pounds pressure. After autoclaving, the flask was placed in a tilted position in an incubator at 37~C until the precipitate settled This required about one hourO The liquid -was then carefully decanted after which the medium was dispersed into tubes and sterilized at 121o7~C for 15 minuteso It was stored in the refrigerator at 4 Co. The procedure for preparation of spore suspensions was as followsC First step! Two ml of a stock culture of PA 3679 spores were transferred into each of three tubes each of which contained 10 ml, of medium along with a small amount of dried pork in the bottom of the tubeo This was stratified with pork agar and incubated at 37~Co Second step Whken good growth was evident, as indicated, by gas production, two ml were transferred into each of six tubes containing 10 ml of medium along with. dried pork particles and a clean iron wire. Th.ese tubes were incubated for one day at 370Co Third step: The contents of three of the above tubes were transferred into two 50 ml flasks containing the same mediu m.o These were then incubated at 370C for two dayso Fourth step! The contents of the two cultures from the third, step were transferred to a flask which contained five li.ters of mediiLum.

-26dried pork and 10 pieces of iron wire. This was incubated at 37~C for a period of one week and then at 30~C for two weeks. After incubation the flask was chilled in the refrigerator and the pork particles were strained off through a thin cotton pado The liquid was centrifuged in order to collect the spores in a small volume. Otherwise harvesting and subsequent treatment of the putrefactive anaerobe spores was essentially the same as that previously described for Clostridium botulinum spores. A difficulty was encountered here that was not present with Clostridium botulinum spores. namely, removal of adhering meat particles. Consequently, more washing and filtrations were necessary in order to obtain a clean spore suspension Preparation of Spore Suspensions for Irradiation In order to minimize any variation inherent within the spores, the same spore suspension was used in all experiments where comparative results were to be madeo All experiments were performed with spores suspended, in sterile M/15 phosphate buffer (Sorensons) at a pH of 7 0o 0 This menstruum was selected because spores have been demonstrated to exhibit maximum heat resistance when suspended in this medium (Esty and Meyer, 1922) and therefore it is widely used in heat resistance studies Solutions of M/15 K2HPO4 and Na2HP04 were mixed in the desired proportions to give a final pH value of 7~05~ They were then sterilized for 15 minutes at 1210~C During sterilization the pH of the solution dropped 0.05 of a unit. After preparation, solutions were stored at 4~C in pyrex flaskso Fresh buffer solutions were prepared every two weekso Prior to preparation of spore suspensions for the experiments, appropriate amounts of stock suspensions were heat-shocked to break their

27dormancyo Spores of Clostridium botulinum. were heated for 15 minutes at 85"C and those of the putrefactive anaerobe, for five minutes at 100 C. These treatments have been shown to be optimum for subsequent germination of these organisms (Reynolds et al., 1945- Desrosier and Heiligman, 1952) After shaking with glass beads in a rotary type shaker to dispense clumpps, stock suspensions containing approximately 108 spores per ml were diluted with cold buffer to a final concentration of 106 spores per mlo The latter concentration was selected because it represented a convenient dilution for subsequent counting. Previous experiments,emonstrated that the sporocidal effect of irradiation is independent of the concentration of spores in the range of 104 to 107 spores per mrri Four ml. of the diluted spore suspension were next aseptizcally transferre.d 1to sterile five ml. Neutraglass vials (Kimble) and cooled in a refrigerator at 4~Co The vials were then quickly sealed. in an oxygen flame. No appreciable heating of the spore suspensions was noted during this sealing. All vials were stored in a refrigerator at 4~C until used. In order to minimize any storage effect, spore suspensions were al.ways prepared. a few hours before each series of experiments and plated immedi.atel'W>y after irradiation The frozen spore suspensions were preparei-, by plunging the sealed. vials indiv';id.uall.y into an ethyl alcohol. dry ice bath maintai.ned. a-t -72, Co The spore suspensions were maintained at this temperature until irradiation was completed. After irradiation the vials were stored in a deep-freeze chest until they were removed and thawed for plating, During storage the temperature rose to -10~C at times. This metbod of freezing ha. no effect

-28on the viability of the spore suspensions as determined by viable countso Actually alternate freezing and thawing of the spore suspensions as much as five times did not decrease the viability of the spores as determined by plating procedures Counting Techniques and Recovery Medium Several anaerobic techniques for enumeration of anaerobic organisms have been described. Among these are~ Brewer Anaerobic Plate (Brewer, 1942), Spray dish (Spray, 1931), modified deep agar shake tube (Miller et alo, 1939), roller tube technique (Saleh and Ordal, 1955) incubation of petri dishes in inert atmospheres and Andersons layer plate method (Anderson, 1951)o For these studies^ the modified deep agar shake tube technique as described by Miller et alo (1939) was selectedo Not only were maximum spore counts obtained by this technique but it was adaptable to handling of a large number of samples. Several different recovery media have been described for counting heated anaerobic spores but so far no agreement exists concerning the best medium for irradiated spores. The pork-extract medium previously described, with the addition of Lo5 per cent agar, was used as the counting mediumo This medium was slightly modified by incorporation. of 0.1 per cent soluble starch and O01 per cent sodium thioglycolateo The former has been shown to increase the recovery of severely heated spores (Olsen and Scott, 1946) while the later assures a sufficiently low redox potential for anaerobic growth. The pork-extract medium, as modified., has been shown in one study to have a definite superiority over media that have been used for recovery of severely heated spores (Frank and Campbell, 1955). A disadvantage of

-29of this medium is the possible variability in the preparation of different lots of pork-extract In order to reduce possible effects of such variability, the same batch of recovery medium was used throughout any one set of experiments The procedure for counting the spores was as follows~ Prior to use, tubes of media were melted cooled at 50~C and held at this temperature. Vials containing irradiated spores were then aseptically opened, and appropriate dilutions of the spore suspensions were made in 99 ml of sterile distilled watero Tubes of media were then inoculated9 rotated to disperse the spores uniformly, and immersed in ice watero After solidification two per cent agar containing 0!1 per cent sodium thioglycolate was added to seal the tubeso Duplicate tubes of triplicate dilutions were usually prepared and incubated at 30 C for three to six dayso Prolonged incubation after this time did not result in an increase in survivorso'Tubes containing between 1.0 to 100 visible colonies were counted w'ith the aid of a Quebec Colony counter Irradiation Facility For irradiation purposes, a cobalt-60 radiation souorce was employedo It consisted of i00 cobalt rods encased in alumnum. jackets. The rods were six millimeters in diameter', 25 centimeters'ong, and were supported in two concentric circles by an aluminum. rack with an inner diameter of 15 centimeterso The source had. 51 rods in the outer and 49 in the inner circle; it was housed in a irradiation room shielded with concretes and could be raised into the room or lowered into a 12 foot well filled. with water by means of a hand operated elevatoro A detailed d.escription of the source has been published (Brownell et alo l 19535 Nehemias et alo. 1954)o

Irradiation Techniques The center well of the cobalt-60 source was used since this position offered both the zone of maximum flux and uniformity of radiationo Because of the design of the radiation faculty, it was necessary to consider several factors in designing the equipment for irradiating samples at controlled temperatures. These were: (1) The geometry needed to be such that each individual sample received the same dosage. (2) A maximum number of samples should be irradiated at one timeo (3) The device needed to be sufficiently simple so that it could be removed when not in use but still be repositioned accurately for each series of individual experimentso (4) A minimum amount of radiation should be absorbed by the system. The above criteria were quite satisfactorily satisfied by the apparatus diagramatically illustrated in Figure 1 and. described belowo The irradiation chamber, which also served as a temperature bath, was fabricated from a 4-1/2 x 6 inch stainless steel beakero It was insulated with alternate layers of glass wool and asbestos sheetso The chamber was positioned in the center well of the radiation source by means of a wooden support which consisted of circular bases separated by a risero When the rack containing the cobalt-60 rods was raised into the irradiation room it was enclosed in a wire cage which protected it against accidental spilling of the rods. This cage also acted as a support for the irradiation chamber in the center wello The lower disc of the chamber fitted snugly into the bottom of the cage, the other supported the chambero A double row brass rack1, each row containing spaces for fourteen vials, held the spore suspensions in proper position in the chamber By proper adjustment of the height of the wooden riser, the irradiation chamber was positioned -30

-315 IT 9i~HEATING COIL FOR BATH 11,, // //,^-INSULATED IRRADIATION CHAMBER -I1 ---- lj- / | ^ -SAMPLE VIALS (28) 2!Xl in BRASS SUPPORT ____ _____ /C08~-~ COBALT -60 RODS, k[ | ^ | ~- ~J ^-' ^ ^ /I 100 PIECES I I0 Vy /l ^- r SSAFETY CAGE I WIRE MESH ao )51 ^0 1 I ~I M S- ELEVATOR ROD a 4 ~ol// 4 1 0 0 I A 00, _ Figure 1. Diagram of Facility for Irradiation of Spore Suspensions with Cobalt-60.

-32in the center well in such a way that one rack of vials was above and one below the mid-plane of the cobalt rods, A spiral coil of aluminum tubing (5/16 ino O.Do) was used to heat or cool the batho This coil fitted into the center of the racks along the vertical axis of the irradiation chamber and was connected by means of insulated pipes to a reservoir of a temperature controlling liquid located outside the irradiation room. During operation, heating or cooling liquids were pumped through the coil in order to maintain the irradiation chamber at a constant temperatureo The vials within the irradiation chamber were completely immersed in the batho After allowing time for the entire system to come to equilibrium, the temperature could be maintained within 2 ~C with this arrangement. For the experiments in which the spores were subjected to heat alone, an insulated temperature bath was constructed. For the heating menstruum, twelve quarts of dibutyl phthalate was employedo Temperature control was maintained by means of a bimetallic thermoregulator and a timedelay-relay systemo The temperature of the bath could be controlled within Oo2~C with this systemo The thermometer used for temperature measurement was calibrated against a Bureau of Standards thermometer. To minimize errors due to extra manipulation, the spore suspensions were heated in the same vials in which they had been irradiated. Under these conditions the time required for the spore suspensions in the vials to come up to the bath temperature after immersion was determined to be two minuteso Measurement of Dose Rates In order to carry out a quantitative study of the lethal effects of radiation on bacterial spores it was necessary to calibrate the radiation

-33field in which the sample vials were placedo Furthermore since the strength of the radiation field was not the same in all positions wi`thin the centerwell, calibration at each spot occupied by a vial was necessaryo Extensive calibration measurements were carried, out initiallyl and whenever required., usually at monthly intervalso The ferrous sulphate method as outlined by Weiss (1952) was used for radiation intensity measurements As he pointed, ou;t dilute ferrous sulphate is oxidized quantitatively by ionizing radiation in the dose range of 0 to 50,000 repo The concentration of ferric ion thus produced can then be determined as a function of ul.tra-violet light absorption of the so.Lution at a wavelength of 305 m.o The procedure and soluthions used for dosimetry stu:,i. es -were as follows Stock Solution (made from reagent grade stock) Ferrous Ammonium Sulfate 3 90 g Sodium Chloride Oo2 g Sulfuric Acid concentrated 2 o20 ml. Triple Distil-led aerated'water 100o00 ml. This stock solution couldi be stored for ahout a month provided it was protected from, lighto Dilute Solution (the actua-l dosimetric solution) Stock Solution LoO ml. Sulfuric Acid o4 ml Triple distilled water 200o0 ml. The dilute solution was aerated, by passing filtered air through it for several. hours prior to useo All solutions were prepared from glassware which ha previously been soaked in acid d.chromatee cleaning solultion thoroughly rinsed and then dried in an ovenO The same type of vials used.

334for dosimetric studies were later used to contain spore suspensions used in the experimental runs. These vials were soaked at least 24 hours in acid dichromate cleaning solution and then thoroughly rinsedo The final rinsing was carried out with, triple distilled watero The vials were thoroughly dried in an oven before useo For calibration runs, four m1. of the dosimetry solution were carefully pipetted into each vial; then the vials were sealedo These dosimeters were positioned in the irradiation vessel in the exact position in which the spore suspensions were later to be exposed; the vessel was placed in the center-well and the source brought into position. The time of exposure was adjusted to give a total dose between 10 and 45 kilorep since the procedure is most accurate in this range The concentration of ferric ion, produced by the radiations, was determined by comparison of the optical densities of the irradiated solution to that of known concentrations of ferric ion, The optical density of the solutions was determined in a Beckman Spectrophotometer model DU at wavelength of 305 mp. and with a slit width of 005 mmo The quartz cuvettes used in the procedure were rinsed four times with triple distilled water and dried with acetone between each reading. For the conversion of ferric ion concentration to radiation dose a value based upon oxidation of 15.4 micromoles of ferric ion per liter per kilorep was employedo This value is based on the absorption of 93 ergs per gram of water exposed to one roentgen of radiation (Weiss, 1952)o It defines the unit, rep, used in this thesiso The standard ferric ion solution used for calibrating the spectrophotometer was prepared as follows~ a 0olN solution of ferric sulphate

-35was dissolved in 0o8N sulfuric acid. The ferric sulphate solution was then standardized by reduction of adequate portions'with. high purity aluminum followed by immediate titration using Ferroin (o-Phenanthroline) solution as an indicator. The standardized ferric solution was diluted to concentrations between 50 and 500 micromoles of ferric ion per liter and the optical density of these samples then determinedo From this a standard curve was prepared which related the total radiation dose to the ferric ion concentration observedo Dosimetry Results The results of the dosimetry study are presented in Table I1 Figure 2 shows the relative dose rate in the vials during August 1954, in the various positions that they occupied during irradiationo TABLE I DOSE FATE AT VARIOUS POSITIONS IN THE IRRADIATION CHAMBER, AUGUST, 1.954 Bottom Row of Rack Dosimeter rep per hour 1 221, 000 2 232,000 3 227,000 4 228,000 5 238,000 6 2355000 7 225 000 8 224,000 9 221,000 10 225 000 11 220,000 12 225,000

-36TABLE I (CONT D) Top Row of Rack Dosimeter rep per hour 1 225,000 2 214,000 3 2153000 4 216,000 5 204,000 6 202,000 7 219,000 8 200,000

-37BOTTOM ROW TOP227 228ROW YQQ^^ Figure 2. Dose Rate in Rep Per Hour x 3 at TOP ROW Chamber.13 \(ZOO)\^ 204 Figure 2. Dose Rate in Rep Per Hour x 103 at Various Positions in the Irradiation Chambe r.

EXPERIMENTAL RESULTS The Effect of Temperature During Irradiation on the Survival of Clo Botulinum and putrefactive anaerobe Spores The object of the first series of experiments was to determine whether the exposure of bacterial spores to gamma radiation. at a wide range of temperature would affect their subsequent survival as determined by colony formation. As outlined in the section on methods, the experimental conditions were so designed that the only variable was the ambient temperature during irradiation. The general. procedure followed was to bring the irradiation bath to a desired temperature, add the vials containing the spore suspensions and then allow them to equilibrate to the bath. temperature. The time for the spore suspensions, within the vial.s to reach the bath temperature was determined to be two minuteso The irradiation chamber was then placed in the radiation field and, samples were removed at time intervals which gave the desired dosage levelso In the experiments in which the spores were:irradiated at temperatures greater than 50~@ the vials were removed as rapidly as possible and, plunged into a container containing ice water. This was necessary to minimize any after effect due to post radiation heating of the sporeso The time for removal of samples was never more'than 30 seconds. The total time for manipulation of the radiation source varied between one to two minuteso After irradiation the samples were stored in a refrigerator until the spores were cultured into the growth mediumo In cases where the spores were exposed to temperatures greater than 30 ~C appropriate temperature controls were usedo In this case the spore suspensions, contained in the -38

-39vials, were exposed at the temperature at which the irradiation was performed for a period of time equivalent to that used for t;he experimental suspensions In the experiments in which the spores were frozen during irradiation, the sample used to determine the initial number of spores was also frozen and, maintained in that condition. during irradiation of the other sampleso The frozen spore suspensions were thawed just before subculturing into the recovery mediumo As can be seen from the temperature controls, freezing and thawing did not hlave a deleterious influence on the subsequent survival of the spores as determined by colony formation.'The previously mentioned procedures were the protocol for this first series of experiments o The data from the experiments for Clo botliinum 21.3-B spores suspend.ed in M/15 neutral phosphate buffer and. irradiatedo in the temperature range from -70~ to 950C are presented in Table II and, Figure 35 As can be observed, the number of organisms surviving radiation exposure can be influenced by the ambient temperature during irradiation. The spores are slightly more resistant in the frozen. condition when compared with the non-frozen stateo There is a trend towards radio-resistance as the temperature is raised above room temperature With this suspension of 213-B spores the maximum number of survivors occurred at approximately 30~C In order to more clearly demonstrate this effect the number of survivors at a constant dosage of 7409000 rep are shown in Figure 4. and Table IIIo The results of a similar experiment, but with spores of putrefactive anaerobe NCA strain. 3679, are presented itn Tab.le IV and7 Figure 5~

-'4oTEMP CODE 2.001- c -70 o -7 A 80 0 A\^ 95 1.00 \ 09 0 --- V'l - - -70 80 - \00 -3.00 213-B Suspended in M/15 Phosphate Buffer at pH 7.0.

TABLE II 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 50 c 300 C " 58~ C Dose* Number of Log Number of Log q Number of Log Megarep Spores Survivors Spores Survivors Spore s Survivors 0 6,200,000 2.000 2,500,000 2.000 3,500,000 2.000 0.185 2,000,000 1.508 ------- -- 1,300,000 1.570 0.370 5,700,000 0.964 900,000 1.560 5,200,000 1.170 0.550 140,000 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 -1.598 ----- - 0.925 3,900 -2.201 150 -2.222 1,500 -1.369 1.017 150 -3.617 85 -3.469 ------- --- 1.110 3 -43.15 ------- _ -- ----- ---- 80~ C 95~ C Heat Control Dose* Number of Log 9 Number of Log % H Megarep Spores Survivors Spores Survivors 8 C 85 0 5,500,000 2.000 2,700,000 2.000 0 5,500,000 2,700,000 0.185 3,200,000 1.765 ------- --- 2 —,000,000 0.370 1,400,000 1.407 2,300,000 1.930 3 -------- 2,300,000 0.550 160,000 o.465 14,000 -0.215 4 -------- 1,500,000 0.740 7,800 -0.848 45 -1L777 5 3,000,000 1,400,000 0.832 3,500 -1.197 0 -- - --------- ------- 0.877 1,400 -1.595 0 - ------ ----- -70~ c -7~ c 27~ C Dose**Number of Log -Number of Log ~ Number of Log Megarep Spores Survivors Spores Survivors Spores Survivors 0 950,000 2.000 520,000 2.000 670,000 2.000 0.227 720,000 1.903 520,000 2.000 530,000 1.898 0.454 350,000 1.522 79,000 1.225 260,000 1.589 0.680 75,000 0.940 9,700 0.314 78,000 1.065 0.794 25,000 o.44o ---- - -- 0.907 12,000 -0.060 1,800 -0.523 71,000 1.025 1.020 5,000 -0.246 200 -1.366 ---- --- 1.134 1,500 -0.773 -_ —- -- ___790 -0.928 Frozen Control 9.0 x 105 *Dosage Rate = 0o185 megarep per hour. **Dosage Rate = 0.227 megarep per hour (vials not immersed in liquid).

-4220 Key -- PA 3679 ----- C. botulinum 213 B Rodiation dose 0.740 megarep i 6 ~ 12 l l|[ —-- so) I" 8 p I I 4 0 W-A -100 -80 -40 0 40 80 100 TEMPERATURE C Figure 4. 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.

-43TABLE III EFFECT OF TEMPERATURE DURING IRRADIATION WITH GAMMA RAYS FROM COBALT-60 ON THE SURVIVAL OF ANAEROBIC BACTERIAL SPORE SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 Percent Survivors Temp, oC Dosage Megarep ___0.550 0.647 O.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 0.06 27 11.6 10.5 30 14.o 4.4 0.88 56 10.0 0.3 58 2.9 0.4 80 2.9 0.14 95 O.16

-442.00 3?7l\ \ 5.} 6 85 -2 3 80 -3 1.00 54 58 - 1 56 -5 6 $ 30 -6 f 0 o3 I > -1.007 6 03 o -2.006 6 -3.00 -4.00 - -- 7 - 0.2 0.4 0.6 0.8 1.0 1.2 MEGAREP Figure 5. Effect of Temperature During Irradiation with Gamma Radiation from Cobalt-60 on the Survival of Spores of PA 3679 Suspended in M/15 Phosphate Buffer at pH 7.0.

-45TABLE IV 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 o5~c 30~c3 56~c Dose,* Number of Log % Number of Log INumber of Log megarep Spores Survivors Spores Survivors Spores Survivors 0 850,000 2.000 280,000 2.000 400,000 2.000 O.185 700,000 1.911 ----- ---- 300,000 1.875 0.370 220,000 1.409 54,000 1.286 18,oo000 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 0.832 ------- ---- 220 -1.103 ------- 0.925 160 -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 580C 80 oc 850 C Dose,* Number of Log Number of Log Number of Log megarep Spores Survivors Spores.Survivors] Spores Survivors 0 480,000 2.000 19100,000 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 --- -- --- - - --- --- 95Co Heat Control Dose, Number of Log PA 5679 megarep Spores Survivors Hr 85-~ C 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 - _ ___

-46The number of spores surviving at a constant dose but irradiated at various temperatures is given in Figure 4 and Table IIIo In this series of experiments the organisms were irradiated in the temperature range of 5~ to 90~Co From the data presented it is observed that the spores of this organism are most sensitive to radiation at a temperature of 5~Co The greater number of survivors were obtained at a irradiation temperature of 90~C. As can be observed from the temperature control, holding the spores of this organism at 900C for five and one half hours did not have an adverse effect on their viabilityo Similarily the experimental results obtained when spores of Clo botulinum 62-A were irradiated in the temperature range of -720 to 870C are presented in Table V and Figure 60 A curve indicating the number of survivors at a constant dose of 860,000 rep at various irradiation temperatures is given in Figure 70 The results show that the spores of this organism exhibit a slight increase in resistance to radiation when irradiation is carried out at -72~C as compared to 4~Co As was observed with the spores of the other bacteria, there is an increase in resistance to gamma radiation as the irradiation temperature is increased, With spores of this bacterial strain the maximum resistance is obtained at about 70 C, A further increase in the irradiation temperature results in a rapid kill of the organisms. This is demonstrated when irradiation is carried out at 87 C. The temperature control at 87~C shows that some heat inactivation of the spores occurs at this temperatureo However., the independent effect of this temperature was not pronounced for the length of time these spores were held at this temperatureo

-473.0- 2.0 1.0 0 -I.0 o_ -J -2.0 X TEMP. CONT. 60~C _\_ V TEMP. CONT. 870~C -5.0 I 0 0.4 0.6 0.8 1.0 1.2 REP X 106 Figure 6. Effect of Temperature During Irradiation on the Survival of C1. botulinum 62-A Spores Suspended in M/15 Phosphate Buffer at pH 7.0.

-48TABLE V EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF Cl. botulinum 62-A SPORES SUSPENDED IN PHOSPHATE BUFFER AT PH 7.0 40~ _ "..4", _ 200 C Dose Number of 0o,Log, Number ofl Log % Megarep Spores Survivors Survivors Spore Svivors Survivors S 0 1,400,000 100.00 2.000 785,000 100.00 2.000 0.218 1,200,000 85.7 1.933 -- - 0.327 660,000 47.1 1.673 - 0.436 360,000 25.7 1.41 465,000 55.4 1.744 0.545 - -- 615,000 21.0 1.322 0.654 - -61,500 7.82 0.893 0.763 4,900.35 -0.456 14,350 1.83 -0.265 0.872 1,100.0785 -1.105 4,350 0.554 -0.257 0.981 410.0293 -1.533 1,500 0.191 -0.719 1.090 24.00171 -2.766 455 0.058 -1.237 1.199 14.00100 -3.000 -- 20 C I43 C Dose Number of % Log % Number of I Log % Megarep Spores Survivors Survivors Spores Survivors Survivors 0 7,250,000 100.00 2.000 5,350,000 100.00 2.000 0.218 - - - 6,650,000 124.00 0.327 - -- - 0.436 2,400,000 33.1 1.54 3,450,000 b4.5 1.82 0.545 - - - 1,800000 33.6 1.528 0.654 230,000 3.17 0.50 850,000 15.8 1.2 0.763 98,000 1.35 0.013 420,000 7.85 0.904 0.872 14,900.205 -0.688 245,000 4.57 0.66 0.981 610 0.0084 -2.075 55,000 1.02 0.0086 1.090 145.002 -2.699 2,850.0533 -1.274 1.199 - -. -... 54~ C, 600 Dose Number of % Log % Number of Log % Megarep Spores Survivors Survivors Spores Survivors Survivors 0. 7,600,000 100.0 2.000 3,550,000 100.00 2.000 0.218 4,500,000 59.3 1.77 2,550,000 71.80 1.856 0.327 - - -. 0.436 4,150,000 54.5 1.73 1,900,000 53.50 1.728 0.545 2,500,000 32.9 1.52 -- - 0.654 860,000 11.3 1.05 485,000 13.65 1.350 0.763 360,000 4.73 0.665 300,000 8.45 0.927 0.872 99,000 1.30 0.114 94,000 3.78 0.578 0.981 58,500 0.77 0.115 37,000 1.04 0.017 1.090 8,950 0.118 0.928 9,600 0.27 -0.569 1.199 - -- -- -

-49TABLE V (CONT'D) Dose Number of I Log % Number of Log Megarep Spores Survivors Survivors Spores Survivors Survivors 0. 5,750,000 100.00 2.000 4,900,000 100.00 2.000 0.218 2,000,000 34.800 1.541 4,600,000 93.9 1.8690 0.327. - 0.436 1,900,000 33.0 1.519 2,300,000 46.8 1.294 0.545 — - - - - 0.654 870,000 15.1 1.179 220,000 4.48 0.281 0.763 700,000 12.2 1.086 255,000 5.20 0.180 0.872 108,000 1.88 0.2742 12,700 0.259 -0.818 0.981 43,000 0.748 -0.126 2,150 0.0438 -1.726 1.090 6,700 0.116 -0.935 - 1.199 - - - _87.50 C -650 C "Dose Number of Log% Number of % Log % Megarep Spores Survivors Survivors Spores Survivors Survivors 0 4,350,000 100.00 2.000 15,250,000 100.00 2.000 0.218 3,700,000 86.00 1.935 - - 0.327 -.-. -. 0.436 520,000 11.9 1.076 4,450,000 29.2 1.465 0.545 110,000 2.53 0.401 - 0.654 7,300 0.168 -0.775 540,000 3.52 0.547 0.763 6,300 0.161 -0.791 285,000 1.87 0.272 0.872 455 0.01445 -1.839 109,000 0.718 -0.145 0.981 7 0.00016 -3.793 29,000 0.190 -0.722 1.090.5 0.0000115-4.939 20,000 0.131 -0.883 1.199 -720 C40C_ Dose Number of Log % Number of Log % Megarep Spores Survivors Survivors Spores Survivors Survivors 0 1,800,000 100.00 2.000 7,150,000 100.0 2.000 0.218 - - 0.327 - 0.436 200,000 11.1 1.045 1,650 000 21.1 13 ~~~0.545 - - ~620 000 8.68 0.938 0.654 55,000 3.05 0.4 245,000 3.4353 0.763 8,500 0.472 -0.326 46,500 0.642 -0.192 0.872 2,400 0.133 0.97615,600 0.218 -0.661 0.981 760 0.0422 -1.375 4,250 0.0594 -1.276 1.090 240 0.0133 -1.877 315 0.0044 -2.355 1.199 - - 126 0.00177 -2.752

-50TABLE V (CONT'D) Temperature Controls - -60 C 7307 C Hours Number of Hours Number of —.Spores ____ Spore 0 3,500,000 0 4,750,000 1 3,450,000 1 5,100,000 2 4,100,000 2 5,000,000 4 3,450,000 4.2 5,850,000 4.5 3,450,000 5 4,900,000..~.C -7o' - - - -- Hour Number Hurs Number of.Spores I Spore s 0 4,900,000 0 4,350,000 3 4,500,000 1 3,450,000 16.2 1,520,000 2 3,400,000 1 2,700,000 4 1,950,000 3 3,450,000 6 1,950,000 4.5 3,750,000

-51Io iC ____ _____ __ L__ -_- - _-=-= 0J<., w z - 0 __ (n 0 d I.01.L o.001 __ _ _,_1___= =!=___'80 -60 -40 -20 0 20 40 60 80 100 120 T E M PE RATUR E- CENTIGRADE Figure 7. Effect of Temperature During Irradiation on the Lethality of Gamma Radiation from Cobalt 60 for the Spores of C1. Botulinum 62A When They are Suspended in M/15 Phosphate Buffer at pH 7.0.

In order to substantiate the above results, a series of experiments was performed using different spore suspensions of the same organism, In this case only a few temperatures were selected in order to strengthen the observation that bacterial spores are more resistant at a high temperature and also when irradiated in the frozen condition at a temperature of -72~Co In this series, spores of putrefactive anaerobes and Cl botulinum 213-B were irradiated at -72~C, lO1C, and 90OCo Another botulinum strain designated as 457-A was also included, The spores of this strain were irradiated at -72~C, 5~C, 25~C, and 900Cb The results from this series of experiments are presented in Tables VI and VIII and Figures 8 through 10, The trend observed in previous experiments is again evident in that irradiation at high temperatures and in the frozen state increases the resistance of the sporeso The Effect of Temperature During Irradiation on the Survival of Bacillus subtilis varo niger Spores In order to determine if the temperature of irradiation has an effect on spores of another genus, spores of the aerobic bacterium, Bacillus subtilis varo niger, were irradiated, In this case the number of spores surviving at irradiation temperatures of 5~ and 65CC was determined, A temperature of 65~C was selected since this represents a sub-lethal temperature for this organism, This can be observed from the temperature control, The results presented in Table IX and Figure 11 show a slight but definite increase in number of surviving spores at all dosage levels at 65~Co The other aspect for which this experiment was performed will be presented subsequentlyo

-533.0. 2.0 1.0 > 0 U) 0 0.4_ 012.. 0\ ~ -2.0 1O 90~C 2.0 650C O_ 4~C + TEMP. CONT. 650C -3.0 -4.0 0 0.4 0.8 1.2 1.4 1.6 REP X 106 Figure 8. Effect of Temperature During Irradiation on Survival of C1. botulinum 213-B Spores Suspended in M/15 Phosphate Buffer at pH 7.0.

-54TABLE VI EFFECT OF TEMPERATURE DURING IRRADIATION ON SURVIVAL OF C1. botulinui-a 213-B SPORES 90~C Number %Log % Dose Rep.__ Of Spores Survivors Survivors 0 7,000,000 100.0 2.00 360,000 290000 24.14 O 0.670 540,000 33,000 0.472 -0.326 630,000 16,000 0.229 -0.649 720,000 4,700 0.0672 -1.173 810,000 3,400 0.0485 -.314 900,000 48 0.000675 -3.171 Temperature Control 90~ C Time Hrs. 0 7,000,000 100.0 2.000 2 910,000 13.0 1.114 4 140,000 2.0 0.301 6 140,000 2.0 0.301 8 200,000 2.89 0.456 65~C 0 10,000,000 100.0 2.000 360,000 3,420,000 34.2 1.534 540,000 2,358,000 23.6 1.369 630,000 1,340,000 13.4 1.127 720,000 482,000 4.82 0.683 810,000 432,000 4.32 0.635 990,000 7,450 0.075 -0.125 25~C 0 6,300,000 100.0 2.000 360,000 3,700,000 58.7 1.386 540, 1,2 000 1,2119. l2 1.156 765,000 117,000 1.81 0.152 900,000 1, 200.162 -0.693 1,080,000 6,300.100 -0.874 cont' d.

TABLE VI (COIT'D) Numb e r Log' Dose Rep... Of Sporss Survivors..Survivors 40 0 3,100,000 100.0 2.000 360,000 955,000 3050 1.4 4 540Oo,0 23.5,000 6.93 0].41 63000 7,000 15000 06 0.704 720,000 32,000 1.03 0.013 ]0, 000 39,000 061 -0.22 990,000 L4,700 0.15 -0. 21 Temperature Control 65~ C Time Hrs. 0 10,000,000 100.00 2.000 3 ~, 000,000 O.00 1.9031 6 1, 00,00o 0.o 1.279031 6 1 sloo 000 10 00 1O ]..27~8

-563.0 - 2.0 1.0 > 0 U' 0 -1.0 0 _J E IOOC REP X I100 Figure ____Effect of Tee 0 90C I -2.0 X TEMP. CONT. 90~C -3.0 -4.0 -. —-—. — 0 0.4 0.8 1.2 1.6 2.0 REP X 106 Figure 9. Effect of Temperature During Irradiation on The Survival of putrefactive anaerobe Spores NCA 5679 Suspended. in M/15 Phosphate Buffer at pH 7.0.

TABLE VII// EFFECT OF IRRADIATION AT 100C AND 900C ON SURVIVAL OF PUTREFACTIVE ANAEROBE JCA 3679 Irradiated at 100 C Irradiated at 900 C Dose Spores per /0 Log % Spores per {0Log Re pml — Survivors Survivors ml Survivors Survivors 0 380,000 100.0 2.000 590,000 100.0 2.000 370,000 150,000ooo 39.5 1.593 340,000 37.6 1.575 555,000 18,000 5.15 0.712 200,000 33.9 1.530 647,500 90,000 15.3 1.185 736,000 800 0.21 -0.678 30,000 5.1 0.708 826,500 260 0.0684 -1.165 25,000 4.23 0.626 925,000 - - 8,400 1.42 0.152 1,175,000 5 0.00131 -2.8827 1,300 0.222 -0.6517 Temperature Control 0 Hrs 590,000 100.00 2.000 2 Hrs 510,000 86.5 1.937 3 Hrs 410,000 69.5 1.842 5 Hrs 380,000 64.5 1.809 6 Hrs 410,000 69.5 1.842

3.0 --- 2.0 1.0 (/) - 1.0 O 250C 302~ 0REX -70-C ~~>_ 0~~~~ ~90C - ------ 0.4 0.8 1.2 1.6 2C Figure 10. Effect of Temperature During Irradiation on the Survival of C1. botulinun 457-A Spores Suspended in M/15 Phosphate Buffer ~-3.0 -- -- — at pH 7.0. X -70~C - -- + TEMP. CONT.90~C -4.0 I 0 0.4 0.8 1.2 1.6 2.0 REP X 106 Figure 10. Effect of Temperature During Irradiation on the Survival of Cl. botulinum 457-A at pH 7.0.

-59TABLE VIII EFFECT OF TEIMPERATUJRE DURIING IRRADIATION ON SURVIVAL OF C1. parabotulinum 457-A SPORES ~90~0~C~~ARC 900C Number % Log % Dose Rep.,...Of Spores... Survivors Survivors 0 3,200,000 100.0 2.000 360,000 660,000 20.6 1.314 540,000 27,000 0.843 -0.074 630,000 3,600 0.112 -0.9508 720,000 17 0.0053 -2.276 810,000 - - 900,000 7 0.000218 -3.773 Temperature Control 90~ C Hours O-0 3,200,000 100.0 2.000 2 3,400,000 106.0 2.025 4 1,600,000 50.0 1.699 6 1,030,000 32.5 1.511 8 30,000 9.36 0.971 25~ C 0 3,600,000 100.0 2.000 360,000 2,100,000 58.4 1.7664 51+, 000 430,000 12.2 1.0864 765, 00 52,000 1.44.154 900,000 3,900.107 -.9706 -.9706 1,108,000 670.0181 -1.6258 -1.6258 c~ C 0 900,000 100.0 2.000 360,000 745,000 82.7 1.9143 540,000 59 000 6 55.8162 630,000 14,000 1.56.1931 720,000 2,100 232 0o.6 45 -0.63/5 810,000 1,450 161 -0.6932 -0.6932 990,000 c40 0104 -1.920 -1.9b20

-60TABLE VIII (CONT' D) Irradiated at -70~C Dose Spores % Log % Rep per ml Survivors Survivors 0 1,900,000 100.0 2.000 Frozen 2,300,000 121.0 2.083 360,000 1,030,000 44.8 1.651 540,000 385,000 16.7 1.223 630,000 123,000 5.4 0.732 720,000 58,000 2.52 0.401 810,000 17,100 0.774 -0.111 900,000 7,700 0.334 -0.476

3.0 2.0 0x^ 0 I Eo 5 C u) I.O 0L 0Q 65 C o 0 rE -2.0 DOSE REP X 106 Figure 11. Effect of Temperature During Irradiation on Survival of Bacillus subtilis var. niger Spores.

TABLE IX EFFECT OF TElPERATURE DURING IRRADIATION ON SURVIVAL OF B. subtilis var. niger SPORES...............TEiPERATURE 50 C 650 C0iP CO -TROL DOSE LOG%% --- L % -- ATREPX06 COUNT SURVIVORS SURVIVOR COUNTi SURVIVORS SURVIVORS 650 C 0 1.0x106, 100.0 2P.000.x0 100.0 2.000 0.031 4.0x105 40.0 1.602 4.6x105 41.8 1.621 i1l.2x 16 i I' l t' 1 i! I i! iO 0.062 1.9x105 19.0 1.279 3.0x105 37.3 1.573 1.lx106 0.124 7.9x104 7.9 0.898 l1.x105 10.9 1.037 1.3x106 J.186 2.4x104 2.4 0.80 5.5x104 5.0 0.699 - 0.248 5.7x103 0.57 -.244 1.5104 1.37 0.137.0lxlO6 0.372 4.9x102 0.049 -1.310 2.0x103 0.182 -.740 0.434 3.0x102 0.030 -1.523 9.0x102 0.082 -1.087.2x106

The Effect of Post-Irradiation Heating on the Survival of Bacterial Spores At the beginning of this study it was anticipated that an increase in the temperature during irradiation would result in a significant reduction in the number of viable spores after such a treatmentO Such a result would be expected if the coagulation temperature of proteins is'lowered by a pre-irradiation treatment as Stedman and Mend.el (1926) have shown occurred with ultra-violet radiationo However the results of the experiments of the present srudy, in which the spores were irradliated at various temperatures, indicated that the reverse was occurring; the spores were more resistant to radiation as the temperature was raised above room temperatureo In the investigations in which photo-sensitization was demonstrated in various li ving cell-7s the experimentors generally used t'emperatu.res which. were lethal for the cellso Therefore it was not clear if a lowering of the inactivation temperature occurred as a resu.lt of the pre —irradiation treatmento A series of experiments was performed to ieterm.ine whether the temperature for spore inactivation was lowered by a pre -rrad ation treatmentt The second objective for this experiment was to letermine whether the sequence of pre-irradiation plus heat wou1ld leai to a n increase in the number of surviving spores In this series of experiments the spores were exposed to various dosage levels of radiation at 4- C and then subjecte d to heat treatment for one hour at various temperatu-.reso The results for the experiment in which Cl. botuiinumn 21.3-B spores were irradiated at various dosage levels at 4 C and. then subjecte +to heat treatment at various temperatures between 50~ and. 100~C are given in Table Xo?63

-64The results for an identical but separate experiment with the same organism are given in Table XIo In order to better illustrate the effect, the results of Table X are plotted in Figure 1.2 The results of a similar experiment but using putrefactive anaerobes are presented in Table XII and Figure 13 o The most significant conclusions which can be derived from these experiments is that the thermal lethal threshold of the spores is not lowered by a pre-irradiation treatment, even at radiation dosages which cause considerable inactivation of the spores. However, a pre-irradiation treatment accelerates the rate of thermal inactivation at lethal temperatureso With Clo botulinum spores no significant inactivation occurs until 90~C is attainedo At this temperature non-irradiated spores are inactivatedo With putrefactive anaerobe spores, a temperature over 100~C must be attained before inactivation occurso However, it is noticed that some inactivation of irradiated spores can occur even at the sublethal holding temperatureso The Effect of Heating Clo Botulinumr and. Putrefactive Anaerobe Spores Prior to Irradiation An important factor to be considered in any study of the effect of heat on bacterial spores is the phenomenon of'dormancy"o Dormancy in relation to bacterial spores refers to a delayed germination of the sporeso It was early recognized that a mild heat treatment, commonly referred to as "heat shock",, could increase the number of Clo botulinum spores germinating in a suitable medium (Burke, 1923) Subsequent investigations have demonstrated that a wide variety of bacterial spores require a mild heat treatment to attain maximum germination (Curran and Evans 1937) In many cases, suitable cultural factors can replace the initial heat treatment

TABLE X SURVIVAL OF C1. botulinum 213B SPORES SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0, WHICH HAVE BEEN IRRADIATED AT 5~C WITH GAMMA RAYS FROM COBALT-60 AND THEN HELD FOR ONE HOUR AT THE INDICATED TEMPERATURE Control 100,000 rep 200,000 rep 400,000 rep Temp Spores Log % Spores Log % Spores Log % Spores Log % ~C per ml Survivors per ml Survivors per ml Survivors per ml Survivors 5 1,050,000 2.000 820,000 2.000 730,000 2.000 370,000 2.000 50 1,050,000 2.000 1,000,000 2.083 630 1940 0,000 1.940 20,000 1.937 60 930,000 1.947 670,000 1.912 970,000 2.124 240,000 1.813 70 960,000 1.961 620,000 1.879 550,000 1.877 450,000 2.083 80 590,000 1.749 620,000 1.879 720,000 1.984 330,000 1.950 90 630,000 1.778 520,000 1.803 240,000 1.513 106,000 1.457 95 310,000 1.450 1 0 11,3 0000 1.5 500.914 2,800 0.013 100 1,700 -0.791 170 -1.894 40 -2.261 0

TABLE 11 EFFECT OF HOLDING IRRADIATED SPORES OF C1. BOTULINUM 213 B FOR ONE HOUR AFTER IRRADIATION Control 400000 Rep Tempo Spores % Log % Spores Log % C per ml Survivors Survivors per ml Survivors Survivors 0 13,000,000 1000l 2 000 5,600,0 1000o 2o000 60 15,000,000 115 0 2.061 5400,o000 96 5 1l984 80 15,000,000 115 0 2o061 4,610,000 73~3 1o865 90 12,000,000 92~5 1l966 2,300,000 41,o 1o613 100 660,000 5.07 0o705 1,400 0o025 - 1.602 110 0 0 0 0 0 800,000 Rep 1,000.000 Rep 0 730,000 100,0 2.000 79500 O00o0 2,000 60 280,000 38.3 1.583 14,000 187.0 2.272 80 360,000 49.3 1.692 7,400 99.0 1.996 90 82,000 11.4 1.057 100 1.46 o,164 100 0 0 - 0 0 110 0 0 - 00 - Exposed for one hour at each temperature

2.00 --— 4 — 1,20 1.00 g 0.00 (0 -1.00 Key o- 0- Control Held for I hour at o I- Irradiated for 100,000 rep each temperature 2- Irradiated for 200,000 rep 106 spores/ml 4- Irradiated for 400,000 rep -2.0 0 ------— i —----— i —----— i —-------------------- *-4 n —----- -3.00 _______L_ 0 50 60 70 80 90 100 TEMPERATURE ~C Figure 12. Survival Curves for Cl. Votulinum. 213-B 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.

TABLE XII EFFECT OF POSTIRRADIATION HEATING FOR ONE HOUR AT VARIOUS TEMPERATURES ON PREVIOUSLY IRRADIATED PA 3679 SPORES Temperature, ~C Spores per ml Percent Survivors Log Percent Survivors a) Nonirradiated Control 860,000 100 2.000 70 650,000 75.5 1.878 80 660,000 76.7 1.885 90 640,000 74.5 1.872 95 500,000 58.2 1.765 100 580,000 67.5 1.829 105 310,000 34.9 1.543 110 0 0 b) Irradiated with 400,000 rep Control 150,000 100 2.000 70 170,000 113 2.053 80 160,000 106 2.025 90 162,000 108 2.033 95 175,000 116 2.065 100 134,000 89.5 1.952 105 3,600 2.40. 380 110 0 0 c) Irradiated with 800,000 rep Control 1,340 100 2.000 70 200 14.9 1.1732 90 305 22.8 1.358 95 560 41.7 1.620 100 65 4.85 0.686 105 0 0 110 0 0

-694.0 C CONTROL 4 IRRADIATED 400,000 REP 3.0 8 IRRADIATED 800,000 REP 2.0 4 4 —44_4 — 4 -.- C,) 0 10 o 0 I -2.0 Th -3.0 --- 50 70 90 110 130 HOLDING TEMPERATURE, ~C Figure 13. Effect of Post-Irradiation Heating for One Hour at Various Temperatures on Previously Irradiated putrefactive anaerobe NCA 3679 Spores.

-70(Curran and Evans, 1937)o Dormancy in bacterial spores has been the subject of extensive investigation in the past decade and has been adequately reviewed (Knaysi,1938; Curran, 1950; Schmidt, 1955; Stedman, 1956; Halvorson and Church, 1957). However, it can be generally concluded that those factors which are capable of supplying energy to the bacterial spores are responsible for the induction of the germination process (Halvorson and Church 1957) Since the present study concerns the simultaneous lethal effect of heat and radiation, the possible activation of bacterial spores by heat must be consideredo The question arises whether the apparent increase in resistance by the spores to gamma radi.ation at the elevated temperatures is due to activation by heat of any dormant spores which may be present in the suspensions, However, the rationale of the experimental procedure rules out this possibilityo All spores used in these experiments were heat shocked prior to each experiment. The time and temperature selected for this treatment was considered maximal for germination of the bacterial strains employedo Not only was this evident from the results of this study but the investigation of Desrosier and Heiligman (1952) is significant in this respecto They demonstrated that a time temperature relationship existed for breaking dormancy of several bacterial species including the putrefactive anaerobe strain 3679~ Their results showed no increase in germination, as measured by a viable count, if the spores were heated for a prescribed time at a given temperature. The maximum number of spores germinated after a heat treatment of five minutes at 950~C With Clo botulinum spores a heat treatment for 20 minutes at temperatures between 70~ and 90~C was sufficient

-71to obtain maximum viability of the spores as determined, by colony forming ability (Reynolds and Lichtenstein, 1949) However it would seem advisable to determine what effect a prolonged heat exposure at a high but sub-lethal temperature before irradiation would have on the resistance of the spores to irradiationo Previous studies with several strains of food spoilage organisms have shown that a preheat treatment at lethal. temperatures and, for time intervals which were sporicidal for a large number of the spores did not affect the resistance of the survivors to gamma radiation (Kan et alo,, 1957; Kempe, 1955; Morgan and Reed, 1954)o For this experiment spores of Clo botulinuLL 213-B contained in vials were held at 65~C for 14 hours. After this heat treatment the spore suspensions were irradiated for various lengths of time at 4~Co The results indicated that a prolonged heat treatment at 65 C did not render the spores more sensitive to radiationo Table XIII and Figure 14o In a similar experiment, spores of PoAo 3679 were preheated at 90~C for seven hours and then subjected to gamma radiationo The number of spores surviving this treatment are shown in Table XIV and Figure 15. Here the results would seem. to indicate that preheating made the spores slightly more resistant, rather than making them more sensitive to subsequent radiation treatment. The Effect of Irradiation on the Subsequent Germination of Bacterial Spores A question which arises relative to heat activation is whether radiant energy can be substituted for thermal energy in breaking dormancy of bacterial sporeso If this were so then the apparent increase in

-723.0 2.0 1.0 (1) o -1 -2.0 E PREHEATED FOR 14 HOURS AT 65~C -3.0 0- NOT HEATED -4.0 --- I I 0 0.4 0.8 1.2 1.6 REP X 106 Figure 14. Effect of Pre-Heating C1. botulinum 213-B Spores at 65~C for 14 Hours Prior to Irradiation.

TABLE XIII EJFECT OF PRE-HEATING C1. botulinurM 213-B SPORES AT 650c FOR 14 HOURS PRIOR TO IRRADIATION Heated Samples* Non-Heated Sam les Dose umber of0Log % Number of'0Log, Megarep ____ Spores Survivors Survivors Spores Survivors Survivors Control 630,000 100.0 2.000 630,000 100.0 2.000 Heated 4 hrs. 535,000 85.1 - - Heated 14 hrs. 525,000 83.4 - - 340,000 rep 250,000 46.7 1.669 172,000 27.3 1.438 510,000 rep 75,000 14.2 1.52 72,000 11.4 1.057 5956000 rep 35,000 6.67 0.824 11,500 1.83 0.263 680 000 rep 7,100 1.32 0.121 6,100.968 -0.141 765 000 rep 2,100 0.400 -0.398 760.121 -0.917 850 000 rep 625 0.119 -0.925 370.0587 -1.231 * heated at 650C for 14 hours

-743.0 - - -- 2.0 1.0 0 0 0 0 I -j I -1.0 -2.0 0 HEATED FOR 7 HOURS AT 900C oE NOT HEATED -3.0 I I 0 0.4 Q8 1.2 1.6 REP X IO Figure 15. Effect of Pre-Heating putrefactive anaerobe Spores at 900C for Seven Hours Prior to Irradiation.

TABLE XIV EFFECT OF PRE-HEATING P.A. 367c SPORES AT 90'C FOR 7 HOURS PRIOR TO IRRADIATION Heated S mples * Non-Heated Samples Dose Number of Log Number of J-Log % Megarep- Spores Survivors Survivors Spores Survivors Survivors Control 860,000 100.0 2.000 860,000 100.00 2.000 Heated 3.0 hrs. 500,000 58.2 - Heated 4.8 hrs. 540,000 62.8 - - Heated 7.0 hrs. 600,000 69.8 - 340,000 rep 235,000 39.2 1.593 365,000 42.4 1.627 510,000 rep 76,500 12.7 1.104 35,100 4.07 0.610 595,000 rep 29,000 4.84.685 24,500 2.85 0.455 680,000 rep 8,900 1.48.170 5,000 0.582 -0.235 765,000 rep 4,350 0.725 -0.140 950 0.110 -0.959 850,000 rep 1,040 0.173 -0.762 240 0.0245 -1.694 * heated at 900C for 7 hours

-76survivors at the elevated temperatures might be due to increased numbers of germinating sporesO To test this possibility with anaerobic spores is difficult since the requirements for their complete and rapid germination without subsequent outgrowth has yet to be determined. Therefore a strain of an aerobic spore former, whose exact germination requirements has been accurately determined, was selected. For this experiment spores of B. cereus varo terminalis were employed. This organism germinates rapidly without outgrowth in the presence of adenosine and 1-alanine after a heat treatment of 15 minutes at 65~C (Church, 1955)o In this experiment nonheated spores were irradiated for one and three hours which correspond to 180,000 to 540,000 rep respectively. After irradiation, aliquots of irradiated spores along with nonirradiated controls were added to the germination solution and heat shocked for 15 minutes at 65~C. The rate of germination at 30~C was followed by changes in optical density of the spore suspensionso Also uptake of methylene by the various spore samples was determined. The results of this experiment are presented. in Table XV. As can be observed, radiation does not induce the spores to germinate as measured by the above criteriao A difference was noted in the straining characteristics of heat and radiation inactivated spores. Botulinum spores, subjected to steam sterilization (10 minutes at 121~C), if subsequently stained by the Gram method will stain entirely gram positiveo However, no differences were noted in the staining characteristics of the spores which had been subjected to high dosages of radiation; although, if the irradiated spores were placed in a complete growth medium,^ incubated and then stained by the

-77TABLE XV EFFECT OF PRE-IRRADIATION TREATMENT ON GERMIINATION OF B. cereus var. terminalis SPORES Germination Optical Density of Spore Suspensions Time Minutes Control Heated* Irradiated 1 hr. Irradiated 3 hr. Irradiated Plus Heat * Plus Heat No Heat 0.564.564.536.536.536 10.536.360.346.324.492 20.522.320.320.300.480 30.516.320.302.292.478 40 516.306.300.282.474 120.500.29.2.286.276.460 *Heat Treatment 15 min. 65 C Germination Solution: 3.2 mgm adenosine, / 2.67 mgm 1-alamine in;067 M pyrophosphate buffer pH 7.3. Number of Spores as Determined by Llethylene Blue Uptake Spores Staining With Methylene Blue Sample Spores Germinated Spores* After Irradiated 1 hour 100.0% After Irradiated 3 hours 100.0 % 0 After Germination 40 minutes Spores Germinated Spores Not Heated 266 — 3a Heated 7 239 Irradiated 1 hour / Heat 16 225 Irradiated 3 hour Heat"* 11 173 Irradiated + No Heat 291 53 ** Germination Time 20 Minutes * Those Staining (with MB)

-78Gram method, a discrete but irregular gram positive area was observed within the spore. It would seem that drastic heating causes complete polymerization of the cellular components responsible for the Gram stain, whereas radiation is more selective in its action on the sporeso The results of Henry and Stacey (1943, 1954) on the mechanism of the Gram stain is pertinent in this respecto They observed the gram positive staining character of Clostridium welchii is due to a complex formed by protein and nucleic acids in the presence of magnesium and formaldehyde. The Effect of Temperature During Irradiation on the Subsequent Heat Resistance of Bacterial Spores Since a pre-irradiation treatment of bacterial spores makes them more susceptible to heat, one could conclude that a common mechanism within the spore, responsible for its' viability, is acted upon by both forms of energyo Onthe other hand the fact that a preheat treatment of the spores does not alter their subsequent sensitivity to radiation would seem to indicate that the same mechanism is not involved. However. this conclusion may not be valid since both radiation and heat could, affect the same mechanism but the manner in which these two forms of energy are operative might be different. It would seem that if less damage occurs to the spores when irradiation is carried out at an elevated temperature, and if the spores are subsequently heated, any difference in the number of surviving organisms would indicate whether a joint mechanism is responsible for the lethal actiono

-79 An experiment was devised, to test whether irradiation at different temperatures would be reflected in differences in t;he sensitivity of the spores to subsequent heat treatmentso In the first experiment spores of putrefactive anaerobe NCA 5679 were irradiated at 95~ and. 5~C at various dosage levelso After irradiation they were heated at 99.5~C for a period. of one houro A control spore suspension, which received no irradiation treatments, was also heated for 5 hours, the length of time needed to deliver th.e maximum radiation dosage usedo The results of this experiment are presented, in Table XVI and Figures 16 and 17o The results indicate that an irradiation temperature which produces a maximum number of survivors is also a temperature at whbich greater numbers of survivors are found. when the organisms are heated, after irradiation. The differences in the number of spores surviving heat treatment after being irradiated at the two temperatures is not great, but the holding temperature of 99~5~C is a sub-lethal temperature for this organismo This can be observed from the temperature control which indicates no inactivation of nonirradiated spores Previous experiments have demonstrated that in order for the maximum expression of the sensitization phenomenon to occur, heating must be carried out at or above the thermal lethal threshold for that particular organismo The results of this experiment indicate that there is a slight reduction in the number of spores at a high but sublethal temperature at all radiation dosage levels. Furthermore, there is a difference in heat sensitivity when irradiation is carried out at two extremes of temperature. That is those spores which exhibit greater resistance during irradiation at the higher temperatures are also the more resistant to heat after irradiation

-803.0 IRRADIATED PLUS HEATING ^2.0 0<O IRRADIATED AT 95~C PLUS,__ \ \__ I HOUR HEATING AT 99~C 1 IRRADIATED AT 5~C PLUS 1.0 I HOUR HEATING AT 99~C 0-3.0 -- -- -- --- ---— __0 0 -4.0 -5.0.. 0 0.4 0.8 1.2 1.4 1.6 REP X 106 Figure 16. Effect of a Combined Treatment Consisting of Irradiation with Gamma Radiation from Cobalt-60 Followed by Heating for One Hour at 99~C on Survival of putrefactive anaerobe Spores Suspended in M/15 Phosphate Buffer at pH 7.0.

-813.0 2.0 o 1. 0 00. U) 3 - -1.0- - -2.0.... KEY - -- - IRRADIATED 0 AT 95OC -3.0 ____AT 5C___ -4.0 -- 0 0.4 0.8 1.2 1.6 2.0 REP X 106 Figure 17. Effect of Irradiation at 5~ and 95~C on the Survival of putrefactive anaerobe NCA 3679 Spores.

-82TABLE XVI EFFECT OF A COMBINED TREATMENT CONSISTING OF IRRADIATION WITH GAMMA RAYS FROM COBALT-60 FOLLOWED BY HEATING FOR 1 HOUR AT 990C ON THE SURVIVAL OF PA 3679 SPORES SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 Dosage, Spores per Log o rep ml Survivors Survivors la Irradiated at 5~C 0 2,700,000 100. 2.00 370,000 460,000 17.0 1.230 550,000 95,000 3.51 0.545 650,000 52,000 1.18 0.0719 740,000 14,000 0.519 -0.2848 833,000 3,200 0.118 -0.9281 1,015,000 340 0.0126 -1.8996 1,100,000 54 0.002 -2.699 lb Irradiated at 5~C and Heated for 1 Hour at 99~C 0 2,300,000 92.0 1.964 370,000 450,000 19.5 1.29 550,000 14,000 0.61 -0.2147 832,000 700 0.0304 -2.5171 1,000,000 2 ooo00008 -4.0605 2a Irradiated at 95~C* 0 1,200,000 100.0 2.00 370,000 900,000 75.0 1.875 550,000 570,000 47.5 1.676 740,000 140,000 11.7 1.068 832,000 51,000 4.25 0.628 925,000 30,000 2.5 0.398 1,017,000 10,400 0.866 -0.063 1,100,000 3,100 0.258 -0.588 2b Irradiated at 95~C and heated 1 hour at 99~C 0 1,100,000 100.0 2.00 370,000 600,000 54.5 1.7364 550,000 120,000 10.9 1.0374 740,000 8,700 0.791 -0.1018 852,000 1,200 0.109 -0.9626 925,000 260 0.0236 -1.6271 1,000,000 36 0.00328 -2.4840 1,100,000 8 000726 -3.1391 * Control Held at 95~C for 5 hours (the time required for 925,000 rep); 1,200,000 spores per ml remain, which is the same number as was originally present.

-83In a similar experiment Bo subtilis varo niger spores were irradiated at 5 and 65 ~C for various dosage levels and then heated.9 after irradiation at 995O~C, for a total of four minutes. The results of this experiment are given in Table XVII and illustrated in Figure 18o As was observed previously, irradiation of this organism at 65~C resulted in. more spores surviving at this temperature than at 50~Co This temperature is not lethal for this organism. The spores which exhibit the greatest resistance during irradiation also show a greater resistance to subsequent heat treatment after irradiationo In order to more clearly demonstrate that spores irradiatedi at different temperatures also exhibit differences during post-irradiation heating another approach was used. In this series of experiments vials of spores were irradiated for a constant dose at two extremes of temperatures and then a thermal inacti.vation curve was determined for the irradiated organisms o In the first experiment spores of Clo botul-inum 213-B were irradiated at 5~C and -70~C for a total dose of 250,000 and 500,000 rep. The vials of spore suspensions were then heated, in an oil bath maintained, at 100~Co The results of this experiment appear in Tables XVIII and XIX Figures 19 and 20o As can be observed. sensitization of the spores occurred when spores were irradiated at 5~ and -70~0C However, post-irradiation heating of the spores indicated differences in thermal sensitivity; the spores irradiated at -70~C being the more resistant. As previously shown in this study, irradiation of these spores at -70~C results in greater numbers of survivorso The degree of rSensitization at two different

-842.0 - o IRRADIATED AT 65~C 1.0 El IRRADIATED AT 5~C 1.0 \ \ HEATING TIME 4MIN. AT 99.5 C C/) > \ \ Do 0 00 I _ o -1.0 I0 - -- — 10 -2.0 -3.0 0 0.1 0.2 0.3 0.4 DOSE REP X 106 Figure 18. Effect of Post-Irradiation Heating on the Survival of Bacillus sublilis var. niger Spores Irradiated at 5~C and 65 "C.

TABLE XVII EFFECT OF POST-IRRADIATION HEATING AT 990C FOR FOUR MINUTES ON SURVIVAL OF B. subtilis var. niger SPORES IRRADIATED AT 50 AND 650C TEMPERATURE 5~ C 65~ C LOG %= LOG t j DOSE COUNT SURVIVORS SURVIVORS COUNT SURVIVORS SURVIVORS 0 4.2x105 42.0 1.623 4.2x105 42.0 1.623 0.031 1.2x105 12.0 1.079 0.062 2.9x103 2.9 0.462.9x104 19.0 1.279 0.124 2.4x102 0.24 1.380 5.2x103 3.2 0.505 low________-. -.620...._....... 0.186 2.8xl01 0.028 2.447.0xl01 0.7 r.845, ____________________-1..552 _- _155 0.246 -.0xl0 0.05 2.699 -1. 301

-863.0 0 CONTROL 2.0- 0 - 72 C ~ ___O'Z'0 +40C 1.0 Crr 0: 0 HEATING TIME IN MINUTES ~ -J — Ii —.0 —,. -, -2.o -0L, —.> on Survival of Cl. botulinum 213-B Spores. on Survival of C1. botulinum 213-B Spores.

TA3BLE XVIIII EFFECT OF PRE-IRRADIATION AT 50 AND -70~C 7ITH SUBSEQUENT HEATING AT 100~C ON SURVIVAL OF C1. bot ulinumr 213B Control Minutes Spores at Per % Log % 100~C ml Survivors Survivors 0 3,600,000 100.00 2.000 20 500,000 13.9 1.143 30 99,000 2.74 0.443 50.2 9,300 0.258 -0.588 60.1 4,500 0.125 -0.903 Irradiated -70~ C* 0 (Cont IrradJ 3,300,000 100.00 2.000 5.3 1,270,000 38.5 1.586 10.0 680,000 20.6 1.314 15.0 250,000 7.58 0.880 20.0 57,000 1.78 0.241 25.2 10,400 0.316 -0.500 30.0 1,300.0394 -1.405 Irradiated 5~C* 0 2,500,000 100.00 2.000 5.3 950,000 38.0 1.580 10.0 390,000 15.6 1.1931 15.0 114,000 4.56 0.659 20.0 7,100 0.284 -0.5467 25.2 870.0348 -1.458 30.0 550.022 -1.658 40.0 270.0108 -1.967 * Irradiated for 250,000 Rep.

-883.0 213 B 2.0- 0 CONTROL 0 5~C - -700C 1.0 0:: 0 Wr 0 0 — 0 - 2.0 --- ------------ -3.0 -4.0 -------- 0 20 40 60 80 TIME IN MINUTES AT 100~C Figure 20. Effect of Pre-Irradiation at 5~C and -70~C with Subsequent Heating on Survival of C1. botulinrun 213-B Spores.

-89TABLE XIX EFFECT OF PRE-IRRADIATION AT 50 AND -72~ C WITH SUBSEQUENT HEATING ON SURVIVAL OF C1. botulinum 213-K3 SPORES Control Not Irradiated Heating Time Spores in Per % Log % Min. ml Survivors Survivors 0 2,800,000 100.0 2.000 10 840,000 30.0 1.447 20 310,000 11.1 1.045 31 89,000 3.17 0.5011 41.5 35,000 1.25 0.0969 50.0 10,000 0.358 -0.4461 60.0 3,500 0.125 -0.9131 70.0 1,700 0.0607 -1.2168 Irradiated at -70~ C 0 (Irrad.) 1,100,000 100.00 5. 830,000 75.2 1.896 10 190,000 17.3 1.238 15 19,000 1.73 0.738 20 1,200 0.109 -0.963 Irradiated at / 5~ C* 0 (Irrad.) 460,000 100.00 2.000 5.2 66,000 14.3 1.553 10.0 4,200 9.15 0.961 15 140 0.030 -1.517 20 18 0.00391 -2.408 30 6 0.0013 -2.886 *500,000 Rep.

-90temperatures when spores are irradiated with 250,000 rep is not greato However, it has been previously reported that the sensitization phenomenon is not significantly manifested until a total dosage of 300,000 rep is delivered to the spores (Kempe, 1955). As can be observed in the radiation survival curve for Clo botulinum spores, significant radiation inactivation does not occur until the spores have received approximately 300,000 repo The results of another experiment with the same organism are presented in Table XX and Figure 21o In this case the irradiation was carried out at 10~ and 90~Co As is apparent from the results, irradiation at 90~C results in greater thermal inactivation than if the spores are irradiated at 10~C. Spores of this organism are the most sensitive when irradiation is carried out at 90~Co In still another experiment, spores of Clo botulinum 62-A were irradiated at -70~ and 50C for a total dosage of 500,000 rep before being heated at 100~Co The results shown in Table XXIand Figure 22 indicate that there is a 10-fold difference in survivors to subsequent heating at 100~C when irradiation is carried out at these two different temperatures The spores irradiated at -700C were the more resistant to heato The results of an experiment in which putrefactive anaerobe spores were treated as described above for Clo botulinum spores are presented in Table XXII and Figure 235 As noted previously, the spores of this organism are many times more resistant to heat than C1. botulinum strainso This is adequately demonstrated in the temperature control for this experimento Heating for three hours had no apparent effect on the viability of these spores. A difference in their heat sensitivity is again demonstrated when

-913.0 - KEY oE NOT IRRADIATED CONT. 2.0 0 IRRADIATED AT 10~C 0 IRRADIATED AT 900C 1.0 e0 -3.0 o -I.0 -2.0 -3.0 -4.0 -- 0 20 40 60 80 TIME IN MINUTES Figure 21. Effect of Irradiation at 10~C and 90~C with Post-Irradiation Heating on 100 ~C on Survival of C1. botulinum 213-B Spores.

TABLE XX EFFECT OF IRRADIATION AT 100~C AND 900~C WITH POST-IRRADIATION HEATING AT 100oO~C ON SURVIVAL OF CLo BOTULINUM 213-B SPORES Control Time (Minutes) Spores per ml Survivors Survivors 0 2,200,000 100 0 2.000 10 1,100,000 50o0 1o699 20 460,000 20o9 1.320 30 85,000 3 86 0,5866 40 22,000 100 0. 000 50 4,400 0,200 -o0699 Irradiated at 10~C 0 230,000 100.0 2 000 10 160,000 69,5 1,842 20 99700 4a21 Oo624 30 1,6oo00 0695 -0o158 40 360 0o156 -0o807 50 30 0 0130 -1l886 Irradiated at 900C 0 600,000 100 o 2000 10 61,oo000 102 1,0086 20 3,200 0,533 -0o273 30 750 0o125 -0o903 40 160 0.0267 -10574 50 13 0,00216 -2.666 -92

-933.0 - 2.0 1.0 C,, 0 0 L I 0 -J -I.0 -- CONTROL ____ __^__-2.0_ 0 IRRADIATED AT-70~C 0 IRRADIATED AT 5~C -3.0 0 20 40 60 80 TIME IN MINUTES AT O10~C Figure 22. Effect of Temperature During Irradiation on the Subsequent Heat Resistance of C1. botulinum 62-A Spores to Heating at 100uC.

-94TABLE XXI EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SUBSEQUENT RESISTANCE OF C. BOTULINUM 62A SPORES TO HEATING AT 100~C 0 0 Pp 0) 0 t3H 0'QH 0J3 *H~-t P4 0 P3 *p CO CO CQ CO CO CO A) (500,000 rep at -70~C) 0 370,000 100.0 2.00 90,000 100.0 2.00 5 58,000 64.5 1.4416 10 220,000 59.5 1.774 16,000 17.7 1.150 15 150,000 40.5 1.6o8 7,700 8.55 o.7161 20 110,000 29.8 1.474 290 0.322 -0.4939 30 26,000 7.03 0.847 40 9,900 2.68 0.428 50 3,000 o.811 -0.091 6o 1,100 0.298 -0.526 B) (500,000 rep at +50C) 0 86,000 100.0 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 0.065 20 5.5 o.o4 -2.

-953.0 3.0. --— Q —-..2.0 1.0 T I cr) 0 0 ~0-~~ ~\ 0 -1.0 0 CONTROL NOT IRRADIATED -2.0 E IRRADIATED AT -70~C 0 IRRADIATED AT +50C -3.0 -o- I I I 0 40 80 120 160 200 TIME IN MINUTES AT 100~C Figure 23. Effect of Temperature During Irradiation on the Subsequent Heat Resistance of putrefactive anaerobe Spores to Heating at 100 C.

-96TABLE XXII EFFECT OF' TEMPERATURE - T!i S UBS U; NTT HEAT R-SISTANCE OF P.A. 3679 SPORES TO T ATIN5 AT 100C C Heating Time Spores Control in Per Spores Log % Min. ml Per ml Survivors Control 34,000 2.00 2.000 51 110,000 303 2.481 90 77,000 226 2.354 136 77,000 226 2.354 170 65,000 190 2.788 Irradiated at -70~ C Control 34,000 Control Irrad. 10,000 100.0 2.000 21 7,300 73.0 1.863 51 6,700 67.0 1.826 70 6,100 61.0 1.785 136 2,200 22.0 1.342 170 2,100 21.0 1.322 Irradiated at 5~ C Control 34,000 Control Irrad. 5,300 100.0 2.000 21 1,800 33.9 1.530 51 1,900 35.8 1.553 70 1,800 33.9 1.530 90 750 1.41 0.149 137 110 0.208 -0.06819 Irradiated for 500,000 rep.

-97the spores are irradiated at two different temperatureso With these spores slight thermal inactivation occurs at 100~C using spores irradiated at -70 ~C Furthermore these results also demonstrate that the spores are slightly heat sensitized by irradiationo Spores irradiated at 5~C and heated for short periods of time show an equivalent degree of resistance at 100~C as do those irradiated at -70~Co However, after heating the spores for two hours, those irradiated at 5~C become more sensitive to heato It would seem that an irradiation treatment, followed by heating of sufficient duration, results in loss of thermal resistance~ The results of the previously outlined experiments all indicate that irradiation at different temperatures results in differences in the subsequent heat resistance of the sporeso This is supporting evidence for the observation, previously reported in this thesis, that spores vary in their resistance to radiation when irradiation is carried out at different temperatures since variation in thermal resistance of these spores also occurs after such treatmento

DISCUSSION Several observations can be made from the results of this investigationo Depending on the ambient temperature during irradiation, the bacterial spores used in this study exhibit a varying degree of resistance to gamma radiationo The degree of resistance is slightly greater when irradiation is carried out at -70~C as compared to the nonfrozen condition at 40Co Furthermore, there is a trend towards radioresistance as the ambient irradiation temperature is increased above room temperature. The maximum degree of resistance to radiation occurs at higher temperatures and is greater just before thermal inactivation of the spores becomes significanto A second observation indicates that a pre-irradiation treatment of the spores, at a dose which is sporocidal for large numbers of the spores, does not significantly lower the temperature threshold necessary for thermal inactivation. These results indicate that radio-sensitization of the bacterial spores is induced by gamma radiation but in order for the phenomenon to be significantly expressed the spores must be heated at their thermal, lethal thresholdo A third observation is that spores irradiated at different temperatures also exhibit differences in thermal resistance Those spores which exhibit the greater resistance to radiation at various temperatures are also the more resistant to heato These results suggest that a common mechanism is responsible for spore survival and radio-sensitization and that this mechanism is acted upon by both forms of energy. An interpretation of the results of the simultaneous effect of temperature and radiation on bacterial spores observed in this study is -98

-99difficult under the proposed mechanisms which have been advanced for the biological effects of radiationo The direct action model assumes no effect of temperature during irradiation. A proposed explanation for a temperature effect during irradiation by this theory has been on the recovery of the cell from the original lesion produced by the radiation treatmento In case of bacterial spores, such an explanation is not plausible since the spores can be considered to be metabolically inerto However, the possibility that temperature may in some way alter the target sites within the cell and thus change their response to radiation can not be ruled outo Also thermal agitation of intracellular molecules could affect the transfer of radiation energy within the cell. A more adequate interpretation of an observed temperature dependency during irradiation on bacterial spores can be made by the indirect and diffusion models. The relatively greater resistance of bacterial spores irradiated at -700C can be interpreted as resulting from a decrease in the hydrogen peroxide concentration, and indirectly to radicals which lead to its production, as has been observed in pure water systems. Similarly, the rate of diffusion of toxic substances, produced by radiation, to the sensitive sites within the cell would be retarded by the frozen condition, However, an explanation of the increase in radiation resistance of the spores at elevated temperatures, as observed in this study, is not possible by the above modelo As pointed out in a previous section, an increase in temperature results in decreased hydrogen peroxide formation during irradiation o If one can project the observations made in pure water systems to

-100the one under consideration, the number of spores surviving radiation exposure at elevated temperatures should thus be less than that observed at lower irradiation temperatureso However, an opposite trend has been observed in this investigation with bacterial sporeso If the temperature during irradiation were acting on an extrinsic mechanism, then the apparent increase in radiation resistance observed with the yarious species of spores would be uniform at the same irradiation temperatureo Then the degree of resistance of the particular organism under study would solely be dependent on its inherent resistanceo The possibility exists that free radicals and peroxides can be produced in molecules present in the cell other than the water moleculeso The detection and importance of these moieties has yet to be ascertained. As the temperature during irradiation is increased one could assume that the decay of these radicals and peroxides would be hastened thus decreasing their effectiveness in causing death of the cello However, any temperature dependency during irradiation on cellular death would still be at the same temperature and would not vary with the organism being studiedo The results of this investigation do not permit identificati.on.: of the model by which radiation energy is dissipated to the target sites within the cello Results of other investigations have indicated that the hydrogen peroxide produced may be the basis upon which gamma radiation exerts its lethal effecto These results show that if catalase, an enzyme capable of decomposing hydrogen peroxide rapidly, is present during irradiation, the lethality of gamma radiations for anaerobic spores is significantly reduced (Williams and Kempe, 1959)~ Also the presence of other

-101compounds during irradiation which may affect the concentration of hydrogen peroxide has been shown to alter the sensitivity of bacterial spores to ionizing radiation (Proctor et al, 1955, 1958; Walls, 1959) A discrepancy is noted in certain published results in that an oxygen effect during irradiation in the absence of moisture has been reported for aerobic spores (Tallentire, 1958)o The investigation by Houtermans (1956) showed a greater sensitivity of Bo subtilis spores to radiation when irradiated at room temperature in the dehydrated condition as compared to wet spores irradiated at the same temperature. It appears that oxygen must have an additional role in inducing radiation sensitivity in certain bacterial spores other than that of increasing hydrogen peroxide formation in water. An alternate approach to that of an effect of temperature on extrinsic factors which may modify the lethal effects of radiation is the involvement of an intrinsic mechanism of the cello A variation in temperature could act in some way on. this mechanism which then would be reflected in a varied sensitivity of the spore to the lethal effects of radiationo The question to be resolved is the nature of the factor within the spore and the manner by which it is affected by changes of temperature in order to vary the resistance of the spore to radiationo In order to arrive at an adequate explanation for the results delineated in this study several aspects of spore physiology in conjunction with radiation induced mechanisms must be consideredo These include the phenomena of bacterial spore dormancy' factors responsible for spore resistance and certain aspects of protein denaturationo Each of the previously named fields has been the object of extensive investigationo A great deal

-102of experimentation will be needed before the involved mechanisms will be elucidated. Nevertheless, certain conjectures can be made at this time in the light of the present knowledge. Therefore, the object of this discussion is to consider certain aspects of each topic which would be of importance in arriving at an interpretation of the results of this study and the pertinent results which have appeared in the literature. Certain factors concerning the heat activation of dormant spores were considered in the section on experimental results. A ramification of dormancy demonstrated with thermal sterilization was the observation that severly heated spores of C1. botulinum present in food substrates would only grow out after prolonged periods of incubation (Burke, 1919, 1923). This phenomenon is probably more in the nature of a recovery from injury rather than delayed germination of the spores. The slow recovery of the spore may come about by the retarded replacement of protein molecules within thecell'lrthe undamaged synthetic mechanisms. Evidence for this is that severly heated spores when added to media which normally support growth of untreated spores need enrichment factors in order for growth to occur without prolonged periods of incubation (Curran and Evans, 1937). It would seem that the presence of all the necessary growth factors in the culture medium would simplify the resynthesis of essential cellular components necessary for the outgrowth of the damaged spore. This would then shorten the incubation time for visible growth to occur. Also incubation at lower temperatures, which is more conducive to synthetic processes, will shorten the time of incubation and increase the outgrowth of drastically heated spores (Williams and Reed, 1942). In this connection it has been demanstrated that a nonspecific factor, soluble starch, added to the recovery

-103medium will also increase the number of survivors following heat treatment (Foster and Wynne, 1948; Olson and Scott, 1946)o However, the exact function of this substance is not knowno Whether radiation-inactivated spores can recover from the damage has not been adequately demonstrated. In any study on the effect of deleterious influences on bacterial spores consideration must be given to the resistance mechanlsmo Although of practical significance in the evaluation of sterilization processes, the basic factors of this mechanism, have not been elucidated~ As evident from the early work on thermal inactivation of bacterial spores, the magnitude of the temperature coefficient of death adds support to the theory that organisms subjected to moist heat are killed by the denaturation of proteino Since protein denaturation occurs most readily in the presence of moisture, and since bacterial spores are capable of withstanding long periods of desiccation, attention was drawn to the relative amounts of water in spores as compared to the vegetative cells as an attempt to explain the resistance mechanismo No doubt the thick wall which encompasses the spore and which distinguishes it morphologically from the vegetative cell was also thought to act as a perm-eabilnity barrier for the water molecules However, early studies on the relative amount of water present in spores and vegetative cells showed no quantative differences in the water content of the two types of cells (Henry and Friedman 19357) Therefore, the concept was advanced that if the total amount of water was the same in the vegetative cells and spores of bacteria^ it must be present in some inert form and not be available for chemical reactions This l.ed to the concept of bound water as an explanation for the extreme resistance of bacterial spores to deleterious agents (Friedman and Henry, 1.936) >

o-104The concept of bound water as a mechanism of resistance is still controversial. One difficulty arises in the accurate determination of the water content of the cell. Recent investigations are contradictory on the absolute amounts of water present in the bacterial spores. The results obtained by Waldhalm and Halvorson (1954), using differences in vapor pressures, indicate that the water content of spores and cells is similar. By another technique, namely differences in the refractive index of spores and cells, the water content of several aerobic spores was observed to be much less than that of the vegetative cells (Ross and Billing, 1957). Another approach has been followed in order to obtain an understanding of the resistance mechanism of the bacterial spore to deleterious influenceso This has been to determine if chemical differences exists which are unique to the spore and not the vegetative cell and which would contribute to its characteristic resistance. However, prior to 19535 it was generally recognized that with a few notable exceptions the chemical constituents of the spores were similar to those of the vegetative cello The most significant exception recognized was in the differences in the inorganic chemical composition of the cells. Curran et alo (1943) observed, by means of spectral analysis of ashed vegetative cells and spores of 14 species of bacteria, a greater amount of calcium in the sporeso The evidence that calcium and another divalent cation, manganese, are involved in the thermostability of Clo botulinum, Bo megatherium and B. coagulans var. thermoacidurans spores is demonstrated by the fact that spores produced in media with sub optimal concentrations of these ions also demonstrated decreased thermal resistance (Sugiyama, 1951; Grelet, 1952; Amaha and Ordal, 1957)o

-105A significant finding in spore physiology was the discovery of dipicolinic acid (DPA) in spores of Bo megatherium by Powell in 19530 This is the first demonstration of a substance unique to the sporeo Subsequent investigations have demonstrated this compound to be present in. all the spores tested but not in the vegetative cellso The fact that dipicolinic acid is involved in spore resistance also seems to be established. During germination, and at a time when heat resistance is lost, there is a release of DPA (Powell, 19435 Woese, 1.958) Also during sporulation of Clostridium roseum an increase in heat resistance is correlated with the synthesis of DPA (Halvorson, 1957) Furthermore, there is evidence that DPA is related in some way to the calcium content of the spore. Powell (1953) originally observed that dipicolinic acid is released as the calcium salt during germinationo However, the exact relation of calcium and DPA needs to be elucidatedo Perry and Foster (1955) observed that DPA and calcium are not present in equivalent amounts in Bo cereuso The mechanism by which the divalent cations, calcium and manganese, increase the resistance of the spore needs to be ascertainedo A suggestion has been made that divalent cations may be capable of combining with proteins in a manner which would impart new strength to their intra-molecular linkage (Sugiyama, 1951) Similarily the mechanism of DPA involvement in spore resistance is unknowno Suggestions have been advanced that it may act as a chelating agent (Harrel, 1957;, Powell, 1957). The relationship to radiation resistance of the factors which alter the thermalstability of bacterial spores has not been establishedo There is a suggestion that ultra-violet resistance preceeds heat resistance

-106which, in turn, preceeds DPA synthesis (Romey and Wyss, 1957). Similarily, it has also been observed that the resistance to gamma radiation by spores of B, cereus var. terminalis precedes heat resistance (Black et al,, 1960), Halvorson (1957) observed that heat resistance preceeded DPA synthesis in Cl. roseum, Also, a certain amount of correlation exists between DPA content and radiation resistance of certain aerobic spores (Woese, 1959). Those spores which exhibit greater resistance to X-radiation have a slightly higher DPA content. An explanation of the exponential order of death of many organisms is difficult without assuming the existence of an essential molecule or, at best, a few molecules which are responsible for cellular deatho The hypothesis has been advanced that the thermal death of a cell is due to a lethal gene mutation (Rahn, 1945), This same concept has been advanced for radiation induced cellular death by Lea (1946)o The validity of the latter hypothesis is based on the observation that bacteria, when exposed to radiation, are still capable of growing, as evidenced by the long filamentous forms present in the growth medium. This would indicate that the cells have lost their capability of reproduction (Lea, 1936), Protein denaturation is any irreversible, non-proteolytic modification of the unique structure of a native protein giving rise to definite changes in its chemical and physical properties, The most commonly observed change in proteins, which have undergone denaturation, is the reduction in solubility which is manifested by the appearance of a visible coagulumn However, other changes can occur such as changes in viscosity, unmasking of sulphydryl groups, etc. These changes can be detected only by special means. In bacteria, denaturation of essential cellular

-107components results in death. Depending on the extent of heat treatment, different degrees of damage can occur because of the differences in the types of cellular protein which have different temperature inactivation coefficients. A theory of protein denaturation has been advanced by Mirsky and Pauling (1936) which is based on their conception of the structure of the protein molecule. Their conception of a protein molecule is one (or more) polypeptide chain which is held in a unique position by means of hydrogen bonds between two electronegative atoms. As a result of an increase in temperature or attack by denaturing agents such as urea, acids, etc., the side chain bonds are broken leaving the protein molecule to assume any of a large number of configurations. If a large number of bonds are broken then the integrity of the molecule is lost and hence, denaturated. Since the energy of activation of the protein denaturation process is equal to 150,000 cal/mole and the strength of the hydrogen bond is equal to 5, - 8,000 cal/mole, Mirsky and Pauling proposed that approximately 30 hydrogen bonds must be ruptured before the molecule loses its native state. The basis of their theory is the high entropy of activation needed for the denaturation process which indicates hydrogen bond disruption, and the fact that compounds which are specific in attacking hydrogen bonds are also responsible for the denaturation of proteins. However, these authors do not imply that hydrogen bond breakage is the sole mechanism of protein denaturation. In any discussion of lethal gene mutation, consideration must be given to the mechanism of hereditary transfer by the cell. It has been well established that hereditary transfer in cells occurs through chromosomes which are composed of nucleoproteins. Furthermore, the evidence is

-108quite conclusive that the hereditary mechanism in bacteria is the deoxyribonucleic acid (DNA) component of the nucleoproteino The evidence for this is the transformation of certain bacterial cells by purified preparations of deoxyribonucleic acid. However the exact nature of the gene is not knowno Since protein denaturation is based on molecular structure, attention must be directed to the structure of deoxyribonucleic acid for an understanding of the mechanism of denaturation of this compoundo On the basis of available information Watson and Crick (1953 ab) proposed a model for the structure of deoxyribonucleic acido The basis of the model is a double strand helix consisting of a phosphate ester linked backbone of pentose molecules with the purine and pyrimidine bases guanine, cytosine, thymine and adenine projecting inward. These.' bases are linked together with hydrogen bonsin the ratio of ll.o The hydrogen bonds link the -Ni=C6(NH2)- groups of adenine or cytosine on one of the chains with the -N(1)H-C60- group of thymine or guanine respectively in the other chaino Guanine and cytosine are linked with three hydrogen bonds while thymine and adenine have only twoo Heat denaturation of DNA solutions can occur by the splitting of the hydrogen bonds holding the two helices together (Thomas, 1954) (ICavalieri, Rosenberg, 1957), the guanine and cytosine bond system being more resistant since three hydrogen bonds are involved (Cox and Peacocke, 1956a, 1956b)o In-',this investigation, with DNA from herring sperm, it was observed that the hydrogen bonds were not broken permanently until a temperature of 75~C was reachedo Heating the solutions for as long as one hour at temperatures up to 75~C did not cleave the bonds for a long

-109enough time so that they could not healo However, above this temperature a critical number were broken irreversibly and denaturation of the substance occurred. A unique characteristic of DNA obtained from a wide variety of sources shows that they must be heated to a specific temperature before any appreciable denaturation occurso This specific temperature varies with the source of DNA and seems to be a reflection of the structure of this substance (Geiduschek, 1958). The effect of gamma radiation on sodium deoxyribonucleate can result in cleavage of the inter-nucleotide-phosphate-ester bonds and the cross linking hydrogen. bonds (Cox et alo. 1955)o However, the number of phospho-ester bonds which are broken along the main chain. are proportionally smallo Preferential breakage of the bonds between the adenine and thymine occurs with gamma radiation from cobalt indicating that the denaturation of DNA by radiation is a non-random process (Cox and Peacocke, 1957). It has been observed that pre-irradiation with 9,000 rep followed by a heat treatment at 100~C markedly changes the sedimentation coefficient of the treated solutions (Shooter et alo, 1956)o However, it was found necessary to heat irradiated DNA solutions at a high temperature before appreciable inactivation would occur (Butler, 1956)o Another concept which must be considered in any study of the effect of temperature on biological systems is the activation hypothesiso This hypothesis was proposed by Erying and Laidler (1940) to account for the high energies required to initiate a wide variety of chemical reactionso They proposed that the reactants needed to overcome an energy barrier or "hump" prior to going to completion The individual reactants are considered to be in an activated state when present in this "hump"o The

-110energy needed to initiate the reaction is localized in this activated complex of reactants. The exact fate of the energy within the activated complex is not known. However, the energy can go into the formation of new intermolecular bonds, healing of ruptured bonds, or in strengthening existing bonds of the reacting molecules (Sizer, 1943; Glasstone et al., 1941). Because of the very transient existence of the activated complex, direct experimental evidence for its existence is meager. The detection of new bonds formed during the decomposition of ethylidene diacetate (and esters of the general formula R-CH(0~CO~R)2) has been demonstrated (Glasstone, Laidler and Eyring, 1941). Because of the high temperature coefficient of most biological reactions such as heat denaturation, enzyme catalyzed reactions, etc, the activation hypothesis has found wide application in biology. The evidence for this theory toawide variety biological reactions has been complied into a review by Johnson, Eyring and Polissar (1954). These authors consider heat activation of dormant spores as an example to which the activation hypothesis can be appliedo On the basis of seemingly unrelated observations concerning spore physiology, protein denaturation and the activation hypothesis, an explanation can be advanced for the results in which a temperature dependency has been observed during irradiation of bacterial spores. As the temperature during irradiation is increased, the molecules within the spore are placed in an activated state. The time-temperature relationship for the maximum expression of the excited state will depend, on the

-Illindividual molecules involved in the spore and will vary with. the organismso Therefore, a different response would be expected at various irradiation temperatures with different organisms. This is reflected in the results of this study in that Clo botulinum and putrefactive anaerobe spores exhibit maximal resistance to radiation at different temperatures during radiationo The putrefactive anaerobe spores are more heat resistant and more resistant to radiation at higher irradiation temperatures than the Clo botulinum sporeso A similar phenomenon has been observed during irradiation with maize seedso Dried maize seeds exposed to X-radiation in the temperature range of -187~ to 66~C exhibit the greatest radiation resistance at 50~ to 60~C (Kempton, 194l1) This temperature is just below the lethal thermal threshold for these seedso A further increase in irradiation temperature results in greater sensitivity of the seeds as measured by subsequent seedling heighto These results would. indicate a similarity of temperature dependency during irradiation which is not limited to bacterial sporeso The idea that bond breakage is involved, in the lethal action of radiation is obtained by inferenceo The energy of a photon from radioactive cobalt when absorbed within the cell is sufficiently high to rupture any of the various types of chemical bonds present in the cello The results of Curran and Evans (1938) on the photosensitization of bacterial spores to heat by ultra-violet radiation suggests that bond rupture is involvedo These investigators observed that it was necessary to irradiate the spores for a sufficiently long time at 2537A0 for the sensitization to be adequately expressedo Irradiation of the spores at 300 - L600A

-112had the greatest effect for heat sensitizing the spores. Also Giese and Crossman (1945) observed that irradiation at 2383A0 was most effective in inducing photo-sensitization in Paramecium. The amount of energy corresponding to a wavelength of 2537A~ is 112 k cal/mole. This amount of energy, if completely absorbed, is just below the threshold needed for irreversible denaturation to occur. Also, most stable atoms and molecules have ionization potentials in the shorter ultra-violet region (below 1000A0). Therefore, if bond breakage is involved in the process, one would not expect that irradiation at 2537A0 or at longer wavelengths to be as effective as higher energy wavelengths in inducing sensitization. The suggestion has been made that the DNA component of nucleoprotein is involved in spore resistance and hence in survival. Furthermore the hypothesis was advanced that both thermal and radiation energy are operative on the same factor within the spore. The results of this study in which the irradiated spores were heated at various temperatures after irradiation resulted in a curve, Figure 12, which is similar to the published results obtained with DNA solutions treated in a similar manner (Butler, 1956). In both cases it was found necessary for heating to be at a sufficiently high temperature in order for inactivation to occur even after the radiation treatmento With spores of Clo botulinum and putrefactive anaerobe this temperature varied. Putrefactive anaerobe spores, which are more heat resistant than botulinum spores needed to be heated at higher temperatures before sensitization was demonstratedo With purified protein solutions, a pre-irradiation treatment with ultra-violet light results in a lowering of the coagulation temperature of the proteins (Stedman and Mendel, 1926). The sensitization of purified proteins to heat by ionizing radiation has not been investigated

-13thoroughly. It was reported that the coagulation temperature of human serum albumen in lowered by irradiation with high speed electrons (Kan et al,, 1957)o In the case in which the coagulation temperature of the protein is lowered, it would be anticipated that the kinetics of the thermal inactivation of the irradiated protein would result in a curve more typical of an Arrhenius ploto This type of curve is different from what has been observed here with pre-irradiated, spores, heated at various sub-lethal temperatureso This can be interpreted on the basis of a difference in the molecular structure of the component being acted upono Several lines of experimentation indicated that the repro.ductive mechanism, hence the nucleoprotein, of the cell is involved in. radiosensitizationo Duggar and Anderson (1939) concluded that nucleoprotein must be involved in the photosensitization of yeast cells since the organisms were incapable of producing visible colonies, but still retained their respiratory capacities. Giese and Crossman (1945) observed that Paramecium recovered. more slowly from the damage when irradiated at a wavelength of 2650A~. They arrived at this conclusion since absorption by nucleoprotein occurs at this wavelengtho It has been recognized for sometime that radiation inhibits the ability of bacterial spores to form colonies but not their ability to germinate (Lea, 1946) o The fact that bacterial spores are capable of germinating even after an irradiation treatment which prevents colony formation indicates that the reproductive function and the mechanism of germination are separate, It can be assumed that other protein components of the cell are capable of being heat sensitized by irradiationo However it appears that this does not impair their

-114subsequent function since the spores are still capable of germinating. However, injury to the germinating mechanism within the spore could be capable of repair or replacement. The irreversible effect of radiaion, which manifests itself in lethality of the spore, is confined within the mechanism which is responsible for the outgrowth and reproduction of the spore. Although photosensitization of other cellular proteins can conceiveably occur, this does not adversely affect certain metabolic functions of the cell. The phenomenon of dormancy, as separate from that induced by heat injury, is related to spore resistance in that those spores exhibiting greater thermal resistance must be heated for a greater length of time before dormancy is broken (Desrosier and Heiligman, 1952)o Furthermore, the spores must be activated before germination can be initiatedo As demonstrated in this study, a pre-irradiation or heat treatment of bacterial spores at sub-lethal temperatures does not cause them to germinate as measured by loss in heat resistance and staining. It is conceivable that radiations of lower energy thresholds, which, when absorbed, would not inactivate the spore, might be substituted for heat in breaking dormancyo The observation reported in the present study showed that heating botulinum and putrefactive anaerobe spores for prolonged periods prior to irradiation did not alter their resistance to radiation, demonstrates that heat alone, without the presence of a substrate, cannot break dormancy. It was anticipated that such a prolonged heat treatment would induce the spores to germinate or would alter them in some way that would decrease their resistance to radiationo It can be inferred from the results of these experiments that heat must be applied simultaneously with radiabtion

-115in order for the observed, temperature effect during irradiation to be demonstrated. If calcium and dipicolinic acid are acting as bonding or chelating agents within the spore, the bonding must be quite tenacious to withstand such drastic heat treatmento As mentioned previously, DPA and calcium have been implicated in the thermal resistance of bacterial sporeso However, the site of action within the spore has not been postulatedo The structure of dipicolinic acid would make it easy for it to function as a bonding agent via hydrogen bonds. Conceivably, DPA could, act as a bridge between the bases of DNA and in this manner increase the stability of the molecule This linkage could be formed with or without the participation of calciumo An observation made with another cell system has implicated the importance of calcium in mutagenesiso Stephansen (1956) observed an increase in the number of chromosomal aberrations produced by radiation in Tradescantia microspores grown in calcium deficient media~ Although divalent cations seem to be associated with nucleoproteins, the function of these substances is not known (Williamson and Grelek, 1944) o The fact that pre-heating does not alter the sensitivity of the spores to radiation can be interpreted on the basis of the differences in the manner of absorption of the two forms of energyo Application of heat will result in the random. absorption of the thermal energy within all the molecular bonds within the sporeo Breakage of molecular bonds will occur once the threshold of energy for a particular bond is attained. In the case of ionizing radiations the absorption of the incident photons must be localized within the sensitive area of the spore. After absorption of a photon irreversible damage occurs to the molecules which are

-116responsible for maintaining the integrity of the cello However, the absorption of insufficient photons within the critical areas results in only partial bond breakage without inducing lethality. Since the theory of protein denaturation, stated in this discussion, maintains that a critical number of bonds must be ruptured before the integrity of the molecule is lost, a pre-irradiation treatment may occasion a decrease in the number of bonds which must be subsequently inactivated by a heat treatment in order to cause denaturation. This view is in accord with the observation that irradiated spores have a lower susceptibility to heato The reverse, a preheat treatment, would not cause the spores to be more sensitive to radiation under the above considerations since irreversible breakage of the molecular bonds occur once the thermal threshold energy is attained.

SUMMARY The object of this investigation has been to study the role of temperature during irradiation on the subsequent survival of bacterial spores in order to arrive at an explanation for some of the conflicting results which have appeared in the literature. The phenomenon of photosensitization of proteins by electromagnetic radiations was also consideredo Previously published work offers no satisfactory explanation for the diverse ways that temperature can affect the lethal effects of ionizing radiationo From the results delineated in this study however, several clarifying observations can be made. Thus, anaerobic bacterial spores, when exposed to gamma radiation in the temperature range of -70 to 95~0C exhibited a varying degree of response to irradiation as determined by their colony forming ability: the spores were slightly more resistant to radiation at -70~ than at 4~C, and the maximum number of spores surviving radiation exposure occurred at a temperature just below the thermal lethal threshold for the particular organism under investigationo The thermal lethal threshold for Clo botulinum spores was found to be approximately 85~C. A further increase above this temperature resulted in rapid inactivation of the spores by radiationo In the case of putrefactive anaerobic spores, the greatest number of survivors were observed in the temperature range of 900 to 1000~C with progressively greater numbers of surv rs being obtained.s the temperature was increased above room temperature. Although radiation increases the sensitivity of bacterial spores to heat, the thermal lethal temperature is not lowered by this treatment. -117

-118This is contrary to the published results which show that the coagulation temperature of purified proteins is progressively lowered by exposure to ultra-violet radiationo The spores which exhibit greatest resistance at the various temperatures during irradiation, also exhibit greater thermal resistance. In the protocol of this investigation, all spores were heat treated in order to break dormancyo The heating time and temperatures employed were considered to be maximal for subsequent germination of the organisms usedo This would rule out the possibility that the apparent increase in survivors at the elevated temperatures was caused by heat activation of any dormant spores present in the spores suspensionso Furthermore, it was observed that gamma radiation from cobalt-60 was not capable of inducing the spores of an aerobic species to germinateo On the basis of the observations obtained in this study and pertinent evidence in the literature, it is postulated that the lethal effects of heat and radiation, as well as the radio-sensitization phenomenon are operative on a joint mechanism within the sporeo It is suggested that the key mechanism is the reproductive mechanism and that nucleoprotein, or more specifically the deoxyribonucleic acid component of nucleoprotein, is critical in this regard. Several practical aspects concerning the application of heat and radiation for sterilization purposes can be delineated from the results of this investigation viz.: an accurate determination of the sterility dose must consider the temperature at which irradiation is carried outo if the sensitization of bacterial spores to heat by radiation is to be utilized, the irradiation must be conducted at the thermal, lethal

-119 threshold of the organism under consideration: the most thermally resistant organism must be used in the evaluation of this method of sterilization: for a combined process where radiation and heat is to be utilized, a preirradiation treatment, followed by post-irradiation heating at a lethal temperature, must be employed. to take full advantage of the sensitization of bacterial spores to heato

BIBLIOGRAPHY 1. Adams, W. R. and Pollard, E. "Combined Thermal and Primary Ionization Effects on a Bacterial Virus." Arch. Biochem. Biophys., 36, (1952), 311-322. 2. Allen, A. 0. "The Yields of Free H and OH in the Irradiation of Water." Radiation Research, 1, (1954), 85-96. 3. Amaha, M. and Ordal, Z. J. "Effect of Divalent Cations in the Sporulation Medium on the Thermal Death Rate of Bacillus coagulans var. thermoacidurans." J. Bacteriol, 74, (1957), 596-604. 4. Anderson, A. A. "A Rapid Plate Method for Counting Spores of C1. Botulinum." J. Bacteriol, 62, (1951), 425-432. 5. Anderson, T. F. and Duggar, B. M. "The Effect of Heat and UltraViolet Light on Certain Physiological Properties of Yeast." Am. Philo. Soc. Proceed, 84, (1941), 661-668. 6. Arnow, L. E. "Effects Produced by the Irradiation of Proteins and Amino Acids." Physiol. Rev., 16, (1936), 671-685. 7. Bachafer, E., Ehret, C. F., Mayer, S. and Powers, E. H. "The Influence of Temperature Upon the Intivation of a Bacterial Virus by X-rays." Proc. Natl. Acad. Sci., 39, (1953), 744-750. 8. Black, S., Hashimoto, T. and Gerhardt, P. "Calcium Reversal of Heat Susceptibility and Dipicolinate Deficiency of Spores Formed Endotrophically in Water." Can. J. Microbiol. 6, (1960), 213-224. 9. Bonet-Maury, P. and LeFort, M. "Formation of Hydrogen Peroxide in Water Irradiated with X- and Alpha-rays." Nature, 162, (1948), 381382. 10. Bovie, W. T. "Heat Sensitization of Egg Albumen by Ultra-violet Light." Science, 37, (1913), 373. 11. Bovie, W. T. and Klein, A. "Sensitization to Heat Due to Exposure to Light of Short Wavelengths." J. Gen. Physiol., 1, (1919), 331-336. 12. Bovie, W. T. and Daland, G. A. "New Experiments on the Sensitization of Protoplasm to Heat by Exposure to Light of Short Wave-length." Amer. J. Physiol., 66, (1923), 55-56. 13. Brewer, J. H. "A New Petri Dish Cover and Technique for Use in the Cultivation of Anaerobes and Micro-aerophiles." Science, 95, (1942), 587. 14. Brownell, L. E., Meinke, W. W. Nehemias, J. V., and Coleman, E. W. "Design and Use of Ten Kilocurie Source of Gamma Radiation." Chem. Eng. Prog., 49, (1953 ) -120

-121l 15. Burke, Go So "The Effect of Heat on the Spores of Bacillus tbotulinus." JoAoMoAo, 72, No, 2, (1919), 88-92, 16. tBurke, Go So "Studies on the Thermal Death Time of Spores of Cl, botulinumo III, Dormancy or Slow Germination of Spores Under Optinum Conditions " Jo Infect. Disea,, 33 (1923), 274-284. 17. Butler, JoA.Vo "Effect of X-Rays and Radiomimetic Agents on Nucleic Acids and Nucleoproteins. o In Ciba Foundation Symposium on Ionizing Radiations and Cell Metabolism, 59-76o Edited by G. EoWo Wolstenholme and Eo Mo O'Conner, Little Brown and Coo, Boston, Masso, (1956), 318, 18o Cavalieri, Lo Fo and Rosenberg, Bo H, "Studies on the Structure of Nucleic Acids, XI The Role of Heat and Acid in Deoxyribonucleic Denaturation." J, Am. Chem. Sci, 79, (1957), 5352-7 19. Chick, Ho and Martin C. J. "On the Heat Coagulation of Proteins," J, Physiol., 40, (1910), 4o4-430o 20. Chick, H. and Martin, C. J. "On the Heat Coag.lation Proteinso Part II The Action of Hot Water Upon Egg Albumen and Influence of Acids and Salts Upon the Reaction Velocityo" Jo Physiolo, 43, (1911), 1-28. 21. Church, B. PhD Thesis, The University of Michigan, 1955. 22. Clark, J. Ho "The Action of Ultra-violet Light on Egg Albumen in Relation to the Isoelectric Point " Amero Jo Physiol., 29, (1922), 72-79. 23. Clark, J. Ho "The Denaturation of Egg Albumin by Ultra-violet Radiation," JO Gen. Physiol,, 19, (1935), 199=210o 24, Cox, R. A,, Overhand, Wo G., Peacocke, Ao R. and Wilson, So "Effects of Gamma Rays on Solutions of Sodium Deoxyribonucleate " Nature, 176, (1955), 919-921o 25, Cox, Ro Ao and Peacocke, Ao Ro "Electrometric Titration of Sodium Salts of Deoxyribonucleic Acidso Part III The Effects of Sodium Chlorideo" Jo Chemo Soc.(1956a) 2499-2512. 26. Cox, Ro A, and Peacocke, A. Ro "Electrometric Titration of the Sodium Salts of Deoxyribonucleic Acids, Part IV Denaturation by Heat in Aqueous Solution," Jo Chem. Soc.,, (1956b) 2646-2651o 27, Cox, Ro Ao and Peacocke, Ao R. "Application of the Titration Method to Studies on the Denaturation of Sodiam. Deoxyribonucleate," Jo Polyo Scio 23, (1957), 765-779. 28. Curran, H. Ro, Brumnstetter, B Co and Myers, Ao T, "Spectrochemical Analysis of Vegetative Cells and Spores of Bacteria," Jo Bacteriol, 45, (1943), 485494,

-12229. Curran, H. R. and Evans, F. R. "The Importance of Enrichments in Cultivation of Bacterial Spores Previously Exposed to Lethal Agents." J. Bacterial, 34, (1937), 179-189. 30. Curran, H. R. and Evans, F. R. "Sensitizing Bacterial Spores to Heat by Exposing Time to Ultra-violet Light." J. Bacteriol, 36, (1938), 455-465. 31. Curran, H. R. and Evans, F. R. "Heat Activation Inducing Germination in the Spores of Thermotolerant and Thermophilic Aerobic Bacteria." J. Bacteriol, 49, (1954), 335-346. 32. Curran, H. R. "The Mineral Requirements for Sporulation." In Spores, Edited by H. Orin Halvorson. Am. Inst. of Biol. Sci. Washington, D. C. (1957), 1-9. 33. Davis, F. L., Jr. and Williams, O. B. "Chromatographic Analysis of the Amino Acid Composition of Bacterial Spores. V Studies on Heat Resistance." J. Bacteriol, 64, (1952) 766-767. 34. Desrosier, N. W. and Heiligman, F. "Heat Activation of Bacterial Spores." Food Research, 21, (1952), 54-62. 35. Duggar, B. M. and Anderson, T. F. "The Physiological Changes Produced in Yeast by Ultra-violet Light and by Heat." Science, 90, (1939), 358. 36. Edwards, R. B., Peterson, L. J. and Cummings, D. G. "The Effect of Cathodode Rays on Bacteria." Food Technol, 8, (1954), 284-290. 37. Esty, J. R. and Meyer, K. F. The Heat Resistance of the Spores of B. botulinus and Allied Anaerobes. XI. J. Infectious Diseases, 31, (1922), 650-663. 38. Forbes, H. S. and Deland, G. A. "Further Studies on the Sensitization to Heat Due to Exposure of Short Wave Lengths." Am. J. Physiol., 66, (1923), 50-54. 39. Foster, J. W. and Wynne, E. S. "The Problem of'Dormancy' in Bacterial Spores." J. Bacteriol, 55, (1948), 623-625. 40. Frank, H. A. and Campbell, L. L., Jr. "The Influence of Recovery Media on Terminal Resistance Values of Spores of a putrefactive bacterium." Appl. Microbiol., 3, (1955), 300-302. 41. Friedman, C. A. and Henry, B. S. "Bound Water Content of Vegetative and Spore Forms of Bacteria." J. Bacteriol, 36, (1936), 99105. 42. Fricke, H. The Chemical-physical Foundation for the Biological Effects of X-Rays. Cold Spring Harbor Symposia on Quant. Biol., 3, (1934), 241-248.

-12343, Geiduschek, Eo, Peter, Holtzer, Ao "Application of Light Scattering to Biological Systems~ Deoxyribonucleic Acid and the Muscle Proteinso" In Advances in Biological and Medical Physics, Edited by Co Ao Tobias and J, Ho Lawrence. Academic Press, Inc,, New Yorko (1958), 431-551. 440 Giese, A, C. and Crossman, Eo Bo "The Action Spectrum of Sensitization to Heat with Ultra-violet Lighto" Jo Gen, Physiol,, 29, (1945), 79-87. 45. Giese, Ao C. and Heath, Ho Do "Sensitization to Heat by X-Rays," J, Gen. Physiol, 31, (1948), 249-257, 46. Glasstone, S,, Laidler, Ko J, and Eyring, H, "The Theory of Rate Processes, New York: McGraw-Hill Book CO,, Inc,, (9419411951),152 47. Goddard, Do Ro "The Reversible Heat Activation Inducing Germination and Increased Respiration in the Ascospores of Neurospora Tetra Spermao" Jo Gen, Physiol,, 19, (1935), 45-60o 48, Grelet, No "Le Determinisme de la Sporulation de Bacillus Megatherium, IV Constituants Mineraux du Melieu Synthetique Necessaires a la Sporulation," Annales Institut Pasteur, 83, (1952), 71-790 49. Gromley, Jo A. and Stewart, Ao Co "Effects of X-Radiation On Ice," Am, Chem, Soco Journo, 78, (1956) 2934-29390 500 Halvorson, Ho Orino "Rapid and Simultaneous Sporulationo" J, Appl, Bacteriol,, 20, (1957), 305-314, 51. Halvorson, H, 0o and Church, Bo "Biochemistry of Spores of Aerobic Bacilli with Special Reference to Germination," Bacteriolo Rev,, 21, No, 2, (1957), 112-131o 52, Harrel, Wo Ko "Stimulation of Glucose Oxidation in Extracts of Bacterial Spores by Dipicolinic Acido" Bacteriolo Proceed,, (1957), 32-33o 530 Henry, Bo So and Friedman, C, A, "The Water Content of Bacterial Spores,' Jo Bacteriol, 33, (1937), 323-329o 54, Henry, Ho and Stacey, Mo "Histochemistry of the Gram-staining Reaction for Microorganisms,," Nature, 151 (1943), 671o 55. Henry, H,, Stacey, Mo and Tice, Eo Go "Nature of the Gram-positive Complex in Microorganismso" Naturee, 156, (1945), 720-721o 56~ Hochandel, Co J, "Effects of Cobalt X-Radiation on Water and Aqueous SystemsO" Jo Phyo Chem,, 56, (1952), 587~

-12457. Hollaender, Ao and Stapleton, Go Eo "Fundamental Aspects of Radiation Protection from a Microbiological Point of View,," Biological Revo, 33, (1953), 77-89. 58, Houtermans, VonTheao "Uber den 7Bwflugz der tempertur auf biologische strablen-wirkungeno 2 Mitto Inktivierung van trockenen und feuchten sporen Yon Baco 6ubtilis durch und rontgenstrahleno" Zeito fur natur forschung, -16 (ll)T (1956) 636-643o 59. Johnson, Fo H., Erying, Ho, and Polissar, Mo J. The Kinetic Basis of Molecular Biology, New York0 John Wiley and Sons., Inc., 1954o 60 Kan, Bo,, Goldblith, S. Ao and Proctor, B, E. "Complementary Effect of Heat and Ionizing Radiation." Food Research, 22, (1957), 509-518o 61. Kempe, Lo Lo "Combined Effects of Heat and Radiation in Food Sterilizationo" Applo Microbiol, 3, (1955), 346-352. 62, Kempe, Lo L., Graikoski, J, T, and Bonventre, Po Fo "Combined Irradiation Heat Processing of Canned Foods. Io Cooked Ground Beef Inoculated with Clostridium Botulinum Spores." Applo Microbiol, 5, (1957), 292-295. 63o Kempe, Lo Lo and Graikoski, J, To "Combined Irradiation - Heat Processing of Canned Foods, II Raw Ground Beef Inoculated with Spores of Clostridium botulinumo" Appl, Microbiol, 6, (1958)9 261-2630 64. Kempe, Lo Lo, Graikoski, Jo T. and Bonventre, Po Fo "Combined Irradiation Heat Processing of Canned Foods, III Cooked Ground Beef Inoculated with Spores of a Putrefactive Anaerobe," Applo Microbiol., 7, (1959), 131-134. 65, Kempe, Lo L., Graikoski, J. T. and Bonventre, Po Fo "Combined Irradiation - Heat Processing of Canned Foodso IV Green Peas Inoculated with Anaerobic Bacterial Sporeso" J. Biochem and Microbiol, Techno Engo,2, (1959)1-8. 66, Kempton, J. Ho and Maxwell, Lo Ro "Effect of Temperature During Irradiation on the X-Ray Sensitivity of Maize Seed," Jo Agrio Research, 62,(1941), 603-618, 670 Knaesii Go "The Endospore of Bacteriao" Bacterial Rev., 12, (1948) 19-77o 68, Laidler, Ko Jo and Eyring, Ho "Effects of Solvents on Reaction Rateso" Anno No Yo Acado Scio, 39, (1940), 303-3300 69o Laidler, Ko Jo The Chemical Kinetics of Excited Stateso Oxford at the Clarendon Press, Londaon, (1955), 140o

-12570. Lea, D. E., Haines, R. B. and Coulson, C. A. "The Mechanism of the Bactericidal Action of Radioactive Radiations." Proc. Roy. Soc. B, 120, (1936), 47-76. 71. Lea. D. E., Haines, R. B. and Coulson, C. A. "The Action of Radiations on Bacteria III X-Rays on Growing and Non-proliferating Bacteria." Proc. Roy. Soc. B, 123, (1937), 1-2. 72. Lea, D. E. Action of Radiations on Living Cells. New York: MacMillan Co., (1947) 402. 73. McLaren, A. D. "Photochemistry of Enzymes, Proteins and Viruses." In Advances in Enzymnology, (1949), 75. Edited by F. F. Nord. Interscience. 74. Miller, N. J., Garrett, O. W. and Prickett. "Anaerobic Technique a Modified Deep Agar Shake." Food Research, 4, (1939), 447-451. 75. Mirsky, A. E. and Pauling, L. "On the Structure of Native Denatured and Coagulated Proteins." Proc. Natl. Acad. Sci. U.S., 22, (1936), 439-447. 76. Morgan, B. H. and Reed, J. "Resistance of Bacterial Spores to Gamma Irradiation." Food Research, 19, (1954), 357-366. 77. Morrison, E. W. and Rettger, L. F. "Bacterial Spores. 1. A Study in Heat Resistance and Dormancy." J. Bacteriol, 30, (1930), 299-311. 78. Muller, H. J. "The Manner of Production of Mutations by Radiations." Ch. 8 (1954), 475-626, Radiation Biology, 1, Part 1 Edited by Alexander Hollaender, New York: Morgan-Hill Book Co., Inc. 79. National Canners Association, Bulletin, Washington, D. C. 80. Nehemias, J. V., Brownell, L. E., Meinke, W. W. and Coleman, E. W. "Installation and Operation of Ten-Kilocurie Cobalt-60 GammaRadiation Source." American J. Physics, 22, (1954), 88-92. 81. Olsen, A. M. and Scott, W. J. "Influence of Starch in Media Used for the Detection of Heated Bacterial Spores." Nature, 157, (1946), 337. 82, Perry, J. J. and Foster, S. W. Calcium Content of Spores of Bacillus cereus var. mycoides in Relation to Heat Resistance and Content of Diplicolinic Acid. Proceed of Texas Branch. Texas Report on Bio. and Med., 13, (1955), 920-921. 83. Pollard, E. C. "Primary Ionization as a Test of Molecular Organization." Advances in Biol. and Med. Phys. 3, (1953), 153-189. Eds. J. H. Lawrence and C. A. Tobias. New York: Academic Press.

-12684. Pollard, E. C., Guld, W. R., Hutchinson, F. and Setlow, R. B. "The Direct Action of Ionizing Radiations on Enzymes and Antigens." (1955), 72-108. In Progress in Biophysics 5. Edited by Pergamon Press, New York, N. Y. 85. Powell, J. F. "Isolation of Dipicolinic Acid Pyridine 2:6 Dicarboxylic Acid from Spores of B. Megatherium." Biochem. J., 54, (1953), 210-211. 86 Powell, J. F. "Biochemical Changes Occurring During Spore Germination in Bacillus Species." J. Appl. Bacterial, 20, (1957), 349-358. 87. Procter, B. E., Goldblith, S. A., Fuld, G. J. and Oberle, E. W. "Radiosensitivity of B. thermoacidurans Under Different Environmental Conditions." Radiation Research, 8, (1958), 51-63. 88. Procter, B. E., Goldblith, S. A., Oberle, E. M. and Miller, W. C.,Jr. "Radiosensitivity of Bacillus subtilis Under Different Environmental Conditions." Radiation Researc-h, 3, (1955), 295-303. 89. Rahn, 0. "Sterilization of Microorganisms." Bacteriol, Rev., 1, (1945), 1-17 90. 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, (1951), 383-408. 91. Reynolds, H. and Lichtenstein, H. "Germination of Anaerobic Spores Induced by Sublethal Heating Bacterial." Proc., 9. 1949. 92. Risse, H. P. "X-Ray Photolysis of Water." Zeit. Phys. Chem. A 140133-157. 93. Risse, 0. "Die physikalischen grundlagen der chemischen wirkungen des lichts und der rontgenstrahlen." Erg. der Physiol, 30, (1930), 242-293. 94. Romey, W. R. and Wyas, O. "Some Effects of Ultraviolet Radiation on Sporulating Cultures of Bacillus cereus." J. Bacteriol, 74, (1957), 386-391. L 95. Rose, K. A. and Billing E. "The Water Content and Solid Content of Living Bacteria Spores and Vegetative Cells as Indicated by Refractive Index Measurements." J. Gen. Microbiol, 16, (1957), 418-525. 96. Saleh, M. A. and Ordal, Z. J. "Studies on Growth and Toxic Production of Clostridium Botulinum in a Pre-cooked Frozen Food. I. Some Factors Affecting Growth and Toxin Production." Food Research, 20, (1955), 332-339. 97. Schmidt, C. F. "The Resistance of Bacterial Spores with Reference to Spore Germination and its Inhibition." Annual Review of Microbiology, 9, (1955), 387-400.

-12798. Shooter, Ko Vo, Pain, R. H. and Butler, JoAoVo "Effect of heat and X-Rays on Deoxyribonucleic Acid." Biochem et Ciophys Acta,, 20, 497-502. 99. Sizer, Io Wo "Effects of Temperature on Enzyme Kinetics in Advances in Enzymolo" Edo by Fo F. Nord and C. H. Werkman, New York, NoYo~ Interscience Publishers, Inc,, (19t3) 35-62, 100. Spray, Ro So "An Improved Anaerobic Culture Dish." J. Lab Clino MeOd, 16, (1931), 203-206. 101, Stedman, Ho Lo and Mendel, Ro L. "The Effects of Radiation from a Quartz Mercury Vapor are Upon Some Properties of Proteins." Am. Jo Physiol., 77, (1926), 199-210. 10.2 - Stedman,- R L o "Biochemical Aspects of Bacterial Endospore Formation and Germinationo" Am, Jouro Pharm,, 128, (1956), 84-97 and 114-130. 103. Steffensen, Do "A Higher Frequency of X-Ray Induced Chromosomal Aberrations in Tradescantia Plants Grown on Sub Optimal Coliciumo" Radiation Research, 5, (1956) 597-598. 104. Sugiyama, H. "Studies on Factors Affecting the Heat Resistance of Spores of Clostridium Botulinum J, ctexiol,:1 1 62-. (1951),81-96. 105o "Symposium on the Biology of Bacterial Spores." Bactoriol Revs,, 16, 89-143o 106. Tallentire, Ao "An Observed Oxygen Effect During Gamma Irradiation of Dried Bacterial Spores," Nature, 182, (1958), 1024-1025o 107 Thomas, R, "Recherches sur la de Naturation des Acidis Desoxyribonucleicque." Biochem et Biophys. Acta, 14, (1954), 231-240' 108. Vera, Ho D. "A Comparative Study of Materials Suitable for the Cultivation of Clostridia. J, Bacteriol, 47, (1944), 59-69. 109o Waldhalm, Do Go and Halvorsonr. Ho 0O "Studies on the Relationship Between Equilibrium Vapor Pressure and Moisture Content of Bacterial Endospores." Appl. Microbiol., 2, (1954), 333-338. 110. Walls, No Jo PhD Thesis. The University of Michigan, 1959. 111 Watson, J. D. and Crick, F.HCo "'Genetical Imp ication of the Structure of Deoxyribonucleic Acid," Nature, 171, (1953a), 964-967, 112. Watson, J. D, and Crick, FHC, "Molecular Structure of Nucleic Acids o" Nature, 171, (1953b), 737-738o

-128113o Webb, Ro Bo, Ehret, Co F. and Powers, Eo L0o'A Study of the Temperature Dependence of Radiation Sensitivity of Dry Spores of Bacillus Megatherium between 50K and 3090Ko" Experientia, 14, fasc 9, (1958), 324-326o 114 Weiss, Jo Eo and Spaulding, Eo Ho "A Simple Method for Obtaining Anaerobiosis," Jo Labo Clino Med,, 22, (1937), 726-728o 115. Weiss, Jo "Radio=Chemistry of Aqueous Solutions," Nature, 153, (1944), 748-750 116o Weiss, Jo "Some Aspects of the Chemical and Biological Action of Radiationo" Trans. Foraday, SoCo 43, (1947), 314-324. 117. Weiss, Jo "Chemical Dosimetry Using Ferrous and Ceric Sulfateo" Nucleonics, 10, (1952), 28-31o 118o Williams, N. J andKempe, Lo L. "The Effect of Catalase on the Lethality of Cobalt Gamma Radiation for Certain Anaerobic Bacterial Sporeso" Appl. Bacteriolo, 5, (1957), 365-3680 119o Williams, 0o B0 and Reed, J Mo "The Significance of Incubation Temperature on Recovery Cultures in Determining Spore Resistanceo" J. Infecto Diseases, 71, (1942), 225-227o 120o Williamson, Mo Bo and Grelek, Ao "The Calcium and Magnesium Content of Mammaleau Cell Nuclei'" Jo Cell and Compo Physiolo, 23, (1944), 77-82, 121o Woese, Co and Morowitz, F0 "Kinetics of the Release of Dipicolinic Acid from Spores of Bacillus Subtiliso" J0 Bacteriol, 76, (1958), 81-83o 122o Woese, Co "Further Studies on the Ionizing Radiation Inactivation of Bacterial Sporeso" Jo Bacteriol,, 77, (1959)9 38-42o 123o Wood, To Ho "influence of Temperature and Phase State on X-Ray Sensitivity of Yeast." Arch. Biochemo and Biophyso, 52, (19I5,) 157-174 124, Zerkle, Ro Eo and Tobias, C. Ao "Effects of Ploidy and Linear Energy Transfer on Radiobiological Survivor Curves." Arch, Biochemo Biophyso, 47, (1953), 282=306o