Radiation survival of bacterial spores in neutral and alkaline ice J. UPADHVAY~ AND N. GRECZ

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Biopflysics Laboratory, Biology Department, Illinois Institute of Technology, Chicago, Illinois Received July 14, 1969

UPADHYAY, J. and GRECZ,N. 1969. Radiation survival of bacterial spores in neutral and alkaline ice. Can. J. Microbiol. 15: 1419-1425. The effect of pH values ranging from 6 to 12 on spores of Clostridiutn bot~ilinurn33A exposed to 0.6-0.9 Mrad Co-60 resulted in a complex wavelike pattern of survival which was influenced by pH, temperature, and dose of radiation. Within the experimental pH range three typical survival patterns could be recognized. Pattern A (at - 190 "C) showed peaks of high survival at pH 7 and 9.5 and troughs of low survival at pH 8 and 10-1 1. Pattern B (at -50 'C) showed a single broad peak of high survival a t pH 8.5 to 9 and troughs of low survival at pH 7 and 10. Pattern C (at 0 OC) showed peaks of high survival at pH 7.5 and 10 and troughs of low survival at p H 6,9, and 11. Between the temperatures showing typical A, B, and C patterns the survival profiles indicated various degrees of transition from A to B and from B to C. These results can be tentatively explained in terms of indirect effects, viz. maximum activity of radicals such as .OH in the troughs and extinction of .OH by reaction with eaa- or He at the peaks.

Introduction There 1s a general paucity of information concerning the effect of pH on radiation survival of bacteria. In this connection, two considerations are important: (i) pH does affect radical yields in water, and (ii) radicals are the primary lethal species to bacteria in aqueous systems. Thus, it would be expected that pH should have an effect on radiation survival of bacteria. Radical reactions in the neutral and alkaline range, especially those of the hydrated electron (esg-) have received much attention from radiation chemists in the past 5-6 years (11). Pulse radiolysis experiments support the idea that there is an increase in e,,- by about 4540% at alkaline pH (5, 21). Furthermore, in the alkaline range a substantial rise in the yield of .OH radicals was reported (9, 10, 17). The lethal effect of . O H radicals on bacteria and phage deoxyribonucleic acid (DNA) has been demonstrated (3, 4, 18, 19). It is known in radiation chemistry that the primary processes in liquid and solidly frozen water are generally identical. However, the diffusion of free radicals in ice is greatly impeded and therefore primary radical interactions supposedly occur within the spur or at very close distances from the spur (22). Living cells which are irradiated in the frozen state (bacterial spores can be used for this purpose) show that the indirect effects of radiation that are due to free radicals in water are greatly reduced (but not 1Present address: Department of Microbiology, Virginia State College, Petersburg, Virginia.

completely eliminated) by the liquid-solid transition at 0 O C (16). In the present study spores of Clostridiuln botulinzim 33A were irradiated in buffers of pH values from 6 to 12 at temperatures of - 196 O C to 0 O C to test the degree and manner of indirect action of radiation in the frozen state. Materials and Methods Microbiological Clostridiurn bot~ilinuntstrain 33A was selected because of its high resistance to y-radiation (1, 24), ultraviolet (uv.) radiation (12), and heat (20). Spores were produced in 5% trypticase - 0.5% peptone (TP-broth) as described by Durban and Grecz (12). The number of viable spores at the beginning of the experiment and after irradiation was determined either by the most probable number (M.P.N.) method (experiment I) or by direct colony counts (experiment 11). In both cases Wynne's broth (25) was used as the basic culture medium. In experiment 11, 1.5% agar was added for solidification. The colonies in oval culture tubes were scored after 2, 4, and 6 days at 30 "C. M.P.N. tubes in experiment I were observed for 6 weeks. Buffer Solutions Wide-range borate buffer (7), of appropriate pH, was made up in 2X concentrations and sterilized in the autoclave. Samples were prepared by mixing equal volumes of spores and buffer to give a range from pH 6 to 12; pH 6-12 had no noticeable effect on viability of unirradiated control samples. Samples of 2.2 ml were pipetted into 10 X 75 mm pyrex tubes, sealed, and immediately frozen in dry ice. The samples were stored in the freezer at -20°C or in dry ice at all times except during irradiation. After irradiation, the samples were melted at room temperature. Serial decimal dilutions were made in 0.067 M phosphate buffer blanks and 1 ml of appropriate dilutions was added to 18 to 22 ml of Wynne's medium for determination of surviving spores. This procedure effectively neutralized the original pH.

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2 "C at - 196 "C and Y1.5 "C at 0 OC using the temperature control system described by Grecz, Snyder, Walker, and Anellis (15).

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Radiation Experiment I was irradiated at the Gamma Process Cobalt-60 Facility, Morton Grove, Illinois, at a dose rate of about 300 krad per hour, whereas experiment I1 was treated at the U.S. Army Radiation Laboratory, Natick, Massachusetts, at a dose rate of about 3600 krad per hour. Temperature control during irradiation was within

Experiments I and I1 Experiment I was a preliminary study with a single sample at each pH, temperature, and radiation dose. Two

0---0 0 . 7 M r a d _1

O---O

O.9Mrad

-150°C

-I0O0C

FIG. 1. Effect of radiation dose on survival profile of spores of Clostridilmz botti/inrrrn 33A: comparison of profiles obtained after 0.7 and 0.9 Mrad in experiment I.

PH

FIG. 2. Families of related survival profiles as affected by pH and temperature during radiation (experiment 11, 0.9 Mrad).

UPADHYAY AND GRECZ: RADIATION SURVIVAL O F SPORES

sets of samples were irradiated, one to 0.7 Mrad and another to 0.9 Mrad. In experiment 11 samples were irradiated to 0.6 and 0.9 Mrad. This experiment consisted actually of two distinct studies each with duplicate samples. One set of samples

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lo6

- 20.c

-30°C

O.9Mrad

pH

FIG.3. Effect of radiation dose on survival profiles of spores of Clostridium botulinirnz 33A: comparison of profiles obtained after 0.6 and 0.9 Mrad in experiment 11.

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was evaluated "horizontally", i.e., samples were selected in units of a single temperature, cutting across pH 6 to 12. The second set of duplicate samples was evaluated "vertically", i.e., samples were selected in units of a single pH cutting across all temperatures from -190 O C to 0 O C . The two experiments were carried out in different laboratories using different spore crops. The initial spore loads were 6.4 X 106 spores per milliliter in experiment I and 1.5 X 10s spores per milliliter in experiment 11. Two different cobalt-60 facilities were used for irradiation. These differences between experiment I and I1 are important for the evaluation of the effect of pH on radiation survival of spores described below.

Results Patterns of Spore Survival The data from experiments I and I1 were summarized in Figs. 1, 2, and 3. The survival points were connected by straight lines, thus yielding complex wavelike survival patterns. On close examination of the data three basic type patterns could be recognized. Figure 4 illustrates and analyzes survival patterns A, B, and C obtained after the more intense radiation dose of 0.9 Mrad. Patter11A (Fig. 4, A) occurred at - 196 "C and - 190 "C. Here two peaks of high survival, peak a, at pH 7 , and the rather broad peak b, at p H 9-10, alternated with three troughs of low survival, through c at pH 6, d at pH 8, and e at p H 10-11. Pattern B (Fig. 4, B) occurred at - 50 "C and was especially pronounced in experiment 11. It showed one extremely flat peak (f) of high survival in the range of pH 8 to 10 and two troughs of low survival, one at pH 7 (trough g), and another at pH 10 (trough lz).

pH

FIG.4. Typical survival patterns A, B, and C.

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Pattern C (Fig. 4, C) occurred at -20 "C (experiment I, Fig. 1) and -2O0C, -1O0C, and 0 "C (experiment 11, Fig. 3). In Fig. 4, C two peaks (i at pH 7-8 and j at pH 10) alternated with three troughs (k at pH 6, 1 at pH 9, and nz at pH 11). Pattern C was especially distinguished by very high peaks and deep troughs. The exception was 0 "C, experiment I (Fig. I), which did not have a deep cut pattern. The breakdown of pattern C in this case may be due to transition from solidly frozen to liquid state at this rather crucial temperature range, viz. 0 1.5 "C.

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Relation of Survival Profiles to pH Since experiments I and I1 were greatly different in many respects, the similarities in the horizontal planes of their survival profiles could be attributed to the radiation chemical effects of pH itself. ~ c o m ~ a r i s of o nappropriate curves in F ~ ~ 1, 2, and 3 revealed relatively close agreement in the general features of survival profile A in experiment I and I1 at - 196 and - 190 "C, as well as agreement in survival profile B at -50°C. Furthermore, the patterns obtained at -20 OC in expts. I and I1 also were in good agreement. Between the temperatures showing typical patterns A, B, and C, the survival profiles indicated various degrees of transition from A to B and from B to C as was especially brought out in Fig. 2. Family A-The survival profiles at - 190 "C and - 150 "C (Fig. 2, A) coincided at all pH values except pH 9 and 10; these points signified departure from typical pattern A because of increase in lethal effectiveness of radiation at - 150 "C. Family B-Curves at - 100 "C, - 50 "C, and -40 "C illustrated the gradual permutation of pattern A into typical pattern B, and eventually at -30 "C the first indications of pattern C (Fig. 2, B). The profile at - 100 "C showed already a predominant B character. Thus, there was one peak of high survival corresponding to peak f and two troughs corresponding to g and h. The most pronounced change in the profiles from - 100 "C to -40 "C was the gradual deepening of trough h, which remained at about pH 10; trough g remained consistently at about pH 7 at all temperatures from - 100 "C through - 30 OC. The profile at -30 "C represented a decisive step in the transition from the single-peaked pattern

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B to the double-peaked pattern C. Here two peaks are outstanding, one at pH 8 (cf. with peak i ) and another at pH I 0 (cf. with peak j, Fig. 2C). Family C-Peaks i and j appeared to shift considerably but could be clearly identified at all temperatures in Fig. 2C. Generally, there was much uncertainty in the location of peaks and troughs in family C, suggesting intensifying interactions between pH and temperature. The overall impression from this family of curves was that at 0 "C outstandingly high radiation protection was achieved at peaks i and j. On the other hand, the troughs k, I, and m were extremely deep. The reasons for these extreme contrasts between maxima and minima in the survival profile at 0 "C are not clear but they may be related to increased radical activity (in the troughs) as well as to increased mutual radical-radical extinction (in the peaks). s. Eflect of Radiatiorz Dose on Survival Profiles Figure I illustrates the relation between survival profiles obtained in experiment I after 0.7 and 0.9 Mrad. This figure demonstrates that the corresponding profiles of spore survival at each temperature appeared to exhibit a great degree of resemblance. The curves at 0.9 Mrad showed generally more contrast between peaks and troughs, indicating increased radical activities at the higher radiation dose. Similar results were obtained within the temperature range -30 "C to 0 "C in experiment IS (Fig. 3). Experiment 11 showed more details in survival profiles because of closer spacing of temperatures and pH. However, in a general way experiment IS (Fig. 3) supported and reinforced the conclusions gained from experiment I.

Discussion Consideratiorz of Radiation Chemistry Lethal radiobiological action on various organisms in water is thought to be due primarily to the action of radicals produced by radiolysis of water. In the frozen state, the ice restricts diffusion and therefore primary radicals recombine very rapidly either within the spur or very close to it. As a result, secondary reactions are substantially suppressed, and consequently most effects are due to primary radicals, such as .OH, Ha, and esq-. It has been reported that reducing species H and eaq- have no appreciable effect

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UPADHYAY AND GRECZ: RADIATION SURVIVAL OF SPORES

on microorganisms. In contrast, the oxidizing radical .OH is highly lethal whether produced by radiolysis of water (3, 4, 18, 19, 23), or by chemical means (2, 8). With respect to the action of pH on radiation survival of spores of C . botulinum 33A, two distinct aspects must be considered: (i) the effect of pH on radiochemical processes in frozen aqueous media, (ii) the effect of pH on radiation susceptibility of biopolymers, particularly on vital biopolymers in the spore itself. Radical Processes in Neutral and Alkaline Ice Most chemical changes in ice can be explained assuming that radiation produces primary and 'OH) and two ions (H20+ and e-) (22). The effect pH is due to reactions of these primary ~ r o d u c t swith the products of electrolytic dissociation of water, and OH-. With respect t' pH, the most Outstanding process is the following equilibrium.

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This chain of reactions has (as a final consequence) two major effects at alkaline pH: (i) it increases the yield of eaq- via reaction 7; here the highly unstable Ha is converted into a relatively stable es,-, and (ii) it increases the yield of .OH radicals by removing the He atoms via reaction 7. If not removed, H . atoms would react with .OH to form water via reaction 6. This sequence of reactions explains the observations that alkaline pH increases the yield of eaq- (13, 14) and .OH radicals (6). A point of special interest to the present paper is the behavior of the hydrogen atom in the alkaline range. The conversion of H. into eaqhas a pK = 9.6; however, reaction 7 exhibits, in most aqueous systems tested to date, a delay until pH 11. This delay is thought to be due to reactions of H. with products of water radiolysis. These reactions presumably with reaction 7, and in this way shift the apparent pK toward DH (6).

~xamination'of our survival profiles A and B representing the highly frozen state showed a [I] *H2+ H' eaq-. consistently high survival around pH 9.6. This OHmay perhaps indicate that radical reactions of relevance to spore protection may somehow be For the present paper, the reactions in the alrelated to pH = 9.6, i.e. the point at which the kaline range are of particular interest. According concentrations of H. and eaq- were theoretically to Dainton and Watt (9, lo), the following equal. changes occur at alkaline pH: The fact that changes in spore lethality in pat[2] H20* (or He and .OH pair) OHterns A and B took place at pH 9.6, not at pH 11, --+ H20OH. seemed to signify that pH-dependent radical reThe product H20- is the same as eaqHzO. actions were extremely rapid and occurred at In alkaline aqueous systems above pH 11 the relatively short distances. This reasoning is in eaq- and not the H-atom is the predominant accord with the view that water radicals in ice do reducing species since any H . present would be not migrate as extensively as those in liquid water (22). rapidly converted to esq-: The shifting of the highly protective condition [3] H - OH- --t H20- = esqH2O. from pH 9.6 toward pH 10.5 in pattern C sugThe above considerations suggest that reaction 2 gests that at - 20 OC to 0 OC radical mobility inin reality takes place in a series of four steps, of creased sufficiently to allow extensive radicalwhich three steps are pH independent and one radical interactions in the water phase. The step depends on pH: possibility of increased radical-radical interactions and consequently radical-radical extincH 2 0 ---t H20* (excited water), [4] tion of lethal species is further supported by the H20* c= H. = .OH, == [51 fact= that = spore survival was generally much higher in pattern C than in pattern B. H . . OH t H 2 0 (deexcitation). [6] The reactions which may be of pertinence to Reactions 4, 5, and 6 are pH independent the effect of alkaline pH on radiation survival of pK = 9.6 spores were summarized, in simple form, in Fig. H. OH- --+ eaq- H20. 171 5. The predominant primary radical species to be (pH dependent)

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CANADIAN JOURNAL O F MICROBIOLOGY.

considered are Ha, eaq-, and - O H (6, 22). Reaction 1 converts H-atoms into eaq-:

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He OH- --t H20- = eaq-; and thus prevents reaction 2: H. .OH -+- H20. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIV on 01/18/17 For personal use only.

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As a consequence, the concentration of .OH available for killing of spores rises. This increase in .OH above the pH 9.6 may explain the rapid decline in spore survival between pH 9.6 and about pH 10.5 in survival patterns A and B. The protection of spores above pH 10.5 is not understood, but may somehow be related to accumulation of eaq- and .OH, which may interact as follows : (Dorfman and Matheson (1 1)). On the other hand, the highly lethal condition in the range from pH 9.6 toward pH 8.5 in patterns A and C and from pH 9 toward pH 7 in pattern B (cf. troughs d, g, and I, Fig. 4) may have resulted from gradual increase in the concentration of He and OH radicals. Biopolymers Radiation susceptibility of biopolymers similar to those found in the spore may be influenced by pH, pK, and the nature of tertiary structure and subunit assembly of complex molecules (2). However, the biopolymer nature of spores is too complex to make any meaningful conclusions at this time.

OH-

e-iq

RADIATION PROTECTION AT HIGH pH

'bq EFFECT ON SPORES UNKNOWN

OH RADICAL LETHAL TO SPORES

FIG. 5. Summary of postulated radical process in the alkaline range.

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In conclusion, typical survival patterns A, B, and C (Fig. 4) indicated that the characteristic shifting of peaks and troughs in survival profiles was the result of a critical balance and dynamic interaction of radical processes in ice. Survival profiles showed a great degree of consistency with respect to pH under a variety of conditions, including the extreme differences in spore crops, radiation dose rate, temperature during irradiation, and microbiological recovery methods in expts. I and 11. This indicated that pH-dependent radical processes in ice were probably the most important reactions responsible for the characteristic wavelike patterns of spore survival. Acknowledgments This research was supported by Public Health Service Grant UI 00138 and P H s Career Development Award 5-K3-AI-21,763. Experiment I1 was irradiated by U.S. Army Natick Laboratories under agreement N Labs No. 212. 1. ANELLIS,A. and KOCH,R. B. 1962. Comparative resistance of strains of Clostridium botrrlitrra~ito gamma rays. Appl. Microbiol. 10: 326-330. 2. BACQ,2.M. and ALEXANDER, P. 1961. Fundamentals of radiobiology. 2nd ed. Pergamon Press, New York. 3. BLOK,J., LUTHJENS, L. H., and Roos, A. L. M. 1967. The radiosensitivity of bacteriophage DNA in aqueous solution. Radiat. Res. 30: 468-482. 4. BLOK,J. and VERHEY, W. S. D. 1968. The attack of free radicals on biologically active DNA in irradiated aqueous solutions. Radiat. Res. 34: 689-703. 5. BROWN,D. M., DAINTON, F. S., KEENE,J. P., and WALKER, D. C. 1964. Solute isotope and pH effects on the primary species produced in the pulse radiolysis of water. Proc. Chem. Soc. 266-268. 6. BUXTON, G. V. 1967. The effect of pH on radical and molecular yields in the radiolysis of water. 112 Radiation Research, Proc. 3rd Int. Congr. Radiation Research, Cortina dlAmpezzo, Italy, June-July 1966. Edited b y G. Silini. John Wiley & Sons Inc., New York. pp. 235-250. 7. CARMODY, W. R. 1961. An easily prepared wide range buffer series. J. Chem. Educ. 38: 559-560. 8. COLLINSON, E., DAINTON, F. S., and HOLMES, B. 1950. Inactivation by hydroxyl radicals. Nature, 165: 267269. 9. D ~ I N T O F. N , S. and WATT,W. S. 1962. The effects of pH on the radical yields in the y-radiolysis of aqueous systems. Nature, 195: 1294-1296. 10. DAINTON, F. S. and WATT,W. S. 1963. p H effects in the y-radiolysis of aqueous solutions. Proc. R ~ y . ~ S o c . A, 275: 447-464. 11. DORFMAN, L. M. and MATHESON, M. S. 1965. Pulse radiolysis. Progr. React. Kinetics, 3: 239-301. 12. DURBAN,E. and GRECZ,N. 1969. Resistance of spores of Clostridium botulinrrm 33A to combinations of ultraviolet and gamma rays. Appl. Microbiol. 18: 44-50.

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UPADHYAY AND GRECZ: RADIATION SURVIVAL OF E. M. and HART,E. J. 1967. Primary radi13. FIELDEN, cal yields in pulse-irradiated alkaline aqueous solution. Radiat. Res. 32: 564-580. 14. FIELDEN, E. M. and HART,E. J. 1968. Primary radical yields and some rate constants in heavy water. Radiat. Res. 33: 426-436. 15. GRECZ,N., SNYDER, 0. P., WALKER,A. A,, and ANELLIS,A. 1965. Effect of temperature of liquid nitrogen on radiation resistance of spores of Clostridium botulinum. Appl. Microbiol. 13: 527-536. 16. GRECZ,N., UPADHYAY, J., and TANG,T. C. 1967. Effect of temperature on radiation resistance of spores of Clostridium botulinum 33A. Can. J. Microbiol. 13: 287-293. 17. HAYON, E. 1965. Radical and molecular yields in the radiolysis of alkaline aqueous solutions. Trans. Faraday Soc. 61: 734-743. 18. JOHANSEN, I. 1965. The contribution of water-free radicals to the x-ray inactivation of bacteria. In Cellular radiation biology. 18th Annu. Symp. on Fundamental Cancer Research, 1964. The Williams & Wilkens Co., Baltimore, Md. pp. 103-106. I. and HOWARD-FLANDERS, P. 1965. 19. JOHANSEN, Macromolecular repair and free radical scavenging in the protection of bacteria against x-rays. Radiat. Res. 24 : 184-200.

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20. Lm, C. A. 1966. Heat resistant toxin in spores of Clostridium botulinum. M.S. Thesis, Illinois Institute of Technology, Chicago, Illinois. 21. MATHESON, M. S. and RABMI,J. 1965. Pulse radiolysis of aqueous hydrogen solutions. I. Rate constant for reaction of e,,- with itself and other .~ . transients. 11. The interikvertibi~it~ of e,,- a d He. J. Phys. Chem. 69: 1324-1335. 22. SPMKS,J. W. T. and WOODS,R. J. 1964. Introduction to radiation chemistry. J. Wiley & Sons,- Inc... New York. 23. SUCHANEK, G., MIURA,T., and GRECZ,N. 1969. Role of Oz and Nz in thermorestoration of hydrated bacterial spores. Radiat. Res. 40:222-232. 24. TOWNSEND, C. T., PERKINS,W. E., BENKO,P., and DURAND, R. R. 1959.Determination of relative resistance of selected strains of Clostridium botulinum to ionizing radiations. U.S. Army Quartermaster Research and Engineering Center, Natick, Mass. Contract D.A. 19-129-QM-1184. Rep. No. 5 (final) Dec. 1959. 25. WYNNE, E. S., SCHMIEDMG, W. R., and DAYE,G. T., JR. 1955. A simplified medium for counting Clostridium spores. Food Res. 20: 9-12. ~

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