Resistance of Micrococcus radiodurans

Transcription

1 APPLIED MICROBIOLOGY, Jan., 1967, pp Copyright 1967 American Society for Microbiology Influence of Culture Media on the Radiation Resistance of Micrococcus radiodurans K. L. KRABBENHOFT,' A. W. ANDERSON, AND P. R. ELLIKER Department of Microbiology, Oregon State University, Corvallis, Oregon Received for publication 29 August 1966 ABSTRACT The addition of NZ-case (a tryptic digest of casein) to a growth medium (PC) consisting of tryptone, glucose, and yeast extract caused a significant decrease in y radiation resistance of Micrococcus radiodurans. The level of radiation resistance was inversely related to the concentration of NZ-case. The LD5o for this organism was approximately 700 krad when grown in tryptone, glucose, yeast extract, and DL-methionine (TGYM) broth, but it was approximately one-half as resistant when grown in a PC medium containing 0.5% NZ-case (PCNZ). The resistance to ultraviolet light was also reduced. Cultures transferred from PCNZ to TGYM media regained the high level of resistance. Micrococcus radiodurans is a nonsporeforming, pink-pigmented tetracoccus which was isolated from irradiated cans of meat (3) and has been recently isolated from other sources (13). It is not pathogenic or heat-resistant, but is resistant to ultraviolet (UV) irradiation (8), and has an LD5o of 500 to 700 krad for y radiation in phosphate buffer. Strains of other species with similar resistance properties have been isolated more recently (6). The unusually high radiation resistance of this organism has been attributed to various factors, including sulfhydryl compounds (4), an extremely efficient repair system for damaged deoxyribonucleic acid (DNA; 21), and unique ultrafine structures in the cell wall (23). The pigments of M. radiodurans have been identified as being of the carotenoid type, but attempts to correlate radiation resistance with the presence of these pigments have not been successful (12, 15) or consistent (17, 18). Preliminary studies from earlier work (13) indicated that a change in the type of culture medium increased the cell yield of M. radiodurans by approximately twofold. The present study was undertaken to determine what influence the culture medium has on the radioresistance of this microorganism. MATERIALS AND METHODS Microorganisms. A culture of M. radiodurans was obtained from the stock culture collection of the I Present address: Department of Biology, New Mexico State University, Las Cruces. 178 Vol. 15, No. 1 Prinzted in U.S.A. Department of Microbiology at Oregon State University, Corvallis. The culture was maintained on slants of TGYM and PCNZ media, which are described below Ṁedia. TGYM medium, which has been used for routine propagation of this microorganism and in earlier radiation studies in our laboratory, consisted of the following (per liter): tryptone (Difco), 5 g; glucose, 1 g; yeast extract (Difco), 1 g; DL-methionine, 20 mg. Subsequent studies indicated growth was superior on PCNZ medium, which consisted of plate count medium (per liter: tryptone, 5.0 g; yeast extract, 2.5 g; and glucose, 1.0 g) supplemented with 0.5%770 NZ-case (Sheffield Chemical, Norwich, N.Y.). The final ph of both media was 7.0. For plate counts, 1.5% agar was added to the growth medium. Culture conditions. Cells harvested from 44-hr TGYM or PCNZ cultures were used in all radiation experiments unless otherwise specified. These were prepared by using a 1% inoculum of a 44-hr culture and incubating at 30 C on a shaker-incubator. Harvesting of cells. Cells were harvested by centrifugation (7,000 X g, 20 min), washed twice, and resuspended in M phosphate buffer (ph 7.0). Cell density was adjusted with buffer to contain 108 cells per milliliter. Radiation. Duplicate 10-ml quantities of cell suspensions were transferred to glass radiation vials and irradiated at room temperature with the cobalt-60 source at Oregon State University. Unless otherwise specified, the exposure was 700 krads with an administered dose rate of 713 krad/hr. The LD5o of the TGYM and PCNZ cultures was determined by use of radiation levels of 100 to 700 krads. Cell suspensions containing 104 cells per milliliter were used for UV exposure (100 ergs per cm2 per sec). Samples of 1 ml in triplicate were uniformly spread over the surface of flat-bottom petri plates by hand

2 VOL. 15, 1967 CULTURE MEDIA AND RADIATION RESISTANCE 179 agitation, and the plates were gently agitated on a rotator (Eberbach Corp., Ann Arbor, Mich.) during the exposure period. Relative effect ofgrowth and recovery medium. It has been previously reported (1, 2, 9, 22) that the difference in radiation resistance of Escherichia coli B and E. coli B/r lay not in the initial damage, but in the metabolic processes after irradiation, including the postirradiation conditions of recovery medium. In the present study, irradiated cells which had been grown in TGYM broth were recovered on TGYM agar for controls and also on PCNZ agar for comparative purposes. In a similar manner, cells initially grown in PCNZ were recovered on PCNZ and TGYM agar. Effect of different levels of NZ-case. A series of PCNZ media were inoculated in duplicate; the media contained the following percentages of NZ-case: 0.01, 0.05, 0.1, , 0.4, and 0.5. Because M. radiodurans has a consistent high level of radiation resistance when grown in TGYM, this medium was used as a control. Growth rate studies. Standard growth curves were constructed by using a 44-hr culture and inoculating the two kinds of media in triplicate. The effect of physiological age on radiation resistance was also investigated. By adjusting the time of inoculation, cultures of five different physiological ages could be harvested and subsequently irradiated simultaneously. Biochemical determinations. DNA and ribonucleic acid (RNA) measurements were made by use of cells washed twice in saline and resuspended in a solution (5 ml per 100 ml of culture) of 0.15 M saline and 0.1 M ethylenediaminetetraacetic acid (tetrasodium salt). DNA and RNA were liberated by hydrolysis with 11.7 N perchloric acid at 70 C for 10 min. The suspension was mixed once for 30 sec with a Vortex Junior mixer during the heating period, and, after cooling, it was centrifuged (3,000 X g, 20 min); the supernatant fluid was decanted. This supernatant fluid was used for quantitative determinations of DNA (7). RNA was measured by the orcinol procedure (16). The precipitate from the acid hydrolysis of cells described above was dissolved with 10 ml of NaOH (3%) and used in the determination of total protein by the biuret method (10). Relative amounts of DNA, RNA, and protein were compared on a dry weight basis. Reversibility of radiation resistance. Initial studies indicated that the degree of resistance could be controlled by altering the NZ-case concentration in the growth medium. To test the possibility that the radiation resistance also could be reversed, 44-hr washed cultures grown in TGYM and PCNZ broth were resuspended in the same or opposite medium for 3 or 44 hr. After washing the cells free of the new medium and resuspending in buffer, their relative resistance to -y radiation (700 krad) was determined. The effect of endogenous metabolism on radiation resistance was demonstrated by using washed cells resuspended in buffer and placed on a shaker (10 hr at 30 C). After centrifugation, the cells were again resuspended in buffer and irradiated (700 krad). Control cultures were handled in a similar manner, except they were not shaken prior to irradiation. RESULTS Effect of culture media on radiation resistance. The influence of two different growth media on the percentage of survival of irradiated M. radiodurans is given in Table 1. Cells grown in TGYM were 10 times more radiation-resistant than those grown in PCNZ broth. Cell cultures grown in PCNZ produced a clear, brown-colored liquid within 44 hr, whereas the TGYM broth was tan. Both media were the same color initially. The color change began to occur after approximately 20 hr of incubation. Centrifuged cells grown in TGYM medium were bright pink, whereas those grown in PCNZ were a faded, dull pink color. The survival of M. radiodurans was not altered when it was grown on one medium and recovered on the same or another medium after irradiation (Table 1). Cells grown in TGYM broth and recovered on TGYM or PCNZ agar after irradiation (700 krad) had approximately 60% survival. On the other hand, cells initially grown in PCNZ broth had approximately 6% survival, regardless of whether they were recovered on PCNZ or TGYM medium. The initial cell concentration was similar in all cases before irradiation. Effects of major component differences of media on radiation resistance. M. radiodurans was grown in three different media to determine the effect of various additives on its radiation resistance. These additives were ones which represented major differences in PCNZ and TGYM media. The addition of DL-methionine to PCNZ broth at the same concentration used in TGYM broth did not produce increased radiation resistance (Table 2). When the yeast extract concentration was increased in TGYM to levels comparable to that in PCNZ medium, the resistance of the organisms was approximately the same as for those grown in the standard TGYM broth. However, TABLE 1. Effect of growth and recovery media on survival of Micrococcus radiodurans exposed to 700,000 rad -y irradiation in phosphate buffer Viable cells/mla Growth medium Recovery medium Before After Sur- vival irradiation irradiation TGYM TGYM 207 X X TGYM PCNZ 189 X X PCNZ PCNZ 123 X X PCNZ TGYM 140 X X a Average of triplicate plates of duplicate irradiated samples from three experiments.

3 180 KRABBENHOFT', ANDERSON, AND ELLIKER APPL. MICROBIOL. TABLE 2. Effect of various growth media on the survival of Micrococcus radiodurans exposed to 700,000 rad -r irradiation while suspended in phosphate buffer Medium Before radiation Viable cells/mla After radiation Survival TGYM X X PCNZ (0.01% NZ) X X PCNZ (0.05% NZ) X X PCNZ (0.1% NZ) X X PCNZ (0.3% NZ) X X PCNZ (0.5% NZ) X X TGYM plus NZ (0.5%))...( 37 X X TGYM plus yeast extract (0.15%) X X PCNZ plus DL -methionine (0.002%) X X j a Average of triplicate plates of duplicate irradiated samples from three experiments. l9 I08 2 ~-8 (I -J 10 -J LZJ U 0 7 Z 10 6 TIME - (HOURS) I00 FIG. 1. Growth curves of Micrococcus radiodurans grown in PCNZ or TGYM broth at 30 C on a shaker. the addition of NZ-case to TGYM medium reduced the resistance to a level comparable for cells grown in PCNZ broth containing 0.5% NZ-case. The higher the concentration of NZ-case in the growth medium, the lower was the observed radiation resistance (Table 2). The survival of cells grown in the absence of NZ-case was 68%, whereas approximately 5% of the cells survived when they had been grown in the presence of 0.5% NZ-case. The least radiation-resistant cultures were also less brightly pigmented. Growth ofm. radiodurans in PCNZ and TGYM media. When M. radiodurans was grown in PCNZ broth, it entered the death phase after 65 hr of incubation, whereas the cells remained in the stationary phase for at least 96 hr when grown in TGYM medium (Fig. 1). The rate of growth in the log phase was essentially the same for the two media. The effect of physiological age on radiation TABLE 3. Effect ofphysiological age onz radiation of Micrococcus radiodurans grown in two different media and irradiated in phosphate buffer Viable cells/ml after exposure to medium Culture vival 0 krad 700 krad TGYM X X X X X X X X X X PCNZ X X X X X X X X X X resistance indicated that cells grown in PCNZ broth were at least 10 times more radiationsensitive than cells grown in TGYM at all ages tested (Table 3). The single exception to this general observation was that the 8-hr culture grown in TGYM broth was only four times more resistant than the PCNZ culture. With that exception, the survival for the TGYM cultures ranged from 35 to 67%, whereas the survival of the irradiated PCNZ cultures ranged from 2 to 6% at the different ages. Effect of altered radiation resistance on certain chemical factors and cell morphology. Chemical determinations indicated that there was 4.5%' RNA, 1% DNA, and 47% protein for both the highly radiation-resistant and the more radiationsensitive cultures. Hence, there was no difference in the relative amounts of these substances between cells grown in PCNZ and TGYM.

4 VOL. 15, 1967 CULTURE MEDIA AND RADIATION RESISTANCE 181 The initial ph of the growth medium was 7.0, and the final ph was 8.0 and 8.2 for a 44-hr culture in TGYM and PCNZ broth, respectively. In addition, the relative dry weight and the packed cell volume of the cells grown in TGYM were 25% less than an equal volume of cells grown in PCNZ broth. Evidence that this increase in cell mass of the PCNZ cultures was because of higher numbers of cells and not because of an increase in cell size or morphology was apparent from the growth curves (Fig. 1) and phase-contrast microscopic studies (Fig. 2 and 3). Radiation dose-level responses. The LD5o values of M. radiodurans grown in the two different media were found to be significantly different (Fig. 4). The LD5o for cells grown in PCNZ broth was approximately 370 krad, as compared to 700 krad for TGYM-grown cells. Effect ofgrowth media on UV resistance. When M. radiodurans was grown in PCNZ broth and suspended in phosphate buffer, its resistance to UV was approximately one-tenth of that of similar cells grown in TGYM broth (Fig. 5). A sigmoidal survival curve was obtained for cells grown in TGYM broth. However, the rate of cellular inactivation was exponential for cells grown in PCNZ broth. Reversibility of radiation resistance. When M. radiodurans was grown in TGYM broth and then exposed to PCNZ for 44 hr, its radiation resistance was found to be 10% of that for cells grown in TGYM and exposed for 3 hr to PCNZ broth (Table 4). On the other hand, the radiation resistance for cells grown in PCNZ broth and then exposed to TGYM broth for either 3 or 44 hr was found to be similar for both exposure periods. The generation times for M. radiodurans in the TGYM and PCNZ media were 50 and 38 min, respectively. This means that a 3-hr incubation period was sufficient for the production of only two or three generations of cells. For controls, cultures were exposed to fresh medium comparable to the initial growth medium. Effect of endogenous metabolism on radiation resistance. When M. radiodurans was grown in PCNZ broth and then shaken in phosphate buffer for 10 hr, its radiation resistance increased more than 10 times (Table 5). Control experiments with cells grown in TGYM broth indicated no significant change in resistance when they were shaken in buffer. Downloaded from on April 12, 2018 by guest FIG. 2. Phase-contrast photomicrograph of Micrococcus radiodurans grown in TG YM broth at 30 C for 44 hr. X 35,000.

5 182 KRABBENHOFT, ANDERSON, AND ELLIKER APPL. MICROBIOL. FIG. 3. Phase-contrast photomicrograph of Micrococcus radiodurans grown in PCNZ broth at 30 C for 44 hr. X 35,000. DIscussioN The addition of NZ-case to plate count agar has been reported (6) to provide an excellent isolation medium for obtaining a radiation-resistant o0ange-brown micrococcus from irradiated haddock tissues. A similar NZ-case medium has been used (13) to investigate possible sources of M. radiodurans. PCNZ medium provided a larger cell yield than TGYM, but the cultures were 10 times more radiation-sensitive and also less pigmented when grown in the former medium. A "recovery medium effect" has been described for radiation-resistant and -sensitive strains of E. coli (1, 2, 9). A richer, more complex recovery medium produced higher counts of the resistant strain and fewer radiation survivors of the sensitive strains. It was concluded on the basis of these results that the difference between the two strains lay not in the initial radiation damage, but in their metabolic processes immediately after irradiation. In the present study, no "recovery medium effect" was observed for M. radiodurans which was grown on TGYM or PCNZ medium and which was recovered on either of these two media. Additional studies indicated there were no effects from the major component differences of the two media on the radiation resistance of this microorganism. When M. radiodurans was grown in PCNZ broth containing various levels of NZ-case, an inverse relationship was observed between the concentration of NZ-case and radiation resistance. In addition to altered resistance, cells grown in higher levels of NZ-case appeared to be the least pignented. This observation suggested that pigmentation and radiation resistance were, in some manner, related and that NZ-case was capable of altering these factors. Studies are presently being conducted to consider these possibilities. Growth characteristics of M. radiodurans in the two different media indicated similar growth patterns. An earlier death phase for the PCNZ culture probably was a reflection of a larger number of cells depleting the available nutrients or producing toxic waste materials more rapidly than a lesser number of cells in TGYM. If unbalanced growth was occurring in one of these media, it was not apparent from the standard growth curves. Further evidence that unbalanced growth did not occur in PCNZ was provided from experiments on the effect of physiological age on radia-

6 VOL. 15, 1967 CULTURE MEDIA AND RADIATION RESISTANCE 183 tion resistance. At all physiological ages tested, M. radiodurans was more resistant when grown in TGYM as compared to PCNZ. The general observation that radiation resistance of M. radiodurans does not change with age was in agreement with previous work by Lee (Ph.D. Thesis, Oregon State University, Corvallis, 1963), although the percentage of survival was about twice as great in the present studies. This may have been due to a difference in growth media. When M. radiodurans was grown in TGYM and irradiated in buffer at different dosage levels, the 100 9YM~Y PCNZ D.\ U) bi 0~ z~~oe RAD x10 I DOSE- RADS x 10O5 FIG. 4. Dose survival curves for Micrococcus radiodurans grown in PCNZ or TGYM broth and exposed to ry radiation while suspended in phosphate buffer. Irradiation was conducted in air at room temperature at a dose rate of 713 krad/hr. survival curve was characterized by a wide "shoulder." However, the survival curve decreased exponentially after 350 krad for cells grown in PCNZ. The wide- "shoulder" sigmoidal survival curves are typically found in radiation-resistant organisms (14, 18) and are often referred to as multihit (multitarget or multiunit) curves, because they suggest a multiplicity of events required to bring about inactivation. The cells grown in PCNZ did not produce a wide "shoulder" in their survival curve, and this suggests that the number of radiation-sensitive sites had been increased (i.e., the greater the number of sensitive sites, the greater the proba- U1) 0 D Uf) z 0- CLJ EXPOSURE TIME - (MIN.) FIG. 5. Survival curves of Micrococcus radiodurans grown in PCNZ or TGYM broth and exposed to UV while suspended in phosphate buffer. Exposure rate was 100 ergs per cm2 per sec. TABLE 4. Change in the -y radiation resistance of Micrococcus radiodurans by different exposure times to TGYM or PCNZ broth. Cells were irradiated in phosphate buffer Viable cells/ml after exposure to medium Exposure time Exposure medium Survival 0 krad 700 krad hr % TGYM 0 TGYM 139 X 10O 81 X PCNZ 0 PCNZ 170 X X TGYM 3 PCNZ 245 X X PCNZ 3 TGYM 145 X X TGYM 44 PCNZ 224 X X PCNZ 44 TGYM 135 X X

7 184 KRABBENHOFT, ANDERSON, AND ELLIKER APPL. MICROBIOL. TABLE 5. Survival rate ofmicrococcus radiodurans grown in PCNZ or TGYM broth and shaken in phosphate buffer for 10 hr prior to irradiation Growth medium No. of survivors after exposure to 0 krad 700 krad Sur- vival PCNZ broth X X PCNZ-shaken in buffer X X TGYM broth X 10e 70 X TGYM-shaken in buffer X X bility of lethal cell damage), or that the number of radioprotective units had been decreased by the growth media. The LD50 was approximately 370 and 700 krad for cells grown in PCNZ and TGYM broth, respectively. A similar alteration in the survival curve was observed when cells from the two types of cultures were exposed to UV while suspended in buffer. The survival curve for the TGYM-grown cells was similar to that previously reported (8). However, cells grown in PCNZ broth were approximately one-tenth as resistant as the TGYM cells. This degree of resistance was now comparable to those reported (8) for nonsporeformers or spores of Bacillus globigii when exposed to UV. There was no difference in the relative amounts of DNA, RNA, and protein, or in the ph of the medium of M. radiodurans when grown in the two different media, and it did not appear that the 10-fold difference in radiation sensitivity was due to quantitative differences in these factors. The ratio of DNA to RNA agrees with literature values (5), and the amount of DNA agreed with that previously reported (20). Phase-contrast microscopic studies indicated no change in the typical tetrad cellular arrangement, and protein measurements indicated that the cell mass was similar for the two different cultures. The response of M. radiodurans to UV was similar in some respects to its response to y radiation. Mechanisms of resistance to UV and to y rays may have some factors in common, and it has recently been suggested that there is a similarity of repair of ionizing- and UV-radiation damage in M. radiodurans (19). Both forms of radiation cause formation of peroxides in the medium (11). UV radiation in some cases causes radiation reactions resembling those of ionizing radiations, but the big difference between the two types of radiation is most frequently depicted as one of ionization instead of excitation of molecules. Evidence that the growth media caused cellular biochemical alterations was demonstrated by showing that the modification in radiation resistance could be reversed by a 3-hr exposure period to a growth medium different from the initial growth medium. Cells grown in TGYM, washed and exposed to PCNZ for 3 hr, were 10 times more radiation-resistant than those exposed 44 hr. On the other hand, cells grown in PCNZ and exposed to TGYM for 3 hr had approximately the same percentage of survival as cells exposed for 44 hr to TGYM. Since cells grown in PCNZ became resistant within 3 hr after transfer to TGYM, then precursors of factors related to increased resistance were probably present in cells originally grown in PCNZ, but the factors themselves could not be synthesized in PCNZ. Additional evidence to support this possibility was provided from experiments in which endogenous metabolism of PCNZ cultures resulted in cells with radiation resistance characteristics comparable to those grown in TGYM medium. Visual changes in pigmentation were also observed to be reversed by exposing the cells to the two different media. Quantitative differences in the pigments from the two different cultures were demonstrated by thin-layer chromatography and will be considered elsewhere (Krabbenhoft et al., in preparation). ACKNOWLEDGMENT This investigation was supported by a National Aeronautics and Space Administration Training Grant. LITERATURE CITED 1. ALPER, T., AND N. E. GILLIES Restoration of E. coli strain B after irradiation; its dependence on suboptimal growth conditions. J. Gen. Microbiol. 18: ALPER, T., AND N. E. GILLIES The relationship between growth and survival after irradiation of E. coli B and two resistant mutants. J. Gen. Microbiol. 22: ANDERSON, A. W., H. C. NORDAN, R. F. CAIN, G. PARRISH, AND D. DUGGAN Studies on the radio-resistant micrococcus. I. The isolation, morphology, cultural characteristics and resistance to gamma radiation. Food Technol. 10: BRUCE, A. K Extraction of the radioresistant factor of Micrococcus radiodurans. Radiation Res. 22: DAVIDSON, J. N The biochemistry of the nucleic acids. John Wiley & Sons, Inc., New York.

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