APPLIED MICROBIOLOGY, Jan., 1967, pp. 178-185Copyright © 1967 American Society for Microbiology

Vol. 15, No. 1Prinzted in U.S.A.

Influence of Culture Media on the RadiationResistance of Micrococcus radiodurans

K. L. KRABBENHOFT,' A. W. ANDERSON, AND P. R. ELLIKERDepartment 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 iny radiation resistance of Micrococcus radiodurans. The level of radiation resistancewas inversely related to the concentration of NZ-case. The LD5o for this organismwas approximately 700 krad when grown in tryptone, glucose, yeast extract, andDL-methionine (TGYM) broth, but it was approximately one-half as resistantwhen grown in a PC medium containing 0.5% NZ-case (PCNZ). The resistance toultraviolet light was also reduced. Cultures transferred from PCNZ to TGYMmedia regained the high level of resistance.

Micrococcus radiodurans is a nonsporeform-ing, pink-pigmented tetracoccus which was iso-lated from irradiated cans of meat (3) and hasbeen recently isolated from other sources (13). Itis not pathogenic or heat-resistant, but is resistantto ultraviolet (UV) irradiation (8), and has anLD5o of 500 to 700 krad for y radiation in phos-phate buffer. Strains of other species with similarresistance properties have been isolated morerecently (6).The unusually high radiation resistance of this

organism has been attributed to various factors,including sulfhydryl compounds (4), an extremelyefficient repair system for damaged deoxyribo-nucleic acid (DNA; 21), and unique ultrafinestructures in the cell wall (23). The pigments ofM. radiodurans have been identified as being ofthe carotenoid type, but attempts to correlateradiation resistance with the presence of thesepigments have not been successful (12, 15) orconsistent (17, 18).

Preliminary studies from earlier work (13)indicated that a change in the type of culturemedium increased the cell yield of M. radio-durans by approximately twofold. The presentstudy was undertaken to determine what in-fluence the culture medium has on the radio-resistance of this microorganism.

MATERIALS AND METHODSMicroorganisms. A culture of M. radiodurans was

obtained from the stock culture collection of theI Present address: Department of Biology, New

Mexico State University, Las Cruces.

Department of Microbiology at Oregon State Univer-sity, Corvallis. The culture was maintained on slantsof TGYM and PCNZ media, which are described be-low.

Media. TGYM medium, which has been used forroutine propagation of this microorganism and inearlier radiation studies in our laboratory, consistedof the following (per liter): tryptone (Difco), 5 g; glu-cose, 1 g; yeast extract (Difco), 1 g; DL-methionine,20 mg. Subsequent studies indicated growth was supe-rior on PCNZ medium, which consisted of plate countmedium (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 ofboth media was 7.0. For plate counts, 1.5% agar wasadded to the growth medium.

Culture conditions. Cells harvested from 44-hrTGYM or PCNZ cultures were used in all radiationexperiments unless otherwise specified. These wereprepared by using a 1% inoculum of a 44-hr cultureand incubating at 30 C on a shaker-incubator.

Harvesting of cells. Cells were harvested by centrif-ugation (7,000 X g, 20 min), washed twice, and re-suspended in 0.067 M phosphate buffer (pH 7.0). Celldensity was adjusted with buffer to contain 108 cellsper milliliter.

Radiation. Duplicate 10-ml quantities of cell sus-pensions were transferred to glass radiation vials andirradiated at room temperature with the cobalt-60source at Oregon State University. Unless otherwisespecified, the exposure was 700 krads with an admin-istered dose rate of 713 krad/hr. The LD5o of theTGYM and PCNZ cultures was determined by useof radiation levels of 100 to 700 krads.

Cell suspensions containing 104 cells per milliliterwere used for UV exposure (100 ergs per cm2 per sec).Samples of 1 ml in triplicate were uniformly spreadover the surface of flat-bottom petri plates by hand

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CULTURE MEDIA AND RADIATION RESISTANCE

agitation, and the plates were gently agitated on a

rotator (Eberbach Corp., Ann Arbor, Mich.) duringthe exposure period.

Relative effect ofgrowth and recovery medium. It hasbeen previously reported (1, 2, 9, 22) that the differ-ence in radiation resistance of Escherichia coli B andE. coli B/r lay not in the initial damage, but in themetabolic processes after irradiation, including thepostirradiation conditions of recovery medium.

In the present study, irradiated cells which had beengrown in TGYM broth were recovered on TGYMagar for controls and also on PCNZ agar for compara-tive purposes. In a similar manner, cells initially grownin PCNZ were recovered on PCNZ andTGYM agar.

Effect of different levels of NZ-case. A series ofPCNZ media were inoculated in duplicate; the mediacontained the following percentages of NZ-case: 0.01,0.05,0.1, 0.2. 0.3, 0.4, and 0.5. Because M. radioduranshas a consistent high level of radiation resistancewhen 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 inoculatingthe two kinds of media in triplicate.The effect of physiological age on radiation resist-

ance was also investigated. By adjusting the time ofinoculation, cultures of five different physiologicalages could be harvested and subsequently irradiatedsimultaneously.

Biochemical determinations. DNA and ribonucleicacid (RNA) measurements were made by use of cellswashed twice in saline and resuspended in a solution(5 ml per 100 ml of culture) of 0.15 M saline and 0.1 Methylenediaminetetraacetic acid (tetrasodium salt).DNA and RNA were liberated by hydrolysis with11.7 N perchloric acid at 70 C for 10 min. The suspen-sion was mixed once for 30 sec with a Vortex Juniormixer during the heating period, and, after cooling,it was centrifuged (3,000 X g, 20 min); the super-natant fluid was decanted. This supernatant fluid wasused 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 proteinby the biuret method (10). Relative amounts of DNA,RNA, and protein were compared on a dry weightbasis.

Reversibility of radiation resistance. Initial studiesindicated that the degree of resistance could be con-

trolled by altering the NZ-case concentration in thegrowth medium. To test the possibility that the radia-tion resistance also could be reversed, 44-hr washedcultures grown in TGYM and PCNZ broth wereresuspended in the same or opposite medium for 3 or

44 hr. After washing the cells free of the new mediumand 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 cellsresuspended in buffer and placed on a shaker (10 hrat 30 C). After centrifugation, the cells were againresuspended 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 onthe percentage of survival of irradiated M.radiodurans is given in Table 1. Cells grown inTGYM were 10 times more radiation-resistantthan those grown in PCNZ broth.

Cell cultures grown in PCNZ produced a clear,brown-colored liquid within 44 hr, whereas theTGYM broth was tan. Both media were the samecolor initially. The color change began to occurafter approximately 20 hr of incubation. Cen-trifuged cells grown in TGYM medium werebright pink, whereas those grown in PCNZ werea faded, dull pink color.The survival of M. radiodurans was not altered

when it was grown on one medium and recoveredon the same or another medium after irradiation(Table 1). Cells grown in TGYM broth and re-covered on TGYM or PCNZ agar after irradia-tion (700 krad) had approximately 60% survival.On the other hand, cells initially grown in PCNZbroth had approximately 6% survival, regardlessof whether they were recovered on PCNZ orTGYM medium. The initial cell concentrationwas similar in all cases before irradiation.

Effects of major component differences ofmediaon radiation resistance. M. radiodurans was grownin three different media to determine the effectof various additives on its radiation resistance.These additives were ones which representedmajor differences in PCNZ and TGYM media.The addition of DL-methionine to PCNZ brothat the same concentration used in TGYM brothdid not produce increased radiation resistance(Table 2). When the yeast extract concentrationwas increased in TGYM to levels comparable tothat in PCNZ medium, the resistance of the or-ganisms was approximately the same as for thosegrown in the standard TGYM broth. However,

TABLE 1. Effect of growth and recovery media onsurvival of Micrococcus radiodurans exposed

to 700,000 rad -y irradiation in phosphatebuffer

Viable cells/mlaGrowth Recovery Sur-medium medium Before After vival

irradiation irradiation

TGYM TGYM 207 X 101 124 X 106 60.0TGYM PCNZ 189 X 106 121 X 106 64.0PCNZ PCNZ 123 X 101 77 X 105 6.3PCNZ TGYM 140 X 106 86 X 105 6.1

a Average of triplicate plates of duplicateirradiated samples from three experiments.

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KRABBENHOFT', ANDERSON, AND ELLIKER

TABLE 2. Effect of various growth media on the survival of Micrococcus radiodurans exposed to700,000 rad -r irradiation while suspended in phosphate buffer

Viable cells/mlaMedium Survival

Before radiation After radiation

TGYM................................... 43 X 107 29 X 107 68.1PCNZ (0.01% NZ)......................... 188 X 106 127 X 106 67.4PCNZ (0.05% NZ)......................... 211 X 106 93 X 106 44.3PCNZ (0.1% NZ).......................... 162 X 106 41 X 106 25PCNZ (0.3% NZ).......................... 197 X 106 226 X 105 11.5PCNZ (0.5% NZ).......................... 162 X 106 76 X 106 4.5TGYM plus NZ(0.5%)).................(37 X 107 38 X 105 1.0TGYM plus yeast extract (0.15%) .......... 47 X 107 29 X 107 61.2PCNZ plus DL -methionine (0.002%) ........ 38 X 107 64 X 105 1.7

a Average of triplicate plates of duplicate irradiated samples from three experiments.

l9

I08j2~-8(I 10-J

-JLZJU0 7Z 10

6I00

TIME - (HOURS)

FIG. 1. Growth curves of Micrococcus radioduransgrown in PCNZ or TGYM broth at 30 C on a shaker.

the addition of NZ-case to TGYM medium re-duced the resistance to a level comparable forcells grown in PCNZ broth containing 0.5%NZ-case.The higher the concentration of NZ-case in the

growth medium, the lower was the observedradiation resistance (Table 2). The survival ofcells grown in the absence of NZ-case was 68%,whereas approximately 5% of the cells survivedwhen they had been grown in the presence of0.5% NZ-case. The least radiation-resistantcultures were also less brightly pigmented.

Growth ofM. radiodurans in PCNZ and TGYMmedia. When M. radiodurans was grown inPCNZ broth, it entered the death phase after 65hr of incubation, whereas the cells remained inthe stationary phase for at least 96 hr whengrown in TGYM medium (Fig. 1). The rate ofgrowth in the log phase was essentially the samefor the two media.The effect of physiological age on radiation

TABLE 3. Effect ofphysiological age onZ radiationof Micrococcus radiodurans grown in two

different media and irradiated inphosphate buffer

Viable cells/ml after exposure to

medium Culture vival0 krad 700 krad

TGYM 8 210 X 106 281 X 105 13.320 105 X 106 52 X 106 49.532 136 X 106 48 X 106 35.344 133 X 106 90 X 106 67.556 127 X 106 46 X 106 36.2

PCNZ 8 228 X 106 85 X 105 3.720 164 X 106 70 X 105 4.332 159 X 106 93 X 105 5.844 182 X 106 51 X 105 2.856 144 X 106 32 X 105 2.2

resistance indicated that cells grown in PCNZbroth were at least 10 times more radiation-sensitive than cells grown in TGYM at all agestested (Table 3). The single exception to thisgeneral observation was that the 8-hr culturegrown in TGYM broth was only four times moreresistant than the PCNZ culture. With thatexception, the survival for the TGYM culturesranged from 35 to 67%, whereas the survival ofthe irradiated PCNZ cultures ranged from 2 to6% at the different ages.

Effect of altered radiation resistance on certainchemical factors and cell morphology. Chemicaldeterminations indicated that there was 4.5%'RNA, 1% DNA, and 47% protein for both thehighly radiation-resistant and the more radiation-sensitive cultures. Hence, there was no differencein the relative amounts of these substances be-tween cells grown in PCNZ and TGYM.

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The initial pH of the growth medium was 7.0,and the final pH was 8.0 and 8.2 for a 44-hr cul-ture in TGYM and PCNZ broth, respectively.In addition, the relative dry weight and thepacked cell volume of the cells grown in TGYMwere 25% less than an equal volume of cellsgrown in PCNZ broth. Evidence that this in-crease in cell mass of the PCNZ cultures wasbecause of higher numbers of cells and not be-cause of an increase in cell size or morphologywas apparent from the growth curves (Fig. 1)and phase-contrast microscopic studies (Fig.2 and 3).

Radiation dose-level responses. The LD5o valuesof M. radiodurans grown in the two differentmedia were found to be significantly different(Fig. 4). The LD5o for cells grown in PCNZ brothwas approximately 370 krad, as compared to 700krad for TGYM-grown cells.

Effect ofgrowth media on UV resistance. WhenM. radiodurans was grown in PCNZ broth andsuspended in phosphate buffer, its resistance toUV was approximately one-tenth of that of similarcells grown in TGYM broth (Fig. 5). A sig-moidal survival curve was obtained for cellsgrown in TGYM broth. However, the rate of

cellular inactivation was exponential for cellsgrown in PCNZ broth.

Reversibility of radiation resistance. When M.radiodurans was grown in TGYM broth and thenexposed to PCNZ for 44 hr, its radiation re-sistance was found to be 10% of that for cellsgrown in TGYM and exposed for 3 hr to PCNZbroth (Table 4). On the other hand, the radiationresistance for cells grown in PCNZ broth and thenexposed to TGYM broth for either 3 or 44 hr wasfound to be similar for both exposure periods. Thegeneration times for M. radiodurans in theTGYMand PCNZ media were 50 and 38 min, respec-tively. This means that a 3-hr incubation periodwas sufficient for the production of only two orthree generations of cells. For controls, cultureswere exposed to fresh medium comparable to theinitial growth medium.

Effect of endogenous metabolism on radiationresistance. When M. radiodurans was grown inPCNZ broth and then shaken in phosphate bufferfor 10 hr, its radiation resistance increased morethan 10 times (Table 5). Control experimentswith cells grown in TGYM broth indicated nosignificant change in resistance when they wereshaken in buffer.

FIG. 2. Phase-contrast photomicrograph of Micrococcus radiodurans grown in TGYM broth at 30 C for 44 hr.X 35,000.

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KRABBENHOFT, ANDERSON, AND ELLIKER

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 agarhas been reported (6) to provide an excellent iso-lation medium for obtaining a radiation-resistanto0ange-brown micrococcus from irradiated had-dock tissues. A similar NZ-case medium has beenused (13) to investigate possible sources of M.radiodurans. PCNZ medium provided a largercell yield than TGYM, but the cultures were 10times more radiation-sensitive and also less pig-mented 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 recoverymedium produced higher counts of the resistantstrain and fewer radiation survivors of the sensi-tive strains. It was concluded on the basis of theseresults that the difference between the two strainslay not in the initial radiation damage, but intheir metabolic processes immediately afterirradiation. In the present study, no "recoverymedium effect" was observed for M. radioduranswhich was grown on TGYM or PCNZ mediumand which was recovered on either of these twomedia. Additional studies indicated there wereno effects from the major component differences

of the two media on the radiation resistance ofthis microorganism.When M. radiodurans was grown in PCNZ

broth containing various levels of NZ-case, aninverse relationship was observed between theconcentration of NZ-case and radiation re-sistance. In addition to altered resistance, cellsgrown in higher levels of NZ-case appeared to bethe least pignented. This observation suggestedthat pigmentation and radiation resistance were,in some manner, related and that NZ-case wascapable of altering these factors. Studies arepresently being conducted to consider thesepossibilities.Growth characteristics of M. radiodurans in the

two different media indicated similar growthpatterns. An earlier death phase for the PCNZculture probably was a reflection of a larger num-ber of cells depleting the available nutrients orproducing toxic waste materials more rapidly thana lesser number of cells in TGYM. If unbalancedgrowth was occurring in one of these media,it was not apparent from the standard growthcurves.

Further evidence that unbalanced growth didnot occur in PCNZ was provided from experi-ments on the effect of physiological age on radia-

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CULTURE MEDIA AND RADIATION RESISTANCE

tion resistance. At all physiological ages tested,M. radiodurans was more resistant when grownin TGYM as compared to PCNZ. The generalobservation that radiation resistance of M. radio-durans does not change with age was in agree-ment with previous work by Lee (Ph.D. Thesis,Oregon State University, Corvallis, 1963), al-though the percentage of survival was about twiceas great in the present studies. This may have beendue to a difference in growth media.When M. radiodurans was grown inTGYM and

irradiated in buffer at different dosage levels, the

1009YM~Y

PCNZD .\

z~~OE RAD x10

U)

bi

0~

I1 3 4 56 7

DOSE- RADS x 10O5FIG. 4. Dose survival curves for Micrococcus radio-

durans grown in PCNZ or TGYM broth and exposed tory radiation while suspended in phosphate buffer. Irradia-tion was conducted in air at room temperature at a doserate of 713 krad/hr.

survival curve was characterized by a wide"shoulder." However, the survival curve de-creased exponentially after 350 krad for cells

grown in PCNZ. The wide- "shoulder" sig-moidal survival curves are typically found inradiation-resistant organisms (14, 18) and are

often referred to as multihit (multitarget ormultiunit) curves, because they suggest a multi-plicity of events required to bring about inactiva-tion. The cells grown in PCNZ did not produce awide "shoulder" in their survival curve, and thissuggests that the number of radiation-sensitivesites had been increased (i.e., the greater thenumber of sensitive sites, the greater the proba-

100

U1)

0

DUf)

z

CLJ0-

0

10

1.0

0.1

0 2 3

EXPOSURE TIME - (MIN.)FIG. 5. Survival curves of Micrococcus radiodurans

grown in PCNZ or TGYM broth and exposed to UVwhile suspended in phosphate buffer. Exposure rate was100 ergs per cm2 per sec.

TABLE 4. Change in the -y radiation resistance of Micrococcus radiodurans by different exposuretimes to TGYM or PCNZ broth. Cells were irradiated in phosphate buffer

Viable cells/ml after exposure to

medium Exposure time Exposure medium Survival0 krad 700 krad

hr %TGYM 0 TGYM 139 X 10O 81 X 106 58.2PCNZ 0 PCNZ 170 X 106 48 X 105 2.8TGYM 3 PCNZ 245 X 106 118 X 106 48.0PCNZ 3 TGYM 145 X 106 110 X 106 75.4TGYM 44 PCNZ 224 X 106 106 X 105 4.5PCNZ 44 TGYM 135 X 106 95 X 106 70.3

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TABLE 5. Survival rate ofMicrococcus radioduransgrown in PCNZ or TGYM broth and shaken

in phosphate buffer for 10 hr prior toirradiation

No. of survivors afterexposure to Sur-

Growth medium vival

0 krad 700 krad

PCNZ broth..... 149 X 106 52 X 105 3.5PCNZ-shaken

in buffer....... 157 X 106 68 X 106 43.3TGYM broth.... 120 X 10e 70 X 106 58.4TGYM-shaken

in buffer....... 311 X 106 53 X 106 40.5

bility of lethal cell damage), or that the numberof radioprotective units had been decreased bythe growth media. The LD50 was approximately370 and 700 krad for cells grown in PCNZ andTGYM broth, respectively.A similar alteration in the survival curve was

observed when cells from the two types of cul-tures were exposed to UV while suspended inbuffer. The survival curve for the TGYM-growncells was similar to that previously reported (8).However, cells grown in PCNZ broth were ap-proximately one-tenth as resistant as the TGYMcells. This degree of resistance was now com-parable to those reported (8) for nonsporeformersor 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 themedium of M. radiodurans when grown in thetwo different media, and it did not appear thatthe 10-fold difference in radiation sensitivity wasdue to quantitative differences in these factors.The ratio ofDNA to RNA agrees with literaturevalues (5), and the amount of DNA agreed withthat previously reported (20). Phase-contrastmicroscopic studies indicated no change in thetypical tetrad cellular arrangement, and proteinmeasurements indicated that the cell mass wassimilar for the two different cultures.The response of M. radiodurans to UV was

similar in some respects to its response to y radia-tion. Mechanisms of resistance to UV and toy rays may have some factors in common, and ithas recently been suggested that there is a simi-larity of repair of ionizing- and UV-radiationdamage in M. radiodurans (19). Both forms ofradiation cause formation of peroxides in themedium (11). UV radiation in some cases causes

radiation reactions resembling those of ionizingradiations, but the big difference between the twotypes of radiation is most frequently depicted as

one of ionization instead of excitation of mole-cules.

Evidence that the growth media caused cellularbiochemical alterations was demonstrated byshowing that the modification in radiation re-sistance could be reversed by a 3-hr exposureperiod to a growth medium different from theinitial growth medium. Cells grown in TGYM,washed and exposed to PCNZ for 3 hr, were 10times more radiation-resistant than those ex-posed 44 hr. On the other hand, cells grown inPCNZ and exposed to TGYM for 3 hr had ap-proximately the same percentage of survival ascells exposed for 44 hr to TGYM. Since cellsgrown in PCNZ became resistant within 3 hrafter transfer to TGYM, then precursors offactors related to increased resistance were prob-ably present in cells originally grown in PCNZ,but the factors themselves could not be synthe-sized in PCNZ. Additional evidence to supportthis possibility was provided from experiments inwhich endogenous metabolism of PCNZ culturesresulted in cells with radiation resistance charac-teristics comparable to those grown in TGYMmedium.

Visual changes in pigmentation were also ob-served to be reversed by exposing the cells to thetwo different media. Quantitative differences inthe pigments from the two different cultures weredemonstrated by thin-layer chromatography andwill be considered elsewhere (Krabbenhoft et al.,in preparation).

ACKNOWLEDGMENT

This investigation was supported by a NationalAeronautics and Space Administration TrainingGrant.

LITERATURE CITED

1. ALPER, T., AND N. E. GILLIES. 1958. Restorationof E. coli strain B after irradiation; its depend-ence on suboptimal growth conditions. J. Gen.Microbiol. 18:461-465.

2. ALPER, T., AND N. E. GILLIES. 1960. The relation-ship between growth and survival after irradia-tion of E. coli B and two resistant mutants. J.Gen. Microbiol. 22:113-116.

3. ANDERSON, A. W., H. C. NORDAN, R. F. CAIN, G.PARRISH, AND D. DUGGAN. 1956. Studies onthe radio-resistant micrococcus. I. The isola-tion, morphology, cultural characteristics andresistance to gamma radiation. Food Technol.10:575-577.

4. BRUCE, A. K. 1964. Extraction of the radioresist-ant factor of Micrococcus radiodurans. Radia-tion Res. 22:155-164.

5. DAVIDSON, J. N. 1950. The biochemistry of thenucleic acids. John Wiley & Sons, Inc., NewYork.

184 APPL. MICROBIOL.

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6. DAVIS, N. S., G. J. SILVERMAN, AND E. B.MASUROVSKY. 1963. Radiation-resistant, pig-mented coccus isolated from haddock tissue. J.Bacteriol. 86:294-298.

7. DISCHE, Z. 1955. Color reactions of nucleic acidcomponents, p. 285-305. In E. Chargaff and J.N. Davidson [ed.], The nucleic acids, vol. 1.

Academic Press, Inc., New York.8. DUGGAN, D. E., A. W. ANDERSON, P. R. ELLIKER,

AND R. F. CAIN. 1959. Ultraviolet exposurestudies on a gamma radiation resistant micro-

coccus isolated from food. Food Res. 24:376-382.

9. GILLIES, N. E. 1961. The use of autotrophicmutants to study restoration in Escherichia coliB after ultraviolet irradiation. Intern. J. Radia-tion Biol. 3:397-403.

10. GORNALL, A. G., C. S. BARDAWILL, AND M. M.DAVID. 1949. Determination of proteins andtheir derivations. J. Biol. Chem. 177:751-766.

1 1. KELNER, A., W. D. BELLAMY, G. E. STAPLETON,AND M. R. ZELLE. 1955. Symposium on radia-

tion effects on cells and bacteria. Bacteriol.

Rev. 19:22-44.12. KILBURN, R. E., W. 0. BELLAMY, AND S. A.

TERNI. 1958. Studies on a radiation-resistant

Sarcina sp. Radiation Res. 9:207-215.13. KRABBENHOFT, K. L., A. W. ANDERSON, AND P.

R. ELLIKER. 1965. Ecology of Micrococcusradiodurans. Appl. Microbiol. 13:1030-1037.

14. LEE, J. S., A. W. ANDERSON, AND P. R. ELLIKER.1963. The radiation-sensitizing effects of N-

ethylmaleimide and iodoacetic acid on a radia-

tion-resistant Micrococcus. Radiation Res. 19:593-598.

15. MATHEWS, M. M., AND N. I. KRINSKY. 1965. Therelationship between carotenoid pigments andresistance to radiation in non-photosyntheticbacteria. Photochem. Photobiol. 4:813-817.

16. MEJBAUM, W. 1939. Determination of pentoses.Z. Physiol. Chem. 258:117-120.

17. MOSELEY, B. E. B. 1963. The variation in x-ray

resistance of Micrococcus radiodurans and someof its less pigmented mutants. Intern. J. Radia-tion Biol. 6:489.

18. MOSELEY, B. E. B., AND H. LASER. 1965. Repairof x-ray damage in Micrococcus radiodurans.Proc. Roy. Soc. (London) Ser. B 162:210-222.

19. MOSELEY, B. E. B., AND H. LASER. 1965. Similarityof repair of ionizing and ultra-violet radiationdamage in Micrococcus radiodurans. Nature206:373-375.

20. MOSELEY, B. E. B., AND A. H. SCHEIN. 1964. Ra-diation resistance and deoxyribonucleic acidbase composition of Micrococcus radiodurans.Nature 203:1289-1299.

21. SETLOW, J. K., AND D. E. DUGGAN. 1964. Theresistance of Micrococcus radiodurans to ultra-violet radiation. I. Ultraviolet induced le-sions in the cells DNA. Biochim. Biophys.Acta 87:664-668.

22. STAPLETON, G. E., A. J. SBARRA, AND A. HOL-LAENDER. 1955. Some nutritional aspects ofbacterial recovery from ionizing radiations. J.Bacteriol. 70:7-15.

23. THORNELY, M. J., R. W. HORNE, AND A. M.GLAUERT. 1965. The fine structure of Micro-coccus radiodurans. Arch. Mikrobiol. 51:267-289.

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