JOURNAL OF BACTERIOLOGYVol. 88, No. 3, p. 545-552 September, 1964Copyright @ 1964 American Society for Microbiology

Printed in U.S.A.

VIABILITY AND METABOLISM OF STAPHYLOCOCCUSAUREUS AFTER FREEZING, LYOPHILIZATION,

AND IRRADIATIONT. H. AHN, H. NISHIHARA, CHARLES M. CARPENTER, AND G. V. TAPLIN

Harry N. Falk Laboratory, School of Public Health, and Laboratory of Nuclear Medicine and RadiationBiology, Department of Biophysics-Nuclear Medicine, Center for Health Sciences, University

of California, Los Angeles, California

Received for publication 2 January 1964

ABSTRACT

AHN, T. H. (University of California, Los An-geles), H. NISHIHARA, CHARLES M. CARPENTER,AND G. V. TAPLIN. Viability and metabolism ofStaphylococcus aureus after freezing, lyophiliza-tion, and gamma irradiation. J. Bacteriol. 88:545-552. 1964.-Reproductive viability and oxidativeactivity on various substrates (alanine, glutamicacid, glucose, lactic acid) were studied in Staph-ylococcus aureus cells subjected to (i) freezingand thawing, (ii) lyophilization, (iii) irradiationin suspension, (iv) irradiation in lyophilized state,and (v) lyophilization and irradiation in the pres-ence of various substrates. Freezing and thawingin dilute albumin solution had no effect on viablecell count and on lactic acid oxidation, but theoxidation rates of alanine, glutamic acid, and glu-cose were decreased. Lyophilization reduced theviable cell count to 25 to 30% of the initial value,and decreased the oxidation rates of glutamicacid and alanine proportionally, whereas decreasein glucose oxidation was less than proportional.The lactic acid oxidation rate was not affected bylyophilization. An irradiation dosage of about40,000 r was required to reduce the viable countof cells irradiated in suspension by one logio unit;lyophilized cells required 140,000 to 170,000 r.Cells irradiated in suspension with dosages suffi-cient to render them completely nonviable onculture continued to respire on lactic acid at about60% the rate of nonirradiated cells, but the resid-ual activities on the other substrates tested wereless than 10 to 12%. Lyophilized cells irradiatedwith sufficient dosages for cultural nonviabilityretained 40 to 80% of the oxidative capacity ofnonirradiated cells on the test substrates. Cellslyophilized and irradiated in the presence of al-bumin generally retained a greater portion of theiroxidative activities compared with cells lyophil-ized and irradiated in buffer. Cells irradiated inthe presence of various oxidizable substrates gavevarying results, depending both on the substratepresent during irradiation and the substrate addedfor the oxidative studies.

Previous studies on the effects of gammairradiation on Mycobacterium tuberculosis andBrucella abortus showed that bacterial cellsirradiated with gamma rays under conditionswhich render them nonreproductive on suitableculture media and noninfectious for experimentalanimals retain their ability to oxidize varioussubstrates (Ahn et al., 1962). In a search for theideal irradiation medium which will aid in render-ing bacterial cells nonreproductive with the leastdamage to other cellular functions, further studieswere carried out with Staphylococcus aureus cells.The present paper reports the results of studieson the effects of the presence of water and varioussubstrates during irradiation on the metabolicactivity and reproductive ability of the S. aureuscells. Inasmuch as most of our studies deal withcells lyophilized prior to irradiation, the resultsof studies on the effects of freezing and lyophiliza-tion are included.The aim of our studies, to delineate a method

of irradiating bacterial cells which will renderthe cells nonreproductive with minimal damage toother cellular functions, was dictated by the ideathat such cells of pathogenic organisms may beusable as vaccines. It was hoped that thesecells would be incapable of causing disease andyet would retain an immunogenic potency equiva-lent to that of the living virulent cell.

MATERIALS AND METHODS

Test organism. Several strains of S. aureus weretested for oxidative activity on various substrates(Table 1), and strain 1503 was selected as arepresentative strain for the studies on the effectsof freezing, lyophilization, and irradiation onreproductive viability and oxidative activity ofthe cells. This strain is sensitive to ten phages,including types 80 and 81.

Preparation of cell suspension. The Staphylococ-545

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TABLE 1. Oxygen uptake of freshly preparedsuspensions of Staphylococcus aureus

resting cells

Substrate*

Buffer ..............

D-Alanine ...........

L-Arginine ..........

L-Asparagine ........

L-Aspartic acid.....L-Citrulline .........

L-Glutamic acid.....L-Histidine .........

L-Lysine ............

DL-Ornithine ........

L-Proline ...........

L-Threonine.........DL-Tryptophan ....

D-Galactose.........Glucose.............Inositol .............

Lactose .............

D-MannitolSucrose .............

Lactic acid.........Pyruvic acid........

Strain

1503

39t3324587531384214040155302414255

563464082

521578412

2253

32207333130834193129135208313075595302977

329495426

F21

3720530383839

362404298250403882488313296343581477

3189

4343350654895

5213938122211484053587424056

455498389

Wood46

30313302829112460303295202303144522303267

481523412

* All substrates were 1% solutions, except lac-tic and pyruvic acids which were 0.5 M solutions.

t Oxygen uptake expressed in microliters perhour per 5 X 109 cells.

cus cells were cultured in large Roux bottlescontaining a 100-ml layer of Brain Heart InfusionAgar (Difco). Each Roux bottle was inoculatedwith cells from a 48- to 72-hr agar slant culturein a test tube (25 by 150 mm) freshly suspended in5 to 10 ml of sterile 0.85% NaCl. The cultureswere incubated for 20 hr at 37 C. The growthfrom several Roux bottles was harvested in 0.85%NaCl, pooled, washed twice, and resuspended insaline in a concentration of approximately 10billion cells per ml. This concentration of cellsdiluted 1:20 showed a light transmittance of 30to 32% at 600 m, on a Coleman Junior spectro-photometer. Live-cell counts were obtained bythe conventional pour plate method.The saline suspension of the cells was distrib-

uted in equal amounts into an appropriatenumber of tubes, and was centrifuged for 30 minat 1,200 to 1,300 X g. The supernatant fluidwas discarded, and the cells were resuspended in

various solutions in a concentration of about 30billion cells per ml, that is, in one-third theoriginal volume. Each suspension was thendistributed into screw-cap test tubes (13 by 100mm), some of which were lyophilized and othersof which were frozen and stored at -20 C.Physiological (0.85%) saline and 2% Dubosalbumin (Difco) solution in distilled water wereused as suspending medium for lyophilization orirradiation, or both, of the S. aureus cells. Alsotested as suspending medium were 1% solutionsof D-alanine, L-glutamic acid, L-proline, glucose,and sucrose, and a 0.5 M solution of lactic acid inSorenson's phosphate buffer (pH 7).

Lyophilization. The cell suspensions for lyoph-ilization were previously frozen in a freezerat -20 C on a slant in screw-capped test tubes.The test tubes were momentarily dipped into anacetone-Dry Ice bath and connected to the portsof a stainless-steel VirTis Bio-dryer with shortlengths of rubber tubings. The vacuum wasprovided by a two-stage Welch pump with aguaranteed vacuum of 0.05 Iu. The cells werelyophilized for 6 hr.Gamma irradiation. Irradiation was carried out

by exposure of the cells in the test tubes to ahighly active cobalt-60 source. The exposuretime for a dosage of 5 X 105 r was 3 min, for 106 r,6 min. Exposure times for the other dosages werein proportion. Cells to be irradiated in suspensionwere kept frozen until just prior to irradiationand were refrozen immediately afterwards forstorage until used. The lyophilized cells werestored in a refrigerator at 0 to 1 C before andafter irradiation. The storage period did notexceed 15 days from the date of lyophilizationand irradiation before use.

Manometric studies of oxygen uptake. To eachtube of cells, Sorenson's phosphate buffer (pH7) was added in an amount (to six times theoriginal volume) sufficient to make a suspensioncontaining approximately 5 X 109 cells per ml.The standard method of Umbreit, Burris, andStauffer (1954) was followed for the manometricmeasurements. The oxidative rates are expressedin microliters of oxygen consumed per hour by 5billion cells. This cell number was selected be-cause it was found to give readily measurableoxidative rates with the various substrates.Twenty substrates in Sorenson's phosphate

buffer (pH 7) were tested in a series of preliminaryexperiments (Table 1); 1% solutions of D-ala-

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GAMMA-IRRADIATED S. A UREUS

nine, L-glutamic acid, and glucose, and a 0.5 M

solution of lactic acid, were selected for the majorstudies. These latter compounds were mostvigorously oxidized by fresh resting-cell suspen-

sions of S. aureus, and are representative of thedifferent chemical groups tested in the prelimi-nary studies. Each Warburg flask contained 1.4ml of Sorenson's buffer and 1 ml of cell suspen-

sion in the main chamber, 0.5 ml of substratesolution in the side arm, and 0.1 ml of 20% KOHsolution in the center well. The temperatureequilibrating time was 15 min, both before andafter tipping in the substrate. Manometricreadings were taken at 30 min and at 1 hr.Duplicate oxygen uptake determinations were

made for each test mixture, and the resultsreported in the tables are the averages of theduplicate determinations.

Cells lyophilized and irradiated in the varioussubstrates were suspended directly in Sorenson'sbuffer for the manometric studies. Attempts atwashing out the substrates in which the cellswere lyophilized and irradiated resulted in toogreat a loss of cells. Warburg reaction mixturesusing these cells, therefore, contained oxidizablesubstrates carried over from the lyophilizationstage in addition to the test substrates added atthe time of the manometric studies. The amountof this carry-over substrate was equivalent toone-third of the concentration of the test sub-strate added.

RESULTS

Metabolism of fresh resting-cell suspension.Glutamic acid was most vigorously oxidized of

TABLE 2. Reproductive viability of Staphylococcusaureus strain 1503 gamma-irradiated

in suspension*

Reproductively viable cell number per mltirradiated in

Irradiationdosage

0.85% NaCl sfbubin 1% Glucose

r

0 4.6 X 109 5.1 X 109 5.0 X 1092.5 X 105 1.3 X 103 1.5 X 103 1.6 X 1035.0X 10' 0 0 37.5 X 10 0 0 0

* Cells were kept frozen in suspension beforeand after irradiation until used.

t Suspensions contained 5 X 109 cells on thebasis of viable-cell counts.

TABi,E 3. Effects of freezing and lyophilization in2% Dubos albumin solution on the oxygen uptake

of Staphylococcus aureus strain 1503 cells

Fresh Frozen and LyophilizedSubstrate suspension thawed and

resuspended

Buffer ............ 39* 53 35 (12) tD-Alanine (1%) 332 258 101 (100)L-Glutamic acid

(1%).421 285 119 (126)Glucose (1%) ...563 385 240 (169)Lactic acid (0.5 M). 578 570 587 (173)

Viable-cell count.. 5 X 109 5.1 X 109 1.5 X 109

* Oxygen uptake expressed in microliters perhour per 5 X 109 cells based on viable-cell count offresh suspension.

t Oxygen uptake expected on the basis of actualviable-cell count of 1.5 X 109 cells.

the amino acids tested by all of the strains ofStaphylococcus studied (Table 1). Alanine andproline were also well utilized. Among the sugars,glucose was most readily oxidized, followedclosely by sucrose. Lactic and pyruvic acids werealso readily utilized by the fresh cell suspensions.

Effects of freezing. Freezing and thawing in0.85% saline reduced the number of viable cellsslightly, but no decrease was noted in the viablecell count on plating cells frozen in dilute albuminor glucose solution (Table 2). Although no de-crease in viable cell count was noted, oxidationrates of alanine, glutamic acid, and glucose weredecreased in cells frozen in the albumin solutioncompared with rates in fresh cell suspension(Table 3). The oxidation rate of lactic acid wasnot affected by the freezing.

Effects of lyophilization. Lyophilization reducedthe viable cell count to 25 to 30% that of freshcell suspension (Table 4). Lyophilization in0.85% saline was slightly more deleterious thanlyophilization from dilute albumin or glucosesolution. Pait of the damage produced by lyoph-ilization in saline is attributable to the freez-ing, but the major damage occurred duringdrying, and the suspending medium had onlyslight, if any, influence in preventing this damageto cell viability.The oxygen uptake rates of cells lyophilized

in albumin solution with alanine, glutamic acid,and glucose were also decreased (Table 3). Thedecreases in the oxidation rates of the amino

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AHN ET AL.

acids were proportional to the decrease in viablecell count, but the decrease in oxygen uptakeon glucose was less than proportional. Theoxidation rate of lactic acid was not affected bylyophilization.No significant difference was noted in the

substrate oxidation rates of cells lyophilized inalbumin and in buffer (Table 5). Cells lyophilizedin the presence of various oxidizable substratesshowed increased oxygen uptake in buffer com-pared with cells lyophilized in buffer, due to thepresence of the substrates carried over with thecells (Table 5). In the reaction mixtures in whichsubstrates were added to cells, lyophilized inoxidizable substrates, the resulting total oxygenuptake rates were generally slightly less than

TABLE 4. Reproductive viability of gamma-irradiated lyophilized Staphylococcus

aureus strain 1503

Reproductively viable cell number per ml*lyophilized and irradiated in

Irradiationdosage

0.85% NaCl 2%Dubos 1% Glucose

r

0 1.2 X 109 1.5 X 10' 1.5 X 1092.5 X 105 1.5 X 106 7.4 X 106 8.0 X 1065.0 X 10' 7.1 X 10' 1.2 X 106 1.5 X 1067.5 X 10' 1.1 X 104 3.3 X 104 8.2 X 104

106 112 970 2,2001.25 X 106 0 12 551.5 X 106 0 0 0

* Suspensions contained 5 X 10' cells on thebasis of viable-cell counts on the freshly preparedoriginal suspension.

would be expected if the results were additive.Thus, the oxygen uptake rates resulting from theadded substrates, approximated by deductingthe volume of oxygen consumed by the cells inbuffer, were, in most cases, less in cells lyophilizedin the presence of the substrates than in cellslyophilized in buffer. The greatest discrepancieswere noted in mixtures in which the added sub-strate was the same as the substrate carriedover with the cell suspension. This indicates thatthe substrate concentration was not the rate-limiting factor.

Effect of gamma irradiation on reproduction. Avery high dosage of gamma irradiation wasrequired to render S. aureus cells incapable ofreproduction. The decrease in viable-cell countwith increased radiation was approximatelylinear on a loglo scale. Cells irradiated in sus-pension required about 40,000 r to reduce theviable-cell count to one-tenth (by one log,o unit)the initial value (Table 2). Approximately threeto four times this dosage was required to renderlyophilized cells nonviable (Table 4). The irra-diation dosage for producing one log,, unit de-crease in viable cell count ranged from 140,000 rfor cells lyophilized in saline to 160,000 to 170,000r for cells lyophilized in albumin and glucosesolutions. Cells irradiated in suspension also ap-peared to be slightly more susceptible in thepresence of saline compared with cells suspendedin albumin and glucose solutions, but the differ-ence is probably not significant.

Effect of gamma irradiation on oxidation ofsubstrates. The oxygen uptake rates of cellsirradiated in suspension on all substrates testeddecreased with increased irradiation (Table 6).

TABLE 5. Oxygen uptake of Staphylococcus aureus strain 1503 lyophilized and irradiatedin different substrates

Substrate

Buffer ..........Albumin ........D-Alanine .......L-Glutamic acid.Glucose.........Lactic acid......L-Proline .......Sucrose .........

Buffer Alanine Glutamic acid Glucose Lactic acid

2 X106 r

21339

169177

0

2030556721628040137

106 r

7208

21186193

94

2 X106 r

15351734177188

0

82909311526030585166

106 r

49785088206233

112

0

9311011610827535895

201

2 X106 r

10491930214203

106 r

40955571

235256

146

2 X106 r

66738070176180

0

240225245251269340255280

106 r

110119120145202255

182

0

557584580570605587580592

2 X106 r

367486378462492470

106 r

481515470510564505

519

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GAMMA-IRRADIATED S. AUREUS

The ability of the cells to oxidize alanine andglutamic acid decreased sharply and only slight,if any, activity for the amino acids was left afterexposure to a dosage sufficient to render all thecells nonreproductive. The rate of oxidation ofglucose also showed a similar, though less sharp,decline with increased irradiation. The enzymesinvolved in the oxidation of lactic acid wereleast susceptible to gamma irradiation in suspen-sion. Cells irradiated with dosages sufficient torender them completely nonreproductive stilloxidized lactic acid at about 60% of the rate ofnonirradiated cells.

TABLE 6. Oxygen uptake of Staphylococcus aureusstrain 1608 gamma-irradiated in 2% Dubos

albumin (Difco) solution

Irradiation dosage (r)Substrate

0 2.5 X 10 5 X 10 7.5 X 10

Buffer...... 53* 5 0 0D-Alanine (1%) 258 33 3 0L-Glutamic

acid (1%) 285 53 20 12Glucose (1%).. 385 108 44 38Lactic acid (0.5M) ......... 570 470 377 315

Viable cell no. 5.1 X 10' 1.5 X 10' 0 0

* Oxygen uptake in microliters per hour per5 X 109 cells (initial viable-cell count).

TABLE 7. Oxygen uptake of lyophilized and gamma-irradiated* Staphylococcus aureus strain 1608

Irradiation dosage (r)Substrate

0 5X10 106 1.5X 106

Buffer......... 35t 29 21 15D-Alanine (1%) 101 93 82 60L-Glutamicacid (1%)... 119 107 98 71

Glucose (1%) 240 134 128 100Lactic acid (0.5M)..........587 550 525 510

Viable cell no.. 1.5 X 109'1.2 X 106 970 0

* Cells were suspended in 2% Dubos albumin(Difco) solution for lyophilization and irradia-tion.

t Oxygen uptake in microliters per hour per5 X 109 cells (initial viable-cell count).

The oxidation systems of cells irradiated in thelyophilized state were less susceptible to gammairradiation (Table 7). Although a much largerdosage was required to render lyophilized cellsnonreproductive, such cells continued to respireat 40 to 80% of the rates of nonirradiated cellson the substrates tested. The lactic acid oxidationsystem of the lyophilized cells, as in the sus-pended cells, was least affected by gamma irradia-tion.The effect of lyophilization and irradiation in

the presence of the substrates on the oxidationsystems of S. aureus varied with both the sub-strate present during irradiation and the sub-strate added for the metabolic studies. Theoxygen uptake rate in buffer of cells irradiated inalanine was no greater than that of cells irradiatedin buffer only, yet the latter cells measurablyoxidized added alanine (Table 5). Cells irradiatedin glucose respired at a significantly higher ratein buffer than cells irradiated in buffer did inadded glucose, although the glucose concentrationwas lower in the former reaction mixture. This ismore clearly illustrated in Fig. 1, which shows theoxidation rates of cells irradiated in the presenceof glucose compared with rates of cells irradiatedin buffer at various total concentrations of glucose.A similar comparison with lactic acid oxidationrates shows that cells lyophilized and irradiatedin the presence of lactic acid are no more activethan cells lyophilized and irradiated in buffer(Fig. 2).Approximations of the oxygen uptake attribut-

able to the added substrates by deduction of theoxygen consumed in buffer show the presence ofalbumin during irradiation may have someprotective effect on the oxidative enzymes,

pi. of02i

200-

lSO

10

8i

Al_

LYOPHIUZEO AND IRRADIATED INBUFFER: 0.106r, e-=xlOrGLUCOSE: Al06Sr, a.ZxlOGra- r , 0.,.0,,0.,7

Ql 0v2 0X 0.+ OA Q8 0QTTOTAL GLUCOSE CONCENTRATION - %

FGo. 1. Glucose oxidation by gamma-irradiatedStaphylococcus aureus.

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AHN ET AL.

,Al. ofo21

0.05 0.1 0.15 0.2 0.25 03 Q35TOTAL LACTIC ACID CONCENTRATION - M

FIG. 2. Lactic acid oxidation by gamma-irradi-ated Staphylococcus aureus.

except for those concerned with the oxidation ofglucose, compared with cells lyophilized andirradiated in buffer. Such approximations of thedata in which mixtures of oxidizable substratesare present, owing to additions of substrates tosubstrates carried over with the irradiated cells,show that cells irradiated in the presence ofglutamic acid tended to utilize oxygen at a

greater than additive rate on added lactic acidand alanine. Cells irradiated in the presence ofany of the substrates tested utilized oxygen atslightly higher than additive rates when glutamicacid was added as substrate.

DiSCUSSION

Effects of freezing and lyophilization. Thedeleterious effects of freezing are postulated to bedue to intracellular ice formation or concentra-tion of solutes from extracellular freezing, or

both (Taylor, 1960; Mazur, 1960). The latterprocess may also produce an effect by dehydratingthe cells. In studies with Aspergillus flavus,Mazur (1960) noted very high survival rates ofcells and spores suspended in distilled water andin high concentrations of salt solutions whenexposed to temperatures as low as -45 C if nofreezing occurred. The critical temperature forfrozen cells was between -10 and -15 C.Below this temperature he noted a precipitousdecrease in the viability of frozen cells. Hepostulated that at the critical temperature,which varies with the microorganism tested,the extracellular formation of ice crystals of justthe right radius for penetration through theaqueous channels in the cell membrane is favored.Such crystals seed the intracellular fluid, causingit to freeze. The protective effect of sugars and

other substances is l)ostulated to be due to theirprevention of this intracellular freezing. Asadditional support for his hypothesis, Mazur(1960) observed that the freeze-drying process oflyophilization was harmful to Aspergillus spores,whereas dehydration in the absence of freezingwas comparatively harmless.Under conditions of our studies, lyophilization

was observed to be injurious to the viability of S.aureus cells also, and simple freezing in 0.85%saline at the temperature of an ordinary freezerproduced a comparatively slight decrease inviability. Mazur's (1960) hypothesis is applicableto our results, if it can be assumed that thelyophilized cells had at some time been exposedto the critical intracellular icing temperature forS. aureus. In the absence of any data for suchan assumption, the decrease in viability onlyophilization can only be attributed to theprocess of dehydration.

Effect of irradiation. The volume and varietyof papers published in the field of radiation andits effects on living systems present a confusingvista for persons seeking clear-cut simple answers.It is currently impossible to formulate an all-inclusive theory to exl)lain the mechanism ofaction of radiation which will encompass thedata reported. Certain observations, however,have been reported repeatedly, and hypotheseshave been advanced to explain them.

It was early noted that radiation dose-effectcurves had no sharl) break indicating a thresholddose. In studies with homogeneous bacterialpopulations, it was thought unlikely that theresults were entirely due to variation in sensi-tivity. To explain this apparent lack of thresholddose for radiation sensitivv, it was lpostulatedthat a "direct hit" was required to lproduce aneffect (Zimmer, 1961). With increasing dosage ofradiation, a larger proportion of individuals inthe population were hit. Later, specific targetswere proposed for these hits to account for theeffect being measured. Then, when it was notedthat sensitivity to irradiation was modified by avariety of conditions, it was hypothesized thatradiation effects were largely mediated throughan indirect mechanism which was influencedby changes in conditions (Kelner et al., 1955).The increased sensitivity in the presence of waterand oxygen was postulated as being due to theadded destructive effect of diffusible agentsformed from water and oxygen by radiation ac-

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8GANINIA-IRRADIATED S. AUJlREUS5

tion, and protective action of various substances,to neutralization of these diffusible agents orradicals. Owing to the diffusion distances in-volved, however, extracellular water was notbelieved to play much of a role, if any, in thisreaction. Radiation inactivation of bacterialcells is thought then to be chiefly a chemicalreaction mediated by ionizing radicals producedin intracellular water. ZimImer (1961) expressedan opinion that "neweer observations are notreadily explicable by the theory of diffusibleagents and no definite contradiction to the hitand target theory is deducible from them."Our results are in accord with the findings of

other investigators on the effects of various typesof irradiation on bacteria. The dose-effect curvefor viability of S. aureus exposed to gammairradiation indicated no threshold dose, andirradiation in suspension had a greater deleteriouseffect on cell viability on culture than irradiationin the lyophilized state.

Stapleton, Billen, and Hollaender (1952) re-l)orted that incubation at 37 C of Escherichiacoli in the presence of oxidizable substratesprior to X irradiation had some protective effecton (ell viability on culture. Gamma irradiationof S. aureus cells in the presence of glucose insuspension and in the lyophilized state withoutincubation appears to have a very slight protec-tive effect. Many reports indicate that cellsirradiated with dosages sufficient to result inloss of colony-forming ability still retain thecap)acity to synthesize and metabolize varioussubstrates (Kelley, 1961). Our studies showedthat from 40 to 80% of the oxidative capacity,depending on the substrate, may be retained bycells no longer capable of multiplying on culture.

Ultraviolet (Kaplan, Rosenbloom, and Bryson,1953) and X-irradiated (Billen, Stapleton, andHollaender, 1953) E. coli suspensions were re-ported to oxidize substrates at essentially normalrates for varying periods of time (1 to 2 hr) afterirradiation, in spite of the decrease in number ofculturally viable cells to 0.05% or less of theinitial number. For practical reasons, our interestwas not in the immediate postirradiation meta-bolic activity of partially viable suspensions, butin the residual activity of completely nonviablecells after a period of storage. S. aureuts cells,rendered completely reproductively nonviableby irradiation in suspension and stored in thefrozen state, were found to retain less metabolic

activity than cells irradiated in the lyophilizedstate and kept at refrigerator temperatures. Itappears that S. aureus cells irradiated in suspen-sion do not long retain their metabolic activityafter loss of reproductive viability. Conditionsof storage as well as conditions of irradiationprobably contributed to the results. It would beof interest to study the metabolic activities ofcells irradiated in suspension and stored lyophi-lized. Mlany reports have been published whichindicate that the reproductive function of bac-terial cells is immediately sensitive to the damag-ing effects of irradiation at a lower dosage whenexxposed in suspension than in the lyophilizedstate. 1lketabolic activity of such cells appearsto be maintained at normal or near normallevel for a short l)eriod after irradiation. Ourgoal may be achieved by searching for conditionsof storage of cells irradiated in suspension, whichwill continue to maintain the metabolic activitypresent in the cells immediately after irradiation,rather than in attempting to modify the condi-tions during irradiation. Further studies of thischaracter are contemplated.

ACKNOWLEDGMENTS

The Staphylococcus strains were graciously sup-plied by Per Oeding of the University of Bergen,Norway. This investigation was supported in partby Public Health Service grant AJ-02468 from theNational Institute of Allergy and Infectious Dis-eases, and in part by a research grant fromPfizer International.

LITERATURE CITED

AHN, T. H., H. NISHIHARA, C. M. CA.RPENTER,AND G. V. TAPLIN. 1962. Respiration of gammairradiated Britcella abortus and Mycobacteriurntuberculosis. Proc. Soc. Exptl. Biol. Med.111:771-773.

BILLEN, D., G. E. STAPLETON, AND A. HOLLAEN-DER. 1953. The effect of x radiation on the res-

piration of Escherchia coli. J. Bacteriol. 65:131-135.

KAPLAN, S., E. D. ROSENBLOOM, AND V. BRYSON.1953. Adaptive enzyme formation in radiationsensitive and radiation resistant Escherichiacoli following exposure to ultraviolet. J. Cell-ular Comp. Physiol. 41:153-162.

KELLY, L. S. 1961. Radiosensitivity of biochem-ical processes. Brookhaven Symposium on

Fundamental Aspects of Radiosensitivity, p.

33-52.

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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.

MAZAR, P. 1960. Physical factors implicated in thedeath of microorganisms at subzero tempera-tures. Ann. N.Y. Acad. Sci. 85:610-624.

STAPLETON, G. E., D. BILLEN, AND A. HOLLAEN-DER. 1952. The role of enzymatic oxygen re-

moval in chemical protection against x-ray

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inactivation of bacteria. J. Bacteriol. 63:805-811.

TAYLOR, A. C. 1960. The physical state transitionin the freezing of living cells. Ann. N.Y. Acad.Sci. 85:595-609.

UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER. 1954. Manometric techniques. BurgessPublishing Co., Minneapolis.

ZIMMER, K. G. 1961. Studies on quantitative radi-ation biology. Oliver and Boyd Ltd., Edin-burgh.

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