APPLIED MICROBIOLOGY, July, 1966Copyright © 1966 American Society for Microbiology

Vol. 14, No. 4Printed in U.S.A.

Preservation of Serratia marcescens by High-VacuumLyophilizationROBERT R. DEWALD1

Physical Sciences Division, U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland

Received for publication 10 February 1966

ABSTRACTDEWALD, ROBERT R. (Fort Detrick, Frederick, Md.). Preservation of Serratia

marcescens by high-vacuum lyophilization. Appi. Microbiol. 14:561-567. 1966.-Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized inan all-glass system capable of evacuation to pressures of less than 5 X 10-6 torr.Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabi-lized washed organisms compared with 10 to 20% when the cells were lyophilizedat pressures of about 2.5 X 102 torr. At the latter pressures, 45 to 65% survivalwas obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspen-

sions before lyophilization. However, the stabilizers failed to increase significantlythe levels of survival compared with water suspension when cells were lyophilizedat pressures less than 10-i torr. The high survival rates obtained by the high-vacuumtechnique may be attributed to the reduction of traces of molecular oxygen whichhas been reported to be destructive of the dried bacteria. Survival of unstabilizeddried S. marcescens after 1-day storage increased markedly with decreasing sealingpressure. Under the highest vacuum attained, survival of the dried bacteria was

not impaired by storage for up to 1 month at Dry Ice temperatures; at higher tem-peratures, viability losses occurred. Exposure of the dried unstabilized bacteria todry air resulted in rapid viability loss. The inactivation could be stopped almost im-mediately by evacuation to pressures of less than 10- torr, but the evacuationfailed to reverse the viability losses that occurred during exposure.

Lyophilization or vacuum freeze-drying is themost commonly employed method for the preser-vation of bacteria by desiccation (7). This methodcan be considered to be a two-stage process in-volving first freezing and then sublimation ofwater from the frozen material. Death of organ-isms may occur during the freezing, drying,storage after drying, or reconstituting processes.

Generally, no serious problems are encounteredduring freezing of bacteria (18). Satisfactory re-coveries have been obtained, even with bacteriawhich are sensitive to freezing, by control oftemperature, rates of cooling, and suspendingmedia (14). Most viability losses are consideredto occur during the drying phase of the lyophili-zation process (19). A number of factors affectingsurvival after lyophilization also have been re-ported (6, 7). The suspending medium has beenconsidered by many investigators as the mostimportant single factor in determining the sur-vival of an organism after lyophilization. Searchesfor protective substances or stabilizers that im-prove the survival of organisms have been highly

1 Present address: Department of Chemistry, TuftsUniversity, Medford, Mass.

successful, and numerous substances have beenreported to have protective action (6, 7). How-ever, Wagman and Weneck (19) pointed out thatoccasionally contradictory claims are made forprotective effects observed for certain stabilizers.Moreover, stabilizers that afford good protectionto one organism are, at times, ineffective in pro-tecting other organisms. The mode of action ofthe stabilizers is unknown, but attempts toanswer this question have been recently reviewedby Heckly (7). The addition of stabilizers limitsthe usefulness of the dried material for subse-quent studies, but freeze-drying from distilledwater suspensions results in dried organisms thatare essentially pure and free from the componentsof any stabilizing media, allowing more validstudies. Equally important in studying the effectsof various stresses on dried organisms is that theviability following drying should be at a highlevel, so that the dried organisms when rehy-drated are characteristic of the original bacterialpopulation.

Limited information has been reported con-cerning lyophilization of washed cells from watersuspensions. Naylor and Smith (15) and Zimmer-

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man (20) reported that poor recoveries (<5%)resulted when unfortified suspensions of Serratiamarcescens were lyophilized. However, Lion andBergmann (10) reported high recoveries (50%)for Escherichia coli dried from distilled water sus-pensions. Lion and Bergmann also reported thatthe dried bacteria died rapidly upon exposure tooxygen, and they listed substances which affordprotection against the lethal effects of oxygen(11). Electron paramagnetic resonance (EPR)studies by Lion, Kirby-Smith, and Randolph(12) showed that free radical formation occurredduring the period of rapid killing due to oxygenexposure. Similar EPR experiments by Dim-mick, Heckly, and Hollis (5) and Heckly, Dim-mick, and Windle (8) showed that free radicalformation occurred for several species of driedmicroorganisms, including S. marcescens, whenexposed to oxygen. Benedict et al. (3) reportedthat lyophilized S. marcescens cells were killedrapidly when exposed to atmospheric oxygen,but certain reducing agents minimized the ex-tent of inactivation. They also pointed out thatrehydration under vacuum is necessary whencertain stabilizers are employed. More recently,Wagman and Weneck (19) reported a study onthe effect of the residual moisture level upon thesurvival of bacteria dried in the absence ofstabilizers. They reported low levels of survivalafter vacuum freeze-drying, but they failed tomention whether their rehydration was per-formed under vacuum or after exposing thedried bacteria to air.A number of investigators (10, 11, 13, 15, 17)

compared survival during storage in vacuo withstorage in atmospheres of various gases. Theyall found that the survival was highest for driedorganisms stored under vacuum and lowest forthose stored in air or oxygen. Christian and Stock-ton (4) studied the influence of sealing pressureon the recovery of S. marcescens and Staphylo-coccus aureus, and noted on increase in survivalwhen the sealing pressure was decreased.The information reported thus far suggests

that the degree of vacuum employed in vacuumfreeze-drying is an important factor affecting re-covery, especiaUy when washed bacteria aredried in the absence of protective additives. Theprimary objective of this study was to determinethe importance of the degree of vacuum em-ployed during freeze-drying S. marcescens inthe absence and presence of protective substances.Other objectives were to detemine (i) the effectof cell concentrations on the survival level afterhigh vacuum lyophilization, (ii) the temperaturedependence during storage under pressures lessthan 10-i torr, (iii) the effect of sealing pressureupon the storage of S. marcescens dried from

distilled water suspensions, and (iv) the effectsof exposure of the dried organisms to dry air,followed by evacuation.

MATERIALS AND METHODSPreparationz of organisms. S. marcescents strains

(Fort Detrick strain 8UK, ATCC strain 14041) usedin this study were prepared by two different methods.In one method, cells were grown and stored as frozenpellets by a process already described (20). Other or-ganisms were grown for 18 hr at 25 C in 3% TrypticaseSoy Broth (BBL) fortified with 2% glucose. Theharvested organisms were washed twice by alternatesuspension and centrifugation in distilled water andwere shaken aerobically at 25 C for 3 hr. The cellswere then centrifuged and suspended in distilled waterto yield suspensions containing the desired number ofviable cells per milliliter. The washed cell suspensionswere refrigerated between experiments. Viable-cellpopulations were determined by the standard surfaceplating technique, with the use of nutrient agar(Difco) and incubation at 37 C for 24 hr.

Assembly for high-vacuum lyophilization. Figure 1illustrates the vacuum assembly used in these experi-ments. A three-stage oil diffusion pump permittedevacuation of the system to pressures of less than 5 X10-6 torr as determined by an ionization gauge. Thehigh-vacuum stopcocks were lubricated with ApiezonN vacuum grease, and the standard tapers were sealedwith Apiezon W wax. The condensable vapors werecollected in liquid nitrogen traps. The freeze-dryingampoules were connected to the high-vacuum mani-fold via standard taper joints. The ampoules duringlyophilization were immersed in a bath containing anethyleneglycol-water mixture held at selected tem-peratures within =1:0.5 C. During the final 2 hr of the16- to 18-hr lyophilization cycle, the cold-temperaturebath was replaced with a water bath that was thermo-statically controlled at 25 C (=10.1).

Lyophilization procedure. Cel suspensions (1 ml)were introduced into 20-ml ampoules containingglass beads (4-mm diameter). The ampoules werethen connected to the high-vacuum manifold and de-gassed by repeatedly pumping the liquid suspensionsuntil the noncondensable gas pressure between thetwo liquid nitrogen traps was less than 10-3 torr.

IONIZATIONGAUGE

TO TRAP AND MclEOD THERMOCOUPLEGAUGE GAUGE

TO OIL DIFFUSIONAND MECHANICAL PUMPS

K At BATH AND STIRRER20ml AMPOULE L (FOR TEMPERATURE CONTROL)

FIG. 1. Assembly for high-vacuum lyophilizationi.

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Next, the suspensions were frozen as a solid plugcontaining the glass beads by immersing them intothe cold bath at -30 to -40 C. The suspensions were

held at that temperature until the pressure betweenthe liquid nitrogen traps was less than 104 torr. Thefrozen suspensions were then warmed to -12 to -8C and held at those temperatures with continualevacuation at noncondensable gas pressures of lessthan 10-6 torr (approximately 14 to 16 hr). The cold-temperature bath was then replaced by a 25 C bath,and evacuation was continued for another 2 hr. Next,ampoules were either sealed off under vacuum orkept on the high-vacuum manifold during the experi-ments. Rehydration was accomplished by a methoddesigned to prevent exposure of the dried organismsto the atmosphere. A piece of rubber tubing, fitted tothe three-way stopcock as shown in Fig. 1 or to theneck of the sealed-off ampoule, was completely filledwith water and clamped-off from the atmosphere.Rehydration was then achieved by either opening thestopcock or breaking the flame-sealed tip of glassampoules with a hammer. The amount of wateradded was determined by the difference in weight be-tween the ampoule containing the rehydrated cellsuspension and the empty dried ampoule. The weightof the dried cells (approximately 4 to 6 mg) was con-sidered negligible, compared with the 2 ml of distilledwater used for hydration.

Influence of dry air on survival after storage. Inthese experiments, dried air at various pressures wasintroduced into the ampoules before sealing. The pres-sures were determined with a McLeod gauge immedi-ately before sealing. The air was dried by passing itthrough a trap containing activated silica gel at DryIce temperatures.

Storage experiments. Samples were sealed at pres-sures less than 10-5 torr and stored at room tempera-ture, at 4 to 5 C in a refrigerator, and at Dry Icetemperatures. Samples were reconstituted afterstorage for various time intervals up to 1 month. Thesamples held under vacuum at various temperatures(-198 C to 100 C) for 1 hr under high vacuum weresealed under vacuum or left on the vacuum line duringthe experiment. These samples were warmed or cooledto 25 C before rehydration.

Residual moisture measurements. High-vacuumlyophilized samples (approximately 0.1 g) were heldovernight under vacuum (2 torr) at 80 C. The mois-ture contents were estimated by weight difference.

RESULTS

To determine the optimal bath temperaturefor maximal survival of S. marcescens subjectedto high-vacuum drying, cell suspensions at twodifferent initial cell concentrations were dried atvarious temperatures for the periods and at thepressures already described in the lyophilizationprocedure. The results of this experiment withtriply washed cells are plotted in Fig. 2. Thetemperatures correspond to those of the bathin which the ampoules were immersed duringthe vacuum drying process. At temperatures

80_

60

40-

520

>~~~~~~~~~~~~~~~

10 _ t'

6 -

- 1.5 x 101 ORGANISMS/ml t

'4~~~1

4 A 3.4 x 10 ORGANISMS/ml

_ _-16 -12 -8 -4 0 4 8

Bath Temperature, C

FIG. 2. Semilog plot ofper cent survival oftwo popu-lations of washed Serratia marcescens after high-vacuum drying versus bath temperature.

TABLE 1. Survival of washed Serratia marcescens

after lyophilization at pressures of less than 10Jtorr and after freezing and thawing of liquid sus-

pensions

Viable cells per ml before Per cent Per centdrying (X l0) survival after freeze-thawing

lyophilization survivala

591 27b 51 7b 86 5b127 12 59 6 84 9

16.8 1.7 62 4 9 80 125.8 0.3 53 4 S 83 5

Survivals for washed suspensions of S. marce-scens frozen to approximately -40 C, warmed to-12 to -8 C, and held at these temperatures forabout 16 hr, and then thawed and warmed to25 C.

b Indicates 95% confidence interval.

higher than -8 C, the recovery appears to de-pend on the initial cell concentration. Below-8 C, where viable recovery tended to leveloff at its maximal value, the survival became lessdependent upon initial cell concentration.Table 1 gives a summary of viable-cell re-

coveries obtained, after vacuum lyophilizationat bath temperatures between -8 and -12 C,from cell suspensions of different initial concen-trations. It should again be emphasized thatthese results were obtained from triply washedorganisms in the absence of added stabilizers.In the temperature range from -8 to -12 C,recoveries were essentially independent of the

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TABLE 2. Effect ofpressure during lyophilization upon the survival of washed and stabilizedSerratia marcescens

Suspending medium Viable cells per ml before Approx pressure during Per cent survivaldrying (X i0"~) drying (torr)

Naylor-Smith/lO................ 3.9 1.3a 2.5 X 10-2 55 i 9aNaCI, 0.05% ................... 3.6 ± 0.9 2.5 X 10-2 46 i 6Distilled water................. 14.6 = 0.4 2.5 X 10-2 12 :4 4Distilled water................. 3.8 0.9 2.5 X 10-2 19 3

Naylor-Smith/lO ............... 2.3 i 1.4 5 X 10-6 59 i 9NaCI, 0.05% ................... 2.3 i0.4 5 X 10-6 58 i 9Distilled water...........|.-|.5 X 10-6 56 ± 8b

a Indicates 95% confidence interval.bMean of values from Table 1.

0

'- 40-

-5 -4 -3 -2 -1 0

Log1o Pressure of Dry Air, torr

FIG. 3. Survival ofdried unstabilized Serratia mar-

cescens after 1-day storage at room temperature insealed ampoules at various dry air pressures. Per centsurvival = N/No (100) where No and N are the num-ber of viable organisms after lyophilization and afterstorage, respectively.

initial cell concentration of the suspensions.During this study, some samples of dried or-ganisms were rehydrated with degassed water.Within the range of experimental error, no dif-ference in recovery could be detected with theuse of degassed water for reconstitution. Also,organism suspensions prepared by the twomethods described above yielded identical re-

sults.The results obtained by lyophilization at higher

pressures (with the use of the same system with-out the diffusion pump) and at a lower pressureare given in Table 2. Also in Table 2, per cent

0.1 _-

N 'l

No0

0.00001

0.000001A-200 -150 -10C

~1I Ill l l~~~~~~~~~~~~~

D -50 0

Temperature, C50 100

FIG. 4. Semilog plot of N/No versus temperatureafter 1-hr storage of dried unstabilized Serratia mar-cescens at pressures of less than 10-5 torr. No andNarethe number of organisms before and after the storage,respectively.

survival of organisms dried from fortified andwater suspensions is compared for two differentpressures during lyophilization. The Naylor-Smith stabilizer and NaCl used in these experi-ments were adjusted to yield the cell-stabilizerratios recommended by Benedict et al. (2). Theresults show that the protective additives had avery beneficial effect upon the level of survivalafter the high-pressure lyophilization, but hadlittle effect compared with water suspensionsafter the lyophilization at a lower pressure.

Moisture determinations by the dry-weightmethod consistently yielded moisture contentsof less than 2%. No vapor pressure could bedetected by an oil manometer when dried samples(approximately 0.5 g) were held at 25 C for 1hr in a closed system. Vapor pressures greaterthan 10- 2torr are easily detected by this method.

564 DEWALD APPL. MICROBIOL.

1

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PRESERVATION OF SERRATIA MARCESCENS

0

0

0

ROO(23

5 10 15 20Storage Time, days

FiG. 5. Log [N/No (100)] versussdried unstabilized Serratia marcescensealed at pressures of less than 10-61are the number of viable organisms im,lyophilization and after storage, respeci

,( DRY AIR INTRODUCED

- III

-I

- I

_ 0

t AIR PUMPED OUT

0 1 2 3Time, hours

FIG. 6. Semilog plot of N/No versuw

unstabilized Serratia marcescens exposmapproximately 15 min and evacuated tctorr. No and N are the number of viablifore and after the exposure, respective}

obtaining high levels of survival after lyophiliza-7

tion of washed organisms.-78 C Figure 4 shows the survival levels alter 1-hr

storage as a function of temperature for dried4 C organisms held at pressures of less than 105

torr. No losses in viability were detected after 1hr at temperatures between 3 and 32 C. When thedried bacteria were held at 0 C or below for 1hr, 40 to 50% losses in viability occurred. Similarlosses occurred when the bacteria were held atsubzero temperatures for only 20 min. The driedsamples originally at 25 C, cooled below 0 C,and warmed to 25 C (by immersing the ampoulein a water bath at 25 C) before rehydration,were, therefore, subjected to a cooling and

'IO warming cycle. When the bacteria were subjectedM TEMP. to three temperature cycles, the resulting viabil-3-26C) ity losses were the same as those observed afterthe first cycle. The viable population of the dried

L bacteria was reduced about 80% when held at25 30 40 C for 1 hr. When the bacteria were subjected

to temperatures between 40 and 75 C for 1 hr,storage time f the viability losses did not increase markedly7S i ampoules with temperature, but at 100 C the losses in via-torr. No and N bility were so large that the extent was difficulttively. to define experimentally.

Survival after storage for as long as 1 monthof dried organisms sealed under pressures of lessthan 10-5 torr is shown in Fig. 5. At Dry Icetemperatures, the only viability loss appears tocorrelate with that found for the cooling andwarming cycle previously mentioned. At 4 C,there was a gradual decrease in viability withtime; the losses became more extensive at roomtemperature.

0-0 Figure 6 is a representation of the typical

viability losses that occur when the dried organ-isms at 25 C are exposed to dry air at atmos-

4 5 pheric pressure for short periods of time. Inthis case, approximately 85% of the dried or-ganisms were inactivated in 15 min. The dried

time for dried bacteria could not be reactivated by continual9dl thodryair for evacuation at pressures of less than 10-5 torrleorganisms be- for periods up to 5 hr, but evacuation stopped theaY inactivation process immediately.

It was concluded, after comparison with re-

ported moisture-vapor pressure data (1), that thesamples had little, if any, residual moisture.Therefore, data reported in Tables 1 and 2 are

considered representative of samples having a

residual moisture content of less than 2%.The influence of sealing pressure on survival

after 1 day of storage at room temperature isshown in Fig. 3. The survival increased markedlywith decreasing pressure. This again indicatesthat degree of vacuum is very important for

DIscussIoN

This study has demonstrated that high levelsof survival can be obtained upon lyophilizationof washed S. marcescens even in the absence ofadded protective substances. The pressuresduring lyophilization and storage were found tobe important factors affecting the survival of thedried bacteria. The results obtained by the high-vacuum technique can best be explained in termsof molecular oxygen which is lethal to the driedbacteria (3, 10, 11). Also, the high vacuum

N

N 0

0.1

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appeared to have no adverse effects on the sur-vival of the dried S. marcescens. This is in agree-ment with the results reported by Portner et al.(16), for the exposure of dried bacteria to ultra-high vacuums. The survival levels for dried S.marcescens after 1-day storage under varioussealing pressures show that the dried bacteriaare sensitive even to traces of dry air.The results obtained from lyophilization at

higher pressures (approximately 2.5 x 10-2torr) emphasize the importance of the degree ofvacuum. Under identical conditions, i.e., time,temperature, and history of organisms, lyophil-ization at higher pressures gave substantiallylower survival levels: 10 to 20% compared with50 to 65% survivals for the high-vacuum tech-nique. The addition of stabilizers (NaCl andNaylor-Smith) to cell suspensions prior to lyo-philization did have a beneficial effect on thesurvival at the higher lyophilization pressure.However, both stabilizers failed to increase thelevels of survival compared with water suspen-sion when lyophilized at pressures less than10-5 torr. Lion (9) suggested that perhaps themain function of stabilizers is to afford protec-tion against the adverse effect of oxygen. Theeffect of pressure during lyophilization found inthis study supports Lion's suggestion.The freezing-thawing recoveries reported in

Table 1 were obtained by taking samples of a cellsuspension through the same temperature cycleas during lyophilization, except that the suspen-sions were not dried. The freezing, thawing, andwarming of the liquid suspension consistentlyresulted in viability losses of 15% (48). Therate of freezing and thawing was found not toeffect these viability losses. This is consistentwith results reported by Heckly (7) for the sameorganism. The dependence of recovery afterlyophilization upon bath temperatures is alsocompatible with the results reported by otherinvestigators, as reviewed by Heckly (7).The 1-hr storage studies under high vacuum

gave an unexpected result below 0 C. No explana-tion is given for the 40 to 60% losses in viabilitythat occurred when the dried organisms weresubjected to a cooling and warming cycle. Theseviability losses were quite reproducible. Noattempt was made to control the rates of coolingor warming in these experiments. Also, the 50 to60% survival resulting after the cooling-warmingcycle corresponds to the 50 to 65% survivalobtained for unstabilized S. marcescens afterlyophilization, in which the dried organisms weresubjected to the warming phase of the cycle.

Considerable viability losses occurred duringstorage after approximately 1 month at 25 C,even under initial sealing pressures of less than

10-5 torr. No explanation can be given at thistime concerning the nature of these losses.Naylor and Smith (15) reported high levels ofstorage survival for the same organism whenlyophilized in the presence of effective stabilizers.It appears reasonable that stabilizers might bebeneficial for long-term storage stability evenwhen organisms are stored under pressures ofless than 10-5 torr. The trend of temperaturedependence of survival during storage reportedin this study is in agreement with results reportedby other investigators (7). Studies of survivalafter storage for longer times are now in progressin this laboratory.The recoveries after lyophilization reported

here are in marked disagreement with those ofWagman and Weneck (19). They reported lowrecoveries for washed S. marcescens when cellsfrom water suspensions were freeze-dried undervacuum and higher recoveries when the organ-isms were freeze-dried in an atmosphere of cir-culating gases. They also stated that the residualmoisture level was a major factor in determiningthe recovery of organisms, and suggested thatover-drying should be avoided. In the presentwork, high levels of recovery (greater than 50%)were obtained even at residual moisture contentsof less than 2%. These results indicate that theremoval of water is not the major factor causingdeath of organisms during lyophilization.

ACKNOWLEDGMENTSI am indebted to Milton Shon, George Hess, and

Leonard Zimmerman for valuable advice during thisstudy. Appreciation is expressed to Eileen C. Camp-bell and Peter Castric for excellent technical as-sistance.

LrrERATURE CITED1. BATEMAN, J. B., C. L. STEvENS, W. B. MERCER,

AND E. L. CARSTENSON. 1962. Relative humidityand the killing of bacteria: the variation ofcellular water content with external relativehumidity or osmolality. J. Gen. Microbiol.29:207-219.

2. BENEDICT, R. G., J. CORMAN, E. S. SHARPE, C.E. KEMP, H. H. HALL, AND R. W. JACKSON.1958. Preservation of microorganisms byfreeze-drying. I. Cell supernatant, Naylor-Smith solution, and salts of various acids asstabilizers for Serratia marcescens. Appl.Microbiol. 6:401-407.

3. BENEDICT, R. G., E. S. SHARPE, J. CORMAN, G. B.MEYERS, E. F. BAER, H. H. HALL, AND R. W.JACKSON. 1961. Preservation of microorganismsby freeze-drying. II. The destructive action ofoxygen. Additional stabilizers for Serratiamarcescens. Experiments with other microor-ganisms. Appl. Microbiol. 9:256-262.

4. CHRISTIAN, R. T., AND J. J. STOCKTON. 1956. The

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influence of sealing pressure on survival ofSerratia marcescens and Micrococcus pyogenesvar. aureus desiccated from the frozen state.Appl. Microbiol. 4:88-90.

5. DIMMICK, R. L., R. J. HECKLY, AND D. P. HOLLIS.1961. Free radical formation during storage offreeze-dried Serratia marcescens. Nature 192:776-777.

6. FRY, R. M. 1954. The preservation of bacteria,p. 215-252. In R. J. C. Harris [ed.], Biologicalapplications of freezing and drying. AcademicPress, Inc., New York.

7. HECKLY, R. J., 1961. Preservation of bacteria bylyophilization. Advan. Appl. Microbiol. 3:1-76.

8. HECKLY, R. J., R. L. DIMMICK, AND J. J. WUIDLE.1963. Free radical formation and survival oflyophilized microorganisms. J. Bacteriol. 85:961-965.

9. LION, M. B. 1963. Quantitative aspects of theprotection of freeze-dried Escherichia coliagainst the toxic effect of oxygen. J. Gen.Microbiol. 32:321-329.

10. LION, M. B., AND E. D. BERGMANN. 1961. Theeffect of oxygen on freeze-dried Escherichiacoli. J. Gen. Microbiol. 24:191-200.

11. LION, M. B., AND E. D. BERGMANN. 1961. Sub-stances which protect lyophilized Escherichiacoli against the lethal effect of oxygen. J. Gen.Microbiol. 25:291-296.

12. LION, M. B., J. S. KIRBY-SMITH, AND M. L.RANDOLPH. 1961. Electron-spin resonancesignals from lyophilized bacterial cells exposedto oxygen. Nature 192:34-36.

13. MAISTER, H. G., V. F. PFEIDER, W. M. BOGART,AND E. N. HEGER. 1958. Survival duringstorage of Serratia marcescens dried by con-tinuous vacuum sublimation. Appl. Microbiol.6:413-419.

14. MAZUR, P., M. A. RHIAN, AND B. G. MAHLANDT.1957. Survival of Pasteurella tularensis in sugarsolutions after cooling and warming at sub-zero temperatures. J. Bacteriol. 73:394-397.

15. NAYLOR, H. B., AND P. A. SMITH. 1946. Factorsaffecting the viability of Serratia marcescensduring rehydration and storage. J. Bacteriol.52:565-573.

16. PORTNER, D. M., D. R. SPINER, R. K. HOFFMAN,AND C. R. PHILLIPS. 1961. Effect of ultrahighvacuum on viability of umicroorganisms.Science 134:2047.

17. ROGERS, L. A. 1914. The preparation of driedcultures. J. Infect. Diseases 14:100-123.

18. SMITH, A. V. 1954. Effect of low temperature onliving cells and tissues, p. 1-62. In R. J. C.Harris [ed.], Biological applications of freez-ing and drying. Academic Press, Inc., NewYork.

19: WAGMAN, J., AND E. J. WENECK. 1963. Preserva-tion of bacteria by circulating-gas freezedrying. Appl. Microbiol. 11:244-248.

20. ZIMMERMAN, L. 1962. Survival of Serratia mar-cescens after freeze-drying or aerosolization atunfavorable humidity. I. Effects of sugars. J.Bacteriol. 84:1297-1302.

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