Reaction of the Sterilant, Ethylene Oxide, on Plastics

J. TESSLER

Plum Island Animal Disease Laboratory, Animal Disease and Parasite Research Division, Agricultural Research Service,U. S. Department of Agriculture, Greenport, Long Island, New York

Received for publication September 16, 1960

Ethylene oxide (ETO) in the form, "Cryoxcide,"' isused to sterilize surfaces and objects that cannot betreated with heat or corrosive chemicals. Cryoxcide isa gaseous formulation consisting of 11 per cent ofethylene oxide, 44.5 per cent of trichloromonofluoro-methane (Freon 11), and 44.5 per cent of dichlorodi-fluoromethane (Freon 12). Cryoxcide is used at thislaboratory to sterilize sensitive equipment that mightbe contaminated with foot-and-mouth disease virus.

Sterilization was performed in the following manner.The contaminated equipment was placed in a gasautoclave at 78 F for 30 min at 40 per cent relativehumidity. Cryoxcide was admitted into the chamberto a pressure of 11 lb per sq in. and the equipmentwas maintained for a 5-hr exposure under these con-ditions.An office calculating machine from a contaminated

I "Cryoxcide" is the trade name of a product made byAmerican Sterilizer Company, Erie, Pennsylvania.

area was exposed to gaseous ETO, and some of theplastic parts were badly damaged and softened. Sam-ples of the thermoplastics used in the machine wereobtained from the manufacturer. The plastics wereTenite, a cellulose acetate butyrate; Styron 480, apolystyrene resin; and Zytel 101, a nylon resin plastic.Samples of these plastics were placed in the gas auto-clave and subjected to the gaseous sterilization pro-cedure described above. The Styron and Tenite sampleswere damaged, but the Zytel sample was not. Plasticsamples were also placed in pure Freon 11 for 5 hr at37 C; the Styron sample was the only one damagedby such treatment. Pure Freon 12 was not tested onthese samples. Other plastic samples were immersedin liquid ETO at 25 C for 4 hr. The Styron and Tenitesamples were damaged severely, but again the Zytelplastic was undamaged.

This information shows the need for testing the effectsof ETO and Freon 11 before using Cryoxcide on plasticmaterials.

Preservation of Microorganisms by Freeze-dryingII. The Destructive Action of Oxygen. Additional Stabilizers for Serratia marcescens.

Experiments with Other Microorganisms'

R. G. BENEDICT, E. S. SHARPE, J. CORMAN, G. B. MEYERS, E. F. BAER,H. H. HALL, AND R. W. JACKSON

Northern Regional Research Laboratory,' Peoria, Illinois

Received for publication September 21, 1960

In an earlier paper (Benedict et al., 1958) experi-ments were presented on cell supernatant, Naylor-Smith solution, and salts of various organic acids asstabilizers for Serratia marcescens subjected to freeze-drying. In this paper, we report the contribution ofatmospheric oxygen to the death of dried S. marcescenscells when exposed to air before rehydration as opposed

' This work was supported by a contract with the ChemicalCorps, Fort Detrick, Frederick, Maryland.

2 This is a laboratory of the Northern Utilization Researchand Development Division, Agricultural Research Service,U. S. Department of Agriculture.

to rehydration under vacuum. We also report on avariety of other substances as stabilizers for S. marces-cens. These include urea and related compounds, dex-tran, mucin, and bovine serum. Upon completion oexperiments with S. marcescens, we proceeded to applysome of the more effective stabilizers to cells of moresensitive organisms: Two yeasts, Saccharomyces cap-sularis strain no. NRRL Y-676 and Eremotheciumashbyii strain no. NRRL Y-1363, and two bacteria,Pseudomonas aureofaciens strain no. NRRL B-1543Pand Leuconostoc mesenteroides strain no. NRRL B-512F.

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Although the two yeasts were usually killed by freeze-drying in bovine serum, a percentage of cells of thetwo bacteria normally survived without difficulty.Bovine serum, which is ordinarily employed as theprotective menstruum in the lyophilization of culturesof the ARS Culture Collection, was used as a standardmedium for comparison. We believe that the techniquesemployed to increase the percentage of dried viablecells or spores of the sensitive strains may be applicableto other cultures.

MATERIALS AND METHODS

S. marcescens strain no. NRRL B-1481, obtained asstrain 8 UK from the Chemical Corps Biological Lab-oratories, was used. The techniques for propagation,dehydration, rehydration, plating, and counting thisorganism were described (Benedict et al., 1958).

S. capsularis Y-676 and E. ashbyii Y-1363 weregrown in MY medium consisting of 0.5 per cent pep-tone, 0.3 per cent malt extract, 0.3 per cent yeast ex-tract, and 1 per cent glucose, with no adjustment ofthe pH. Counts of viable cells were made on platesof this medium with 2 per cent agar.

P. aureofaciens NRRL B-1543P was cultivated inshaken flasks in a medium consisting of 2 per centpeptone, 1 per cent glucose, and 0.5 ml each of solutionsof Speakman salts A and B per 100 ml, adjusted topH 7.7. Speakman salts are usually made up in theform of two solutions as follows: solution A: K2HPO4,10 per cent and KH2PO4, 10 per cent; solution B:MgSO4*7H20, 4 per cent; NaCl, 0.2 per cent;FeSO4 7H20, 0.2 per cent; MnSO4-4H20, 0.2 per cent.The medium for L. mesenteroides NRRL B-512F con-tained 0.5 per cent yeast extract, 0.25 per cent tryptone,0.5 per cent glucose, and 0.5 per cent K2HPO4, ad-justed to pH 7.4. Strain B-1543P colonized well on theplating medium for S. marcescens; strain B-512F wasplated on the medium employed for shaken flasksexcept that sucrose was substituted for the glucose andthat the medium was solidified by the addition of 2per cent agar.

Fifty milliliters of medium seeded with culture ma-terial from a stock slant were incubated on a recipro-cal shaker for 24 hr and used to inoculate 500-mlquantities of media in plain Fernbach flasks. Thebacterial cells were harvested after 24-hr growth on thereciprocal shaker; the yeasts, after 48 hr. Amounts(2 or 3 ml) of the cell concentrate-stabilizer mixture in15-ml serum bottles were freeze-dried for 18 hr in anNRC unit no. 3501,3 at a final chamber pressure of15 to 25 . Unless otherwise noted, the standard dryingschedule described in the preceding paper wasemployed.

3 National Research Corporation, Newton, Massachusetts.The mention of trade name products does not imply endorse-ment of these over others of equal quality.

RESULTS

Proof that the ratio of viable cells of S. marcescensto stabilizer level prior to drying markedly affects thepercentage of cells that survive drying was establishedin our previous paper. Naylor-Smith solution wasemployed as the stabilizing agent. Briggs et al. (1955)made a similar observation with regard to the survivalof Lactobacillus acidophilus and Lactobacillus caseidried in horse serum plus 8 per cent glucose. Furtherevidence of this dependence was obtained as we pur-sued our investigation. For example, water-washedcell preparations of S. marcescens with initial countsof 192 X 109, 68 X 108, and 33 X 107 viable cells perml suspended in bovine serum gave survivals of dryingof 56, 100, and 100 per cent, respectively.

Effect of oxygen on death rate of dried cells. MIostinvestigators agree that preservation of cultures byfreeze-drying under high vacuum, with subsequentsealing of vials or tubes at a pressure of 0.01 mm Hgor lower, gives the best results. Storage under nitrogenis less desirable according to Rogers (1914) and Naylorand Smith (1946). Storage in air or in oxygen leads torapid killing of dehydrated cells. Losses amounting to74 per cent in viability of S. marcescens stored under"treated nitrogen" for 49 days as reported by Naylorand Smith (1946) seemed excessive, since nitrogen isinert. We chose an alternative method of studying thedeath rate of dehydrated cells of S. marcescens. Therubber stoppers in the bottles were punctured withhypodermic needles, so that the contents were exposedto air for varying periods of time prior to rehydration.The volume of spun-frozen, cell-stabilizer mixturewas reduced to 2 ml to give a thin layer in each bottle.Cells stabilized in 0.05 M Na-acetate and 14 per centsupernatant culture liquor were dried and thereafterexposed to air at intervals ranging from 0 to 40 min;the temperature was 75 F, and the relative humidity(RH) 50 per cent. The cells proved to be extremelysensitive to these conditions (table 1).When the percentage survivals are plotted against

exposure time on log-log paper, a straight line is ob-tained. An experiment was set up to determine theloss of viability of dried cells in dry air at 0 per centRH, in laboratory air at 50 per cent RH with and with-out antioxidants, with antioxidant without exposure toair, and under N2 gas at 50 per cent RH. Tests weremade in tight, dry boxes each containing a Serdexhygrometer as an RH indicator. Gloved ports enabledthe operators to work in a dry atmosphere, puncturestoppers, and rehydrate cells after 10-min exposure.The results (table 2) indicate that exposure to dry

air at 0 per cent RH under the stated conditions causedthe same loss in viability as exposure to air at 50 percent RH. On the other hand, exposure to wet nitrogengave survivals equal to those of the unexposed controls.When the cells were protected from oxygen daTnage

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by the inclusion of antioxidants, survival was the sameas in the unexposed control. Oxygen appears to con-tribute most, if not entirely, to the death of cells andhumidity, little or nothing. Nitrogen at 50 per centRH was inert. When dried preparations are coveredwith water (solution), apparently the water protectsthe cells by excluding excessive amounts of oxygen.

Urea and related compounds as stabilizers. Whenprevious study indicated that thiourea contributed amajor portion of the drying protection afforded by

TABLE 1Effect of various times of exposure to air at 75 F and 50 per cent

RH on viability of unwashed Serratia marcescenscells after drying*

Viable Cell SurvivalCount and 95% Mean Obtained,

Treatment Confidence Survivalt Taking theLimits before Control as

Drying 100%

X 109/ml % %

Control cells-rehydratedimmediately after drying,without exposure.......... 206 i 25 44.6

Cells exposed 5 min prior torehydration ............... 206 i 25 10.8 24t

Exposed 10 min ............. 206 ± 25 5.4 12Exposed 20 min ............. 206 4 25 2.2 5Exposed 40 min ............. 206 4 25 0.8 2

* Cells stabilized with 0.05 M Na-acetate and 14 per centsupernatant liquor prior to drying.

t The values are means of 5 replicates. Each bottle con-tained 2 ml of cell-stabilizer mixture.

t Approximately 22.2 X 109 per ml viable cells left out of92 X 109 per ml in the control, or 24 per cent alive, 76 per centkilled.

TABLE 2Effects of oxygen, moisture, and antioxidant on viability

of unwashed Serratia marcescens cells after drying*

Treatment

Control cells rehydrated immediatelyafter drying........................

As in control above, but exposed to airfor 10 min at 75 F and 50% RH.....

As in control, plus 0.5% each of Na-thiodipropionate and morpholino-hexose reductone, not exposed to air..

As above, but exposed for 10 min to airat 75 F and 50% RH................

As in control, but exposed to dry air(RH = 0%) for 10 min.............

As in control, but exposed to N2 with50% RH for 10 min.................

Viable CellCount and 95%

ConfidenceLimits before

Drying

X 109/ml

166 ± 14

166 ± 14

172 ± 13

172 ± 13

166 ± 14

166 ± 14

MeanSurvivalt

47

5.7

56

58

6.6

46

Naylor-Smith stabilizer,4 a trial was made with 0.5per cent urea as the drying protectant for washed cells.The recoveries averaged 78 per cent. Apparently ureaalone compares favorably with NS. Several runs werethen made with various concentrations of urea andrelated compounds as stabilizers (table 3). Acetamide,guanidine, and semicarbazide in 1 per cent solutionseemed about as effective as urea. Also, urea was sup-plemented with each of several compounds, includingglucose and salts of several acids. Although none ofthe supplements alone had permitted more than 50per cent survival of cells after drying, the percentagesurvivals obtained with the combinations ranged from82 to 88 per cent. However, no combination gave highersurvival than did urea alone.

Glucose and glucose polymers as stabilizers. Inasmuchas glucose had been found by us and others to be fairlyeffective in protecting S. marcescens subjected to drying,we extended our trials to some polymers of glucose

4Naylor-Smith stabilizer (NS) contained 0.5 per cent eachof thiourea, ascorbic acid, and NH4Cl. Half strength is des-ignated herein as NS/2, etc.

TABLE 3Effect of urea and related compounds on viability ofwashed cells of Serratia marcescens after drying

Mean Survival* with Stabilizer Concentrations, in Per Cent

Viable CellCount and 95% Gudine- Semi-Confidence Urea HCI carbazide- Acetamide

Lixmits before HC ICDrying

1.0 0.5 0.25 1.0 1.0 1.0 0.5

X 109/ml % % % % % % %236 i 22 80 85 50255 ± 22 83 77 -

232 ± 22 - 78 81 62 57168 i 18 96 -160 + 12 - 85 - 92 - -

* The values are means of 5 replicates. Each bottle con-tained 3 ml of cell-stabilizer mixture.

TABLE 4Glucose and glucose polymers as stabilizers of unwashed

cells of Serratia marcescens after drying

Viable Cell SriaCount and 95sf SRvialg MaStabilizer Employed Confidence tRange SMervvanLimits before DryingDrying

X 109/ml % %

Control, 75% supernatantand 25% water.214 19 35-49 43

Control plus2% glucose. 214 19 47-68 59Control plus 2% isomaltose 214 ± 19 48-59 52Control plus 2% isomalto-triose .214 ± 19 28-38 33

Control plus 2% dextran of22,500 mol wt.214 ± 19 26-32 28

* These values are means of 6 replicates. Each bottle con-tained 3 ml cell-stabilizer combination.

* Cells stabilized with 0.05 M Na-acetate and 14 per centsupernatant liquor prior to drying.

t The values are means of 6 replicates. Each bottle con-tained 2 ml cell-stabilizer combination.

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(table 4). Tests were made with 2 per cent glucose or

its polymer in 75 per cent supernatant which itselfsupported a mean survival of 43 per cent. Addition ofglucose raised the figure to 59 and isomaltose to 52,whereas isomaltotriose gave only 33 per cent. Dextran,having a molecular weight of 22,500, was used but gave

a lower survival than the control. A negative result wasalso obtained in an additional experiment with gastricmucin. Dextran and mucin were of particular interestbecause Miller and Goodner (1953) had proposed a

theory that a substance could protect the cell by render-ing the cell wall impermeable.

Comparative survivals of selected yeasts stabilized withbovine serum, urea, NS, and supernatant liquor. Tofacilitate preservation and storage of large numbersof cultures in our laboratory, undetermined quantitiesof spores, mycelial fragments, or vegetative cells are

thoroughly mixed with 0.1 to 0.15 ml of sterile bovineserum, dispensed into small Pyrex tubes, frozen, anddried under vacuum. The manifold-type drier describedby Wickerham and Flickinger (1946) and by Haynes,Wickerham, and Hesseltine (1955) is employed. Al-though the majority of yeast and mold strains with-stand freeze-drying quite satisfactorily, certain culturesmanifested poor survival; i.e., no viable cells were

present immediately after drying, or after drying andlimited storage. We selected for further tests two yeastcultures which were difficult to freeze-dry.Unwashed cell concentrates of S. capsularis NRRL

Y-676 and E. ashbyii NRRL Y-1363 (arthrospores)were processed with bovine serum, urea, NS solution,and supernatant liquor as previously employed forS. marcescens. Survivals of S. capsularis and E. ashbyiiwere extremely poor in all instances (table 5).

Effect of rates of freezing and thawing on viability.Hutton, Hilmoe, and Roberts (1951) had found thatwarming, i.e., increase in the ice-film temperaturebetween time of freezing and initiation of the dryingprocess, markedly reduced the percentage recovery ofBrucella abortus (strain 19). One set of bottles, frozenat -30 C and warmed to a predrying temperatureof -10 C, contained only 6 per cent viable cells afterdrying; whereas duplicate sets, warmed to a predryingtemperature of -25 C, averaged 54 per cent recovery

of viable cells. Comparable results were reported forEscherichia coli by Wolff (1952) and for spores ofAspergillus flavus by Mazur (1953). The data suggestedthat we should alter our drying procedure to eliminatepossible increase in ice-film temperature. To investigatethis point and to ascertain whether the initial freezingtemperature should be modified, we set up an experi-ment to determine the sensitivity of the cells and ofarthrospores to freezing and thawing at various tem-peratures. Concentrates of the 2 yeast cultures in 3different fluids were slowly frozen at -25 C, and alsorapidly frozen at -25 C and -65 C in Dry Ice-methyl

TABLE 5Effect of various stabilizing agents on survival of unwashed cells

of Saccharomyces capsularis and Eremothecium ashbyiiafter drying

Mean Survival*

Stabilizer S. capsularis E. ashbyliNRRL Y-676 NRRL

(134.9 X Y-1363 (140 X107/mlt) 106/mlt)

Bovine serum ................0...... .4 0.0007Urea, 1.0% .....................0... .04 0.0007Urea, 0.1% ............. 0.1 0.0003Urea, 0.01% ....................... 0.6 0.0007NS/2t ............................. 1.5 0.0007NS/20 ............................ 1.0 0.0007NS/200 ........................... 0.2 0.0007Supernatant liquor .. .............. 0.05 0.0007

* The values are means of 5 replicates. Each bottle con-tained 2.0 ml cell-stabilizer mixture spun-frozen at -45 C.

t Viable cell count before drying.t NS designates Naylor-Smith solution containing 0.5%

each ascorbic acid, thiourea, and NH4Cl.

TABLE 6

Effect of rates of freezing and thawing on viability of unwashedcells of Saccharomyces capsularis and Eremothecium

ashbyii in serum Naylor-Smith solution (NS),and water

Mean Survival* with SuspendingFluid

Rate ofThaw-

Bovine NS Distilledserum H20

S. capsularis NRRL Y-676Slow freeze, -25 C Rt 100 50 50

S 95 33 84

Fast freeze, -25 C R 73 50 60S 70 35 30

Fast freeze, -65 C R 2.5 1.0 0S 2.0 0 1.0

E. ashbyii NRRL Y-1363tSlow freeze, -25 C R 82 54 71

S 68 54 89

Fast freeze, -25 C R 80 68 83S 73 57 76

Fast freeze, -65 C R 89 63 100S 56 19 54

* Viable cell count before freezing was 187 X 107 per mlfor Y-676 and 71 X 106 per ml for Y-1363. One milliliter ofcell-stabilizer was frozen per 15-ml serum bottle.

t R (rapid) = 55 C per min, except when thawed at -65 C,R = 95 C per min; S (slow) = 5 C per min, except when thawedat -65 C, S = 10 C per min.

t Approximately 95 to 98 per cent arthrospores from shakenflask cultures.

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Cellosolve baths. After a short holding time the mixtureswere thawed out both slowly and rapidly, and thepercentage viability determined (table 6).

S. capsularis was extremely sensitive to freezing at-65 C regardless of the rate of thawing. It was notsurprising that there was low survival of this organismin the previous experiment. E. ashbyii survived in allthe trials and was far less sensitive to freezing and thaw-ing at -65 C than was S. capsularis. It is concluded,therefore, that the major losses experienced in freeze-drying E. ashbyii occurred during the drying operation.To get satisfactory survival of the two sensitive

yeasts in the freeze-drying operation, we tried driedskim milk and glycerol as stabilizers, reduced theamount of sample, and modified the method of pro-cessing. One milliliter of cell concentrate plus stabilizerwas frozen in each bottle at -25 C, and the bottleswere immediately transferred to the chilled platen at-25 C to avoid warming at this point. As soon as thechamber pressure was reduced to 100 j., the platenwas rapidly warmed to 30 C for Y-676 and to 39 C forY-1363. After 5-hr drying the cultures were rehydratedand diluted for counting. The results are shown intable 7.

It appears that changes in processing technique werebeneficial because survival in bovine serum was atleast severalfold better than that reported in table 5.Addition of 2 per cent glucose to the bovine serumincreased survival of S. capsularis and E. ashbyiianother fivefold, to 11.5 and 2 per cent, respectively.Survivals of 4.5 and 8 per cent were obtained with themedium containing 5 per cent skim milk and 2 per centglycerol. Skim milk was definitely improved by theaddition of glycerol.Although Fry (1954), in his review of freeze-drying

TABLE 7Effect of various stabilizers on viability of unwashed cells of

Saccharomyces capsularis and Eremothecium ashbyiiafter drying*

Mean Survivalt

Stabilizer S. capsularis E. ashbyiiNRRL Y-676 NRRL Y-1363

(153 X (89.3 X107/ml:) 106/ml+)

Bovine serum ...................... 2.1 0.34Bovine serum + 2% glucose......... 11.5 2.05% Skim milk solids + 2%, glycerol.. 4.5 8.056%,X Skim milk solids + 1% glycerol. 3.1 2.557% Skim milk solids + 0.5%o gly-

cerol ............................ 2.1 0.75% Skim milk solids alone ......... 1.8 0.007Distilled H20 ...................... 0.4 0.005

* Drying for 5 hr instead of usual 18 hr.t These values are means of 5 replicates. Each bottle con-

tained 2 ml of cell-stabilizer mixture.

of cultures, makes no mention of the effect of freezingtemperature, we suggest that the survival of sensitiveforms may be markedly improved by a critical exami-nation of the survival of cells with stabilizer afterfreezing and thawing at various temperatures. Forexample, if it were found that freezing at -65 C fol-lowed by rapid thawing killed 80 per cent of the cellsas opposed to only 20 per cent if they were frozen at-25 C, the preferred temperature would be -25 C.Warming was also found to have a deleterious effecton the survival of the sturdy cells of S. marcescens.When stabilized in supernatant liquor, frozen at -50C, and immediately freeze-dried, cells of strain B-1481survived to the extent of 78 per cent as opposed to 33per cent survival when they were allowed to warm upslowly to -25 C overnight in a deep-freeze cabinetprior to drying. A repeat experiment produced similarresults.

Survivals of P. aureofaciens NRRL B-1543P and L.mesenteroides NRRL B-512F freeze-dried with variousstabilizers. These bacteria were not as difficult to freeze-dry as the yeast or yeastlike cultures previously dis-cussed. Cell concentrates of P. aureofaciens and L.mesenteroides were stabilized with bovine serum andwith both urea and NS solution at various concentra-tions (table 8).

Survivals under comparable conditions were inter-mediate of those found for S. capsularis and E. ashbyiion the one hand and for S. marcescens on the otherhand. Best results were obtained with bovine serum,next with urea, and poorest with NS solution.

DISCUSSION

The value of antioxidants in protecting dried cellsexposed to dry or moist air seems evident from the

TABLE 8Viability of unwashed cells of Pseudomonas aureofaciens and

Leuconostoc mesenteroides when dried in bovine serum,urea, or Naylor-Smith stabilizer (NS)

Mean Survival*

StabiE zer P. aureofaciens L. mesenteroidesNRRL B-1543P NRRL B-512 F

(131 X (669 X109/mlt) 107/mlt)

Bovine serum ...................... 27.5 57.9Urea, 1% .......................... 8.6Urea, 0.5% ........................ 29.3 12.2Urea, 0.25% ....................... 35.5 40.5Urea, 0.025%.. 49.2NS ............................ 4.9NS/2 ............................ 1.1 10.8NS/10 ............................ 3.9 10.2NS/20/. 19.3

* These values are means of 5 replicates. Each bottle con-

tained 2 ml of cell-stabilizer combination.I Viable cell count before drying. t Viable cell count before drying.

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experiments done on that phase of the problem. Anantioxidant might prove of value as a stabilizer com-ponent for sensitive organisms that are dried in twostages between which the vacuum is broken as employedby Briggs et al. (1955). Although they did not obtainmore than 10 per cent survival of certain lactobacillistabilized with NS solution, the number of lactobacillidried was low, i.e., 1 X 109 per ml, and the ratio ofcells to NS levels was not investigated. Extrapolationof the NS graph in figure 3 of our preceding papersuggests that we should not expect more than approxi-mately 7 per cent survival of S. marcescens with fullconcentration of NS for 1 X 109 cells per ml. Haskinsand Anastasiou (1953) also reported on drying micro-organisms in a two-stage system. They suggest thatcertain mold spores survive drying better with wateralone as adjuvant, but our experience indicates thatwater alone does not protect bacteria or yeasts duringfreeze-drying. Although dextrin-free NS solution gaveexcellent protection to S. marcescens during freeze-drying, it poorly protected the other cultures used.The excellent protection afforded S. marcescens by

its culture liquor, which we noted earlier, was notduplicated in experiments with the two sensitive yeastsand their respective supernatants. Although urea waswithout effect as a drying protection for the two yeasts,our results suggest that it has much promise as a stabi-lizer for some bacteria. Apparently maximal protectioncan be attained, as for other materials, only by deter-mination of the amount needed for the given populationof a given organism.The popular concept that protective colloids should

contribute to survival does not appear to be valid forour experiments with S. marcescens, L. mesenteroides,and P. aureofaciens.A wide variety of chemical compounds protects S.

marcescens strain B-1481 during the drying process.Sharp lines of structural demarcation between chemi-cals supporting 70 to 90 per cent survival after dryingand those giving only 1 to 10 per cent survival are notreadily perceived. Although many factors are involvedin the freeze-drying process, most low molecular weightsubstances appear to protect the cells from damageduring the freezing operation by their cumulativeeffect on the freezing point, the number and size of icecrystals, and their rate of formation, both inside andoutside the cells. Meryman (1956) has pointed out thatadditives may pass into the cell, bind free water, andreduce crystallization velocity during rapid freezing.Substances such as glucose, urea, and sodium citrateminimized intracellular crystal formation in the rapidfreezing of erythrocytes.

Losses of viability in freeze-drying microorganismscan be attributed to: (a) losses during initial freezing,(b) damaging effects of warming prior to placing in thedryer, (c) deleterious action during the drying process

itself, and (d) losses occurring at the moment of rehy-dration of the dried cells. Losses listed under (a), (b),and (d) can be minimized by carefully controlling thesestages of the operation.

SUMMARY

The oxygen of air was found to kill 95 per cent ofdried Serratia marcescens in 10 min. Certain reducingagents prevented the action of the oxygen. Humidityseemed to play no role in the phenomenon. Urea andseveral of its derivatives protected S. marcescens duringthe drying process.

Glucose and isomaltose improved the protectingaction of supernatant liquor; isomaltotriose and dextran(and mucin) did not.Attempts to freeze-dry Saccharomyces capsularis and

Eremothecium ashbyii gave very poor results. A studyof these two organisms subjected to freezing and thaw-ing without drying showed that S. capsularis wasmostly destroyed when frozen at -65 C, perhaps be-cause of its large size. Otherwise, rapid freezing andrapid thawing gave best results.

Apparently the best conditions for survival of or-ganisms by freeze-drying are freezing at moderatelylow temperature, and drying for a short period at anelevated platen temperature.

REFERENCES

BENEDICT, R.G., CORMAN, J., SHARPE, E. S., KEMP, C. E.,HALL, H. H., AND JACKSON, R. W. 1958 Preservation ofmicroorganisms by freeze-drying. I. Cell supernatant,Naylor-Smith solution, and salts of various acids as stabi-lizers for Serratia marcescens. Appl. Microbiol., 6, 401-407.

BR1GGS, M., TULL, G., NEWLAND, L. G. M., AND BRIGGS, C.A. E. 1955 The preservation of lactobacilli by freeze-drying. J. Gen. Microbiol., 12, 503-512.

FRY, R. M. 1954 The preservation of bacteria. In Biologicalapplications of freezing and drying. Ch. 10, pp 215-252.Edited by R. J. C. Harris. Academic Press, Inc., NewYork, New York.

HASKINS, R. H. AND ANASTASIOU, J. 1953 Comparison of thesurvivals of Aspergillus niger spores lyophilized by variousmethods. Mycologia, 45, 523-532.

HAYNES, W. C., WICKERHAM, L. J., AND HESSELTINE, C. W.1955 Maintenance of cultures of industrially importantmicroorganisms. Appl. Microbiol., 3, 361-368.

HUTTON, R. S., HILMOE, R. J., AND ROBERTS, J. L. 1951Some physical factors that influence the survival ofBrucella abortus during freeze-drying. J. Bacteriol., 61,309-319.

MAZUR, P. 1953 Studies on the effects of low temperatureand dehydration on the viability of fungous spores. Ph.D.thesis, Harvard University, Cambridge, Massachusetts.

MERYMAN, H. T. 1956 Mechanics of freezing in living cellsand tissues. Science, 124, 515-521.

MILLER, R., JR., AND GOODNER, K. 1953 Studies on thestability of lyophilized BCG vaccine. Yale J. Biol. Med.,25, 262-283.

NAYLOR, H. B. AND SMITH, P. A. 1946 Factors affecting the

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viability of Serratia marcescens during dehydration andstorage. J. Bacteriol., 52, 565-573.

ROGERS, L. A. 1914 The preparation of dried cultures. J.Infectious Diseases, 14, 100-123.

WICKERHAM, L. J. AND FLICKINGER, M. H. 1946 Viability

of yeast preserved two years by the lyophil process.

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HYDROXYLATION OF FLUOROHYDROCORTISONE

ACKNOWLEDGMENTS

The authors wish to thank H. Bishop and M. Darkenfor supplying the natural isolates of Streptomyces roseo-chromogenes used in this study. The technical assistanceof Mary Matrishin, Mrs. M. May, and Mrs. E. Sleezeris gratefully acknowledged.

LITERATURE CITED

ASAI, T., K. AIDA, T. TANAKA, E. OHKI, T. MATSUHISA, Y.TAKEDA, AND T. INUI. 1959. Microbiological hydroxylationof steroid. IX. Hydroxylation of steroid by Syncephalas-trum racemosum. J. Gen. Appi. Microbiol. 5:127-137.

DODSON, R. M., A. H. GOLDKAMP, AND R. D. MUIR. 1957Microbiological hydroxylation of C19-steroids at positionsC-1 and C-2. J. Am. Chem. Soc. 79:3921.

DODSON, R. M., A. H. GOLDKAMP, AND R. D. MUIR. 1960.Microbiological transformations. V. la- and 2,-Hydroxyla-tions of Cig-steroids. J. Am. Chem. Soc. 82:4026-4033.

DULANEY, E. L., AND E. 0. STAPLEY. 1959. Studies on the trans-formation of 11-deoxy-17a-hydroxycorticosterone with a

strain of Curvularia lunata. Appl. Microbiol. 7:276-284.GOODMAN, J. J., AND L. L. SMITH. 1960. 16a-Hydroxy steroids.

IX. The effect of medium composition on isomerization of9a-fluoro-16a-hydroxyhydrocortisone and 9a-fluoro-16a-hydroxyprednisolone (triamcinolone) during microbio-logical fermentations. Appl. Microbiol. 8:363-366.

GREENSPAN, G., C. P. SCHAFFNER, W. CHARNEY, H. L.HERZOG, AND E. B. HERSHBERG. 1957. Transformation ofsteroids by fungi. Introduction of lt- and 2,3-hydroxylgroups into Reichstein's Compound S. J. Am. Chem. Soc.79:3922-3923.

HERZOG, H. L., M. J. GENTLES, E. B. HERSHBERG, F. CARVA-JAL, D. SUTTER, E. CHARNEY, AND C. P. SCHAFFNER. 1957.Microbiological transformation of steroids, 2,B-hydroxyla-tion. J. Am. Chem. Soc. 79:3921-3922.

IIZUKA, H., A. NAITO, AND M. HATTORI. 1958. Microbiological

hydroxylation of steroids. II. Microbiological hydroxyla-tion of progesterone by Aspergillus. J. Gen. Appl. Micro-biol. 4:67-78.

SHIRASAKA, M., M. TSURUTA, AND M. NAKAMURA. 1958. Micro-biological hydroxylation of Reichstein's Compound S(17a-hydroxydesoxycorticosterone) by Sclerotinia sp.;2#-hydroxylation. Bull. Agr. Chem. Soc. Japan 22:273-274.

SHIRASAKA, M., R. TAKASAKI, R. HAYASKI, AND M. TSURUTA.1959. Microbiological hydroxylation of progesterone and17a-hydroxyprogesterone by Sclerotinia libertiana;2ft-hydroxylation. Bull. Agr. Chem. Soc. Japan 23:245-246.

SMITH, L. L., T. FOELL, R. DEMAIO, AND M. HALWER. 1959.16a-Hydroxy steroids. II. Partition chromatography oftriamcinolone and related steroids. J. Am. Pharm. Assoc.,Sci. Ed. 48:528-532.

SMITH, L. L., M. MARX, J. J. GARBARINI, T. FOELL, V. ORI.GONI, AND J. J. GOODMAN. 1960a. 16a-Hydroxy steroids.VII. The isomerization of triamcinolone. J. Am. Chem.Soc. 82:4616-4625.

SMITH, L. L., H. MEDELSOHN, T. FOELL, AND J. J. GOODMAN.1961. 16a-Hydroxy steroids. X. 2j6-Hydroxylation of9a-fluorohydrocortisone by Streptomeyes roseochromogenws.J. Org. Chem. (in press).

TAKEDA, R., I. NAKANISHI, J. TERUMICHI, M. UCHIDA, M.KATSUMATA, M. UCHIBAYASHI, AND H. NAWA. 1959. Trans-formation of Reichstein's Substance S to prednisolone byPseudomonas. Tetrahedron Letters No. 18:17-19.

TANABE, K., R. TAKASAKI, R. HAYASHI, AND M. SHIRASAKA.1959. Steroid series. I. Microbial oxidation of steroids bySclerotinia libertiana. Chem. and Pharm. Bull. (Tokyo)7:801-804.

THOMA, R. W., J. FRIED, S. BONANNO, AND D. GRABOWICH.1957. Oxidation of steroids by microorganisms. IV. 16a-Hydroxylation of 9a-fluorohydrocortisone and 9a-fluoro-prednisolone by Streptomyces roseochromogenus. J. Am.Chem. Soc. 79:4818.

VISCHER, E., and A. WETTSTEIN. 1958. Enzymic transformationof steroids by microorganisms. Advances in Enzymol. 20:237-282.

RRATUM

In Scientific Articles, "Reaction of the Sterilant, Ethylene Oxide, on Plastics,"J. Tessler, Appl. Microbiol. 9: 1961, errors appear on page 256. The corrections are asfollows:The "Cryoxcide" is a trade name of a product supplied by the American Sterilizer

Company, but is not manufactured by them. In addition to ethylene oxide, the productcontains 79 per cent Freon 11 and 10 per cent Freon 12, and not 44.5 per cent of eachFreon as reported. The sterilization procedure used was developed for Foot-and-MouthDisease Virus inactivation and should not be construed as the recommended conditionsstipulated by the American Sterilizer Co. for sterilization of plastic materials.

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