EFFECT OF OZONE ON SURVIVAL AND PERMEABILITY OFESCHERICHIA COLI

D. B. McNAIR SCOTT AND E. C. LESHERDepartment of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication 5 October 1962

ABSTRACTSCOTT, D. B. McNAIR (University of Pennsyl-

vania, Philadelphia) AND E. C. LESHER. Effectof ozone on survival and permeability ofEscherichia coli. J. Bacteriol. 85:567-576. 1963.-Escherichia coli cultures in the logarithmic phaseor resting were treated with various concen-trations of ozone in saline solution. Approxi-mately 2 X 107 molecules of ozone per bacteriumkilled 50% of the cells. Ozone caused leakage ofcell content into the medium, and lysis of somecells. Low concentrations of ozone did not reactwith the glutathione within the cells, althoughreaction with glutathione in solution was im-mediate and stoichiometric. The effect on nucleicacid within the cells was to change the solubilityand to cause the release of ultraviolet-absorbingmaterial into the medium. Ozone attacked thering structure of the base or the carbohydrateonly when the substance was in the medium.Nucleic acids released into the medium were re-absorbed by cells which were not lysed. Viablecells resumed growth immediately, and grew atrates determined by the nutrients either addedto the medium or which resulted from leakageand lysis of nonviable cells. It is postulated thatthe primary attack of ozone was on the cell wallor membrane of the bacteria, probably by re-action with the double bonds of lipids, and thatleakage or lysis of the cells depended on the extentof that reaction.

Ozone is formed by electric discharge throughoxygen or air. It is also formed by ultraviolet(UV) radiation at 2,000 to 2,100 A, especially inthe higher atmosphere (40 km or 15 to 30 miles).The concentration at the surface of the earth isabout 10-8 (parts by volume). Ozone absorbsUV at 2,537 A and is decomposed. The deleteriouseffect of ozone on life is advantageous to man;ozone kills bacteria and is used for this purposein sewage-disposal plants and in preservation of

meat during the tenderizing process, etc. In thislatter instance, a part of the effect of UV lampsin sterilizing is considered to be due to the ozone(0.1 ppm) produced by the wavelengths of 1,750to 2,000 or 2,100 A (Nagy, 1959). The concen-tration of ozone which kills bacteria has beenvariously reported to be 0.04 to 0.1 ppm (volume),whereas the toxicity for small animals is 3 to 12ppm (Stockinger, 1959). Humans experienceheadache, and dryness and irritation of thethroat, respiratory passages, and eyes at 0.1 ppm.At that concentration, the odor of ozone is dis-agreeable, and most people can detect the odorat 0.02 to 0.04 ppm.

Davis (1959) has shown that ozone is alsomutagenic, in fact, one of the most potentmutagens known. Perhaps some of the mutageniceffects of UV irradiation may be attributed tothe ozone produced by the shorter wavelengths.The exact mode of action of ozone in killingbacteria has not been determined. Ozone is sucha strong oxidizing agent that it reacts with manysubstances of biochemical importance. It hasbeen postulated that the primary bactericidal re-action is the oxidation of SH- to S-S (Barron,1954).

It was to test this hypothesis that our experi-ments were undertaken. We found that, on thecontrary, the SH- concentration of bacteria wasnot decreased until it leaked out or the cells werelysed. The attack of the ozone seemed to be atthe cell surface, with alteration of the perme-ability of the membrane. It is postulated thatthe primary attack of ozone is on the doublebonds of unsaturated lipids in the cell membrane.

MATERIALS AND METHODS

Ozone was produced by a Welsbach modelT-23 laboratory ozonator at a voltage of 100 and8-lb dry oxygen pressure, with a flow rate of 0.2ft3 per min. The ozone was passed directly throughthe bacterial culture in some experiments. In themajority of experiments, the ozone was passed

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through saline solutions for a chosen time (from20 sec to 5 min). Saturation time was between 2and 5 min. The solution was then added im-mediately to suspensions of bacteria in saline andalternately to solutions of alkaline 10% KI. Fortreatment of solutions of pure chemicals, such asthe nucleic acids, nucleotides, etc., the ozone wasbubbled through doubly distilled water sur-rounded by an ice bath. To obtain stable solutionsof ozone and reproducible results for the concen-tration of ozone in the solutions, it was necessaryto use glass-distilled water and to rinse all theglassware well with the same. It was found thatthe solutions of ozone in glass-stoppered cylinderswere very stable at 0 to 4 C, whereas there was aslow loss of ozone at room temperature. Thebacterial cultures were treated with ozone so-lutions at room temperature to avoid cold shockto the bacteria. The uncertainty of ozone concen-tration (calculated per 109 bacteria) due to thisfactor and to the variable loss of material whichcould react with ozone, from the bacteria intothe suspending media, was minimized by workingas fast as possible and reproducing the sameconditions in each experiment.

Escherichia coli B was cultivated in salts-glucose medium in an aeration tower at 36 C,as described previously (Scott and Chu, 1958).Turbidity of the cultures was determined in aKlett-Summerson photometer. The total bacteriawere counted microscopically in a Petroff-Hauserbacterial counter. The viable bacteria were de-termined by the number of colonies which grewon plates of nutrient agar (overnight), or salts-glucose agar (72 hr), after spreading of suitabledilutions of the cultures, before and after treat-ment with ozone.

E. coli B (0.01 ml of a weekly culture takenfrom a slant) was inoculated into 200 ml of saltsmedium containing glucose (2 mg/ml). Afterincubation overnight in a water bath at 37 Cwith aeration, the turbidity with a 420-m, filterwas approximately 300 Klett units, growth beinglimited by exhaustion of glucose. The restingculture was centrifuged, and the cells werewashed and resuspended in 6 ml of saline; 1 mlof the suspension was added to each of four glass-stoppered centrifuge tubes containing volumes ofsaline calculated to give a final volume of 25 ml.Three different volumes of ozone in saline so-lution were added alternately to tubes containingculture and to tubes containing the same volume

of KI in saline. Samples of the treated cultureswere then plated on salts-glucose and nutrientagar plates in several dilutions and incubated. Toobtain growing cultures, a portion of the restingculture was inoculated into an aeration towercontaining salts-glucose medium to give a tur-bidity of 50 and a volume of 150 ml. After 2 hrof aeration at 37 C, the cells were harvested andtreated with ozone in the same way as theresting cells.The ozone concentration was determined by

acidification of the alkaline KI solution with 3 Macetic acid and measurement of the opticalabsorbance at 352 m,u, according to the methoddescribed by Davis (1959).For determination of nucleic acids in the bac-

teria, 1-, 2-, or 5-ml samples of the cultures werecentrifuged. The bacterial pellets were treated asfollows. The low-molecular-weight nucleotidesand nucleosides were extracted by treatmentwith 1 M NaCl or with cold 2% perchloric acid.Approximately one-half of the ribonucleic acid(RNA) was extracted with cold 5% perchloricacid overnight. The remaining RNA and thedeoxyribonucleic acid (DNA) were extractedwith hot (90 C) 5% perchloric acid for 5 min.In some experiments, the total nucleic acid wasextracted with hot 5% perchloric acid. Thenucleic acid contents were determined in thesefractions, in whole unfractionated cells, and inthe residues after extraction with cold or hotperchloric acid.DNA, deoxynucleotides, and deoxynucleosides

were determined by the reaction of the deoxy-ribose attached to the purine moiety with di-phenylamine, according to the procedure ofBurton (1956). Similarly, RNA, the ribonucleo-tides, and ribonucleosides were determined byreaction of the purine-bound ribose with orcinol,according to the procedure of Miller, Golder,and Miller (1951). The spectra of the solutionsof nucleic acids, nucleotides, nucleosides, bases,and of the media, before and after treatment withozone, were graphed from measurements in aBeckman model DU spectrophotometer.

Glucose was determined by the glucose oxi-dase method with a kit supplied by WorthingtonBiochemical Corp., Freehold, N.J.The glutathione content of the bacteria was

determined by amperometric titration of extractsobtained by treatment with cold (-15 C) 70%ethanol. The titration procedure was a modifi-

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OZONE EFFECTS ON E. COLI

V2

X2X

-j -30

Oz0(.'

-5 ~~~~~~~~~x

-7 0

M52

2 4 6 8 10 12 14

)JG 03/109

FIG. 1. Survival of logarithmic-phase Escherichiacoli after treatment with ozone.

cation of that described by Benesch, Lardy, andBenesch (1955).The ethanol extracts were also treated with

N-methylmaleimide and submitted to chroma-tography, as described by Benesch et al. (1956).

RESULTSSurvival. The effect of treatment with solutions

of ozone is shown in Fig. 1 for growing culturesand in Fig. 2 for resting cultures. In both figures,the lines have been drawn through the medianvalues of the individual slopes; the mediansdiffered very little from the mean values. Thesetwo values were about 20% higher than thecalculated regression coefficients which weightedthe data in favor of the higher concentrations ofozone. Lower bacterial counts suffered fromgreater variability. The regression calculationsalso gave appreciable values of "a" in the termy = a + bx. For example, the largest a valuewas for growing cultures plated on syntheticmedium, i.e., y = -0.47 + (-0.55)x. The a

values may result from the counts of untreatedcultures being lower on the synthetic mediumthan on nutrient broth, or from the obviousshifts in the effects of ozone with increasingconcentrations. These effects are shown in Table1, in which are listed the survival rates and theratios of growth on salts-glucose and nutrient

o 0t - ° - -

DIRECT

x

wQCX x

o \xx x

SiS

2 4 0 1 82

NO~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~

-4 0

-t

(-6

2 4 6 8 10 12 18s20

)JG 03 /109

FIG. 2. Survival of resting Escherichia coli aftertreatment with ozone.

broth plates, and a calculation of excess "depletedcells." This term indicates the percentage ofbacteria which would grow on nutrient brothand not on synthetic medium, corrected for thissame factor shown by untreated cells. There was

a shift in survival to less than 10% at about 1 ugof 03/109 bacteria with both resting and growingcultures. There was another shift to less than 1%survival at about 5 ,ug of 03/109 bacteria.The concentration of ozone producing 50% or

more "cexcess depleted cells" among the survivorswas approximately 1.5 ,ug/109 with growingcultures and between 2 and 4 ,ug/109 with restingcultures. With survival rates of less than 1 %,most of the survivors seemed to be "depleted."

Microscopic inspection and counting of culturestreated with solutions of ozone indicated thatthere had been only slight immediate lysis ofcells, less than 50% even at the higher concen-

trations. After treatment with concentrationsgreater than 1 Ag/ml, the bacteria were no

longer observed to be motile. After ozone was

bubbled through the cultures, no bacteria were

intact and most had lysed, leaving "ghosts." Noozone was detectable in the media after treatmentof cultures either by ozone in solution or afterbubbling was stopped.

Effect on glutathione. By chromatography,glutathione was found to be the only sulfhydryl-

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TABLE 1. Survival and depletion after treatment ofgrowing Escherichia coli with ozone

Un- Ozonetreated

Expt Os M521 Survival 5X Excessno. NB Survival_ M52_"de-

X 100 NB pletedOn NB On M52 X 100 cells"

jig/lO9 % % % % %

V 0.25 80 72 40 56 24IV 0.59 83 61 72 120 0III 0.65 94 40 34 85 9III 1.35 94 4.2 2.5 60 34V 1.66 80 1.9 0.45 24 56

VII 1.9 6.8 3.0 45 40tVIII 1.9 6.0 1.9 32 53tVI 2.0 109 1.9 1.6 87 13IV 2.7 83 3.1 1.0 33 50IX 3.0 6.7 3.5 52 33tIII 3.6 94 0.39 0.42 110 0V 3.7 80 4.9 0.45 9 71IV 4.5 83 1.6 1.0 67 16II 5.3 85 0.04I 6.1 0.0016 0 loot

II 10.3 85 0.001I 14.0 0.00001 0 l0t

* M52, salts + glucose medium; NB, nutrientbroth.

t Estimated.

containing compound in detectable amounts inthe 70% alcohol extract of the bacteria. Theconcentration determined by titration of thealcohol extracts of untreated bacteria was vari-able, according to the stage in the growth cycleof the culture. With duplicate samples, the meanvariation from the mean was 8% in the experi-ments reported here. The content of glutathioneof untreated E. coli was found to vary widely(from 2 to 14 m,umoles/109 cells). If growingcultures of E. coli were held at 6 C in syntheticmedium without glucose for 1 hr, as is done toproduce synchrony of division, the glutathionecontents were 7 to 10 m,umoles/109. Warming to37 C might or might not lower the values. Ad-dition of glucose caused the values to drop to 2to 4 m,umoles, with a slow increase to 5 or 6m,umoles/109 before doubling of the bacterialcount at 60 min. Addition of nitrogen mustard(Scott, Lesher, and Rosenbaum, unpublisheddata) or N-methylmaleimide (Stern, 1960) inamounts which prevented or delayed division,without effect on turbidity, caused increases of

TABLE 2. Survival and depletion after treatment ofresting Escherichia coli with ozone

OzoneUn-

treatedExpt 03 M52* Survival MB 1Excessno. NB M52_________ "de-

x 100 NB pletedOn NB On M52 X 100 cells"

'AgI/l % % So So %

V 0.36 80 69 44 63 17IV 0.67 48 90 67 74 0III 0.86 110 36 36 100 0III 1.7 110 2.0 2.0 100 0V 2.4 80 1.3 0.13 10 70IV 3.1 48 2.0 2.7 135 0VI 3.25 75 1.05 0.7 67 8

I 3.3 60 24 1.9 8 62III 3.6 110 1.8 0.07 3.8 96II 4.2 32 5.0 0.5 10 22V 5.2 80 0.64IV 5.3 48 1.0 0 100VI 8.7 75 0.07 0.009 13 62

I 8.7 60 0.00014 0 100II 10.0 32 0.0037 0.00007 1.9 30II 18.0 32 0.0001 0 100I 20.0 60 0 0 0

* M52, salts + glucose medium; NB, nutrientbroth.

glutathione content to as much as 10 to 12m,umoles/109 bacteria.There were no SH- groups available to react

with silver on the surface of whole untreatedE. coli. Mixtures of solutions of glutathione andozone resulted in a loss of titrable SH- pro-portional to the ozone added. Table 2 lists theresults of four sets of titrations of the alcoholextracts of bacteria, untreated or treated withozone. After treatment with high concentrationsof ozone, very little or no SH- was detectable inthe extracts. Lower ozone concentrations usuallydecreased the SH- content but, at times, nochange or even an increase was found in the SH-of extracts of resting cultures, the viability ofwhich might be less than 10%.

Effect on nucleic acid. When ozone was allowedto bubble through a bacterial suspension, theabsorption (at 2,600 A) measurable in the super-natant fluid was increased (Fig. 3), with lessabsorption after 30 than after 10 sec. In the sameexperiment, suspensions of growing cells at halfthe count were treated with bubbling ozone forthe same time; after 30 sec of ozone, the super-

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OZONE EFFECTS ON E. COLI

2.0

1.4

1.2

w

z

mon0

(I)

4%

1.0

.800

.600

.400

.200

R 3

220 240 260 280 300 320

WAVELENGTH IN M)

FIG. 3. Effect of bubbled ozone on nucleic acids inthe medium. Resting cells were treated by directbubbling of ozone through a suspension of 1.7 X109 cells/ml in 25 ml of saline for R 1 = control;R 2 = 5 sec; R 3 = 10 sec; and R 4 = 30 sec. Thecells and protein were sedimented by treatment withperchloric acid (2.5%) and centrifugation. Spectrawere read on the supernatant fluid in a Beckmanmodel DU spectrophotometer. The number of viablecells on nutrient agar were R 1: 1.7 X 108; R 2:3.4 X 108; R 3: 4.0 X 108; R 4: 3.6 X 106. On salts-glucose, R 1: 1.1 X 109; R 2:2.0 X 108; R 3:1.2 X108; and R 4: not determined.

natant had no peak at 260 m,, and after 10 sec

the peak was lower than after 5 sec.

In a similar experiment, the protein releasedinto the medium was 53 ,g/ml from resting cellsand 31 ug/ml from growing cells after passage ofozone for 30 sec.

After treatment with solutions of ozone, therewas an increase in absorbance of the medium withas little as 0.18 Mg of 03/ml (3.75 X 10-1 M).With concentrations of 3 ,g/ml (6 X 10- M) or

.200

I'. 4

\0i+ ^ IN MEDIUM

0 A

\\OtooI A

.100 \ -%t\'s \

\0 IANa%PAW

0/ A~~~~~

100

220 240 260 280 300 400WAVELENGTH IN M)J

FIG. 4. Effect of ozone solutions on nucleic acids.Cultures of growing Escherichia coli were harvestedand suspended in saline with a final volume of 25ml and a count of 7 X 108 cells/ml. The ozone solu-tions added were 1: none; 2: 0.18 ag/ml; 3: 1.27,ug/ml; and 4: 2.5 ,ug/ml. Viable bacteria by platingon nutrient agar were 1: 6.7 X 108; 2: 4.8 X 108; 3:1.3 X 107; and 4: 3.3 X 107. On salts-glucose agar,1: 5.5 X 108; 2:2.7 X 108; 3: 3.0 X 106; and 4: 3.0X 10g. Spectra were read on untreated media (pH7.6) and on the two combined 1-ml washings with2% perchloric acid of the pellet from 2 ml of culture.

higher, there was less absorbance than at lowerconcentrations. Concomitant with the increase ofabsorbance in the medium was a decrease ofabsorbance in the material removed by washingwith 2% perchloric acid (Fig. 4). The release ofmaterial into the medium was usually greater bythe growing cultures than by resting cultures.The medium and various fractions of the bacteriawere analyzed for DNA and RNA by the sugarreactions with diphenylamine and orcinol, re-spectively. Figure 5 shows the increase in DNAin the medium and the decrease in the DNAdetermined in whole cells, in cold perchloricacid-extracted cells, and in the hot perchloricacid extracts, after treatment of resting cultureswith increasing concentrations of ozone. The

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SCOTT AND LESHER

'5

c 10

z

5

0

20

I~~~ ~ ~ ~ ~ ~ ~~~~XI0WECLE CELLS

OOLD POAaZXT- CT-

_~~~~~~~~~~~,,XT"I,__ __~~~~~~~~~

4.5 10

PG 03/10 BACTERIA

20

FIG. 5. Effect of ozone on deoxyribonucleic acid(DNA) of resting cultures. To resting-cell suspen-sions of 3.7 X 108 bacteria per ml were added ozonesolutions to give concentrations of 1.7, 3.7, and 7.4,pg/nil of ozone. DNA was determined by the di-phenylamine reaction in the medium, the whole cell,the cells extracted with cold 5% perchloric acid, andthe hot 5% perchloric acid extract.

pattern is similar after ozone treatment ofgrowing cultures.The effect of the ozone solution on the RNA

fractions appears in Fig. 6. Orcinol-reactingmaterial was increased in the medium and alsoin the fraction extractable with cold 5% per-chloric acid. The latter seemed to be derivedfrom the solubilization of RNA which is notusually extracted by cold perchloric acid and isshown as the residual RNA in cold-extractedcells or as that extracted with hot perchloricacid after cold extraction. Growing cells con-tained at least twice as much RNA as restingcells, distributed in about the same proportionsin the fractions. With ozone treatment, there wasmore RNA lost into the medium and less increasein that extractable with cold perchloric acid,while the decrease in the residual RNA wasapproximately the same in amount, althoughless, proportionately, than in resting cells.

It is evident that more than 10 Ag of ozone/109 bacteria were necessary to produce a loss ofDNA into the medium, whereas 4.5 ,ug of ozonecaused a significant increase in RNA in themedium and a change in solubility in 5% per-chloric acid of the RNA inside the cells.

Recovery after ozone treatment. Cultures treatedwith about 2 ,ug of 03/109 bacteria were dilutedinto various media, and the resumption of growth

15

I-120

.-g

z

5

0

VIK 0aLG

NOT PGAazv"^cT

wee1""

COL P0AUtXTRAG

OOLD PEXTRACTEDCILL

N XL 0.4EXTRACT

4.5 10 20

PG 03/109 CELLS

FIG. 6. Effect of ozone on ribonucleic acid (RNA)of resting cultures. Samples of the same suspensionsof bacteria as shown in Fig. 5 were analyzed forRNA by the orcinol method. Solutions are as forFig. 5 and, in addition, RNA was determined inthe cold 5% perchloric acid extract, and the 0.5 MN'aCl extract.

was investigated by determination of directcounts, turbidity, the uptake of nutrients fromthe medium, and the content of nucleic acids inthe hot perchloric acid extract. Figure 7 showsthe initial loss and then increase of turbidityby ozone-treated cultures diluted into nutrientbroth, into salts-glucose medium, into salts-glucose medium fortified with casein hydrolysate,and salts medium with casein hydrolysate alone.Also shown are the glucose uptake by the twocultures containing glucose, and the uptake ofribose-containing material by the culture innutrient broth. After ozone treatment, 6.8%of cells were viable on nutrient broth plates and3% of cells were viable on salts-glucose plates.After 25 min in all three media, by direct count35% of the cells had lysed. The loss of RNAfrom the cells at that time was 26, 43, and 51%in glucose + casein hydrolysate, glucose, andcasein hydrolysate, respectively. These resultswould indicate that in the casein hydrolysatemedium some cells which had not lysed had lostRNA, whereas in glucose + casein hydrolysatesome cells had taken up from the medium someof the RNA released from lysed cells.

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OZONE EFFECTS ON E. COLI

/ LUCOSE UPTAKC

/

/I~~~~~~a ICA-* 0 _--o GLAMOSE UPTAKEf *YX

/ __ *te UPTAKE BY NO

/ // / / O ~~~~~COUNT Ng

7- TU MsOITY NS9/ / T~~~~~~~~~~~~~URtIOlITY!

, / , e~~~~~~~OUNT . .CH

1/// / _o ~~~~~~~~TURBIDITY 0O/ / s @ ~~~~~~~COUNIT 6

~~/- ~̂~~> ~~ a~ COUNT CH

0 30 so so Ito Poo Igo too

T U X 11 N U T E 8

FIG. 7. Growth after ozone treatment. Escherichiacoli were harvested after 2 hr of growth in salts-glucose medium and suspended in 40 ml of salinemixed with 60 ml of saline through which ozone hadbubbled for 1 min. Cell count was 1.76 X 109 bac-teria/ml, and ozone concentration was 6 ,ug/ml;25 ml of the treated cultures were added to each offour towers with a total volume of 100 ml. Towercontained 24 ml of saline, 50 ml of double-strengthM52, and 2 mg/ml of glucose (0, G). Tower 2 con-

tained 23 ml of saline, 50 ml of double-strengthM52, glucose (2 mg/ml), and 1 mg/ml of caseinhydrolysate (A, G + CH). Tower 3 contained 26ml of saline and 60 ml of double-strength nutrientbroth (Difco); (0, NB). Tower 4 contained 24 ml ofsaline, 60 ml of double-strength M52, and mg/mlof casein hydrolysate (X, CH). The towers were

incubated with aeration at 37 C. Count is the directmicroscopic cell count.

For a similar experiment, Fig. 8 shows theincrease of RNA and DNA extracted with hotperchloric acid. It would seem that, when thebacteria took up nutrient from the medium, theystarted to grow again; the lag was shorter andgrowth faster the richer the nutrient medium.There was loss of RNA from the bacteria untilthe time when some were able to take up nutrientand start growing. Approximate values fornucleic acids, nucleotides, and nucleosides inthe cell-free media, obtained by measurement ofthe absorbance at 255 to 300 m,u at pH 2, in-dicated increases of 10 and 12 ,Ag/ml during thefirst 40 min of culture in salts + glucose and salts+ glucose + casein hydrolysate, respectively.During the same time, the sedimented bacterialost 11 and 7 ,g of RNA/ml, respectively. From98 to 177 min, while the RNA in the bacteria

100

A RNA(G#CH)Z RNA (NB)

~10

30 0/

30

I3 DNNA (NB)

Z- DNA(G.CH)

010

0~o 0 DNA (G)

o~~~~~~~~oS 6

C] 4_T gNlEN)ULJA

> 3 / T(G.CH)

~~~~~~~~~~~~~~~OT(G)

G0rt5lE g * [ s

30 60 90 120 I50 ISO 210

M NUTES

FIG. 8. Nucleic acids during growth after ozone.

Nucleic acids were determined in hot perchloric acidextract of samples of the cultures described in Fig. 7.T represents turbidity in Klett units with a 420-m,ufilter.

was increasing 20 ,ug/ml in the culture withglucose alone, the absorbance of the mediumdecreased equivalent to approximately 3.5 ,ug/mlof nucleic acid. Between 67 and 100 min, thefaster-growing culture in salts medium contain-ing glucose + casein hydrolysate showed an

increase of 16 ,g of RNA/ml in the bacteria anda decrease of 5.6 ,ug of nucleic acid/ml in themedium. Thus, the viable bacteria both removedUV-absorbing material from the medium andsynthesized RNA within the cells.

In the upper part of Fig. 9 are plotted thedirect counts of bacteria at times during theexperiment. However, from the results of plating,only 2% of the culture was able to grow on salts-glucose and only 6% on nutrient broth. In thelower part, the numbers of dead bacteria in theculture were subtracted from the direct counts,and the numbers of viable cells were plotted.It can be seen that, in 2 hr, four generations ofcells in nutrient broth produced a viable popula-tion equal to that before ozone treatment; the

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TOTAL CELL COUI

-A 0-0-S

CALCULATED VIABLE

IA I

IX~~~ ~ ~ ~ ~

r~~~ ~ ~ ~

,.1"' ,,,,0'~~~~~

0 30 60 90

MINUTES

FIG. 9. Total and viable cells dozone treatment. The cultures we?ment set up in the same way as

concentration was 1.9 Ag/109 bacculture was slated immediatelu. an

NT NB decomposition products of the nonviable bacteriain the medium.

/ G.&H Mutagenesis. Since after ozone treatment atconcentrations of 2 to 5 ,ug of 03/109 bacteria

a/o °__- G approximately twice as many bacteria wereviable in nutrientbroth as in salts-glucose me-dium, it might be suspected that half the viable

CELLS bacteria were "mutants." Because of the effect,"CH of casein hydrolysate noted above, several

attempts were made to isolate amino acid-re-d. quiring mutants. Several cultures were obtained,

all of which required methionine which was notA - replaceable by vitamin B12.

Effect of ozone on nucleic acids. We also in-vestigated the effect of ozone on purified nucleicacids and on the nucleotides, nucleosides, andpurine and pyrimidine bases. The effects at lowconcentrations were attacks on the pyrimidinerings and loosening of the bond between thepyrimidine base and the sugar moiety, so that

120 150 180 the latter could react with diphenylamine ororcinol in the usual analytic procedures. Athigher concentrations, our results were similar to

uring growth after those of Christensen and Giese (1954), in thatre from an experi- th pyiidn an puiernsweeboe nFig. 7. The z thepyromzne and purine rmgs were broken andteria. The treated the sugars decomposed. The results of thesend the viabe, rount studies will be reported elsewhere in detail.

graphed at 0 time. The nonviable cell count was sub-tracted from the direct counts (upper graph) atthe indicated times to give the plotted calculatedviable counts in the lower graph.

doubling time was 30 min. On salts-glucose,six generations with a doubling time of approxi-

mately 45 min would have reached the same

population at about 270 min if the experimenthad been continued. The usual division time ofE. coli in salts-glucose under the conditions inour laboratory is 55 to 60 min. With addition ofcasein hydrolysate to the salts-glucose, thedoubling time was reduced to 40 min, and thepopulation was replaced in approximately 160min. It must be concluded that the early perioddid not represent a lag phase in the usual sense

for all the bacteria in the culture. During thistime, the nonviable bacteria (more than 90%)were losing ribonucleotides or ribonucleosidesand protein into the medium and probably othercell substance which we did not measure. Approxi-mately 50% were lysing completely, while theviable cells started growth immediately at ratesdetermined by the added nutrients and the

DISCUSSION

It seemed evident that, in the treated cultures,the ozone attacked the primary structure ofnucleic acids or their decomposition productsonly after they had been released into the mediumby leakage or lysis. The effect of ozone on thenucleic acids within the cells may have beenindirectly on the states of aggregation of thenucleoproteins, and not directly on the nucleicacids themselves. That the ozone did not seem

to penetrate the cells is indicated also by theinability of even fairly high concentrations ofozone (up to 40 times the glutathione equiva-lents) to oxidize all the SH- of glutathionecontained within the cell. The loss of protein andnucleic acids by the ozone-treated cells impliesthat the primary locus of activity of ozone was

the bacterial-cell surface, as suggested byChristensen and Giese (1954). Since we have notbeen able to detect any SH- groups by ampero-

metric titration of whole bacteria, we concludethat there are no SH- groups available on thecell surface to react with ozone.

Other substances with which ozone reacts

5x 109

IX109

z 5X1080C-) x

< X 109u1JH-10

m

IX108

5XIC7'

IX 107

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OZONE EFFECTS ON E. COLI

rapidly and completely include unsaturatedfats. Hann (1950), quoting Harries (1910) andothers, reported that ozone reacted with oleicacid rapidly even at temperatures as low as-78 C to produce ozonides, and that hydrolysisor reduction of the ozonides broke the C-Cbond, producing two fatty acids of nine carbonseach.According to Salton (1960), 15% of the dry

weight of E. coli is cell wall. Of the cell wall, 23%is lipid and 13% is phospholipid. Phospholipidmakes up 20% of cytoplasmic membrane (Mohan,persona.l communication).An iodine number for this lipid has not been

found in a search of the literature. Grylls (1961)gives the iodine number for yeast lipid as 130.Assuming that E. coli lipid has approximatelythe same iodine number, we may calculate thatthe lipids in the cell wall of one bacterium willhave 108 double bonds.At a concentration of ozone which kills 50%

of a culture, the calculated number of moleculesof ozone per bacterium is approximately 2 X 107;for 1% survival, the number is approximately4 X 10g.

Thus, the number of lipid double bonds in thecell wall may be of the same order of magnitudeas the number of molecules of ozone which killone E. coli bacterium. It is also of the sameorder as the number of molecules of iodine whichare removed from solution by one bacterium(approximately 7 X 107; Scott and Lesher,unpublished data). It is also of interest that thisis the same order of magnitude as the number ofmolecules of nitrogen mustard (bischlorethyl-aminoethane) per bacterium which will kill 50%of a culture. So it may be postulated that theprimary attack of ozone, of iodine, and perhapsof nitrogen mustard is on the double bonds of thefatty acids in the cell wall and membrane of thebacterium.Nathan (1961) reported experiments on induc-

tion of oxalacetate decarboxylase synthesis inLactobacillus plantarum by malic acid in thepresence but not in the absence of chlorproma-zine. It was suggested that chlorpromazinealtered the cell membrane so that it was permeableto malic acid, at drug concentrations which didnot affect viability. Nathan and Friedman (1962)indicated that the site of action of chlorproma-zine may be a lipid.Whether the primary site of attack of ozone

on the cell membrane of E. coli was on theunsaturated lipid or not, the end result wasincreased permeability. Since the ozone causedleakage of the contents of cells that were notlysed, it is probable that some of the cells wereso depleted that they could not resume growthon salts and glucose alone but were able to growon media that supplied more complicated organicmaterials, which might act as "pump primers."This might explain the observation that thehigher the concentration of ozone and the fewersurvivors there were, the greater was the propor-tion of them which grew on the enriched medium.Davis (1959) found very high rates of mutation

from streptomycin dependence to nondependence,but slight increase in frequencies of phage re-sistance, after exposure to ozone. It is difficultto conceive of the mutagenesis of ozone on thebasis of a primary reaction with lipid. However,ozone was found to react with nucleic acids re-leased into the medium, especially with thepyrimidine bases (thymine being more easilyattacked than cytosine and uracil). If thesechanged bases were then assimilated by the still-viable bacteria and built into the nucleic acids,we might expect to find mutants among theoffspring of the survivors. We were able to pickout only a few mutants, all methionine-requiring,when we tested for amino acid mutants. Themethod for isolating mutants involved centrifuga-tion and washing of the bacteria before theywere suspended in salts-glucose medium con-taining penicillin. Thus, the products of leakageand lysis, including those changed by ozone,may have been removed before the viable cellshad a chance to absorb them. In other attemptsto isolate mutants, an ozone-treated culture wasallowed to grow in nutrient broth before treat-ment with penicillin. All colonies picked asmutants grew on minimal medium when platedagain, so no true mutants were obtained.

In the work reported here, the nucleic acidsin the medium were not isolated to determinewhether they were RNA, DNA, mono- orpolynucleotides, or nucleosides. However, littleor no DNA was lost from cells treated with lessthan 10 ,ug of ozone per 109 bacteria, so thenucleic acid lost to or taken up from the mediumwas mainly RNA.That chemically altered RNA may be mu-

tagenic for a RNA-containing virus was shownby Gierer and Mundry (1958). They produced

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SCOTT AND LESHER

mutants of tobacco mosaic virus by treatment ofits separated RNA with sodium nitrite. Theyfound that, if the RNA was treated for a timewhich reduced the incidence of infection to 50%,19.5% of the infectious units were mutants ofone kind, which produced necrotic rather thanchlorotic lesions on Java tobacco leaves. Whentested on Samsun tobacco, 33 of 60 (55%) ofthe progeny virus isolated produced a variety ofsymptoms different from the untreated TMWvirus or RNA, whereas 20 of the 60 were nolonger viable (Mundry and Gierer, 1958).Litman and Ephrussi-Taylor (1959) reported

that isolated DNA of pneumococci, which wastreated with nitrous acid, produced mutants fordrug resistance at 10% of the rate of mutationproduced by transforming DNA. Ultravioletirradiation of the DNA produced only inactiva-tion and no mutants.

It may be that the cells which we at firstisolated as mutants were temporarily modified byassimilation of altered RNA, whereas Davis'smutants for streptomycin nondependence mighthave resulted from uptake of DNA or deoxy-ribosides altered by ozone. Certainly, furtherwork is required to elucidate the mutagenicaction of ozone.

ACKNOWLEDGMENTSThis work was carried out under U.S. Public

Health Service grant no. CY-2189.We wish to acknowledge the assistance of

Harold Wilson and Warren Williams, who workedwith us during the summer of 1960 under aNational Science Foundation Program grant.A part of this work was reported at the meeting

of the American Chemical Society in New YorkCity, September 1960.

LITERATURE CITEDBARRON, E. S. G. 1954. The role of free radicals and

oxygen in reactions produced by ionizingradiations. Radiation. Res. 1:109-124.

BENESCH, R., R. E. BENESCH, M. GUTCHO, AND L.LAUFER. 1956. New color test for thiols andthiolesters. Science 123:981-982.

BENESCH, R. E., H. A. LARDY, AND R. BENESCH.1955. The sulfhydryl groups of crystallineproteins. I. Some albumins, enzymes, andhemoglobins. J. Biol. Chem. 216:663-676.

BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimation of deoxyribonu-cleic acid. Biochem. J. 62:315-323.

CHRISTENSEN, E., AND A. C. GIESE. 1954. Changesin absorption spectra of nucleic acids and their

derivatives following exposure to ozone andultraviolet irradiation. Arch. Biochem. Bio-phys. 51:208.

DAVIS, I. 1959. The survival and mutability ofEscherichia coli in aqueous solutions of ozone.Ph.D. thesis, University of PennsylvaniaMedical School, Philadelphia.

GIERER, A., AND K. W. MUNDRY. 1958. Productionof mutants of tobacco mosaic virus by chemi-cal alteration of its ribonucleic acid in vitro.Nature 182:1457-1458.

GRYLLS, F. S. M. 1961. The chemical compositionof yeasts, p. 1050-1053. In E. Long [ed.],Biochemists handbook. D. Van Nostrand Co.,Inc., Princeton, N. J.

HANN, V. A. 1950. "Tonnage ozone" for chemicalprocessing. Chem. Industries (Philadelphia)67:386-389.

HARRIES, C. 1910. tber die Einwirkung des Ozonesauf organische Verbindung. Ann. Chem.374:288-368.

LITMAN, R. M., AND H. EPHRUSSI-TAYLOR. 1959.Inactivation et mutation des facteurs geneti-que de l'acide d6soxyribonucldique du pneu-mocoque par l'ultra violet et par l'acide ni-treux. Compt. Rend. 249:838-840.

MILLER, G. L., R. H. GOLDER, AND E. E. MILLER.1951. Effect of varying proportions of com-ponents of Bial's color reagent. Anal. Chem.23:903-905.

MUNDRY, K. W., AND A. GIERER. 1958. Die Erzeu-gung von Mutationen des Tabakmosaikvirusdurch chemische Behandlung seiner Nuclein-saure in vitro. Z. Vererbungslehre 89:614-630.

NAGY, R. 1959. Application of ozone from steri-lamp in control of mold, bacteria, and odors,p. 57-65. In Ozone chemistry and technology.American Chemical Society, Washington,D.C.

NATHAN, H. A. 1961. Alteration of permeabilityof Lactobacillus plantarum caused by chlor-promazine. Nature 192:471-472.

NATHAN, H. A., AND W. FRIEDMAN. 1962. Chlor-promazine affects permeability of restingcells of Tetrahymena pyriformis. Science135:793-794.

SALTON, M. J. R. 1960. Microbial cell walls.John Wiley & Sons, Inc., New York.

SCOTT, D. B. McN., AND E. CHU. 1958. Synchro-nized division of growing cultures of Escherich-ia coli. Exptl. Cell Res. 14:166-174.

STERN, H. 1960. Biochemical sequences in mitosis,p. 135-165. In D. Rudnick [ed.], Developingcell systems and their control. 18th GrowthSymposium, Ronald Press Co., New York.

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