CO2. The effect was far less than with CO. They also experimented extensively with the effect of gases

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THE NATURE OF THE EFFECT OF CO2 UNDER PRES-SURE UPON BACTERIA

JUDSONS. SWEARINGENANDI.M. LEWIS

Department ofChemistry andDepartment of Bacteriology of the University of Texas,

Austin, Texas

Received for publication, October 19, 1932

D'Arsonval and Charrin in 1893 reported that fifty atmos-pheres of CO2 destroyed Bacillus pyocyaneus in two hours. Several investigators had difficulty in duplicating these results. In 1917 Larson, Hartzell, and Diehl obtained reliable results showing that air at 6000 atmospheres hydrostatic pressure de-stroyed non-spore-forming bacteria and that 3000 atmospheres or 44,000 pounds per square inch had no effect on any of those tried, including B. typhosus, B. coli, B. tuberculosis, B. proteus, B. subtilis, staphylococci, streptococci, and pneumococci.

They also experimented extensively with the effect of gases under pressure upon bacteria, and reported no effect with nitrogen at 120 atmospheres pressure, and 10 to 40 per cent fatality in twenty-four hours using hydrogen at 120 atmospheres pressure withB. coli. With C02, 50 atmospheres woulddestroy B. typho-sus, B. coli, B. tuberculosis, etc., in one and one-half to two and one-half hours. They made colorimetric pH determinations of the solution while under pressure and found it tobealittle under 4. Theoretically, it should be 3.15 in pure water. They then acidified cultures to this pH and noticed very

slight

effect upon standing. Cultures were also acidified and sodium chloride added up to the molality existingwhen under 50 atmospheres of CO2. The effectwasfar lessthan with CO.

The investigators suggested that this result may be due to a combination of effects, especially to the quick expansion of the gas, and indicated that theoneand one-half to two and one-half hours was the time necessary for the bacterial bodies to become saturated with the gas.

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Valley andRettgerin 1927reported anextensive piece of work showing that bacteria are comparatively unaffected by CO2 at one atmosphere.

In 1932, David Crowther, who assisted Larsoin, Hartzell and

Diehl during their work in 1917, reported that 800 pounds per

square inch pressure of CO2 in one and one-half to two and

one-half hours is effective in destroying bacteria if the gas is allowed

toexpandsuddenly.

There seems tobesome confusion as towhy CO2has this lethal

effect upon bacteria. The purpose of this work was to study separately the individual influences at work so as to get amore

definite answertothe question.

Since the action on all bacteria is likely to be the same, our

experiments were confined to the use of Escherichia coli no. 463

furnished by thislaboratory. Thisorganismwas chosen because ofits similarity to certain pathogenic bacteria, and because of its ease of cultivation.

First for consideration, was the effect of sudden expansion

while the bacterial bodies were saturated with the gas under pressure, which was expected to have an effect analogous to

plasmoptysis, namely that of rupturing the cell walls.

Fortheuse ofCO2, anapparatuswasbuilt asshown in figure 1, whereby the culture could be saturated with the gas and allowed to expand suddenly into avacuum.

The portion of the apparatusA was shakenwhile theCO2

pres-sure was being applied so as to facilitate saturating the solution.

The stopcock b was then closed and the apparatus allowed to stand for fifteen minutes. A closed armmanometer was used to

measure the pressure. This apparatuswas suited only for

pres-sures under three atmospheres.

The flaskFwas evacuated to a pressure of 20 mm.

At the end of fifteen minutes the apparatus A was raised to a

vertical positionso that thesteel ball sdroppedon thethin-walled portion of the culture container c, breaking it and allowing the

culture saturated with CO2to expand suddenly intothe vacuum.

The data in table 1 wereobtained onplating out samples before

and after treatment.

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EFFECT OF CO2 UPON BACTERIA

The pressure was obviously not high enough to prove destruc-tive. A calculation was made to find the pressure necessary to overcome the effect of surface tension in a bubble whose diameter is approximately one-tenth the diameter of a bacillus.

For a gas in pure water at room temperature the result was 400

pounds per square inch, and for a bubble about the size of a bacterium, it was 40 pounds. We knowof no substance that is

F 7oCO2 -) - Topressure Cylinder manometer FIG. 1 - r Glass I Rubber tubing Chlorine valve FIG.2

likely to be present in the bacterial body that would alter this result appreciably so the pressure that is effective in rupturing the cell wallmustbe onewhich isin excess ofthis.

A new apparatus for

applying

greater pressure was then built asshowninfigure2. Theinclosedglasscontainerwas so

designed

that the unit maybe inverted or shaken without spilling its

con-tents. The lower valve through which samples are taken is a

JOURNALOFBACTERIOLOGY,VOL.XXVI,NO.2

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chlorine valve andis not subject to corrosion. Its outer opening

is wide andis easily washed with a wash bottle while the valve is closed.

The writers wish to express their appreciation to MIr. W. IL.

Benson, IAiechanician in the Department of Chemistry, whose

help in the construction of apparatusgreatly facilitated the work. With this apparatus an experiment was performed using a

similar culture to that employed in the first experiment but CO2

TABLE 1

NVimber of baicteria present

OIlGINAL TItEA'T'ED

39 X 10 44 X 107

45 X 107 45 X 107

TABLE 2 Bacteria prese.nt

ORIGINAL TIIEATED PERCENT SURVIVING

At400 pounds per square inch gauge for twenty minutes

370 X 106 8X 106 1

400X 106 6X 106 1 8

At 200 pounds per square inch gauge fortwenty minutes

400 X 10 115- X 106

480 X 106 100 X 106 24

wasapplied here athigherpressures. The resultsgivenintable 2 were obtained.

The apparatus containing the liquid andthe gas under pressure

was well shaken so that diffusion limited only the saturation of

the bodies themselves. In showing thatfifteen minutes is ample

time for the gas to saturate the cell bodies, particularly with

substances asuniversally soluble as CO2 and

02,

we call attention to a homely comparison. By mathematical analysis it may be shown that the time of diffusion varies asthesecond power of the radiusfor ahomogeneous sphere, for similar final states of

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EFFECT OF CO2 UPON BACTERIA

tion. This means that a bacterial cell after fifteen minutes is as saturated as one the size of a baseball would be after 50,000 years I

Now, using oxygen, which is comparatively inert and not so soluble that its molal concentration is detrimental, the results shown in table 3 were obtained using a fifteen-minute period of detention and then sudden expansion through the valve.

The temperature was kept at 0°C. to increase solubility, and at 1200 pounds the liquid should have contained at least three volumes ofthe gas. Ifthe cell was to be exploded, it surely would have occurred here. The per centsbelow a hundred were

proba-TABLE 3

Pressure gauge. Original 300 500 700 900 1,200 Platecount. . 325 X 106 350 X 106 375 X 101 250 X 106 250 X 106 325 X 106 Plate count. 375 X 106 350 X 106 325 X 106

Per cent

sur-viving 100 100 71 71 93

TABLE 4

TIME OF PERCENT

NIUMBER APPLICATION TIMEOFSTANDING TIME OFRELEASE SURVIVING

1 Suddenly 20minutes Suddenly 5

2 Suddenly 20minutes 20minutes 1.4

3 20 minutes 20minutes 20minutes 1.0

bly due to errors in dilutions and plate counts. These cells

showed a tendency to agglutinate.

A trial was made with CO2, using 350

pounds

maximum pres-sure with sudden

application

of pressure, twenty minutes

stand-ing, then sudden

release;

then another with

gradual

release of pressure over a periodof twenty minutes; and athird where the pressure was gradually applied and also

gradually

released. As shown in table 4, the latter was most effective and the former least effective. These trials required twenty, forty, and

sixty

minutes respectively, and long exposure should account for the

results.

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We conclude therefore, that the explosion of the bacterial bodies by suddenly releasing the pressure when the cells are

saturated with a gas is not possible except where relatively

enormous amounts of the gas are dissolved as in the case of CO2

near itspoint ofliquefaction.

The effect of time is illustratedinthe data of table 5. Samples

were taken at intervals and the outer opening of the valve was washed out with sterile water before taking each sample. The

counts ran sohigh that this simple washingwassufficient. Time

of exposure to CO2 then seems to be the controlling factor in the

process and in view of the rapidity with which diffusion takes

place through microscopic distances, a chemical change must be proceeding.

TABLE 5

Time... Original 3 minutes 7minutes 20minutes

Plate count... 130 X 106 100 X 106 90 X 106 55 X 10,

Plate count... 100 X 106 100 X 106 65 X 106

Per cent surviving 87 83 52

The hydrogen ion may easily be related to the reaction, so considerable attention was given to the bearing of pH upon the process.

The Mass Law Principle was made use of, whereby the pH was controlled by addition of HCl or NaHCO3 in proper amount as found in the following manner.

Consider a titrationcurve in which

log1o

(H+) isplotted

against

the logarithm of the amount in liters of N/i acid added to 1 liter of solution.

Also (Lewis and Randall, 1923):

(H+) X (HCO3-) = (H2C03)k1 = kPco

Basing calculations on data given by Lewis and Randall for 300

pounds

per squareinch pressure, we

get

(H+) X (HC03-) = lo- 5 or

log (H+) + log (HCO3-) = -6.5 ₍₁₎

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EFFECT OF C02 UPON BACTERIA

Uponapplying CO2 to the solution, it acts as a monobasic acid H(HCO3) so that the above titration curve (fig. 3) for the strong acid is followed up until the conditions of (1) are satisfied. Since the reaction with H(HCO3) leaves an HCO3- for each H+ used,

FIG. 3

wemay substitute (H+) for

(HCO3-)

in (1) andbe correct within experimentalerrorfor dilute solutionsasis thecase here. We get

log (H+) + log (acid added) = -6.5 (2) Plotting' thisequation onthesamegraph asthe titrationcurve (fig. 3) gives a graphical solution of the pH

prevailing

when CO2

at300

pounds

per square inch is

applied.

IThe purpose ofplotting logarithms is to have the curve for (2) become a

straightline. Correspondingtodifferent pressures, otherstraightlinesparallel

tothisonemayconvenientlybe drawn.

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If NaHCO3 or HC1 had previously been addedto the solution,

the titration curve would have been in a different position and

would have cut the curve for (2) at a different point indicating a

higher or lower resulting pH, respectively. Adding acid moves the curve to the left andaddingsoda movesit to the right; so, to

obtain a desired resulting pH, observe the difference in acid corresponding to the abscissae of the two curves at the desired hydrogenion concentration and add the difference in acidorsoda as the case may be. In case ofsoda, this is the amount for final unitvolume,notperunit volume of culturemedium.2

I-Ef

fect ofpH

A/one

lI--Ef

fect of

pHi

Upon 60 S :rthe Act/onofCO2

Per-cent

4OSurvIvin9

-2°-

.VI9I1

-

-C

~F

5 45 4 35 3 2.5

FIG. 4

A number of experiments were conducted, the results of a typical one of which is shown in figure 4. The portion of the

2The volume v of NaHCO3 forfinal unit volume of solution is given by the

equation

b v a

v =-+

X-c 1-v c

a = molsacidrequired to raisepHof unit volume of culture torequired value.

b = difference in abscissaeofthe two curves in figure 3 at the required pH.

c = concentration ofNaHCO3solution.

( X issmall andwe arewithin the range of experimental errorin neg-levc

lectingit.

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EFFECT OF C02 UPON BACTERIA

curve BCD is typical, but the part AB depends upon the history of the culture with respect to the mineral constituents of the cul-ture medium. If it originally contained practically no salt, the curve will assume a form shown by A'B. We consider this as a complication connected with the large amount of NaHCO3 necessary to be added in order to reach the higher pH values, and we are notconcerned with studying it in detail.

In most of these experiments all of the liquids with which the bacteria came in contact had total ion concentrations equal to that of 0.86 per cent NaCl solution. This was so that HCI or NaHCO3 solutions could be added without increasing the ion concentration of the solution.

The effect of the pH alone is shown by the curve EF. Obvi-ously more acid must be added to these than to the ones that are treated with CO2 and thereby attain the same hydrogen ion concentration.

As seen from figure 3, the pH attained without addition of other reagents is about four, higher for more buffer and lower for lessbuffer, such as protein constituents. Inthis vicinity the curve (fig. 4) is quite flat showing that the hydrogen ion has nothing to do with the process as long as its concentration is not excessive. As previously mentioned, it was noticed that increased salt content such asisproducedbyadding a large amount ofNaHCO3 to the culture in order to reach the higher pH values, made the CO2more effective. From this we would conclude that the com-bined action is precipitation of the colloidal particles in the cell body. This is about the only assistance this harmless salt could give. There is no direct relation between this effect and any of the ionic ormolecular concentrations resulting from the increased concentration of NaHCO3. Also, there is no changeinthe fugac-ity ofCO2or H2CO3as longas the solution is in equilibrium with the CO2 atmosphere which is at constant pressure. Results analogous to those obtained with alkalies byLevine et al. (1928)

were notencountered.

Hydrogen ion is a more active precipitator of colloids due to

its smallmass. Ithas asimilar andmuch more prominenteffect, becoming quite noticeable when its concentration is as high as N/1 ,000.

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This, and the phenomenon of time and a more or less critical concentration of C02, all are in keeping with experience with colloidal systems.

The conclusion reached from the results of our experiments therefore is that CO2 (or H2C03 which is thoughttobein equilib-rium with it) at concentrations attained using pressures of the order of 15 to 30 atmospheres, is active in precipitating colloidal bodies within the bacterial cells. With higher pressures, such enormous amounts of the gas aredissolved that the pressure may be released with sufficient suddenness to explode many of the cells before too much gas is lost by diffusion.

SUMMARY

1. An apparatus for treating liquid cultures with a gas is described.

2. The individual effects of suddeni application of pressure, sudden release ofpressure, time, andhydrogen ion concentration upon the bacteria-destroying power of CO2 under pressure are given.

3. The action is indicated to be a

precipitationi

of certain colloidal systems within the cellbody.

REFERENCES

CROWTHER, DAVID 1932 Ind.Eng.Chem., NewvsEd., 10, 43. D'ARSONVALANDCHARRIN 1893 Compt. rend. Soc.Biol., 467, 764.

LARSON, W. P., HARTZELL, T. B., AND DIEHL, HAROLD S. 1917 Jour. Infect.

Dis., 22, 271.

LEVINE, MAX, TOULOUSE, J. H., ANDBUCHANAN, J. IH. 1928 Ind. Eng. Chem.,

20,179.

LEWIS,G. N.,ANDRANDALL,MERLE 1923 Thermodynamics and the Free Energy of Chemical Substances. McGraw-Hill Book Company, New York. Pp. 312.

VALLEY,GEORGE,ANDRETTGER,LEO F. 1927 Jour. Bact.,14, 101.

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