P.jb.asm.org/content/40/5/601.full.pdf · THE PRODUCTION OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN: THEROLE OF AGAR, E. P. CASMAN AbingtonMemorialHospital, Abington, Pa. …

THE PRODUCTION OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN: THE ROLE OF AGAR,

E. P. CASMANAbington Memorial Hospital, Abington, Pa.

Received for publication April 30, 1940

For the production of high titer staphylococcus toxin,2 the ad-dition of agar (Burnet 1930) to the medium, and incubation underan atmosphere containing added carbon dioxide (Parker, Hopkinsand Gunther, 1925-1926) have been found to be essential inmost instances. Shallow layers of semi-solid nutrient agar (0.3to 0.5 per cent) are inoculated with the staphylococcus and in-cubated under an atmosphere containing measured amounts ofcarbon dioxide in oxygen or air. Incubation is continued as arule for 48 to 72 hours and the toxin is obtained after removalof the agar and organisms by filtration. Toxin produced by thisprocedure has been of much greater potency than that formed influid media.Walbum (1922), studying the formation of a "hot-cold"

hemolysin for goat cells in a fluid medium, noted that for optimum"hemolysin" production the best results were obtained when themedium contained magnesium, from 0.1 to 0.3 per cent peptoneand a diminished amount of meat extractives and sodium chloride.The speed of production and deterioration of toxin was greaterwith the smaller amounts of peptone than with the larger.McLean (1937) found that by immersing a cellophane bag

containing saline, in broth, for 24 hours and then inoculating thecontents of the bag with a toxin-producing strain of staphy-lococcus (Wood 46), he could obtain a toxin of a titer (Lh 0.1)which compared favorably with that produced by the semi-solidagar technique (Lh 0.08). He attributed the increase in toxin

I Aided by a grant from the Eleanor Widener Dixon Fund for Medical Research.2 In this paper the alpha-hemolysin of Staphylococcus is referred to as the toxin.

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E. P. CABMAN

formation through the use of agar and the cellophane bag to theadsorption of substances inhibiting toxin production. Thecellophane bag procedure, however, could not be applied to largevolumes of medium for the elaboration of potent toxin.McIlwain (1938) attempted to determine the constituent of

agar that was responsible for the increase in toxin productionwhen added to the chemically defined medium of Gladstone(1938). He found that toxin formation was affected unfavorablyby the calcium of the agar, and that the remainder of the agarmolecule served in a toxigenic manner. The stimulating prop-erty was associated with the gelling power of the agar. Mc-Ilwain also suggested that this favorable action of agar was dueto the adsorption of inhibiting substances.Attempts were made by the present author to make the gases

concerned more available to broth cultures of bacteria withoutadding such extraneous substances as agar or cellophane to themedium. To accomplish this, two procedures were employed:(1) Bubbling the gases through the culture (Casman 1938) and(2) continuous rocking. With both procedures a markedenhancement of toxin production was obtained.

BUBBLING GASES THROUGH THE CULTURE-METHODSAND MATERIALS

The medium used in the experiments described below wasusually veal infusion (500 grams per liter of distilled water) con-taining 2 per cent proteose peptone (Difco) and 0.7 per centsodium acetate. Occasionally 0.5 per cent sodium chloride wasincluded. The pH, except where otherwise noted, was 6.8.For the bubbling of gas through the culture a modification of

the apparatus of Plastridge and Rettger (1929) was used. Thegas was forced through a trap containing sulphuric acid, thenthrough the culture tube at a rate of approximately 100 ml.per hour. The culture tube measured 1.0 by 8j inches, and wasaerated by means of a glass tube having a bulb at one end. Thebulb was perforated with seven or eight small holes. In laterexperiments a sintered glass gas-diffusing tube was employed.Using this procedure, 50 ml. of medium were inoculated; during

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the aeration process samples were removed at various intervalsfor toxin titration, pH determination and estimation of amount ofgrowth by matching with turbidity standards (McFarland, 1907).The toxin-producing strain used in the "bubbling" experiments

was #2081, obtained from the Sharp & Dohme Laboratories.It was usually added as one drop of a saline suspension (onebillion organisms per ml.) of a 24-hour beef-infusion agar slantculture.The toxin was titrated with staphylococcus antitoxin prepared

in horses by the Sharp & Dohme Laboratories at Glenolden, Pa.,and checked with standard antitoxin obtained from the NationalInstitute of Health. The Lh values were determined by mixingvarying amounts of toxin with a fixed amount of antitoxin(usually one-half international unit). After preliminary in-cubation at 37.50C. for a hour, indicator cells in the form of anequal volume of 2 per cent washed rabbit erythrocytes wereadded. The dilution showing 50 per cent hemolysis after anotherhour's incubation at 37.50C. was taken as the end point. Lfdeterminations were sometimes made.

RESULTS

In order to compare different combinations of gases, the follow-ing gas mixtures were used in an aeration (bubbling) experiment:A, oxygen; B, 95 per cent 02 plus 5 per cent C02; C, 80 per cent02 plus 20 per cent C02; D, air. A veal-infusion medium of pH7.2 containing 1 per cent proteose peptone and prepared accordingto Wright's method (1933) was employed. Samples wereremoved and studied after 24, 72 and 120 hours of aeration.From figure 1 it is evident that, with an initial mixture of

80 per cent 02 and 20 per cent C02, the Lh value at 120 hours was0.1 ml. Aeration with ordinary air resulted in the production ofno appreciable amount of toxin, while the mixture of 80 per cent02 and 20 per cent C02 was markedly superior to either oxygenalone or a mixture of 95 per cent 02 and 5 per cent C02.

' When plate counts were made the values were much less than those obtainedby the turbidity-matching procedure. The latter technique was used, however,because of its convenience and probable greater accuracy in determining relativeamounts of growth.

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E. P. GASMAN

Since Walbum (1922) had been able to accelerate toxin pro-duction by diminishing the sodium chloride, meat extractives andpeptone content of his medium, a study was made of the effect ofdilution of the medium on toxin production while aerating with80 per cent O and 20 per cent CO2 by the bubbling procedure.The basic medium was veal infusion containing 2 per cent proteosepeptone, 0.7 per cent sodium acetate and 0.5 per cent sodiumchloride. The undiluted medium and portions diluted withwater to give 75, 62.5 and 50 per cent medium concentration were

10 c

7.5-t.itstoxin

a1.5

2.5

l 2 3 4 5DayS

A Oxygs 5% carbon dioxiae in oxygenc 20% a Ul" ,

FIG. 1

aerated for 113 hours. Samples were removed at the end of 24,48, 72 and 96 hours and titrated for their toxin content. Intitrating these samples the supernatants obtained after centrifuga-tion were used.The pH, Lh, and in some instances the Lf, values are presented

in table 1. It is evident that by dilution of the medium theaeration and incubation time may be shortened to 72 and 96hours and that potent toxin may be obtained (Lh 0.04). It isalso evident that toxin production is associated with a rise in pH.When the "bubbling" aeration procedure was applied to a

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large volume of medium in a Florence flask, no appreciableamounts of toxin were obtained. It was observed that the gasevolved in the aeration tube would rise almost in a straight lineto the surface and escape without aerating the medium. Byconstant stirring of such a large volume of medium and by aera-tion with very small bubbles of gas it was possible to obtain anappreciable increase in the amount of toxin. By diluting thebasic medium described above with an equal quantity of distilledwater, and with the use of a stirring device, it has been possibleto obtain an Lh value of 0.1 ml. in 48 hours in a large volume ofmedium (3500 ml.). A Berkefeld V filter was used in order toobtain bubbles of suitable size.

TABLE 1Effect of dilution of the medium on toxin production and pH in the "bubbling

aeration" procedure

24 HOURS 48 HOURS 72 HmouR 96 HOUS 113 HOURSPER CUNTMEDIUM

Lh pH Lh Lf pH Lh Lf pH Lh Lf pH Lh Lf pH

100 0.5 6.4 .16 6.60.1 6.8 .08 .125 7.3 .060.1 7.375 .24 6.4 0.1 .125 6.7 .06 .08 7.2 <.06 .07 7.5 .06 .075 7.762.5 .22 6.4 .09 .09 6.8 .06 .06 7.3 .04 .05 7.6 .06 .045 7.750 0.2 6.5 .08 .075 7.0 .06 .065 7.3 <.06 .055 7.7 .06 .055 7.8

In numerous "bubbling" aeration experiments with the dilutedveal infusion medium and with media consisting of yeast extractand acid digests of casein or gelatin there was a moderate toxinyield in from 24 to 48 hours. On continued aeration, however,there was a loss of potency. An example of this is evident intable 1 in which the results obtained with diluted veal-infusionmedium are presented. Here the Lh values for the diluted mediaare greater at the end of 113 hours than at 96 hours.Although toxin production was markedly improved by the

"bubbling" aeration procedure, it did not equal that obtained bythe agar method (Lh 0.025 to 0.03).

AERATION BY CONTINUOUJS ROCKING

In experiments in which aeration by rocking was practiced,veal-infusion medium was employed, as before. Shallow layers

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. P. CAs

of the medium (5 ml.) in 50 ml. Erlenmeyer flasks were inoculatedwith one drop of a saline suspension containing approximatelyone billion organisms per ml. of a washed culture of a suitablestrain of staphylococcus. The flasks were closed with sterilehollow rubber stoppers through which sterile hypodermic needleswere passed. The hubs of the needles were plugged with cotton.The flasks were placed in a container which was evacuated andthen filled with the desired gaseous mixture. The needles werewithdrawn from the stoppers, and the flasks attached to a woodenrocker (figure 2) by means of adhesive tape and thumb tacks orby tying them into tin bases which were attached to the rockerby means of screws. The rocking mechanism carried the flasksthrough an arc of 45 degrees every three seconds.Four good toxin-producing strains were used: #1, isolated at

autopsy from the duodenum of a case of acute hepatocellulardisease; #2081, from Sharp and Dohme Laboratories; S 24MA,from Connaught Laboratories, Toronto, Canada; Wood 46,from Connaught Laboratories, Toronto, Canada.

Comparison with the agar method was made by adding 0.5 percent Difco agar to the medium and incubating without rocking.The amount of medium, the size of the flask and the method offilling the flask with the gas mixture were the same with thefluid and the semi-solid agar cultures. In the inoculation of theagar medium 0.05 to 0.1 ml. of bacterial suspension was used.The inoculum was distributed over the surface of the semi-solidmedium by careful tipping of the flask.

RESULTS

In order to compare the results of the rocking aeration pro-cedure with those of stationary incubation and to determine towhat extent the addition of agar to the medium influenced theproduction of toxin, an experiment was performed in which mediacontaining 0.03 and 0.5 per cent Difco agar and 0.03 and 0.5 percent potassium agar (prepared from Difco agar according to theprocedure described by Mcflwain in 1938) were compared to thesame medium minus the agar. The addition of 0.03 per centagar resulted in no apparent decrease in the fluidity of the

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PRODUCTION OF ALPHA-HEMOLYSIN 607

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....

mill~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

_TM

! -~gm

...........

.......... .. .

FiG,2

asemi-solid consistency''".. On :e ffudcutrswsrcecontnuouly, hil ante wa incuate w .:ithout: rokng h

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608 E. P. CABMAN

latter set was gently shaken by hand one or two times daily. Thesemi-solid cultures were incubated without any rocking orshaking. A gas mixture of 20 per cent carbon dioxide in oxygenwas employed.

TABLE 2Comparison of results obtained with stationary semi-solid agar medium and rocked

and stationary liquid (agar-free and low agar) media

STAIN

#1#1#1S1S1

S 2081# 2081S 2081S2081# 2081

A24MA* 24MA# 24MA# 24MAS24MA

Wood 46Wood 46Wood 46Wood 46Wood 46

AMOUNT OF AGAR

0.00.03% Ca0.03% K0.5% Ca0.5% K

0.00.03% Ca0.03% K0.5% Ca0.5% K

0.00.03% Ca0.03% K0.5% Ca0.5% K

0.00.03% Ca0.03% K0.5% Ca0.5% K

ROCKED

Lh

0.040.0360.046

0.0220.0220.022

0.0140.0180.01

0.0160.0220.02

Note: Ca, ordinary agar containing calcium;expressed in billions of organisms per ml.

Growth

1213.512

910.57.5

12129

121818

Lh

0.120.140.140.0240.02

0.120.160.120.0260.04

0.070.080.40.0080.008

0.080.070.090.0240.016

K, potassium agar.

Following 66 hours of incubation the cultures were harvested.After estimation of the amount of growth and then centrifugation,the supernatants were titrated for their toxin. The results ofthis experiment are presented in table 2. It is evident thatrocking results in an increase in toxin production and growth.The addition of 0.03 per cent agar to the medium did not result

STATIONARY

Growth

3.555

3.55.53.5

566.5

2.544

Growth

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in an increase in toxin production. With two strains (# 1 and* 24MA) the addition of 0.5 per cent agar resulted in the pro-duction of a more potent toxin than was obtained by the "rock-ing aeration" technique. With strain # 2081, however, therocking procedure proved to be slightly superior to the semi-solidagar technique. The Wood 46 strain cultured on ordinary agarproduced less toxin than when grown on the potassium agar. Onthe latter medium the toxin yield was equal to that obtained bythe "rocking aeration" procedure.

TABLE 3The effect of different gaseous atmospheres on toxin production in fluid and in

semi-solid mediums

STAPH 24MA STAPH WOOD 46

PEE CENTCOs0 .7per cent Fluid medium 0.7 per cent Fluid medium

Lh Lh Growth Lh Growth

10 0.01 0.014 12 0.02 0.016 1520 0.01 0.011 9 0.018 0.016 936 0.012 0.01 3 0.012 0.016 1544 0.01 0.011 2.5 0.012 0.012 1254 0.01 0.012 2.7 0.012 0.012 963 0.016 0.016 3 0.014 0.016 4.580 0.026 0.05 4 0.09 0.038 18OR 0.008 0.03 6 0.02 0.034 4.5

Note: Growth is expressed as billions of organisms per ml.R = gaseous atmosphere renewed at 24 and 48 hours.

Using the "rocking aeration" procedure for fluid cultures andstationary incubation for semi-solid potassium agar, toxin pro-duction was studied after incubating the cultures for sixty hoursunder the following amounts of carbon dioxide in oxygen: 10,20, 36, 44, 54, 63 and 80 per cent. For incubation in 80 per centcarbon dioxide in oxygen, two sets of cultures were prepared;one set was incubated in the usual continuous manner, while withthe other the gaseous atmosphere of 80 per cent carbon dioxide inoxygen was renewed after 24 and 48 hours of incubation.

In table 3 are presented the results obtained with strains 24MAand Wood 46. It was observed that the amount of growth varied

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directly with the concentration of oxygen in the atmosphere.While the determination of the amount of growth in the fluidmedium was complicated by a tendency of the organisms (espe-cially 24MA) to undergo autolysis, the observed relationshipbetween the amount of growth and the concentration of oxygenwas definitely evident in the agar cultures in which less autolysisappeared to take place. Renewal of the gaseous atmosphere of80 per cent carbon dioxide and 20 per cent oxygen resulted in adefinite increase in the amount of growth with both strains andin both types of media.Exmination of the Lh values indicates that with increased

carbon dioxide tension there was an increase in toxin production,provided that there was at the time sufficient oxygen for thesupport of growth. With diminished growth as a result of smallerconcentrations of oxygen, there was a diminution in the amountof toxin produced. When the oxygen was renewed in the pres-ence of as much as 80 per cent carbon dioxide, excellent toxinproduction was obtained. Under the conditions prevailing inthis experiment, an atmosphere of 45 per cent carbon dioxide and55 per cent oxygen appeared to be best for the production oftoxin by these strains.

In order to compare the rocking procedure with the semi-solidagar technique under a more optimal gaseous atmosphere,undiluted fluid medium was inoculated and rocked, while thesame medium containing 0.5 per cent Difco (calcium) agar or0.6 per cent potassium agar was subjected to stationary incu-bation. The gaseous atmosphere was 40 per cent C02 in oxygen.The initial pH of the medium was 6.9. Strains #1, #2081,S24MA and Wood 46 were used and the incubation periodswere 26, 48 and 68 hours.The results presented in table 4 again show that with strains

24MA and Wood 46 the rocked broth culture procedure is asgood as the semi-solid agar technique for toxin production. Withboth strains 100 Lh units of toxin per ml. were obtained. Withstrain # 2081 the rocked broth procedure was only slightly inferiorto the semi-solid agar technique. Strain #1, however, producedmore toxin when the semi-solid agar procedure was used than it

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PRODUCTION OF ALPIA-IIEMOLYSIN 611

did with the rocked broth technique. No marked differencebetween the amounts of toxin production in the calcium andpotassium agars was evident.

In order to determine the effect of dilution of the medium ontoxin production in the closed system of the "rocking aeration"procedure, the basal medium was diluted with water to give thefollowing concentrations of medium: 100, 75 and 62.5 per cent.To portions of these diluted media 0.5 per cent NaCl was added.

TABLE 4Comparison of results of "rocked broth" and "semi-solid agar" procedures, using a

gaseous atmosphere of 40 per cent CO, in 0,

LhVXA MUDIUM-

28 hours 48 hours 68 houn

#1 Broth 0.034 0.034 0.028#1 Ca agar 0.018 0.016 0.018#1 K agar 0.018 0.02 0.018

X 2081 Broth 0.026 0.022 0.04#2081 Ca agar 0.022 0.02 0.023S2081 K agar 0.02 0.02 0.018

024MA Broth 0.014 0.01 0.009%24MA Ca agar 0.011 0.01 0.011#24MA K agar 0.011 0.011 0.048

Wood 46 Broth 0.011 0.01 0.011Wood46 Ca agar 0.014 0.012 0.014Wood 46 K agar 0.011 0.014 0.019

Strains #1, #2081 and # 24MA were employed. For compar-ison with the semi-solid agar culture procedure, the undilutedbasal medium (without added salt) to which 0.6 per cent potas-sium agar was added was also used. The gaseous atmospherewas 40 per cent carbon dioxide in oxygen and the incubationperiods were 24 and 45 hours.From table 5 it is evident that after 24 and 45 hours of incuba-

tion no improvement in the -amount of toxin production wasobtained as a result of dilution of the medium. With strain #1

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the semi-solid agar culture technique gave a higher toxin yieldthan did the rocked fluid culture. With strains #2081 andX 24MA the toxin yields with the two methods were about thesame.

TABLE 5

Effect of dilution of fluid medium on toxin production, using the rocking procedure

TYPOF PzzCuM P1 CGROWTH

LSTRAZN vJUrnu BASAL NaWIMUDIUM 24 hours 45 hours 24 hours 45 hours

x1 Broth 100 0.0 16 15 0.028 0.028Si Broth 75 0.0 12 9.5 0.036 0.035x1 Broth 62.5 0.0 9 9.5 0.04 0.05SI Broth 100 0.5 18 18 0.04 0.032Si Broth 75 0.5 14 9.5 0.038 0.033xi Broth 62.5 0.5 10.5 4.5 0.038 0.035SI Agar 100 0.0 0.02 0.014

S2081 Broth 100 0.0 10.5 12 0.024 0.02202081 Broth 75 0.0 9.5 9 0.03 0.028%02081 Broth 62.5 0.0 7.5 6 0.04 0.036#2081 Broth 100 0.5 14.5 18 0.024 0.042S2081 Broth 75 0.5 12 9 0.028 0.026S 2081 Broth 62.5 0.5 6 7.5 0.032 0.0302081 Agar 100 0.0 0.024 0.034

024MA Broth 100 0.0 15 12 0.014 0.011S24MA Broth 75 0.0 15 9 0.018 0.018S24MA Broth 62.5 0.0 12 9 0.02 0.024S24MA Broth 100 0.5 12 12 0.013 0.012%24MA Broth 75 0.5 15 9 0.014 0.014#24MA Broth 62.5 0.5 13 7.5 0.02 0.021%24MA Agar 100 0.0 0.011 0.01

Note: Growth is expressed in billions of organisms per ml.

Since renewal of the gaseous atmosphere resulted in an in-creased toxin yield (see table 3), an attempt was made to deter-mine to what extent the volume of the gaseous atmosphereinfluenced toxin production. To this end, 21, 6- and 12-oz.cylindrical soft glass jars having approximately the same diam-eters, but varying in height, were used instead of Erlenmeyer

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PRODUCTION OF ALPHA-HEMOLYSIN 613

flasks. The 24 oz. jars measured 2 inches in diameter while the6 and 12 oz. jars had a diameter of 2- inches and differed inheight only. To the 21 oz. jar, 8 ml. and to the 6 and 12 oz. jars,

100

50

100

I

50

100

50

0-*---'I--

12 oz. jar

6 oz. jarKey

#2081Wood 46

24 oz. jar20 40 60 80 100

% carbon dioxide

FIG. 3

10 ml. of semi-solid 0.5 per cent Difco agar medium were added(in order to obtain approximately the same surface-area to volume*relationship). The medium was inoculated in the usual mannerand the jars filled with the different mixtures of carbon dioxide

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and oxygen. Lh determinations were made after stationaryincubation at 37.5C0. for the following periods of time:

skwan Hours Incub&ted#1............................................................... 602081..... 8024MA.... 66

Wood 46.. 80

The results of these studies are presented in figure 3. It isapparent that the optimum mixture of carbon dioxide and oxygenfor toxin production depends on the volume of gas mixtureavailable to the culture. For small volumes, the oxygen tocarbon dioxide ratio employed should be greater than for largervolumes of gas. This dependence of toxin production on thevolume of available gas mixture is especially noticeable in theresults obtained with strain S 24MA. In a 21 oz. jar containing20 per cent C02 and 80 per cent 02, this strain produced 125 unitsof toxin per ml. In a 12 oz. jar containing the same gas mixture,90 units of toxin were produced per ml. On the other hand, whena mixture of 80 per cent C02 and 20 per cent 02 was employedthe results were reversed. In a 21 oz. jar, 60 units of toxin perml. were produced while in the 12 oz. jar, 125 units per ml. wereobtained.

DISCUSSION

The elimination of agar from media used in the preparation ofstaphylococcus alpha-hemolysin has obvious practical advantagesand should facilitate study of the mechanism of toxin productionby this organism.The studies described here indicate that the function of agar is

chiefly one of making more available to the staphylococcus thegaseous atmosphere under which it is cultured rather than theadsorption of substances deleterious to the production of alpha-hemolysin. Perhaps in some of the less complex media like thatof Gladstone, in which the presence of substances inhibitory totoxin formation may play a greater r6le, agar may act by ad-sorbing these substances. Our own experiences with such a

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medium" have been too irregular to permit conclusions in thisrespect.

McClean's results with the cellophane bag technique may beexplained as due to an increased accessibility of the gaseousatmosphere to the bacteria as a result of limitation of growth tothe surface or subsurface portions of the broth. Although wehave been able to obtain potent toxin with strain #2081 byemploying the "cellophane bag procedure," such results have beenfar from consistent. McClean's use of the Wood 46 strain mayaccount for the consistency of his results. This strain appears togive a better yield of toxin in a shallow layer of fluid medium thando most toxin-producing strains of staphylococcus. The Lhvalues reported by McClean with both cellophane bag and agarprocedures (0.1 and 0.08 ml. respectively) are approximatelythe same as the value obtained by us in the stationary shallowfluid culture of this strain (see table 2).

Gladstone (1938) suggested that his failure to obtain toxin bythe "bubbling aeration" procedure with a chemically definedmedium was due to the destruction of the toxin in such a mediumby denaturation. He suggested that the discrepancy betweenhis results and ours (Casman 1938) might be due to the protectiveaction exerted by colloids in the veal-infusion proteose-peptonemedium used by us. This suggestion is supported by our resultswith diluted veal-infusion proteose-peptone media and withmedia prepared with an alcoholic extract of yeast and an aciddigest of gelatin (or casein). The superiority of the "rockingaeration" procedure over the "bubbling aeration" procedure maybe due to the elimination of this destructive action in the formermethod.The studies on the influence of different volumes of combina-

tions of oxygen and carbon dioxide indicate the importance ofthe gaseous atmosphere and especially of carbon dioxide in theproduction of staphylococcal toxin. It is interesting to note, inthis respect, that while most investigators recommend the use of

4A modification of Gladstone's medium using an acid digest of gelatin enrichedwith cystine, tryptophane, glucose, nicotinic acid, thiamine chloride, Fe, Cu, Mgsalts and phosphate.

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a mixture of from 20 to 40 per cent carbon dioxide in oxygen orair, Leonard and Holm (1935) employing a relatively large tankfor their cultures, found a mixture of 80 per cent carbon dioxideand 20 per cent oxygen most favorable for toxin production bystaphylococcus. It is to be borne in mind that the gaseous atmos-pheres indicated as optimal for toxin production are the initialatmospheres of the closed systems employed. Since oxygen isutilized and carbon dioxide produced as a result of the growth andmetabolism of the staphylococcus a concentration of carbondioxide higher than the initial one is obtained on incubation.

SUMMARY

1. Aeration of fluid cultures of staphylococcus by bubbling gasmixtures through them resulted in an enhancement of toxin pro-duction. The toxin so produced did not equal in potency thatobtained by the semi-solid agar procedure.

2. The early work of Walbum, in which dilution of the mediumwas found to accelerate toxin production, was confirmed when the"bubbling aeration" procedure was employed. Such accelerationwas not evident in the "rocking aeration" experiments.

3. Aeration of shallow fluid cultures of four strains of staph-ylococcus by continuous gentle rocking resulted in an even moremarked enhancement of toxin production than when the cultureswere aerated by bubbling. With three of these strains the toxinproduced by the former method equalled in potency the toxinobtained by the semi-solid agar technique.

4. The optimal initial mixture of oxygen and carbon dioxidefor the production of alpha-hemolysin in a closed system varieswith the total gas volume under which the organism is grown.

CONCLUSIONS

Some of the active alpha-hemolysin-producing strains ofstaphylococcus elaborate as much alpha-hemolysin by suitableaeration of fluid (agar free) cultures as they do by the use of thesemi-solid agar technique.The author wishes to acknowledge his indebtedness to Mrs. L.

T. Schermerhorn and Miss M. E. Carmichael for valuable tech-

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PRODUCTION OF ALPHA-lIRTMOLYSIN 617

Mcal assistance; to Doctor John Eiman, Director of the labora-tories, for his support and. cooperation; and to the Sharp andDohme Laboratories of Glenolden, Pa., for the furnishing ofliberal supplies of staphylococcal antitoxin.

REFERENCESBURNET, F. M. 1930 The production of staphylococcal toxin. J. Path. Bact.,

83, 1-16.CASMAN, E. P. 1938 The production of Staphylococcus toxin in fluid media. J.

Bact., 35, 13.GLADSTONE, G. P. 1938 The production of staphylococcal alpha-hemolysin in

a chemically defined medium. Brit. J. Exptl. Path., 19, 208-226.LEONARD, GEORGE F., AND HoLm, AUGUST 1935 A method for the production

of Staphylococcus toxin and toxoid. J. Immunol., 29,209221.McCLxAN, DOUGLAS 1937 Staphylococcus toxin: factors which control its pro-

duction in a fluid medium. J. Path. Bact., 44, 47-70.McFARLAND, JOSEPH 1907 The nephelometer. J. Am. Med. Assoc., 49, 1176-

1178.McILwAN, HENRY 1938 The effect of agar on the production of staphylococcal

alpha-hemolysin. Brit. J. Exptl. Path., 19, 411417.PARKER, J. T., HOPKINS, J. G., AND GUNTHER, A. 1925-26 Further studies on

the production of Staphylococcus aureus toxin. Proc. Soc. Exptl. Biol.Med., 23, 344-346.

PLASTRIDGE, WAYNE N., AND RETTGER, LEO F. 1929 Studies on carbon dioxide.IV. The influence of gaseous environment on growth and toxin pro-duction of C. diphtheria. J. Bact., 18, 1-41.

WALBum, L. E. 1922 Studien ueber die Bildung der Bakteriellen Toxine. I.Mitteilung. Staphylolysin. Biochem. Z., 129, 367-443.

WRIGHT, H. D. 1933 The importance of adequate reduction of peptone in thepreparation of media for the pneumococcus and other organisms. J.Path. Bact., 37, 257-282.

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