JOURNAL oF BACTERIOLOGY, Nov. 1967, p. 1327-1333Copyright © 1967 American Society for Microbiology

Purification and Properties of Staphylococcala-Hemolysinl

I. Production of 5-Hemolysin

RICHARD A. MURPHY AND RIAZ-UL HAQUE

Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60680

Received for publication 18 August 1967

Concentrated preparations of staphylococcal 6-hemolysin were obtained bygrowing selected hemolytic colonies from the 146P strain of Staphylococcus aureus on

dialysis membranes laid over Brain Liver Heart agar plates at 37 C for 20 hr under10% CO2 and harvesting the growth from five such membranes in 1.0 ml of deion-ized distilled water. Incubation in a humid environment facilitated this harvestingprocedure. Incubation longer than 40 hr or incubation under CO2 higher than 10to 20% gave lower yields of 5-lysin. Addition of a sugar, fermented by the organ-isms, resulted in lower yields of 5-hemolysin. Agar, although separated from thegrowing cells by the dialysis membrane, did potentiate 5-hemolysin production.Addition of0.1% agar to the inoculum further enhanced this potentiation. 5-Hemoly-sin produced in broth or semisolid cultures was excessively diluted with the media.Dialysis membranes prevented this dilution and thus yielded concentrated prepa-

rations of 3-hemolysin.

Staphylococcal 5-hemolysin, capable of he-molyzing human erythrocytes, is considered toplay a significant role in the pathogenesis ofstaphylococcal disease. Precisely what this rolemight be is not known and, perhaps, cannot beadequately studied unless this hemolysin is ob-tained in pure form.

Purification of 5-hemolysin has been handi-capped by the difficulties involved with itsidentification, the lack of availability of a strainof Staphylococcus aureus capable of producinglarge quantities of 6-hemolysin, and the lack ofknowledge of the physiological conditions whichfavor its in vitro production.

Difficulties in identifying 8-lysin were recentlyresolved by Haque (3), who has devised an

electrophoretic localization technique for theidentification of various diffusible products ofS. aureus on the basis of their electrophoreticmobilities and biological activity. 5-Lysin of S.aureus was thus described as a hemolytic moietywhich migrates 11 mm towards the anode whenelectrophoresed for 2 hr in agar gel at 5 ma/cmand lyses human, horse, and rabbit erythrocytes.We were also fortunate in isolating a strain of

S. aureus which produces good yields of 6-lysin.

'Taken in part from a thesis submitted by R.Murphy to the Graduate College of the Universityof Illinois in partial fulfillment of the requirementsfor the M.S. degree.

This allowed us to study the cultural conditionsmost conducive for the large-scale productionof 3-hemolysin so that attempts to purify 6-lysincould eventually be made. This report dealswith the production of 5-lysin. Studies con-cerning the purification of 3-lysin will be reportedseparately.

MATERIALS AND METHODS

Strain of S. aureus. The Foggie strain of S. aureus

used by Yoshida (15) for the production of 5-hemoly-sin was not used in this study since it produced largeamounts of ,8-hemolysin (3). Instead, a strain of S.aureus designated 146P, which we isolated from aurine specimen, was used in this investigation. Itproduced large zones of complete lysis on humanerythrocyte agar plates and produced both a- and5-hemolysins when tested by the electrophoreticlocalization technique of Haque (3). It also pro-duced both free and bound coagulase, deoxyribonu-clease, ribonuclease, phosphatase, lipase, caseinase,gelatinase, catalase, and lysozyme.The strain. when grown in broth, regularly formed

a small percentage of nonhemolytic variants. Theculture was routinely maintained on Heart InfusionAgar (HIA; Difco) slants. Prior to use, the organismwas streaked on human erythrocyte agar plates andonly hemolytic colonies were employed for theproduction of 8-hemolysin.

Production of 8-hemolysin. Since this study wasdesigedn to investigate the effect of various environ-mental factors on the production of a-hemolysin, the

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conditions of growth and incubation were frequentlyaltered. In general, the following technique adaptedfrom Birch-Hirschfeld (1) and Marks and Vaughan(10) was used for the production of 5-hemolysin.Discs (87 mm in diameter), cut from dialysis casing[3.25 inch (8.3 cm) inflated diameter; Visking Corp.,Chicago, Ill.J, were thrice-washed in deionized dis-tilled water and were placed between alternate layersof moist filter paper discs in a large (150 mm) petriplate. A batch of 25 dialysis membranes were thusplaced in the plate, and, after adding 100 ml ofdeionized distilled water, the assembly was sterilizedby autoclaving at 15 lb for 30 min. The membraneswere then separately laid over the surfaces of appro-priate agar plates. The plates were left at room tem-perature overnight to detect possible contaminants,and then the surfaces of the membranes were inocu-lated with the test organism. Generally, a set of fiveplates was used for each experiment.The inoculum consisted of a suspension, in de-

ionized distilled water, of five hemolytic coloniesobtained by streaking the 146P strain of S. aureus ona human erythrocyte agar plate. These plates wereprepared by adding 3% thrice-washed human eryth-rocytes to sterile HIA. The opacity of the bacterialsuspension was adjusted to that of a McFarlandnephelometer tube no. 1, and a 0.1-ml portion of thesuspension was spread over each plate with a sterileglass spreader which was finally used to inoculate ahuman blood-agar plate. This last step was introducedas a necessary control to determine the homogeneityof the inoculum.

Incubation. The plates were generally incubated at37 C for 20 hr in a humid incubator containing 10%CO2. When this method of incubation was notfeasible, e.g., when the effect of various concentrationsof CO2 or the effect of temperature on the productionof 5-lysin was studied, the atmosphere containing theneeded concentration of CO2 was produced accordingto the method of Wadsworth (13) by use of sodiumcarbonate and sulfuric acid. An atmosphere of 100%CO2 was produced by flushing a Brewer's anaerobicjar containing the inoculated plates for 1 min withcarbon dioxide.

Harvesting 8-hemolysin. After incubation, themembranes from a set of five plates were removed,and the growth adhering to them was recovered in1.0 ml of deionized distilled water. This step wasfacilitated by repeatedly agitating each membrane in apetri dish containing 1.0 ml of deionized distilledwater and then pulling the membrane over the edge ofthe dish so that the fluid adhering to the membranewas recovered. The plate was then tilted, and thebacterial suspension was pipetted directly into thebottom of a conical glass centrifuge tube. The pipettewas allowed to drain slowly to recover the maximalamount of the suspension. The supernatant fluid,recovered by centrifugation (2,000 X g, 20 mmn), wasfiltered through a 0.22 membrane filter by use of aMicro Syringe Filter Holder (Millipore Corp., Bed-ford, Mass.). The filtrate, generally 0.8 ml, wastitrated for hemolytic activity. When not in use, itwas kept frozen at -8 C.

Quantitation of 6-hemolysin. A conventional tubetitration procedure and an agar-well procedure were

used for the quantitation of 5-hemolysin. For the tubetitration procedure, serial twofold dilutions of thesample were prepared in 0.5-ml quantities withphosphate-buffered saline (Difco HemagglutinationBuffer), pH 7.2, as the diluent. A single pipette wasused to make a series of dilutions, and 0.5-ml portionsof a 1% suspension of thrice-washed human red bloodcells were added to each tube. The tubes were incu-bated for 15 min at 37 C and then refrigerated over-night. The highest dilution showing at least 50%hemolysis was recorded as the titer of the sample.Lysis of 50% was determined by visual comparisonof the test tubes with a control (0.25 ml of red bloodcell suspension and 0.75 ml of distilled water).

For the agar-well method of quantitation of 8-hemolysin, wells (0.5 mm in diameter) were punchedinto phosphate-buffered human erythrocyte agarplates and were filled with the culture filtrates. Theplates were then incubated at room temperature for48 hr, when the diameter of the zone of lysis surround-ing each well was measured. The human erythrocyteagar plates used in this procedure were prepared byadding 3% thrice-washed human erythrocytes tosterile phosphate-buffered saline agar (Hemagglutina-tion Buffer) and pouring exactly 15.0 ml ofthe mediuminto sterile flat bottom petri plates.

The culture filtrates showing the highest hemolyticactivity against human erythrocytes were furtheranalyzed by the electrophoretic localization techniqueof Haque (3). This was done to confirm that theincreased hemolytic activity was due to 8-hemolysin.

Units of6-hemolysin. The total units of 5-hemolysinwere calculated by multiplying the reciprocal of thetiter of the sample by the volume of the harvestedfluid.

RESULTS

Production of 3-hemolysin in broth and semi-solid cultures. Quantities of 20 ml of HeartInfusion (HI) Broth (Difco) and semisolidHIA (0.3% agar) were separately inoculatedwith 0.1 ml of the inoculum and were pouredinto sterile petri plates. After incubation for 24hr under 8% C02, the cultures were centrifugedfor 20 min at 2,000 X g, and the supernatant fluidswere filtered through membrane filters (0.22 ,A;Millipore Corp., Bedford, Mass.) and titrated.Both filtrates failed to produce any lysis ofhuman erythrocytes at a 1:10 dilution.

Production of 8-lysin on dialysis membranes.Initial trials, by use of the procedure of Birch-Hirschfeld (1), yielded a titer of 1:20 againsthuman erythrocytes. In subsequent trials, mem-branes from sets of two, four, and five plateswere harvested in 1.0 ml of fluid. This procedureyielded preparations of 6-hemolysin with titersof 1: 40, 1:160, and 1:320, respectively. Sinceharvesting five membranes per 1.0 ml of fluidwas easily accomplished, all subsequent experi-ments were performed in sets of five plates each.

Effect of duration of incubation. Growth fromseparate sets of five HIA plates incubated at

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37 C under 10% CO2 was harvested at 4-hrintervals, and the filtrates obtained were titrated.Traces of 6-hemolysin were detected as earlyas 4 hr of incubation (Table 1). Highest yieldsof 6-hemolysin were obtained after 20 hr ofincubation. This appeared to be the optimaltime for production of B-hemolysin and was

used for all subsequent experiments. Incubationfor periods longer than 40 hr gave low titers.

Effect of carbon dioxide. Separate groups offive HIA plates overlaid with dialysis membraneswere inoculated and incubated at 37 C for 20hr in jars containing various concentrations ofcarbon dioxide (referred to as closed) and inatmospheres of 0 or 5% added carbon dioxidein either an air or CO2 incubator (referred to as

open). Incubation of cultures in a concentrationof 10% carbon dioxide was optimal for theproduction of 5-hemolysin (Table 2) and was

used for all subsequent experiments. Platesincubated in 80 or 100% carbon dioxide yieldedvery low amounts of 6-hemolysin. Plates incu-bated in 10 and 100% CO2 had final pH valuesof 8.5 and 7.0, respectively.

Effect ofpH of the medium. Separate groups offive plates of HIA buffered weekly with a phos-phate buffer to pH 6.0, 6.4, 6.8, 7.0, 7.2, 7.6, and8.0 were employed for this experiment. Theinitial pH of the medium did not influence 6-lysin production, as no apparent differences were

noted in the titers of various filtrates. The finalpH of the filtrates varied from 7.0 to 8.0.

Effect of a fermentable sugar. Separate sets offive HIA plates containing 1%, dextrose or 1%,raffinose were used for this experiment. Oneset of HIA plates was used as a control. Theincorporation of dextrose, which was utilized bythe organism, resulted in a lower titer of the6-lysin preparation (1:80). Raffinose, on theother hand, was not utilized by the organism, andthe yield of 6-lysin was not affected by its pres-ence. The final titer was equal to that of thecontrol (1:320). The pH values of the filtratesobtained from media containing dextrose andraffinose were 6.0 and 8.0, respectively.

Effect of the temperature of incubation. Sep-arate groups of five HIA plates overlaid withdialysis membranes were inoculated and incu-bated for 20 hr under 10% CO2 at temperaturesof 28, 35, 38, and 41 C. The filtrates obtainedupon harvest from plates incubated at tempera-tures of 35 to 41 C yielded equal titers of 6-he-molysin (1:320). Incubation at 28 C showed a

marked decrease in both cellular growth andhemolysin production (1: 80).

Effect of various commercial media. Separategroups of five plates of Brain Heart Infusion(Difco), Brain Liver Heart (Difco), 2% Casein

TABLE 1. Effect of length of incubation on theproduction of B-hemolysin onl HIA at 37 C

under 10% C02

Length of Reciprocal of Duration of Reciprocal ofincubation titer incubation titer

hr hr

0 0 32 3204 <10 36 3208 40 40 32012 160 44 16016 320 48 8020 640 52 8024 320 56 8028 320 60 80

TABLE 2. Effect oJ incubation in atmospheres con-taining various concentrations ofcarbon dioxide

on the production of 5-lysin

Concn of C02 (%) Incubation Reciprocal of titer

0 Closeda 805 Closed 32010 Closed 64020 Closed 32040 Closed 32080 Closed 80100 Closed 800 Openb 405 Open 160

a Closed means plates were incubated in sealedjars containing the desired concentration of CO2.

b Open means plates were incubated in a CO2incubator with a constantly flowing C02-air mix-ture.

Hydrolysate (Nutritional Biochemicals Corp.,Cleveland, Ohio), C T A Medium (BBL), EugonAgar (Difco), Nutrient Agar (Difco), Tryptic SoyAgar (Difco), and HIA (Difco), containing afinal concentration of 1.5% Agar (Difco), wereutilized for this experiment. Results of the tubetitration procedure and the agar-well method ofestimating the hemolytic capabilities of the cul-ture filtrates are shown in Table 3. 6-Hemolysinwas produced on all the media tested. The highestyield was obtained from Brain Liver Heartmedium. Brain Heart Infusion and Eugon Agarwere also found superior to HIA for the produc-tion of a-hemolysin.

Effect of varying the amount ofmedium. Sets offive plates containing 10, 15, 20, 25, and 30 ml ofthe normal concentration of HIA and one setcontaining 15 ml of twice the normal concentra-tion of HI broth with 1.5% Agar added wereoverlaid with dialysis membranes. The plates wereinoculated and incubated as usual. Results shownin Table 4 indicate that increasing the volume or

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concentration of the medium led to a stepwiseincrease in the volume of cells harvested and aconcomitant increase in production. This in-crease in 5-lysin production was more evidentwhen the agar-well method of determining5-lysin activity was used.

Effect of agar. Plates of HIA containing HIBroth (Difco) and 0.4, 0.8, and 1.5% Agar(Difco), 1.5% Difco-Special Agar (Noble), 1.5%Agar Agar (BBL), and 1.5% Ionagar No. 2(Oxoid) were employed for this experiment.For the production of 6-hemolysin in broth,

two sets of five plates each, one containing enoughglass beads (5 mm diameter) to form a monolayerand the other containing 15 filter paper discs(9.0 cm), were sterilized by autoclaving. A 20-mlquantity of sterile HI Broth was then poured intoeach of the plates, and the dialysis membraneswere placed over the glass beads and the filterpaper discs.To test whether filter paper would have any

effect on the production of 5-hemolysin, a set offive HIA plates (1.5% Agar) were first overlaidwith one filter paper each and then the dialysismembranes were laid over the filter papers.

All the plates were inoculated and incubated asusual. Results shown in Table 5 indicate thatagar potentiated the production of 6-hemolysin.

TABLE 3. Production of 8-lysin on variouscommercially available media

Medium Reciprocal Zone ofof titer lysis (mm)

Brain Liver Heart ........... 1,280 6.5Brain Heart Infusion........ 640 5.5Eugon Agar................. 640 5.5Heart Infusion Agar......... 320 4.5C T A Medium .............. 320 4.5Casein Hydrolysate.......... 80 3.0Tryptic Soy Agar............ 80 3.0Nutrient Agar. 40 Trace

TABLE 4. Effect of the amount and concentration ofthe HIA on the production of 8-hemolysin

Volume ofao eirclomedium in Concna of HI titer Zone of lysis

eachplatetie

ml mm

10 1X 160 3.015 1X 640 4.020 1X 640 5.025 1X 640 6.030 1X 1,280 6.515 2X 1,280 6.5

a Relative to normal concentration.

Concentrations of agar as low as 0.4% were aseffective as higher concentrations. All brands ofagar tested, with the exception of Ionagar No. 2,potentiated the production of 6-hemolysin to anequal degree.

In these experiments, the agar was not in directcontact with the growing cells. Incorporation of0.1% agar into the inoculum yielded a furthertwofold increase in the titer of the lysin prepara-tion (Table 5).

Effect of dialysis membranes on the produc-tion of 6-hemolysin. To determine whether thedialysis membranes potentiated hemolysin pro-duction or merely prevented the diffusion of8-lysin into the media, and thus yielded concen-trated preparations, the bacteria were grown in10-, 5-, and 1-ml volumes of semisolid (0.3%agar) media each containing 0.5 g of dehydratedHI broth. This resulted in a 2-, 4-, and 20-foldincrease in the concentration of the medium. Thetotal amount of nutrients available to the cellswas thus kept constant, and the reduced volumeserved to minimize the dilution of the hemolysinproduced. 6-Lysin was also produced on mem-branes laid over HIA plates containing 20 ml ofthe normal concentration of HI (0.5 g/20 ml).These served as controls. The growth from thesedialysis membranes was harvested as usual in 1.0ml of deionized distilled water. The fluid fromsemisolid agar cultures was harvested by freezingthem overnight and then thawing them (11). Itwas centrifuged (2,000 x g, 20 min) and, afterfiltration through a membrane filter, was titrated.The total units of hemolysin produced per plate(0.5 g of media) were used in comparing theefficiency of hemolysin production.

Results shown in Table 6 indicate that the totalunits of 6-lysin per 0.5 g of dehydrated mediawere the same whether the cells were grown insemisolid cultures or on top of the dialysis mem-branes. The dialysis membranes, therefore, appearto merely prevent the diffusion of 8-lysin into themedium and thereby yield concentrated prepara-tions.

DISCUSSION

The production and subsequent purification ofstaphylococcal 5-hemolysin can be amply facili-tated provided a strain of S. aureus capable ofproducing 6-lysin is available. As far as possible,the strain should not produce diffusible productscapable of interfering with or potentiating theactivity of the 5-hemolysin. Since ,3-hemolysin isable to act synergistically with 5-hemolysin (14),strains capable of producing large amounts of,8-hemolysin should not be used for the produc-tion of 5-lysin. Haque (3) has shown that the

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TABLE 5. Effect of agar on the production of 8-hemolysin

Inoculum Membrane support Brand of agar Agar (70) Reciprocal Zone ofoftte Jysis (mm)

Aqueous cell suspen- Agar Difco 1.5 640 5.0sion 0.8 640 5.0

0.4 640 5.0Aqueous cell suspen- Agar Difco Special Agar 1 .5 640 5.0

sion (Noble)Agar Agar (BBL) 1.5 640 5.0Ionagar No. 2 1.5 320 4.0

(Oxoid)Aqueous cell suspen- Agar + filter paper Difco 1.5 640 5.0

sion Filter paper None 0 40 1.5Glass beads None 0 40 1.5

Aqueous cell suspen- Agar Difco 1.5 1,280 6.0sion with 0.1%o agaradded

TABLE 6. Effect ofdialysis membranes on the production of8-hemolysin

Unitsb ofCocf Volume of Plates 5-lysin perMedium CHja medium per harvested Reciprocal of titer Recoveor plate (0.5 gplate ~~~~~~~~~~~~ofHI)

ml ml

HI semisolid................... 2X 10 1 10 5.8 58HI semisolid................... 4X 5 1 20 3.3 66HI semisolid................... 20X 1 1 No growthHIA + membrane.............. iX 20 5 320 0.95 61

a Relative to normal concentration.b The units of 5-lysin = reciprocal of the titer X

Foggie strain of staphylococci, used by Yoshida(15) for the production of 6-lysin, producedlarge amounts of ,B-hemolysin. This strain istherefore considered undesirable for the produc-tion of 6-hemolysin. Strain 146P, on the otherhand, did not produce ,B-lysin under the culturalconditions used in the present investigation forthe production of 5-lysin. This strain is thereforerecommended for the in vitro production of8-lysin.The strain 146P, however, undergoes dissocia-

tion and nonhemolytic variants appear frequentlyin the culture. Since such variants would competewith the hemolytic members of the population forthe available nutrients, they will materially reducethe harvest of 6-lysin. To avoid reduced yields of6-lysin, it was felt desirable that only hemolyticcolonies be used as inoculum. This problem of theemergence of variants in staphylococcal culturesis much more widespread than has been hithertorealized. Attention to this effect has now beenfocused by Parisi (12), Haque and Baldwin (5),Gross (M. S. Thesis, Univ. of Illinois, Chicago,1966), and Kjems (7), and it would be advanta-

the volume of the filtrate.

geous to keep this fact in mind while attempting toproduce diffusible products of S. aureus.Although initial studies indicated that detect-

able quantities of 5-lysin were not produced inbroth or semisolid heart infusion cultures, laterfindings through use of concentrated semisolidagar cultures and broth overlaid with dialysismembranes revealed that this lysin was, in fact,produced in these media. The hemolysin, how-ever, was excessively diluted with the medium,and extensive concentration procedures wouldhave to be used before this lysin could be sub-jected to purification procedures. As most pro-cedures of concentration are laborious and sufferfrom the disadvantage that they may result indenaturation of the product, it is suggested thatproduction of 3-lysin in broth and semisolidcultures should be avoided.The adaptation of the procedure of Birch-

Hirschfeld, on the other hand, eliminates the needfor concentration of 6-lysin before purification.The crude &-lysin from five plates can be easilyharvested in as little as 1.0 ml of an aqueousdiluent. To accomplish this, it is necessary that

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the cultures be incubated in a humid environment.Otherwise, the growth of S. aureus on the dialysismembranes becomes extremely dry and difficultto harvest with such a small volume of fluid.The 6-lysin preparations obtained by the dialy-

sis membrane technique are not only sufficientlyconcentrated with regard to their 6-lysin content,but are also free from the macromolecules con-tained in the medium. As such, this procedure aidsin the subsequent purification of 6-lysin.

Careful attention to such factors as the dura--tion and temperature of incubation, the presenceof added C02, absence of fermentable carbo-hydrates, presence of agar, and the nature of themedium used can materially enhance the yieldsof 6-lysin. Although the production of 6-lysincan be detected as early as 4 hr, maximal yieldswere obtained after 20 hr of incubation. Longerperiods of incubation were not needed and were,in fact, deleterious. Incubation should never beprolonged beyond 40 hr. The reasons for the lossof 6-lysin upon prolonged incubation were notdetermined. It cannot, however, be ascribed to thetemperature of incubation since 6-lysin is re-portedly heat-stable, and our experience, whichwe will report elsewhere, substantiates the heatstability of 5-lysin.

Incubation of cultures in an atmosphere con-taining 10% carbon dioxide yielded higher titersof 6-lysin than incubation in higher or lowerconcentrations of this gas. The production ofa- and f3-lysins of S. aureus are also stimulatedby CO . The reason for this stimulation has neverbeen determined and, although speculations thatit acts by regulating the hydrogen ion concentra-tion have been made, they have never been sub-stantiated. In our studies, the 5-hemolysin wasfound to be produced in high titer when the finalpH of the medium was as high as 8.5 or as low as7.0. It would appear, then, that the function ofcarbon dioxide in the production of 6-hemolysinis not that of pH regulation. A likely possibilityis that CO2 is used as a metabolite for the pro-duction of 6-lysin, and we suggest that radio-isotope tracer studies should be done to deter-mine the applicability of this hypothesis.The addition of a fermentable sugar to the

medium resulted in low titer preparations of6-lysin. Although this finding suggests that fer-mentable sugars should be excluded from themedium, it does not indicate in any way the modeof action of the fermentable sugars. The pH ofthe cultures containing fermentable sugar was6.0, but whether the pH affected production ofthe lysin or its activity or stability was not deter-mined. The possibility that the organisms usealternate pathways in the presence of fermentablesugars or their products should be explored, as it

may pave the way toward understanding thepathway to 6-lysin production.

Various commercial media supported produc-tion of 6-lysin to a different extent. This findingsuggests that either the media contained differentlevels of metabolites essential for 6-lysin produc-tion or that they contained different levels ofinhibitors of 6-lysin production. The use of con-centrated media, however, increased 5-lysinproduction, which appears to favor the hypothesisthat different media contain different levels of oneor more required metabolites. The nature of thesemetabolites is not yet known, but the fact thatlimited amounts of 6-lysin were produced incasein hydrolysate opens avenues for the prepara-tion of synthetic media to study the effect ofvarious metabolites on the production of 6-lysin.The mode of action of agar in stimulating the

production of hemolysins has been a controversialissue. McClean (8) and Haque and Baldwin (4)found that the exposure of broth to agar beforeinoculation had no effect on the production ofa- and fl-hemolysins. Mcllwain (9) suggested thatagar adsorbed metabolic products deleterious tohemolysin production, but not to growth. Cas-man (2), however, believed agar acted by makingthe atmosphere more available to the staphylo-cocci rather than by adsorbing deleterious sub-stances. Haque and Baldwin (4) believed thatagar protected the hemolysin molecule fromdenaturation. In the studies reported here, thepotentiating effect of agar on 6-lysin productionwas not eliminated when the cells were separatedfrom the agar by a dialysis membrane. Agar,therefore, does not merely protect the 6-hemoly-sin molecule, nor does agar simply make theatmosphere more available to the cells. If thiswere the case, similar hemolysin productionwould have been obtained when the dialysismembranes were supported by filter paper discs,glass beads, or agar. The agar, however, maycontain a dialyzable component which stimulateshemolysin production, or the agar may adsorb adialyzable product of bacterial metabolism whichis inhibitory to 6-lysin production. The latterinterpretation will agree with that suggested byMcllwain (9). However, our finding that IonagarNo. 2 gave low yields of lysin seems to indicatethe former possibility. Ionagar No. 2 is a morepurified agar, and the needed dialyzable metabo-lite may be either missing or present in a lowerconcentration in this brand of agar. Special Agar(Noble), however, which is also a purified agar,did not reduce the yield of 5-lysin. Dialysates ofvarious brands of agar should be tested for thiseffect on 5-lysin production. Such experimentswere not carried out at present.

Contact of the cells with 0.1% agar further

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potentiated the production of 5-lysin. This mayindicate a simultaneous protective effect of agar.It is not known whether higher concentrations ofagar would produce an even greater yield ofhemolysin, and experiments to determine theoptimal concentration of agar, which, being indirect contact with the cells, may yield more8-lysin, were not undertaken.The titration procedure, due to its inherent

error of 100%, failed to detect minor differencesin the quantity of 8-lysin produced under differ-ent cultural conditions. This problem was elim-inated by devising the agar-well method for assayof hemolysin. The method has the advantage ofease and simplicity. The size of the hole is wellcontrolled, the volume of the hemolysin prepara-tion tested is quite constant, and the method givesreproducible results. The accuracy can be furtherimproved by filling the wells with a microsyringe.This, however, does not appear to be necessary.It is suggested that this method be adapted forcomparing the hemolytic activities of variouspreparations The method has wide applicationand can be used to detect other biochemicalactivities of bacteria.

ACKNOWLEDGMENT

This investigation was supported by grant 2-41-33-36-2-16 to R. Haque from the Trust Graduate Re-search Funds of the University of Illinois.

L1TERATURE CrrED1. BIRCH-HIRSCHFELD, L. 1933-34. Ueber die Wirk-

samkeit der Extrakte von auf Zellophanagargezuchteten Staphylokokken. Z. Immunitaets-forsch. 81:260-285.

2. CASMAN, E. P. 1940. The production of staphy-lococcal alpha-hemolysin: The role of agar.J. Bacteriol. 40:601-617.

3. HAQUE, R. 1967. Identification of staphylococcal

hemolysins by an electrophoretic localizationtechnique. J. Bacteriol. 93:525-530.

4. HAQUE, R., AND J. N. BALDWrN. 1964. Purificationand properties of staphylococcal beta-hemoly-sin. I. Production of beta-hemolysin. J.Bacteriol. 88:1304-1309.

5. HAQUE, R., AND J. N. BALDWIN. 1964. Types ofhemolysins produced by Staphlylococcus aureus,as determined by the replica plating technique.J. Bacteriol. 88:1442-1447.

6. JACKSON, A. W., AND R. M. LI-rrLE. 1958. Staph-ylococcal toxins. III. Partial purification andsome properties of 5-lysin. Can. J. Microbiol.4:453-461.

7. KJEMS, E. 1963. Two variants of Staphylococcusaureus Wood 46 (NCTC 7121) differing inrespect to alpha toxin production. J. Bacteriol.86: 1127-1128.

8. MCCLEAN, D. 1937. Staphylococcus toxin:Factors which control its production in a fluidmedium. J. Pathol. Bacteriol. 44:47-70.

9. MCILWAIN, H. 1938. The effect of agar on theproduction of staphylococcal a-hemolysin.Brit. J. Exptl. Pathol. 19:411-417.

10. MARKS, J., AND A. C. T. VAUGHAN. 1950. Staphy-lococcal 6-hemolysin. J. Pathol. Bacteriol. 62:597-615.

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