PRODUCTION AND CHARACTERIZATION OF PSEUDOMONAS AERUGINOSAHEMOLYSIN

RICHARD S. BERK

Department of Microbiology, Wayne State University, College of Medicine, Detroit, Michigan

Received for publication June 20, 1962

ABSTRACTBERK, RICHARD S. (Wayne State University,

Detroit, Mich.). Production and characteriza-tion of Pseudomonas aeruginosa hemolysin. J.Bacteriol. 84:1041-1048. 1962.-Hemolysin fromPseudomonas aeruginosa was obtained by eitherextraction of blood agar media after bacterialgrowth or by saline washing of cellophane plate-grown cells, hemolytic activity being greatest inthe latter preparations. Except for variations inheat stability, both types of preparations ap-peared to be similar if not identical. Completehemolysis of red blood cells was observed over awide pH range, although the optima appeared tobe below 6.0. Acidification of agar extracts re-sulted in a coarse precipitate which appeared tobe a hemolysin-agar complex. Partial purifica-tion of hemolysin present in cellophane washingswas obtained by fractionation with ammoniumsulfate or by gel filtration. Further characteriza-tion of hemolysin is described.

Although it has been known for several yearsthat Pseudomonas aeruginosa produces a hemoly-sin when grown on blood agar media, hemolysinproduction has not been demonstrated in brothcultures. Consequently, the study and characteri-zation of this toxin has been somewhat retarded.A survey by Liu (1957) of several different spe-cies of Pseudomonas grown on cellophane-cov-ered agar medium indicated that many organismsexhibit hemolysin production. In addition,hemolytic preparations are toxic for mice. Otherworkers (Bullock and Hunter, 1900) found thatfiltrates of 3- to 4-week-old cultures possesshemolytic activity, but extracellular secretion ofhemolysin does not occur in young cultures. Ac-tivity in aged culture filtrates is presumably dueto autolysis. More recently, Graf (1958) extracteda peptide (mol wt about 7,000) from 5-day-oldcells of P. fluorescens which exhibited bothhemolytic and protozoa-lytic properties. The

peptide was reported to be composed of six aminoacids and was thought to be an anionic surface-active agent. Preliminary studies in our labora-tory have indicated the presence of an intracellu-lar hemolysin in cells of P. aeruginosa growneither on blood agar medium or Tryptose brothin the absence of blood.

Previous studies by Berk and Nelson (1961)demonstrated that various intracellular fractionsof P. aeruginosa exerted a variety of toxic effectson the metabolism of phagocytic cells. However,the toxic effects of extracellular hemolysin werenot studied, owing to its incomplete characteriza-tion. Consequently, the purpose of this paper isto describe various techniques and procedures forobtaining hemolytic preparations and to charac-terize the properties of extracellular hemolysinof P. aeruginosa.

MATERIALS AND METHODS

Organism. The organism used in these studieswas obtained from a patient who had undergonesurgery for ulcerative colitis, and was identifiedas P. aeruginosa. The organisms were withdrawnfrom a blind loop of the terminal ileum, whichopened into the surface of the abdomen. The or-ganism was cultured and purified by streakingTryptose blood agar medium. Another strain ofP. aeruginosa was obtained from the departmentalstock culture collection. All of the studies de-scribed herein were performed with the organismisolated from the patient; however, many of thestudies, such as determination of optimal pH,were corroborated with the departmental strain.Media. Cultivation of large quantities of or-

ganisms was accomplished by inoculating Tryp-tose broth containing 0.1% glucose (pH 7.0).Blood agar plates consisted of either Tryptose orTryptone media (Difco) supplemented with 10%citrated sheep or human blood and varying con-centrations of glucose, depending on the type ofstudy planned. Lytic preference for one species

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of red blood cell over another was not noted,regardless of the red cell species used in thegrowth medium. However, human blood wasusually used in growth media.

Buffers. The phosphate buffers were preparedfrom stock solutions of 0.1 M acid and basic phos-phate. They were made isotonic by addition of3.5 g of NaCl per liter.

Protein. Determination of protein was basedon a modification of the Lowry method (Zak andCohen, 1961).

Pigment. Separation of bacterial pigment fromhemolytic extracts was accomplished by gel filtra-tion, using a column of Sephadex G-25, dialysis,or chloroform extraction.

Gel filtration. Sephadex gel filtration of cello-phane washings of P. aeruginosa was used toseparate hemolytic activity from pigmentation;3.5 ml of both the sample volume and effluentwere used. The flow rate was approximately 1 mlper hr through a column (1.0 by 16.0 cm) ofSephadex G-25. The majority of hemolytic ac-tivity was found in the third fraction, which alsohad the highest protein concentration.

Determination of hemolytic activity. Most studiesemployed sheep red blood cells. The cells werecollected once a week in heparinized saline andrefrigerated. Daily cell suspensions were preparedby washing cells three times with 0.85% salineprior to use. A stock cell suspension of 1% wasused in all assays. The tests were carried out in8-ml spectrophotometer tubes. Cells (0.5 ml) wereadded to 1 ml of saline, 1 ml of phosphate buffer(pH 5.9), and 0.5 ml of hemolysin; the pH was ad-justed to 5.9. After mixing the suspension, thetubes were incubated at 37 C for 2 hr, the unlysedcells centrifuged down at 4,000 X g, and theamount of hemoglobin in the supernatant fluiddetermined with a Bausch and Lomb spectro-photometer at 540 m,u. The per cent hemolysiswas determined by comparison with a standardcurve of varying concentrations of red cells lysedwith water to give 100% hemolysis. With highlypigmented agar extracts, it was preferable tomeasure directly the degree of hemolysis byturbidimetric measurement of remaining redblood cells at 700 m,u or by resuspension of un-lysed cells in saline after centrifugation.

Titration of hemolysin. Hemolytic titers weredetermined by twofold dilutions in buffered sa-line (pH 5.9); 0.5 ml of a 1% suspension of redcells was added, and the final readings were madeafter 2 hr of incubation at 37 C. The reciprocal of

the highest dilution showing complete hemolysiswas taken as the number of hemolytic units perml present in the preparations.Membranes. Various cellophane and plastic

membranes, having diverse pore size and thick-ness, were examined to determine the best mem-brane for obtaining maximal hemolysin titers.After assessing the results, routine harvesting ofcells and hemolysin was performed by usingdialyzing cellophane D300. Other membranestested were D3 and Cupriphane. All three mem-branes were purchased from the Technicon Co.,Chauncey, 'N.Y. Glass paper was obtained fromW. & R. Balston Ltd. Plastic membranes, asfollows, were obtained from E. I. du Pont deNemours & Co., Inc., Wilmington, Del. Polyflexplastic 010, 005, 0075; Mylar 100-A; Teslar20-P, 88 CA-43; and Dylan polyethylene 1350.In some cases, removal of the surface charge ofdialyzing membranes was attempted by brieflyimmersing the membranes in antistatic com-pound no. 79 (Merix Chemical Co., Chicago,Ill.), diluted 1-1.

Cellophane technique for hemolysin production.The method of Birch-Hirschfeld (1934) wvas usedfor the harvesting of Pseudomonas hemolysin.Tryptone blood agar supplemented with 1%glucose vas found to be most suitable for ade-quate hemolysin production. A sheet of auto-claved dialyzing membrane was placed on thesurface of the blood agar medium and inoculatedwith a few drops of P. aeruginosa from a brothculture. The organisms were then streaked overthe surface of the membrane with a sterile cottonswab. Growth on the membrane surface was asrapid as on the surface of blood agar. After incu-bation at 37 C for 24 to 48 hr, the membraneswere placed in a beaker and the organisms werewashed off with 3 to 5 ml of phosphate buffer(pH 5.9). The cells were centrifuged at 30,000 Xg for 30 min, and the resultant supernatant fluidcontaining hemolysin was decanted and refriger-ated; the bacterial sediment was discarded.Hemolysin extraction from agar. The complete

surface of Tryptose blood agar medium (0.1%glucose) was swabbed with P. aeruginosa andincubated at 37 C. After 24 to 48 hr of incubation,the organisms were scraped off the surface of theagar with a glass rod and a few milliliters of salineand saved for another study not reported here.The blood agar medium, which was always com-pletely hemolyzed, was extracted by one of twomethods. One method employed the disruption

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of blood agar with a Waring Blendor and 8 to 10ml of either hot or cold saline. The second methodwas to remove the bacteria after growth and thenfreeze the agar medium for 24 to 48 hr. WVhen thefrozen agar was allowed to thaw at room tem-perature, hemolytic fluid was expressed out ofthe contracted medium. Application of pressureto the agar with a spatula enhanced the expressionof fluid. The resultant fluids were separated fromsolid material by centrifugation at 7,500 X g for15 min. All preparations exhibited a pH valuebetween 8.0 and 8.5 and were adjusted to optimalvalues prior to usage. In general, agar extractsdid not possess hemolytic titers as high as thosefound in cellophane washings, but could be in-creased somewhat by using 1% glucose in themedium.

RESULTS

Initial experiments were designed to determinethe conditions necessary for hemolysin pro-duction. An attempt was made to obtain produc-tion of extracellular hemolysin from broth cul-tures. Tryptose broth was inoculated with P.aeruginosa and incubated at 37 C. Examinationof the broth freed of organisms after growth in-tervals of 24, 48, 72, and 96 hr indicated the ab-sence of extracellular hemolysin. Activation ofhemolytic activity with 10 to 100 ,ug of glu-tathione was unsuccessful. Negative results werealso obtained when citrated blood was added tothe broth medium, at pH 5.9 and 7.2 after 24 and48 hr of growth. The final concentration of bloodin the medium was 10%.

Other attempts to demonstrate production ofan extracellular hemolysin centered on the useof bacterial cells removed from the surface ofTryptose or Tryptone blood agar media aftercomplete hemolysis of blood in the media wasobtained. Saline washings of the removed bac-teria were assayed for hemolysin in the presenceof fresh sheep or human red cells. However,neither the washings nor resting cells were ableto exhibit hemolytic activity. In addition, resting-cell suspensions of organisms obtained from brothculture with or without 10% blood were inac-tive. The results seemed to indicate that restingcells of Pseudomonas were unable to elicit hemoly-sin in the presence of red cells despite the factthat the bacteria were grown on blood agar me-dium. Additional experiments employed bloodagar-grown cells incubated with red cells underconditions employing close cellular contact. After

growth, the organisms were washed once, resus-pended in saline with red cells, centrifuged, andthe resultant packed sediment was incubatedovernight in saline. However, no lysis of red cellswas observed after 12 hr of incubation.

Further studies employed the following tech-nique. P. aeruginosa was grown as a solid lawnon the surface of Tryptose agar in the absence ofblood for 24 hr. A few drops of citrated blood werethen added to the surface of the agar, which wasexamined periodically for signs of hemolysis.Hemolysis was not observed on the surface ofthe medium after 4 hr, but was apparent after 16hr. Saline washings of the medium surface andorganisms yielded hemolytic extracts of very lowtiter (4 to 8 units per ml). However, the organismswere not able to secrete additional hemolysinwhen removed from the saline washings and incu-bated with fresh red-cell suspensions. Similarstudies, employing the flooding of growing cul-tures on Tryptose agar with excess blood (10 mlper plate), inhibited hemolysin production.

Since the organism used in most of these studieswas originally obtained from weekly samplingsof a patient's intestinal blind pouch, it was ofinterest to determine whether hemolysin wassecreted extracellularly in vivo. A 10-ml sample,having a milky appearance, was withdrawn andcleared of organisms and other materials bycentrifugation at 10,000 X g for 15 min. Pseudo-monas and Vibrio were usually the predominantorganisms found in the samplings. Examinationof the clear fluid at various pH values indicatedan absence of hemolysin.Agar extracts. Initial studies with cellophane-

Tryptose blood agar medium proved to be un-successful; consequently, other methods of ob-taining hemolysin were contemplated. Sinceblood agar medium exhibited complete hemolysiswhen a lawn of Pseudomonas was grown on itssurface, initial attempts were made to extractthe hemolysin from the agar medium. Hemolyticpreparations were obtained by disrupting bloodagar media after 24 hr of growth and subsequentremoval of the surface growth. Extracts exhibitedhemolytic activity from pH 6.0 to 8.0, with themaximal rate of hemolysis occurring at pH 6.0,although 100% hemolysis was apparent at allof these pH values after 2 hr of incubation. Simi-lar results were obtained with the departmentalstrain of P. aeruginosa. Values below 5.9 or above8.0 were not used, to avoid spontaneous hemoly-sis due to the acidity or alkalinity of reaction mix-

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tures. No hemolytic activity was obtained fromuninoculated blood agar.

Titration of blood agar extracts yielded lowvalues. Most Waring Blendor-treated prepara-

tions seldom exceeded 8 to 16 units per ml; prepa-

rations obtained by freezing and thawing the agar

medium seldom yielded titers exceeding 8 unitsper ml. A comparison of hemolytic activity atvarious pH values can be seen in Table 1. Maxi-mal activity occurred at pH 6.0, with completehemolysis apparent after 60 min at pH 6.3, 6.6,and 6.9 when incubated at 37 C. After 3 hr ofincubation, complete hemolysis was obtained atalmost all pH values tested. Although the pHoptimum of P. aeruginosa hemolysin has notbeen previously reported, the acid optimum ofpH 6.0 or lower and the wide pH-activity range

were unexpected.Temperature studies were performed at pH

5.9 to 6.0. The results in Table 2 demonstrate thecatalytic effect of 48 C over lesser incubation tem-peratures. After 10 min of incubation, hemolysiswas noted at 48 C, but not at 37, 25, or 4 C. Tem-perature controls at 48 C indicated an absence ofspontaneous lysis of red cells. In addition, a

small amount of hemolysin was used, to slowdown the rate of hemolysis to a point where itcould be accurately measured. Occasionally, un-

diluted preparations of hemolysin exhibitedhemolytic activity at temperatures below 20 C.However, no hemolysis was noted after 24 hrof incubation at 4 C.During the performance of the pH optima

studies, it was noted that, after complete hemoly-sis was observed, additional supplements of redblood cells were also lysed. The continual lysis ofcells signified that no loss or binding of hemolysinfrom previous reactions had occurred. However,these results should be expected, since hemolysinwas extractable from blood agar media which hadexhibited complete hemolysis of the red cells.Continued lysis of cells occurred at all pH valuestested in Table 1.

Titer comparisons of identical agar extractsprepared by extraction with hot (70 C) and cold(25 C) saline indicated that hot extraction pro-

cedures yielded preparations of higher potency(Table 3). Some hot saline-agar preparationspossessed twice as much hemolytic activity as didcold extracts. The results in Table 3 also indicatethat both cold and hot agar extracts exhibitedreduced hemolytic rates when the extracts were

boiled for 5 min. In each case, complete hemolysis

TABLE 1. Effect of pH on hemolytic activity ofblood agar extracts at 37 C

Per cent bemolysispH 20 min 20 min 60 min 180 min

(control)

5.6 100 100 100 1006.0 10 100 100 1006.3 0 95 100 1006.6 0 90 100 1006.9 0 45 100 1007.4 0 0 75 1008.0 0 0 25 95

TABLE 2. Effect of temperature onhemolysin activity*

Per cent hemolysisTemp

10 min 90 min

C

4 0 025 0 1437 0 9048 95 100

* Reaction performed at pH 5.9 to 6.0. Reactionmixture was composed of 0.2 ml of lysin, 0.5 mlof red cells, and 2.0 ml of buffered saline (pH6.0).

was obtained but the rate of hemolysis was al-tered, suggesting a partial inactivation ofhemolysin.The effect of red blood cell concentration on

activity can be seen in Fig. 1. With a fixed con-centration of hemolysin, the rate of hemolysis wasrelatively proportional to the cell concentration.Little or no lag was noted with 0.3 ml of a 1% red-cell suspension, and a relatively linear curve wasobtained. As the cell concentration increased, aconcomitant lag period occurred prior to rapidhemolysis. Little or no retardation of hemolyticactivity occurred as the reaction proceeded to-wards completion, as noted in previous studiesby Berk, Ross, and Watson (1954) with strepto-lysin 0, unless hemolytic preparations wereeither of a very low titer or were diluted accord-ingly, as seen in Table 3 (unheated, cold extracts).The sigmoidal nature of most hemolytic curvesobserved in other systems is based on a lag priorto hemolysis, and a somewhat rapid hemolysisfollowed by a retardation prior to completion ofhemolysis. Similar curves can be obtained bykeeping the red-cell conceitration fixed, but

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TABLE 3. Hemolytic activity of hot and coldsaline-agar extracts*

Per cent hemolysis

Time Hot extract Cold extract

Unheated Heatedt Unheated Heatedt

min

0 0 0 0 010 28 0 0 013 100 0 0 030 0 2 032 27 44 035 58 77 240 100 94 450 98 3053 100 4665 100

* Hot extracts were prepared by disruption ofblood agar with 10 ml of hot saline (70 C) and aWaring Blendor for 1 min. Cold extracts wereprepared with saline at 25 C.

t Heated extracts were boiled for 5 min beforeassay.

varying the levels of hemolysin. Undilutedhemolysin produces a very short lag which isdifficult to measure, followed by rapid hemolysiswhich occurs in the matter of a few seconds.When hemolysin was tested against red blood

cells obtained from sheep, rabbits, and humans,identical titers were obtained with all three cellpreparations. No preferential activity for sheepcells was noted when sheep cells were incorpo-rated into the growth medium. Conversely, whenhuman cells were used in the medium, no lyticpreference for human cells was noted.

Dialysis of agar extracts for 24 hr against aphosphate buffer (pH 5.9) reduced titers from 16to 8 units. Passage of hemolysin through themembrane appeared to be difficult, since a coarseprecipitate of some hemolytic material occurredinside the dialysis membrane and did not re-solubilize until the pH was raised above 6.0. Re-moval of the particulate matter by centrifugationat 15,000 X g for 20 min yielded both particulateand soluble fractions with hemolytic activity.The aggregate-free supernatant exhibited a titerof 2 units, and the sediment exhibited 8 units.Hemolysin-agar aggregates were not damagedwhen their pH was adjusted to pH 2.0 for 5 minprior to re-examination at pH 6.0. In addition,when undialyzed preparations were adjusted toa pH of 2.0 and the particulate fraction removed,

Per centHemolysis

0.3 0.6 1.0 1.5 ml RBC100-r5

80-

60-

40 -

20-

05 10 15 20 25 30 35

TIME IN MINUTES

FIG. 1. Effect of various red blood cell levels onthe hemolytic rate of activity.

no hemolytic activity remained in the super-natant. Since these precipitates were very coarsein appearance, it would seem that their originwas from the agar rather than from the bacteria.

Since hemolytic titers of agar extracts were notparticularly high, addition of glucose to Tryp-tose blood agar medium was increased from 0.1 to1%. An increase in titer was obtained in thismanner, although no differences in the hemolyticcharacter of hemolysin were noted. However,comparisons of Tryptose and Tryptone bloodagar containing 1% glucose suggested that thelatter extracts seemed somewhat more heat-labile.

Cellophane washings of blood agar-grown cells.Since many properties of the hemolysin foundin cellophane washings were identical with agarextracts, they will not be described again. How-ever, the similar properties exhibited by bothpreparations will be briefly itemized here: sta-bility of activity at 4 C for several weeks, pHoptima, reduction in activity during dialysis,acid-precipitable hemolytic fractions, activityproportional to concentration, temperature re-sponse, continual lysis of repeated supplementsof red cells, and hemolysis of sheep, human, andrabbit cells.

Initial attempts to obtain hemolysin fromwashings of cells grown on cellophane-coveredblood agar were without success. However, theproblem was re-examined later and successfulpreparations were obtained. Many of the earlydifficulties encountered were found to be relatedto the growth medium and type of membraneused. Highest titers were obtained by using 1%

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glucose-Tryptone blood agar medium and har-vesting cells after 48 hr of growth.Hemolysis of the blood in the agar medium was

usually apparent in most growth studies usingdialyzing membranes within 24 to 48 hr. Occa-sionally, no hemolysis occurred and it is difficultto account for this anomaly. Consequently, Itried to prevent diffusion of the hemolysinthrough the membranes by use of an antistatic or

surface charge-removing agent. However, con-

sistent results were not obtainable. Extraction ofthe diffused hemolysin from the agar medium bypreviously described techniques proved to be un-

successful. No hemolytic activity was ever re-

covered, despite repeated efforts and the obviouspresence of hemolysis.

Dialysis studies of cellophane washings were

initiated in an effort to determine whetherhemolysin was lost as in growth studies and alsoto determine whether the hemolytic agent ex-

hibited cofactor requirements. Initial dialysisstudies utilized 1 ml of hemolysin in the dialysismembrane against 3 ml of phosphate buffer(pH 6.0) for 24 hr. Little or no loss in activitycould be detected, nor was there any hemolyticactivity in the buffer. However, by use of largevolumes of dialysis bath, a reduction in hemo-lytic activity from 32 to 16 units occurred after 48hr of dialysis against buffer (corrected for volumechanges). The buffer volume of 50 ml was muchtoo high for testing hemolytic activity. To de-termine whether the hemolysin had a cofactorrequirement, 0.5 ml of a saturated solution ofethylenediamine tetraacetate was added to 0.5ml of hemolysin and incubated for 5 min. After-wards, addition of red cells yielded the same rateof hemolysis as did untreated hemolysin.The pH optimum of cellophane extracts ap-

peared to be about 5.9 or below, as noted withagar extracts. However, the heat stability ofcellophane extracts proved to be quite strikinglydifferent. No loss in hemolytic activity or titerwas noted by boiling the washings for 60 min.However, occasional precipitation of protein or

other material appeared after 30 min of boilingand was subsequently removed by centrifugationat 4,000 X g for 20 min without loss of activity.Consequently, a partial purification was ob-tained by this process. The protein concentra-tion of one preparation was reduced 15% by theheat and centrifugation treatment, resulting ina gain of specific activity (titer/protein concen-

tration) from 2.52 to 2.96. However, it should be

pointed out that a great deal of variation in ex-tracts seemed to occur, since some extractsexhibited little or no heat-precipitable material.

Titers of cellophane washings were usuallymuch higher than those of agar extracts. In addi-tion, hemolytic activity was much easier toquantitate spectrophotometrically, owing to theabsence of breakdown products of red cells whichwere normally present in agar extracts and whichwere difficult to remove by chemical means.Most cellophane washings exhibited titers rangingfrom 16 to 64 units, depending on the age of thecells when harvested. Maximal activity was ob-tained at 48 hr despite diffusion of the hemolysininto the agar medium.

Stability of hemolysin at extreme pH valueswas investigated and found to be markedly stable.Addition of equal amounts of 20% KOH to cello-phane washings and subsequent boiling for 5 minhad no effect on hemolytic activity after the pHwas readjusted to 6.0 and the washings assayed.Similarly, addition of HCl to hemolytic prepara-tions did not alter activity. At pH 1.85, con-siderable precipitation occurred, resulting inhemolytic activity in both supernatant andparticulate fractions when tested separately atpH 6.0. The fractions were separated from eachother by centrifugation at 8,000 X g for 15 min.No activity was lost by boiling crude extractsat pH 3.5 or 5.0 for 30 min. Similar fractionationof activity was noted upon vigorous shaking of ex-tracts with ehloroform (1:1) for 5 min. A titer of 4hemolytic units per ml was found in the solubleas well as the particulate fractions, although theoriginal extract titer was 20 hemolytic units perml. Extraction of cellophane washings with chlo-roform without vigorous shaking resulted in theremoval of Pseudomonas pigment, without lossin hemolytic activity.

Although it is not the purpose of this paper todescribe the purification of hemolysin, certainstudies along these lines are included since theyyield information on some of the properties ofhemolysin. In general, cellophane extracts seemedto contain from 1 to 10 mg of protein per ml, de-pending on how much buffered saline was used towash off the organisms from the membranes.Treatment of cellophane washings with ammo-nium sulfate at 25% saturation did not result inprecipitation of protein. However, at 50% satura-tion, all hemolytic activity was found in the pre-cipitated material, which was removed by centrif-ugation at 12,000 X g and redissolved in phosphate

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Hemolyticunits/ml

-116

jug protein/ml

600Protein

--- HemolyticActivity

500

400 I-

300

200

100

0I,

-____

2 3 4 5 6 7SAMPLE NUMBER

FIG. 2. Gel filtration of untreated cellophane wash-ings (pH 5.9) through Sephadex G-25.

buffer (pH 6.0). No residual activity was detectedin the supernatant freed of precipitate. The heatstability of the reconstituted hemolysin remainedunaltered when boiled for 30 min. However,addition of absoluite alcohol to crude extracts,resulting in a 50% concentration, yielded a non-

hemolytic precipitate after overnight refrigera-tion.

Gel filtration studies of crude cellophane wash-ings (pH 5.9) were initiated by use of a columnpacked with Sephadex G-25. A 3.5-rl samplewas passed through the column with distilledwater. Eight 3.5-ml samples were collected (Fig.2). The majority of the activity was found in thethird sample, which also exhibited opaquenessand the highest protein concentration. This frac-tion contained 16 hemolytic units per ml; 2 unitswere found in samples 4 and 5. Visible pigmenta-tion was absent in the first six samples, since thepigments migrated through the column muchslower than the hemolysin. Pigment separationoccurred during passage through the column, andresulted first in a narrow green-yellow bandassumed to be fluorescin, followed sometime laterby an easily separable wide blue band assumedto be pyocyanin. No pyorubin was noted(Chmura and Pelczar, 1959). Some protein was

present in both pigment fractions. Samples of3.5 ml were collected for in vivo assays with re-

gard to another study. Column chromatographyand gel filtration studies are now in progress,

using hemolysin partially purified by ammoniumsulfate fractionation.

DISCUSSION

One of the striking properties of P. aeruginosahemolysin found in cellophane washings was itsunusual stability to heat, acid, and alkali. Prop-erties of this nature would seem to rule out thepossibility that hemolysin is an enzyme, althoughthere are reports of unusually heat-stable peroxi-dases (Callow, 1926) and inorganic pyrophos-phatases obtained from bacteria (Blumenthal,Johnson, and Johnson, 1962). However, thehemolysin's dependence upon pH and tempera-ture would seem to rule out the possibility of itbeing a nonspecific surface-active agent. In addi-tion, its presence in protein fractions obtained by50% ammonium sulfate fractionation or by gelfiltration suggests the possibility of it being eitherprotein or protein-linked material combined insome loose form allowing it to pass through adialyzing membrane into the growth medium.This latter possibility might explain why Liu,Abe, and Bates (1961) were unable to produceneutralizing antibody to their hemolysin prepara-tions.The most disturbing fact about the appearance

of hemolysis in blood agar after growth ondialyzing membranes was the inability to ex-tract hemolysin from the medium by either agarextraction technique described herein. Althoughno direct evidence is available, the possibility oftwo distinct extracellular hemolysins present incellophane washings should not be overlooked.This would explain both the partial reduction intiters during dialysis and the presence of hemoly-sin in the growth medium and on the membranesurface.

ACKNOWLEDGMENTS

The author wishes to thank Fred L. Rights,George N. Eaves, and Mrs. L. Gronkowski fortheir interest and suggestions concerning thiswork. In addition, the author greatly appreciatesDr. Zak's generosity in providing the variousmembranes, and thanks A. Orten for providingthe culture of P. aeruginosa.

This project was supported by the iNationalFund for Education.

LITERATURE CITED

BERK, R. S., AND E. L. NELSON. 1961. Effect ofmicrobial fractions on the metabolism of

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liver and monocytes from mice. J. Bacteriol.81:459-463.

BEREK, R. S., J. Ross, AND D. W. WATSON. 1954.A quantitative assay of streptolysin 0. Bac-teriol. Proc., p. 29.

BIRCH-HIRSCHFELD, L. 1934. Ueber die Wirksam-keit der Extrackte von auf Zellophanagargezuechtete Staphylokokken. Z. Immunitats-forsch. 81:260-285.

BLUMENTHAL, B. I., M. K. JOHNSON, AND E. J.JOHNSON. 1962. Distribution of the heatlabile and heat stable inorganic pyrophos-phatases amongst the bacteria. Bacteriol.Proc., p. 104.

BULLOCK, W., AND W. HUNTER. 1900. Ueber Pyo-cyanolysin, eine haemolytische Substanz inCulturen des Bacterium pyocyaneum. Centr.Bakteriol. Parasitenk., Abt. I, Orig. 28:865-876.

CALLOW, A. B. 1926. Heat stable peroxidases ofbacteria. Biochem. J. 20:245-252.

CHMURA, N. W., AND M. J. PELCZAR. 1959. En-hancement of pseudomonad pigment produc-tion by Serratia marcescens. J. Bacteriol.77:518-519.

GRXF, W. 1958. Ueber Gewinnung und Beschaffen-heit protostastisch-protocider Stoffe aus

Kulturen von Pseudomonas fluorescens. Arch.Hyg. u. Bakteriol. 142:267-275.

Liu, P. V. 1957. Survey of hemolysin productionamong species of pseudomonads. J. Bacteriol.72:718-729.

Liu, P. V., Y. ABE, AND J. L. BATES. 1961. Theroles of various fractions of Pseudomonasaeruginosa in its pathogenesis. J. InfectiousDiseases 108:218-228.

ZAK, B., AND J. COHEN. 1961. Automatic analysisof tissue culture proteins with stable Folinreagents. Clin. Chim. Acta 6:665-670.

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