STUDIES ON THE GROWTH OF HISTOPLASMA CAPSULATUM1II. GROWTH OF THE YEAST PHASE ON AGAR MEDIA

LEO PINEWith the technical assistance of CARL L. PEACOCK

Laboratory of Infectious Diseases, National Microbiological Institute, Bethesda, Maryland

Received for publication March 7, 1955

Individual cells of the yeast phase of Histo-plasma capsulatum grow poorly or not at all onmost culture media, although a suitable mediumsupports good growth of a heavy inoculum. Theinitial paper in this series describes a liquidmedium in which 100 cells of the yeast phase,after settling to the bottom of the tube, growunder conditions of stagnant incubation at 37 C(Pine, 1954). When 1.5 per cent agar is added tothis medium and the inoculum is spread overapproximately 20 sq cm of agar surface 107 to108 cells are required to obtain a significantnumber of colonies.Some of the factors which may be responsible

for the failure of an agar medium to supportgrowth of small inocula as yeast have beeninvestigated and are reported here.

MATERIALS AND METHODS

Strain and culture techniques. The strain(no. 6,617) of H. capsulatum used, the preparationof the inoculum, the procedure for determiningthe growth responses in liquid media and theprocedure for determining the concentration ofcells in heavy suspensions have been described(Pine, 1954). In experiments using small inoculathe inoculum was measured by counting cells ina Levy hemacytometer and response to addedgrowth factors was measured by counting coloniesarising from the inoculum spread on the agarsurface. In experiments using large inocula thenumber of cells in the inoculum and the growthresponse were estimated by measuring lightabsorption of a suspension of cells with a model BBeckman spectrophotometer (Pine, 1954).

Yeast phase of the fungus was grown at 37 Cin 25- or 50-ml Erlenmeyer flasks having 5 and

1 From the U. S. Department of Health, Edu-cation, and Welfare, Public Health Service,National Institutes of Health, National Micro-biological Institute, Bethesda, Md.

10 ml of medium, respectively. In preparingsuspensions for growth measurements the follow-ing simple procedure was devised. To each flask,2 ml of water and a short length (approximately1 cm) of 17-gauge soft iron wire were addedwith the usual bacteriological precautions. Theflask was then placed on a magnetic stirrer until,by manipulation of the flask, the rotating wireloosened and homogenized the surface growth.This usually required 15 to 30 seconds. Thismethod of removing cells from an agar surfaceis rapid, it eliminates some of the hazards ofconventional methods, a 1.5 per cent agar sur-face remains unbroken and a uniform suspen-sion is obtained. Growth response was estimatedby diluting an aliquot of the suspension andrecording growth units, i.e., optical density X 1/dilution. The sample must be taken promptlybecause added water is absorbed rapidly by anagar medium which has been incubated at 37 C.

Media. The 3-amino acid (cysteine-glutamic-aspartic) medium containing starch or albuminpreviously described (Pine, 1954) was used inthe preparation of agar media. Both starch andagar were purified by methanol extraction for24 to 72 hours in a Soxhlet apparatus. Total ex-tract from a weighed amount of each materialwas concentrated to 4 or 5 ml on a steam bathand was quantitatively transferred to a 12-mlgraduated centrifuge tube. A sample was taken,evaporated to dryness on a weighed aluminumplanchet under an infra-red lamp, and weighed.This material was soluble in ether upon acidi-fication, gave a strong hydroxamic acid test(Feigl, 1947) for fats and fatty acids and pre-cipitated from ether solution upon neutraliza-tion. Agar, starch, and crystalline bovine albumin contained 1.60, 0.85, and 0.90 per cent,respectively, of methanol extractable material.When these extracts were used in a mediumthey were added in ether solution in amountsequivalent to those present in the crude bio-

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logical materials which would have been usedin the medium. The ether was then driven offby heating. In general, the liquid medium wasprepared double strength and an equal volumeof agar solution was added. When the 3-aminoacid medium was prepared with 1.5 per centagar, flasks were inoculated within 3 hoursafter preparation. When a semi-solid medium(0.3 to 0.5 per cent agar) was prepared, it wasallowed to solidify overnight at room tempera-ture before inoculation.Whole blood was obtained by heart puncture

of adult rabbits, citrated, and stored at 5 Cuntil used (maximum of 24 hours). Fractiona-tion of the blood was done in a cold room at5 C. The formed elements were separated fromthe plasma by centrifugation in graduated

5I I5.0 ~~A B

4.0-3.0 - * BASAL * BASAL o

0 BASAL + ALBUMIN 0 BASAL + STARCH2.0 * BASAL + ALBUMIN * BASAL+ METHANOL

+ AGAR EXTRACT EXTRACTED STARCH

A BASAL+ EXTRACTED1.0 - STARCH+EX

TRACT

150.40-

zn .30

.20

10

.05-

.04-

.03

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DAYS

Figure 1. The effect of methanol extracts on thegrowth of the yeast phase of Histoplara capsula-tum in the cysteine-glutamic-aspartic liquidmedium in the presence of albumin or starch.6.25 X 107 cells of a 4-day-old culture of 6617 was

the inoculum for 25 ml of medium. Cultures were

incubated at 37 C on a rotary shaker (New Bruns-wick Co.) operating at 160 cycles per min. A:24-hour methanol extract equivalent to 0.5 per

cent agar was tested in the presence of 1.0 percent crystalline bovine albumin. B: 0.05 per centequivalent of a 72-hour methanol extract of Argostarch was tested in the presence of 0.05 per centstarch.

centrifuge tubes. The cells were then washedthree times with two volumes of 0.85 per centNaCl and resuspended in physiological salineto give a final volume equal to that of the initialwhole citrated blood. Part of the first washingof the blood cells was added to the plasma frac-tion to make the plasma fraction equal in volumeto that of the original whole blood. Red cellswere lysed by resuspending in water rather thansaline. Equal volumes of these preparations ofred cells, lysed red cells, plasma, and wholeblood were then used to compare their bio-logical activity in stimulating the growth of H.capsulatum. Serum was obtained by allowingthe blood to clot and was added directly withoutdilution. Rabbit albumin was isolated from theplasma fraction (undiluted) by the procedureof Pillemer and Hutchinson (1945). On the basisof the results obtained by Rhiel (1943) it wasassumed that the rabbit albuimin constitutedapproximately 6.85 per cent of the plasma. Foruse, the isolated albumin was dissolved in salineand sterilized by filtering through a Selas 03filter.Throughout this work the blood-glucose-

cystine agar medium, pH 7.3, described byRowley et al. (1954) was used to determine theviability of the yeast inoculum and as a standardfor evaluating other media, since it has con-sistently given the maximum rate of growth andthe highest colony counts when inoculated with100 cells.

RESULTS

The addition of many organic substancesfailed to stimulate growth of scattered cells onthe surface of an agar medium. This suggestedthat the agar contained a toxic factor. Methanolextraction of agar yielded a substance havingthe characteristics of a fatty acid (see Methods).This extract inhibited growth of Histoplasma(figure 1A), but 1.5 per cent agar, even afterextraction, did not support growth of smallinocula. However, when the agar content wasreduced to 0.3 to 0.5 per cent, and 0.07 percent starch or 0.5 per cent crystalline bovinealbumin was present, numerous colonies ap-peared from an inoculum of 100 to 1,000 celLs.The use of extracted or unextracted starch oragar or any combination of these did not affectthe number of colonies obtained from a smallinoculum as long as 0.5 per cent agar was used.

BASAL + AGAR EXRACT _ BASAL + STARCH EXTRACT

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No growth was obtained in the presence of0.5 per cent agar when starch or albumin wasomitted.When the concentration of the extracted or

unextracted agar was increased to 1.5 per centand the amount of starch or albumin increasedproportionally, an inoculum of 105 cells wasneeded to obtain a significant number of colonies.Direct comparison of the total colony counts orof growth rate in the presence of varying con-centrations of starch, crystalline bovine albumin,and bovine albumin, Fraction V, showed thatthe optimal concentration of these substancesin the 3-amino acid medium having 1.5 percent agar was 0.2 to 0.4, 3.0, and 1.5 per cent,respectively. The corresponding figures for thesemisolid media were 0.07, 1.0, and 0.5 to 0.7per cent, respectively.The results of experiments obtained in liquid

culture (figure 1) suggested that one function ofthe starch or albumin in an agar medium is todetoxify fatty acids present in the agar. When amethanol extract of starch or agar was addedto the liquid 3-amino acid medium, growth wascompletely inhibited. The addition of starch oralbumin with such extracts reversed the inhibi-tion. Albumin appeared to have an additionalstimulating effect (figure 1A) and therefore itwas preferentially used to detoxify fatty acidsin solid and semisolid media.The effect of pH on the growth of a 1,000-cell

inoculum in the semisolid medium containingbovine albumin, Fraction V, is given in table 1.At pH 4.5 the albumin coagulated. The resultant

TABLE 1Effect of pH on the growth of a 1000-cell inoculumon the cysteine-glutamic-aspartic acid mediumcontaining 0.6 per cent methanol extracted agarand 0.5 per cent crystalline bovine albumin

pH Colony County per Flask Average

4.5 250, 180, 156, 150, 160, 180, 150, 170150

5.0 115, 96, 105, 147, 105, 100 1115.5 29, 46, 35, 43, 47, 33, 82, 75 406.0 2, 20, 39, 49, 38, 40 336.5 1, 0,10, 0, 0, 0, 0 2

Blood 205, 200, 205, 175, 100, 200, 154, 168agar* 125,150

* Medium of Rowley, Haberman, and Emmons(1954).

TABLE 2Effect of various organic additions on the growth of

small inocula of Histoplasma capsulatum on thecysteine-glutamic-aspartic acid medium con-taining 0.6 per cent bovine serum albumin,Fraction V, and 0.6 per cent agar, pH 5.2*

Control mediaCasamino acid medium,25 Ct..................

Blood agar...............

Additions to basal mediumCarbon dioxide§..........Glutathione..............Liver extract.............Beef extract..............Autolyzed yeast..........Hemin...................Egg yolk.................Acid hydrolyzed yeast ex-

tract...................Potassium fumarate......Sodium pyruvate.........

Size of Inoculnm

1000 cellsSOO celsi 100 cells

Coloniest

>200 135>200 25

0>200

19>200

21377

31>200

4

0782

760450

3551

368

01303050

020

* All additions were tested at a concentrationof 0.1 per cent and at 37 C unless designated other-wise.

t Average of 10 plates.t This medium at 25 C has been shown to give

a reliable count of viable yeast cells which growas mycelia (Rowley and Huber, 1955).

§ A small pellet of dry ice was added to thedesiccator containing the flasks.

opacity of the medium and the large number ofyeast colonies obtained in a small area made thecolony counts at this pH inaccurate. Since thealbumin very often coagulated at pH 5.0, themedium was adjusted to pH 5.2 or 5.3 in furtherexperiments. Table 2 shows the colony countsobtained when certain organic additions weremade to the medium at pH 5.2. The addition ofglutathione, beef extract, hemin, and fumarateall stimulated the growth of inoculum to thepoint where the total number of colonies ob-tained was equal to that obtained with theblood medium. Furthermore, the rate of growthobtained when glutathione or beef extract wasadded was equal to that obtained on the wholeblood medium.

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.5

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0.51-

2 4 6 8 10 12

PERCENT WHOLE RABBIT'S BLOOD

LEO PINE

14

Figure B. The effect of whole rabbit blood on

the growth of the yeast phase of Histoplasmacapsulatum. The basal medium was the cysteine-glutamic-aspartic acid medium containing 1.5 percent agar. 25-ml Erlenmeyer flasks containing 5 mlof medium were inoculated with 10' cells of a

4-day-old culture of strain 6,617, and incubated 5days at 37 C.

As will be discussed later, this demonstrationof additional growth requirements suggestedthat the -SH form of a growth factor such as

coenzyme A was required. Therefore, a crudepreparation of coenzyme As and beef extractwere compared for their effect on growth of a

1000-cell inoculum in the 3-amino acid mediumcontaining 1.5 per cent agar and bovine serum

albumin, Fraction V. When 0.5 per cent beefextract was added the colonies appeared ap-proximately two days later than those on theblood medium but gave comparable colonycounts. The addition of 0.1 per cent beef ex-

tract or 5 Lipmann units/ml of the coenzyme Awere less effective, since the number of colonieswhich appeared was less than that on the bloodmedium and the colonies appeared approxi-mately 3 to 4 days later. However, the 0.1 per

cent beef extract medium and the coenzyme Amedium were equal in their ability to promotegrowth of the inoculum. When neither the beefextract or crude coenzyme A preparation was

added, the number of colonies which appeared2 The author is indebted to Dr. Simon Black for

this preparation which was 20 per cent pure.

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were few and were visible 7 days later than thoseon blood.

Fractionation and testing of blood. Throughoutthis investigation, whole rabbit blood added tothe synthetic medium or in the medium of Rowleyet al. (1954) supported the maximum rate ofgrowth observed and promoted the growth ofsmall inocula of the yeast phase in a mediumcontaining 1.5 per cent agar. The following experi-ments were done to obtain information regardingthe growth stimulating factors in whole blood.The addition of whole citratrd rabbit blood

to the 3-amino acid medium containing 1.5 percent agar resulted in a rate of growth propor-tional to the amount of blood added until a

TABLE 3Effect of blood fractions or treatment8 of bloodfractions on the growth of the yeast phase of

Histoplasma capsulatum on solid media*

Experi- Fraction Per Centment Activity

1 10 per cent whole blood 100Serum 28Whole blood, heated 15 min at 0

100 CWhole blood, heated 60 min at 60C 16

2 Plasma 33Formed elements 51Plasma + 2 per cent formed ele- 54ments

Plasma + 6 per cent formed ele- 92ments

Plasma + 10 per cent formed ele- 90ments

Plasma + 2 per cent formed ele- 16ments, lysed

Plasma + 6 per cent formed ele- 16ments, lysed

Plasma + 10 per cent formed ele- 24ments, lysed

3 Formed elements 52Plasma 40Plasma + formed elements 93

* All fractions added to the cysteine-glutamic-aspartic acid medium, pH 7.3, containing 1.5 percent unextracted agar in volumes equivalent to10 per cent whole blood unless designated other-wise. 25-ml flasks containing 5 ml of medium wereinoculated with 10' cells of a 3-day-old cultureof the yeast phase of strain 6617 and incubated5 days at 37 C.

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GROWTH OF HISTOPLASMA CAPSULATUM 379

concentration greater than 10 per cent wasreached (figure 2). Further addition of bloodshowed no significant increase in the rate ofgrowth. The effects of various treatments ofthe blood or blood fractions are given in table 3.Heating the blood greatly decreased its growth-stimulating properties. Although the plasmafraction contained considerable activity, agreater amount of stimulation was obtainedfrom the formed elements. Recombination ofthe two fractions had an additive effect and therecombined blood had approximately 90 percent of the activity of whole citrated blood. Thatthe activity of the formed elements is due pri-marily to the presence of the intact red cell issuggested by the fact that use of lysed red cellsresulted in reduction of growth to the levelobtained with the plasma fraction alone. Figure3 indicates that the activity of the plasma frac-tion is due to the albuimin or some contaminantassociated with it, inasmuch as the addition ofisolated rabbit albumin gave approximately 90per cent of the stimulation obtained with theentire plasma. Addition of 0.5 per cent crystal-

12 RABBIT PLASMA

1- / > R~~ABBIT ALBUMIN

z

o CRYSTALLINE BOVINE

2 ALBUMIN2

O 1 I .0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

PERCENT ALBUMIN IN THE MEDIUMFigure S. The effect of albumin in the presence

of red cells on the growth of the yeast phase ofHistoplasma capsulatum. The basal medium wasthe cysteine-glutamic-aspartic acid medium con-taining 1.5 per cent agar and 10 per cent wholeblood equivalents of washed formed elements.Based on the results of Rhiel (1943) it is assumedthat the rabbit plasma contains 6.85 per centalbumin. 25-ml Erlenmeyer flasks containing 5ml of medium were inoculated with 10$ cells ofa 4-day-old culture of strain 6,617, and incubated5 days at 37 C.

10.0

D.40- RTO

I l BOVINE ALBUMIN,.05 FRACTION -Z

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2 3 4 5 6DAYS

Figure 4. Effect of pH on the rate of growth onagar media in the presence and absence of wholeblood. The basal medium was the cysteine-glutamic-aspartic acid medium containing 1.5per cent agar. 10 per cent whole rabbit blood wasused. In the absence of whole blood, 1.5 per centbovine serum albumin, Fraction V, was added.50-ml Erlenmeyer flasks containing 10 ml of me-dium were inoculated with 105 cells of a 4-day-old culture of strain 6,617 and incubated at 37 C.Points represent the average of 3 flasks, harvestedat each time.

line bovine albumin caused an immediate lysisof the rabbit red cells and a definite inhibitionof growth was observed. The lytic effect could bedue to the toxic effect of the fatty acids presentin the crystalline bovine albumin (Davis andDubos, 1947). In this experiment the totalamount of growth obtained with the individualaddition of 10 per cent equivalents of plasma,0.5 per cent rabbit albumin, or 0.5 per centcrystalline bovine albumin in the absence ofthe cellular components was 0.42, 0.22, and 0.05growth units, respectively.

It was further noted that, in the presence ofwhole blood, the rate of growth of H. capsulatumwas constant from pH 5.5 to pH 7.5. In theabsence of whole blood, but in the presence ofbovine albumin, the rate of growth increasedwith an increase of pH to the point where the

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rate at pH 7.5 appeared to equal that obtainedwith the whole blood medium except that a4-day lag period occurred (figure 4). Theseresults are in accord with the results obtainedin liquid media (Pine, 1954). However, on thesolid 3-amino acid medium containing bovinealbumin, the lag period is definitely shorter atpH 5.5 than that obtained at pH 7.5 (figure 4).This might be expected from the effects, re-ported above, of low pH on the growth of smallinocula. It would therefore appear that the pHindependence of this strain in a whole bloodmedium is due to the presence of the whole redcell since the cysteine-glutamic-aspartic acidmedium contained added albumin.

DISCUSSION

Evidence has been presented which suggeststhat the fatty acids present in agar inhibit thegrowth of a small inoculum of H. capsulatum.The toxicity of fatty acids for H. capsulatum hasbeen reported (Pine, 1954). Ley and Mueller(1946) reported the isolation from agar of fattyacid inhibitors of Neiseria gonorrheae and, withuse of methanol-extracted agar, growth of theorganism occurred in their medium. Our ex-tracted agar at a level of 0.5 per cent did notsupport growth of 102 to 103 cells unless starchor albumin were added. The albumin or starchpresumably functions to detoxify and to supplyrequired fatty acids (Pine, 1954). McVickar(1951) has also shown that albumin detoxifiessubstances in peptone media used for growingthe yeast phase of H. capsulatum. When theconcentration of agar was increased to 1.5 percent, the addition of starch or albumin was nolonger sufficient to permit growth and otherorganic additions were required. A simple ex-planation for the inhibitory effect of solid agaris not readily apparent. Salvin (1947) has alsoreported the beneficial effect of a semisolid agarmedium.The general requirement of H. capsulatum for

-SH groups has been suggested previously(Salvin, 1949; Pine, 1954). Much of the datamight be interpreted on the basis of such arequirement. For example:

(a) Better growth of small inocula occurs at alow pH. At a low pH the oxidation of cysteineis minimized.

(b) The rate of growth is stimulated by thewhole red cell and not by a red cell lysate or

heated whole blood. Evidence supporting ahypothesis that the "living" red cell can supplya constant source of -SH groups may be ob-tained from the papers of Meldrum (1932),Meldrum and Tarr (1935) and Francoeur andDenstedt (1954).

(c) Growth and growth rate of the yeastphase are not affected by a change of pH from5.5 to 7.5 in the presence of the red cell. In theabsence of the red cell, the initial growth of theyeast phase is inhibited at pH 7.5, and the rateof growth is decreased at pH 5.5. Presumably,then, the red cell can maintain a sufficient -SHconcentration at either pH.

(d) The addition of glutathione, beef extract,or a preparation of coenzyme A stimulatesgrowth of small inocula in the presence of addedcysteine. These additions may serve to eithermaintain or supply -1SH forms of growthfactors. Brown and Snell (1954) have presentedevidence that required growth factors for thestrict aerobic Acetobacter suboxidan2 are the-SHform of coenzyme A or its precursors, not thecorresponding disulfides.The presence of -5H groups cannot be shown

in liquid shake cultures after several hours' in-cubation or in liquid stagnant deeps after 2days. It would appear that the presence of -51His required merely to initiate growth or thatextremely small amounts are needed continu-ously. It is also possible that, once the organismis actively growing, it is capable of reducingthe disulfide to -SH and could maintain theconcentration of the sulfhydryl group in itsimmediate vicinity. Therefore, growth of a smallinoculum in a stagnant liquid culture may bestimulated by environmental conditions at thebottom of the tube where the total inoculumis aggregated or by growth factors carried overin the cells of the inoculum. When the sameinoculum is spread on an agar surface not onlyare the cells exposed to toxic substances in theagar, but the mutual protection or stimulationof adjacent cells is lost. In addition, the use ofthe solid medium creates the problem of supply-ing and maintaining the required sulfhydrylgroup on the agar surface for periods sufficientlylong to initiate growth.

SUMMARY

Evidence is presented which suggests that:(1) Agar contains fatty acids which inhibit

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growth of the yeast phase of Histoplasma capsu-latum.

(2) The use of solid media creates a technicalproblem of supplying and maintaining -SHgroups for periods sufficiently long to initiatethe growth of the organism under aerobic con-ditions.

(3) Unknown growth factors are required topromote the growth of a small number of cellswhen they are separated on the surface of theagar-containing medium.

(4) The results of experiments with bloodand blood fractions show that the stimulatingeffect of whole blood is due to the presence ofthe whole red cell and the albumin fraction. It isconsidered possible that the red blood cell func-tions to maintain. a constant source of -SHgroups required for the growth of the organism.Secondly, the presence of albumin of the wholeblood presumably supplies and maintains anontoxic source of fatty acids.

(5) A simple, safe, and rapid procedure forthe removal of the growth of certain pathogenicmicroorganisms from the surface of agar mediais described.

REFERENCESBROWN, G. M., AND SNELL, E. B. 1954 Panto-

thenic acid conjugates and growth of Aceto-bacter suboxidans. J. Bacteriol., 67, 465-471.

DAVIS, B. D., AND DuBos, R. J. 1947 The bind-ing of fatty acids by serum albumin, a pro-tective growth factor in bacteriological media.J. Exptl. Med., 86, 215-228.

FEIGL, F. 1947 Qualitative analysis by spottests. Elsevier Publishing Co., New York,New York.

FRANCOEUR, M., AND DENSTEDT, 0. F. 1954Metabolism of the mammalian erythrocyte.VII. The glutathione reductase of the mam-malian erythrocyte. Can. J. Biochem. andPhys., 32, 663-669.

LEY, H. L., AND MUELLER, J. H. 1946 On theisolation from agar of an inhibitor for Neis-

seria gonorrhoeae. J. Bacteriol., 52, 453-460.

MELDRUM, N. E. 1932 C. The reduction ofglutathione in mammalian erythrocytes.Biochem. J. (London), 26, 817-828.

MELDRUM, N. U., AND TARR, H. L. A. 1935XV. The reduction of glutathione by theWarburg-Christian system. Biochem. J.(London), 29, 109-115.

MCVrCKAR, D. L. 1951 Factors important forthe growth of Histoplasma capsulatum in theyeast cell phase on peptone media. J. Bac-teriol., 62, 137-143.

PILLEMER, L., AND HUTCHINSON, M. C. 1945The determination of the albumin and globu-lin contents of human serum by methanolprecipitation. J. Biol. Chem., 158, 299-301.

PINE, L. 1954 Studies on the growth of Histo-plasma capsulatum. I. Growth of the yeastphase in liquid media. J. Bacteriol., 68, 671-679.

RHIEL, J. 1943 Vergleichende Blutuntersuchun-gen. Viscositats-und refraktometrische Be-stimmungen des Bluts, Plasmas und Serumsvon Pferden, Rindern, Schweinen, Schafen,Ziegen und Kaninchen nebst Hamoglobin-bestimmungen. Pflugers Arch. ges. Physiol.,246, 709-727.

ROWLEY, D. A., HABERMAN, R. T., AND EMMONS,C. W. 1954 Histoplasmosis: Pathologicstudies of fifty cats and fifty dogs fromLoudoun county, Virginia. J. InfectiousDiseases, 85, 98-105.

ROWLEY, D. A., AND HIUBER, M. 1955 Patho-genesis of experimental histoplasmosis inmice. I. Measurement of infecting dosages ofthe yeast phase of Histoplasma capsulatum.J. Infectious Diseases, 96: 174-183.

SALVIN, S. B. 1947 Cultural studies on theyeastlike phase of Histoplasma capsulatumDarling. J. Bacteriol., 54, 655-660.

SALVIN, S. B. 1949 Cysteine and related com-pounds in the growth of the yeastlike phaseof Histoplasma capsulatum. J. InfectiousDiseases, 84, 275-283.

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