Nutrition of Systemic and Pathogenic Fungi · NUTRITION OF PATHOGENIC FUNGI termine the types and...

BACTERIOLOGICAL REVIEWS, Sept., 1965 Vol. 29, No. 3Copyright © 1965 American Society for Microbiology Printed in U.S.A.

Nutrition of Systemic and SubcutaneousPathogenic Fungi

GERALD L. GILARDIMicrobiology Department, Hospital for Joint Diseases, New York, New York

INTRODUCTION.................................................................. 406NUTRITION OF PATHOGENIC YEASTS............................................ 407Candida...................................................................... 407

Vitamin requirements....................................................... 407Amino acid requirements.................................................... 407Factors influencing dimorphism ............................................. 407Agents demonstrating beneficial or deleterious effects on growth ................ 408Assimilation patterns....................................................... 408

Cryptococcus ..................................................... 408Vitamin and amino acid requirements ....................................... 408Agents demonstrating beneficial or deleterious effects on growth ................ 408Assimilation patterns ..................................................... 409

NUTRITION OF DIMORPHIC FUNGI ............................................... 409Blastomyces ..................................................... 409

Vitamin requirements ..................................................... 409Amino acid requirements.................................................... 409Factors influencing dimorphism............................................. 409Physical requirements ..................................................... 410Agents demonstrating beneficial or deleterious effects on growth ................ 410Assimilation patterns ..................................................... 410Similarity of the species .................................................... 410

Coccidioides..................................................... 411Vitamin and amino acid requirements ....................................... 411In vitro cultivation of parasitic form......................................... 411Agents demonstrating beneficial or deleterious effects on growth ................ 412Vaccine preparation ..................................................... 412Metabolic properties of spherules ............................................ 412Assimilation patterns....................................................... 412

Histoplasma..................................................... 413Vitamin requirements....................................................... 413Amino acid requirements.................................................... 413Physical requirements ..................................................... 413Factors influencing dimorphism ............................................. 413Agents demonstrating beneficial or deleterious effects on growth ................ 413Assimilation patterns .................................................. 414

Sporotrichum..................................................... 414Vitamin requirements....................................................... 414Amino acid requirements.................................................... 414Factors influencing dimorphism ............................................. 414Agents demonstrating beneficial or deleterious effects on growth ................ 414Assimilation patterns ..................................................... 415

NUTRITION OF DEMATIACEOUS FUNGI ........................................... 415NUTRITION OF ALLESCHERIA (MONOSPORIUM) .................................... 416CONCLUSIONS ................................................................... 416LITERATURE CITED............. 421

INTRODUCTION While nutritional investigations with bacteriahave been progressing rapidly, comparativelyMany studies on the nutritional behavior of few recent studies have been carried out with

bacteria have appeared in theleast few decades fungi. The earliest nutritional studies with fungiThe reasons for these studies are apparent: to were performed primarily on saprophytic yeastsexamine the accessory growth factors essential and molds, with little attention paid to thefor bacteria and to increase knowledge of the pathogenic fungi. At first, nutritional studiesbasal nutritional requirements necessary for their concerned with pathogenic fungi consisted ofdevelopment. studies with complex nonsynthetic media to de-

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NUTRITION OF PATHOGENIC FUNGI

termine the types and abundance of growth ob-tained and efficiency of the media in isolation.Later, nutritional investigations with pathogenicfungi were concerned with defining basal nutri-tional requirements and accessory growth factorsnecessary to replace media containing complexmixtures of organic substances of unknownchemical composition. This has resulted in culti-vation of most pathogenic fungi on much simpler,chemically defined culture media.

This review is concerned with those studieswhich define the essential growth factors andnutritional characteristics of fungi responsiblefor the systemic and subcutaneous mycoses ofman. Hence, the areas of primary concern coveredby the review include vitamin and amino acidrequirements, basal media required for growth,effect of chemical and physical agents on growth,studies of carbon and nitrogen assimilation, anddifferences in nutritional requirements betweenthe mold form and the yeast form of dimorphicfungi.

NUTRITION OF PATHOGENIC YEASTS

Candida

Vitamin requirements. Most strains of thegenus Candida do not grow in a glucose-ammo-nium-inorganic salts medium (GASM) unlessthey are supplemented by yeast extract. Vita-min-requirement studies show that C. albicans iscompletely deficient for biotin (9, 13, 14, 23, 24,26, 34, 42, 43, 56, 57, 60, 61, 95) and partiallydeficient for thiamine; that is, thiamine has anadditive effect, enhancing growth (13, 14, 34, 42,57, 60, 61, 95). Koser et al. (34) found panto-thenic acid to be required or stimulatory forthree strains of C. albicans. Various biotin ana-logues replace biotin, including biocytin, N-biotinyl-f3-alanine, N-biotinyl-L-aspartic ethylester, desthiobiotin, biotin-D-sulfoxide, homobio-tin, and oxybiotin; aspartic acid partially re-places biotin, and biotin sulfone, biotin diaminesulfate, biotinol, and norbiotin show little ac-tivity (26, 42).Amino acid requirements. Several of the vita-

min-requirement studies have been performed ina glucose-asparagine-inorganic salts medium (13,14, 57), and one in a more complex medium con-taining casein hydrolysate, several amino acids,purines, and pyrimidines (34). But C. albicansdoes grow in a simple GASM containing biotinwith no exogenous supply of any amino acidrequired, indicating that all amino acids can besynthesized in the presence of biotin (1, 60).

Miyashita, Mliwatani, and Fujino (61) foundthat, in the presence of suboptimal amounts ofbiotin, six amino acids (aspartic acid, glutamic

acid, arginine, proline, serine, and alanine) havea biotin-sparing effect.

Factors influencing dimorphism. Extensive in-vestigations have been concerned with thosefactors which influence yeast (Y) form, pseudo-hyphae, and chlamydospore development inyeastlike fungi, including C. albicans. No attempthas been made here to cover the literature in thisarea since there are many fine reviews on di-morphism (25, 63, 65, 69, 70, 93, 100, 101, 107),including the more recent reviews by Skinner andFletcher in 1960 (101) and Nickerson in 1963(65).

In general, the conditions favoring Y-formdevelopment include a fermentable carbohydratesource such as glucose (30, 32, 67, 68, 100, 103),the presence of cysteine (67, 68, 70, 93), thepresence of glutathione (67), increased oxygentension (93, 100), and the absence of such growthfactors as potassium, phosphorus, and biotin(56).The conditions favoring pseudohyphae de-

velopment include an assimilable but not soreadily fermentable carbohydrate such as sucrose(56), the presence of polysaccharides (67, 68, 93),the presence of certain growth factors such aspotassium, phosphorus, and biotin (56), reducedoxygen tension (30, 93, 100), starvation media(30, 100), and liquid media in general (56, 100).From the review of the literature there seems

to be little agreement among the various investi-gators concerning the influence of pH (30, 33,56, 93, 100) and temperature (30, 56, 93, 100).Scherr and Weaver (93) reported that the Yform is present at pH 7.0, and pseudohyphae arepresent at lower pH levels. Skinner (100), how-ever, found pseudohyphae formation at high pHlevels. This was confirmed by Johnson, Guzmon,and Agurlera (30) at pH 7.4 to 9.6. They foundthe Y form at pH 2.5 to 6.4. McClary (56) saidthat extreme pH ranges almost invariably pro-duce the Y form, and that a pH of 5 is optimalfor pseudohyphae development. In the review bySkinner (100), high temperature (37 C) wasstated to favor pseudohyphae production. John-son et al. (30) found both Y forms and pseudo-hyphae developing at 37 C. McClary (56) foundtemperatures of 25 to 30 C to favor pseudohyphaeproduction, and temperatures of 37 to 40 C tofavor Y-form development.

Chlamydospore production has been attributedto the presence of polysaccharides as the carbonsource (67, 68, 93), the absence of reducing sugars(67, 68, 79), the presence of aminopterin (67),a deficiency in available phosphorus (56), theabsence of sulfhydryl groups (68, 70), and ex-treme pH ranges (56).Media have been developed for demonstrating

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the production of spores by chlamydospore-producing species of Candida (C. albicans, C.stellatoidea). Nickerson and Mankowski (68) ob-tained abundant chlamydospore production in amedium containing purified polysaccharides (freefrom reducing sugars) such as glycogen, solublestarch, and dextrin. A chlamydospore-producingmedium containing zein, the basic protein ofcorn meal, was developed by Reid, Jones, andCarter (79). They concluded that a partial ex-planation for enhanced formation in the zein-agar lies in the absence of reducing sugars in thismedium. Enhanced chlamydospore productionhas been observed in a semisolid medium con-taining polyoxyethylene sorbitan monooleate(Tween 80) (96) and on Loewenstein's mediummoistened with Old Tuberculin (50).

Investigations have been directed towards anunderstanding of metabolic differences betweenthe Y form and the pseudohyphae form of growth.On the basis of the evidence obtained from theseinvestigations, summarized by Morris (63), Ward(107), Skinner and Fletcher (101), and Nickerson(65), pseudohyphae production results from animpaired cell-division mechanism resulting froman apparent breakdown of intracellular sulfhy-dryl maintenance. Conversely, a specific disulfide-reducing process must be maintained if the bud-ding phase is to occur.

Agents demonstrating beneficial or deleteriouseffects on growth. The addition of chemical agentsto basal media has been noted to exert either astimulatory or inhibitory effect on growth offungi. Marwin (53-55) found four nonionic sur-face-tension reductants, Nonisol 100, Polyethyl-ene glycol 400 monolaurate, Pluronic L 64, andMulsor 224, to have a stimulatory effect on thegrowth of several pathogenic fungi, including C.albicans. p-Hydroxymethyl benzoic acid (108) andthiourea (22) are fungistatic to C. albicans,whereas hydrogen sulfide (7), sodium acetate(74), and ascorbic acid (73) have fungicidal ef-fects.

Assimilation patterns. Most authorities agreethat C. albicans assimilates glucose, galactose,sucrose, and maltose, but not lactose or potassiumnitrate (23, 44, C0). Other studies reveal thatraffinose (23) and starch (56) are not assimilatedand that fructose, mannose (56, 60), ethyl alcohol,glycerol, and succinic acid (56) are assimilatedas sole sources of carbon and that ammoniumsulfate, asparagine, and urea (23) are assimilatedas sole sources of nitrogen. Kapica and Blank(32) reported that keratin is assimilated as thesole source of nitrogen. They pointed out thatthis may not be due to keratinolytic activity,since C. albicans might grow on a nitrogen-freemedium by fixing atmospheric nitrogen. They

further questioned whether the breakdown ofkeratin is due to acid hydrolysis, rather thanenzymatic activity which might supply the yeastwith soluble forms of nitrogen.

Miyashita et al. (61) showed that amino acidsutilized as sole sources of nitrogen include as-partic acid, glutamic acid, arginine, proline,serine, alanine, histidine, methionine, and tryp-tophan. They found that the amino acids utilizedas sole sources of carbon include aspartic acid,glutamic acid, and arginine, the latter threebeing utilized as the sole source of both carbonand nitrogen. In addition to the amino acids sofar mentioned, Johnson et al. (30) found thatthe following amino acids are assimilated as thesole source of nitrogen: phenylalanine, amino-acetic acid, tyrosine, leucine, lysine, threonine,norleucine, isoleucine, and valine. They foundthat cystine, homocystine, hydroxyproline, andornithine are not assimilated.

CryptococcusVitamin and amino acid requirements. The

growth requirements of Cryptococcus neoformanshave been determined in a chemically definedmedium by several investigators. In a review onthe vitamin requirements of fungi, Robbins andKavanagh (80) stated that the yeast grows wellin the presence of thiamine but can also grow,although poorly, in a glucose-asparagine-am-monium lactate-inorganic salts medium withoutthe addition of thiamine. Reid (78) and AreaLeao and Cury (2) have grown the yeast in aglucose-asparagine-inorganic salts medium con-taining thiamine. But several investigators (27,41, 94, 105) have grown the yeast in a simpleGASM to which they added thiamine. AreaLeao and Cury (2) found an absolute require-ment for thiamine, and Littman (41) an absoluterequirement for thiamine and its moieties, thia-zole and pyrimidine.

According to Schmidt et al. (94), amino acids,purines, and pyrimidines are not essential forgrowth, with amino acids exerting merely aslight stimulatory effect on growth.

Agents demonstrating beneficial or deleteriouseffects on growth. The nonionic surface-activeagents described as having a stimulatory effecton growth of C. albicans have a similar effect onC. neoformans (52-54). In studies on the inhibi-tion of growth of C. neoformans, p-hydroxymethylbenzoic acid (108) and thiourea (22, 104) werefound to be effective. Tager, Hales, and Danow-ski (104) found that the fungistatic activity ofthiourea is reversed upon the addition of -SH-containing compounds such as cysteine. Theyconcluded that thiourea inactivates the -SHradical through some process such as oxidation,

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thus suppressing growth of the organism, andthat this is reversed upon the addition of com-pounds which replace or reconstitute -SHgroupings.

Assimilation patterns. Most authorities agreethat C. neoformans assimilates glucose, maltose,sucrose, and galactose, but not lactose and potas-sium nitrate (5, 6, 44). But Kao and Schwarz(31) showed that potassium nitrate and lactoseprovide sufficient nutriment for slight growth.This is at variance with the opinion of Benham(5), who reported no growth on media containingthese substances as the sole source of carbonand nitrogen. The assimilation of amino acidsand carbohydrates as the sole source of carbonhas been studied by Littman (41). Of the aminoacids and derivatives investigated, he found thatthe yeast assimilates members of the glutamicacid family: glutamic acid, glutamine, proline,serine, and asparagine. His studies show that 25other sources are not assimilated. In his investi-gation of carbohydrates, all hexoses, a number ofpentoses (ribose, xylose), disaccharides (maltose,sucrose, trehalose, cellobiose), and polysaccha-rides (dextrin, starch) are assimilated. Othercompounds assimilated include ethyl alcohol,glyceraldehyde, glucuronolactone, inositol, glu-cosamine, and the Krebs cycle intermediate,a-ketoglutaric acid. Compounds which he foundare not assimilated include 13-glycerophosphoricacid, glucose-i-phosphate, glutaric acid, glycogen,and lactose.

NUTRITION OF DIMORPHIC FUNGIBlastomyces

Vitamin requirements. Vitamin requirementstudies of the genus Blastomyces have producedvariable results. Halliday and McCoy (29) foundboth the Y and mold (M) forms of B. dermatitidiscompletely deficient for biotin. They observedthat optimal growth of recently isolated strainsis obtained with less biotin than is required foroptimal growth of old laboratory strains. Sub-stances which have been implicated in biotinmetabolism, including oleic acid, aspartic acid,methionine, and pimelic acid, were found to haveno effect on growth; desthiobiotin can be replacedfor biotin. Other investigators (2, 28, 40, 66, 88,102) found no growth-factor requirements neces-sary for growth of either form of B. dermatitidis.

Area Ledo and Cury (2) found two strains ofthe M form of B. brasiliensis (Paracoccidioidesbrasiliensis) to be completely deficient and onestrain partially deficient for thiamine, butNickerson and Edwards (66), Salvin (88), andGilardi and Laffer (28) found no vitamin require-ments for either form. In those cases where vita-

mins are not required, it is also noted that thevitamins are not stimulatory for growth (28, 88).

Halliday and McCoy (29) pointed out thatother investigators have performed vitaminstudies with media solidified with agar in cotton-plugged tubes, which could allow contaminationwith biotin from the cotton plug or agar.They used a liquid medium in aluminum-

capped test tubes for their studies. Salvin (88) ob-tained good growth in sealed tubes, with a semi-solid medium containing washed agar. Gilardiand Laffer (28) used a liquid synthetic mediumin metal-capped tubes, confirming the results ofSalvin (88).Amino acid requirements. Many nutritional

studies have been performed with media contain-ing organic nitrogen sources. The M form of B.dermatitidis has been grown on media containingneopeptone plus Tryptone (58), peptone, caseinhydrolysate (40), ammonium acetate, acetamide(102), asparagine (2), and glycine (66). But the Mform does not require an organic nitrogen source,and can be grown on a GASM (40, 66, 102). TheM form of B. brasiliensis has been cultured on amedium containing asparagine (2) and glycine(66), but has also been grown on a simple GASM(66, 88).The Y form of these fungi has been grown pri-

marily on solid media, because difficulty has beenencountered in obtaining Y-form growth in liquidmedia (40). Y-form growth has been obtained insemisolid media (88) or liquid media (29, 66,89) to which vitamins (29, 66, 89) and organicnitrogen sources (66, 89) have been added, butrecent studies show that the Y form grows in aliquid GASM without the addition of organicnitrogen or vitamins (28). The various studiesindicate that amino acids are not required, butare stimulatory for growth of both the Y and Mforms (1, 28, 40).

Factors influencing dimorphism. Dimorphismin Blastomyces occurs on a minimal medium, andis apparently independent of nutrition and a func-tion only of the temperature of incubation (40,64, 66, 88). However, Bullen (11) found that anincreased concentration of CO2 appears to beessential for the development of the Y form. Thechange in morphology upon conversion to theM form was considered by Nickerson andEdwards (66) to result from the selective inhibi-tion of cell division, without simultaneous inhibi-tion of growth, with selective inhibition beingdependent only on temperature. They furtherconsidered that there is competition for a commonsubstrate between the enzymes responsible forM-form development and the enzymes responsi-ble for Y-form development. They assumed thata normally higher affinity of the mold-enzyme

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system for the common substrate is offset athigher temperatures by an increasing rate in itsreversible thermal inactivation, thus explainingthe observed dependence of the cell-divisionmechanism on the maintenance of an elevatedtemperature.

Physical requirements. The optimal tempera-ture for growth of the M form is stated to be 25 C(91) and 31 to 33 C (40); for the Y form, 35 to37 C (40) and 37 C (66, 91). Optimal pH forgrowth of the M form is 4.5 to 7.5 (40); for the Yform, 5.5 to 8.5 (28, 40). The fungi grow onlyunder aerobic conditions (28).

Agents demonstrating beneficial or deleteriouseffects on growth. Gilardi and Laffer (28) noticeda tendency of the Y form to produce a granulartype of growth and to grow along the side of theculture tube above the liquid medium, suggestingthat the yeast requires a surface or some particu-late matter on which to grow. They found thatadding agar to the medium does not enhancegrowth nor does it minimize the granulation orthe growth along the side of the tube. The in-corporation of agar into a liquid medium wasfound by Salvin (88) to enhance growth.The addition of Tween 80 as a dispersing agent

does not minimize the granular effect, but it hasan inhibitory effect retarding growth. Hallidayand McCoy (29) found Tween 80 to stimulategrowth in the presence, but not the absence, ofbiotin, which is required for growth of theirstrain.

These investigators further observed that inshake cultures the growth still appears along theside of the culture tube above the liquid medium,and, because of the shaking, is more pronouncedthan in quiescent cultures; but the granular typeof growth evident in quiescent cultures is virtuallyeliminated. They noticed that more luxuriantgrowth occurs in a shorter period of time in shakecultures compared to quiescent cultures, and insome cases compared to agar slant cultures. Salvin(89) also noticed luxuriant growth when cultureswere kept in constant rotation.The nonionic surface-active agents which are

stimulatory for C. neoformans are also stimulatoryfor B. dermatitidis (53-55). Oxaloacetic acid (29),thiourea (22, 28), p-aminobenzoic acid (8), p-hy-droxymethyl benzoic acid (108), as well as sulfur-containing compounds such as cysteine, gluta-thione, methionine, and thioglycolate (28), havean inhibitory effect on growth of Blastomyces. Itwas found that the Y form, in the presence oftyrosine, is converted to the M form at 35 C (28).

Assimilation patterns. Stewart and Meyer(102) found that the M form of B. dermatitidis isable to utilize ammonium acetate or acetamideas the sole source of both carbon and nitrogen in

liquid medium. Archibald and Reiss (1) foundthe M form to be capable of assimilating thefollowing amino acids as sole sources of nitrogenon solid medium: cysteine, glutathione, glycine,alanine, leucine, valine, glutamic acid, asparticacid, asparagine, serine, threonine, arginine,histidine, and phenylalanine. They observed poorgrowth with isoleucine, glutamine, lysine, tyro-sine, tryptophan, proline, and hydroxyproline.They obtained no growth with methionine orp-aminobenzoic acid. In these studies, theypointed out that the agar was not purified andthe mycelial inoculum was not washed.

Salvin (88) found that the Y form of B. derma-titidis gives slight growth with ammonium sulfate,potassium nitrate, tyrosine, and arginine, andgood growth with glycine, alanine, serine, valine,aspartic acid, glutamic acid, proline, and hy-droxyproline as the sole source of both carbonand nitrogen in semisolid medium. He found thatthe Y form of B. brasiliensis gives good growthwith glycine, serine, glutamic acid, aspartic acid,proline, and hydroxyproline as the sole sourceof both carbon and nitrogen. He found no carbo-hydrate to be required for growth of the Y formof B. dermatitidis.

Gilardi and Laffer (28) found that the Y formof both B. dermatitidis and B. brasiliensis assimi-lates the same 21 carbon and 25 nitrogen sub-strates in liquid shake cultures. They found thatthe only carbohydrates assimilated as sole sourcesof carbon are the hexoses. They further foundthat glutamic acid, the imino acids proline andhydroxyproline, some aliphatic amino acids(alanine, glycine, serine, and valine), as wellas the substituted amino acid asparagine, theamino acid relative 0-alanine, and the amino acidmetabolites pyruvic acid and lactic acid areassimilated as sole carbon sources. They alsofound three intermediates of the Krebs cycle(a-ketoglutaric acid, oxaloacetic acid, andsuccinic acid) to be assimilated.They found that the only inorganic nitrogen

compounds assimilated as sole sources of nitrogenare ammonium salts. They further found thatsome aliphatic amino acids (glycine, alanine,valine, serine, and isoleucine), the imino acidsproline and hydroxyproline, the acidic aminoacids aspartic acid and glutamic acid, and thebasic amino acids histidine, arginine, and lysineare assimilated as sole nitrogen sources. Theamino acid relatives citrulline, ornithine, and3-alanine, the substituted amino acids asparagineand glycylglycine, and the carbohydrate deriva-tive glucosamine, as well as urea, were also foundto be assimilated.

Similarity of the species. A comparison of theresults obtained from studying these fungi shows

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that there is no difference between the two spe-cies; any variation which the investigators couldsee occurred only at the culture level. It is con-cluded that there is no justification for placingthe two species into separate genera, as proposedby some investigators; the fungi are close phylo-genetically and belong in the same genus (28, 88).

CoccidioidesVitamin and amino acid requirements. Coccidi-

oides immitis does not require any vitamin oramino acid supplement (2, 4, 81, 102), and willgrow in a simple GASM (4, 102).In vitro cultivation of parasitic form. Nutritional

studies have been primarily concerned with thein vitro cultivation of the spherules (parasiticform) of C. immitis. Media that have been em-ployed contain complex organic substances suchas coagulated egg albumin (36) and coconut milk(12). Conant and Vogle (16) obtained spherulesin cultures transferred to Sabouraud mediumafter exposure to Tween 80. Lubarsky andPlunkett (48) obtained spherules in Tyrode'ssolution, containing chick embryo extract androoster serum and aerated periodically withmeasured amounts of 02 and CO2. Larsh, Hinton,and Silberg (37) produced spherules in HeLatissue cultures containing human serum. Northeyand Brooks (72) developed a medium consistingof pyridoxine and a 2% solution of Edamine, alactalbumin hydrolysate containing variousamino acids and peptides, which enhances con-version to the parasitic form.

Other studies have been concerned with ob-taining a chemically defined medium for spheruleproduction. Baker and Mrak (3) obtainedspherules on a solid acetate-ammonium-inorganicsalts medium containing cupric sulfate. The firstproduction of spherules in a liquid chemically de-fined medium, containing glucose, ammoniumacetate, and inorganic salts, was obtained byConverse (17). Converse (18, 19) and Converseand Besemer (20) noticed that most satisfactoryproduction is obtained from young (7- to 14-day)cultures in a medium containing 0.34% solids(total nutrient content) with optimal pH be-tween 6.0 and 8.0, incubated at 37 C.

Converse (18) pointed out that shaking has amarked effect on spherule production. He noticeda decrease in yield in shake cultures, whereasstationary incubation produces a high yield ofspherules. Many other investigators obtainedgood yields of spherules in shake cultures (38,39, 45, 46, 72). Lones and Peacock (45) andNorthey and Brooks (72) demonstrated thatshaking is essential for spherule production.

Increased temperature (between 34 and 40 C)induces spherulation (10, 17, 18, 19, 20, 46, 48,

72), but Converse and Besemer (20) stated it isnot the only factor influencing development. Thestudies by Breslau and Kubota (10) also indi-cated that temperature is a critical factor, butnot the only factor for spherulation. They foundthat, below 40 C, the hyphae remain viable andgrow, but at 40 C the hyphae degenerate (yet,the spherules survive). Whereas arthrosporesgerminate at lower temperatures, they found thatarthrospores transform directly into spherulesat 40 C, and the few viable hyphae quickly de-generate.The addition of chemical agents to basal media

enhances the conversion and maturation of thespherules. Some of these agents include pyr-idoxine (72), sodium and calcium ions (20), so-dium bicarbonate (18, 20), sulfhydryl compounds(20), various metabolic inhibitors including so-dium fluoride, sulfaguanidine, lithium chloride(20), and copper sulfate (3, 20), and hemoglobinand its derivatives such as hemin, biliverden, andbilirubin (72).A marked stimulation of spherules has been

noticed in cultures containing oleic and linoleicacids (20), Tamol N (19, 20, 45, 72), and Tween80 (16, 20, 72). Converse and Besemer (20)stated that the stimulatory action may resultfrom a change in the permeability of the spherulewall or may be due to the fact that these sub-stances serve as growth factors or as detoxifyingagents. Northey and Brooks (72) demonstratedthat Tamol and Tween 80 enhance maturation ofspherules, as reported by others (19, 20, 45), butmaturation is found to be possible without theaddition of a surface-active agent and to benearly equal to that obtained with Tamol. Con-verse (19) was able to obtain an essentially pure(hyphae-free) culture of the spherules and to per-petuate them through several serial transfers, byinoculating arthrospores into his basal mediumsupplemented with Tamol N and incubating ona shaker for 72 hr at 34 C. Spherules have beencontinuously maintained for more than 4 yearsby Breslau and Kubota (10) in a medium lackinga surface-active agent. They used a modifiedConverse medium (less NaCl) diluted 1:25, in amodified Lubarsky and Plunkett (1955) culturetube incubated at 40 C. For continuous cultures,the investigators removed old medium and re-placed it with fresh medium previously bubbledwith a mixture of 20% CO2 and 80% air.

Investigations have demonstrated the favor-able effects of metabolic or bubbled CO2 on thegrowth of the parasitic form (10, 45, 48).Lubarsky and Plunkett (48) were able to obtainspherules in tissue culture media supplementedwith various animal sera aerated periodicallywith 23% 02 and 77% CO2. Lones and Peacock

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(45) showed that Converse's medium, supple-mented with Tamol N and 10% CO2 and incu-bated on a rotary shaker at 35 C, enhancesspherule production. They noticed that enhancedspherulation is demonstrated with large inocula(2.0 mg per ml), without bubbled C02, but notwith small inocula (0.2 mg per ml) unless CO2 issupplied. They conclude that the favorable effectof the larger inoculum is due to a greater concen-tration of metabolic CO2. Breslau and Kubota(10) showed that a modified Converse mediumbubbled with 20% CO2 and incubated at 40 Cenhances spherule production. They observed that,when arthrospores are incubated without CO2,the inoculum germinates and the hyphae grow;with C02, the inoculum transforms into spher-ules. Furthermore, they observed that, whenspherules are subcultured into fresh medium with-out bubbled CO2, they germinate, and that, whena part of the old medium is removed and re-plenished with C02-bubbled fresh medium,germination is eliminated. In contrast to the pre-vious studies, Northey and Brooks (72) foundthat a medium containing increased CO2 hasneither a beneficial nor a deleterious effect. Con-verse (18), in earlier studies, stated that the in-creased yield in stationary cultures may be dueto an accumulation of CO2 in the medium, andthat the addition of sodium bicarbonate to themedium can be a substitute for shaking, with thesame results. But, in later studies by Converseand Besemer (20), a requirement for added CO2was not demonstrated. They observed optimalspherulation to occur in atmospheres containing20% 02, in the absence of added CO2. Further-more, they found that a concentration of 10%CO2 has no effect, but a CO2 concentration greaterthan 10% inhibits spherulation. According toLones and Peacock (45), the explanation thatadded CO2 is not required for spherulation may bein differences in the size and metabolic activityof the inoculum and volume of the culture.

Converse and Besemer (20) found that sulfhy-dryl inhibitors inhibit spherulation, but that theinhibition is reversed by the addition of gluta-thione. Because of this observation, they sug-gested that the disulfide-sulfhydryl metabolicprocess similar to that associated with other di-morphic fungi may be operative in C. immitis.

Converse and Besemer (20) summarized thefindings of their studies by saying that spheruleproduction may be due to one or several factorsand that the tonicity of the growth environmentand the presence of sulfhydryl groups may be themost critical factors. They emphasized that thebasic mechanism underlying conversion has notyet been elucidated.

Agents demonstrating beneficial or deleterious

effects on growth. Various agents have been foundto have a deleterious effect on the growth of C.immitis. Thiourea (22) and p-hydroxymethylbenzoic acid (108) are fungistatic to the arthro-spores of C. immitis. The addition of supple-mental lactose or galactose to a basal medium en-hances conversion of arthrospores to spherules,but not maturation, whereas glucose inhibitsmaturation (72). The addition of cysteine resultsin the formation of very large abortive spherulesthat fail to mature; methionine and thioglycolateinhibit spherulation (20). Northey and Brooks(72) found that, although the addition of thio-glycolate to the original inoculum retards con-version, addition to a 12-hr-old culture enhancesmaturation of spherules. The sulfhydryl inhibi-tors, p-chloromercuribenzoate and iodoacetate,inhibit spherulation, but the inhibition is re-versed by the addition of glutathione (20).

Vaccine preparation. With the formulation ofchemically defined media for the growth of theparasitic form, it has been possible to develop acoccidioidal endospore-spherule vaccine. Levine,Cobb, and Smith (38, 39) described the prepara-tion of such a vaccine by use of a modified Con-verse medium, containing 25% more acid phos-phate, inoculated with arthrospores, and incu-bated at 37 C for 2 to 3 days. They harvested thecrop of endospores by filtration, and used thefiltered endospores as inoculum for fresh cultures.By repeating this procedure several times, theyobtained a relatively hyphae-free endosporephase.

Metabolic properties of spherules. Lones andPeacock (46), in an examination of the metabolicproperties of washed spherules, showed thatrespiration is increased by the addition of glucoseand that most of the glucose utilized is assimi-lated. In contrast, stimulation of respiration ofthe hyphal form by added glucose is obtainedonly after starvation of the cells. The organismis capable of anaerobic metabolism, with theformation of CO2 and ethanol, but this activity,which is stimulated by starvation, is present in amuch lower degree in hyphae than in spherules.

Assimilation patterns. Ammonium salts andurea, but not nitrates, serve as the sole source ofnitrogen; glucose and acetate, but not urea orformic acid, serve as the sole source of carbon(102). Acetamide, as well as all amino acids stud-ied, serve as the sole source of both carbon andnitrogen. Baker and Smith (4) found the follow-ing utilized as the sole source of carbon: allhexoses, xylose, maltose, cellobiose, trehalose,raffinose, a-methyl glucoside, salicin, amygdalin,starch, dextrin, inulin, ethyl alcohol, glycerol,erythritol, mannitol, sorbitol, acetate, propio-nate, caproate, lactate, pyruvate, succinate,

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fumarate, malate, glycine, alanine, cystine, acet-amide, and asparagine. They observed no growthwith arabinose, sucrose, lactose, melizitose, cellu-lose, inositol, formate, butyrate, oxalate, tartrate,gluconate, citrate, or urea. The organic acidswhich do not support growth are not toxic, sincethese investigators observed that the sporesgerminate and develop until the reserve food ma-terial in the spore is exhausted. Baker and Smith(4) found the following utilized as the sole sourceof nitrogen: nitrate, in contrast to the results ofStewart and Meyer (102), ammonium chloride,acetamide, asparagine, glycine, alanine, glutamicacid, tyrosine, and cystine. They observed thatnitrite is not utilized. Lones and Peacock (47)found that the spherule form assimilates glucose(as the sole source of carbon) but not mannitol, asdoes the filamentous form. They found thatneither the spherules nor the filamentous formutilizes citrate or sorbitol. However, Baker andSmith (4) found that sorbitol is assimilated bythe filamentous form.

HistoplasmaVitamin requirements. Histoplasma capsulatum

has been grown on comparatively complex solidand liquid media containing meat infusion (84),blood (15, 84), potato flour plus egg (35), neo-peptone, and Tryptone (11, 58, 59, 85). Many ofthese and other complex media have been usedfor the conversion and maintenance of the Yform (11, 15, 35, 59). Larsh et al. (37) obtainedY-form conversion in HeLa tissue cultures con-taining human serum. Zarafonetis (109) obtainedthe Y form in Dubos medium supplemented withalbumin.

Nutritional studies have been concerned withobtaining a better understanding of the factorsinfluencing growth and dimorphism by studyingthe basal nutritional requirements. Vitamin-re-quirement studies of H. capsulatum show variableresults. The M\ form has been found to require novitamins (2, 77, 87), to require thiamine (2, 90),biotin (87), and pantothenate (83), and to bepartially deficient for biotin and niacin (90). TheY form has been found to require thiamine (77),biotin (77, 87), and thioctic acid (77).Amino acid requirements. The amino acid re-

quirements of the M form have been reported tobe complex and variable for different strains (83).However, Salvin (87) has grown the M form withammonium as the sole nitrogen source, but the Yform requires a sulfide or sulfhydryl group, suchas in cysteine. Other investigations also find thatthe Y form has a requirement for sulfhydrylgroups (75, 76, 91, 92). In addition to a require-ment for cysteine, Pine (75) found that asparticacid and glutamic acid are stimulatory for growth.

Glutathione is able to replace the -SH require-ment but not cysteine, so Pine suggests thatcysteine is not a requirement for -SH groupsalone.The simplest basal medium for the growth of

the M form is the GASM used by Salvin (87),with biotin added for biotin-requiring strains.The Y form is grown on a glucose-cysteine-inor-ganic salts medium containing biotin (87, 91,92).

Physical requirements. The Y form grows bothaerobically and anaerobically (85), but Pine(75) found both the Y and M forms to be obligateaerobes. The optimal pH for growth of the Yform is between 6.3 and 8.1 (85).

Factors influencing dimorphism. Dimorphismin Histoplasma is a function of temperature (11,59, 77, 85, 91, 92), vitamins (77, 87, 89), and sulf-hydryl groups (75, 76, 87, 91, 92). The optimaltemperature for Y-form development is 37 C(11, 59, 77, 85, 91, 92), but some strains willmaintain the Y form at 25 C (77, 91, 92). Y-formdevelopment also depends on the presence ofvitamins such as biotin (77, 87), thiamine, andthioctic acid (77). In addition to vitamins, Salvin(87) found -SH groups to be necessary forY-form development. Salvin demonstrated thatthis requirement is best fulfilled with an organicmolecule of small size, such as an amino acid. Hefound that if the molecular size is too great, asin the tripeptides, some hyphae accompany theY growth. Pine (75) stated that cysteine is re-quired to initiate growth of the Y form; the -SHgroups maintain the viability of the fungus untilgrowth commences. Pine (77) found that the Yform will grow at 25 C when cysteine is presentin the medium. He stated that, given the correctmedium, both forms might grow under ap-parently optimal and identical conditions at25 C. Hence, he believed that the Y form is notmerely a morphological expression of an increasedtemperature. He explained this by suggestingthat two different metabolic reactions occur foreach form, reactions which are competitive butnot necessarily mutually exclusive. Scherr (92)also found that the Y form is maintained at 25 Cwhen cysteine is added to the medium. He statedthat the concentration of cysteine is more criticalfor the maintenance of the Y form than the tem-perature of 37 C; the role of the temperature ap-pears to be the maintenance of optimal conditionsfor the activity of enzymes which direct M to Ytransition.

Agents demonstrating beneficial or deleteriouseffects on growth. Chemical and physical factorsare known to enhance the growth of the Y form.Pine (75) found that albumin stimulates growth.He observed that a high concentration of oleic

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acid inhibits growth, but this effect is reversed bythe addition of albumin. This is attributed to thefact that albumin is able to detoxify fatty acids(59, 75, 109). Rowley and Pine (83) found thatthe addition of KH2PO4 inhibits germination ofthe Y form, but this is reversed upon the additionof albumin or starch.Rowley and Huber (82) did not find any del-

eterious effect upon the addition of agar to theirbasal medium, but Pine (76) observed that theaddition of agar to basal medium retards growthof the Y form. He found that the addition of al-bumin or starch reverses the inhibition, and hesuggested that agar also contains a toxic sub-stance such as a fatty acid. Salvin (85) found thatno growth will occur in a liquid medium unlessthe viscosity is increased by 0.175% agar or silicagel. He attributed this to the fact that a growthsubstance is provided or a specific 02 or CO2 ten-sion is produced. McVickar (58) stated that agardetoxifies the environment and enhances Y-formgrowth. Scherr (92) believed that agar or silicagel reduces the oxidation-reduction potential tooptimal conditions for growth of the Y form.Carbon dioxide is reported to enhance growth

(11, 75), but Salvin (85) reported that CO2 ten-sion is of no importance. Without making anyaddition to the basal medium, Salvin (89) ob-tained extensive growth of the Y form when hisliquid medium was kept in constant rotation.

Studies have also been concerned with the effectof various agents on the M form. Various nonionicsurface-active agents, when incorporated intobasal medium, have been observed to enhancegrowth of the M form (53-55). Diethylthiourea(87), thiourea (22), and p-hydroxymethyl benzoicacid (108) have been reported to be fungistaticto the M form of H. capsulatum.

Assimilation patterns. Scheff (90) found thatthe M form assimilates glucose, maltose, sucrose,lactose, fumaric acid, and succinic acid, but notstarch, as the sole carbon source. He observedthat a carbohydrate is not essential, since growthtakes place in its absence, with asparagine, fu-maric acid, or succinic acid being used as thecarbon source. He found that the M form assimi-lates ammonium sulfate, aspartic acid, as-paragine, succinic acid, and fumaric acid as thesole source of nitrogen. Archibald and Reiss (1)observed that the M form can not grow withmethionine as the sole nitrogen source and givesonly poor growth with cysteine, lysine, histidine,tryptophan, phenylalanine, and p-aminobenzoicacid. Sixteen other amino acids in their investiga-tion were assimilated as the sole nitrogen source.

Salvin (87), in an extensive survey on theability of the Y form to assimilate various inor-ganic and organic nitrogen compounds as the sole

source of nitrogen, found that none of the inor-ganic compounds and only three organic com-pounds, cysteine, cystine, and glutathione, areassimilated. He also noted that the M formassimilates inorganic ammonium, but not nitrateor nitrite compounds.

SporotrichumVitamin requirements. Only a few investiga-

tions of Sporotrichum schenckii have been re-ported. Robbins and Kavanagh (80) stated thatthe M form grows in the presence of thiamine,and can also grow, but to less degree, in a liquidglucose-ammonium lactate-asparagine-inorganicsalts medium without thiamine. Other investiga-tions indicate that both the Y and M forms re-quire thiamine (2, 14, 51, 52), and that the Mform requires no vitamins (49).Amino acid requirements. Most nutritional

studies have been performed with media contain-ing complex organic nitrogen sources such as amixture of proteose-peptone, neopeptone, andTryptone (86), neopeptone plus Tryptone (85),asparagine (2, 14, 52), Casamino Acids plus cys-tine, caseinate (49), casein hydrolysate (51, 52),and individual amino acids (1). The AI form hasbeen grown on a medium containing ammoniumas the sole nitrogen source (51), but the Y formrequires organic nitrogen. In addition to cultiva-tion on agar-containing media, both the Y and Mforms have been grown in liquid media by severalinvestigators (49, 51, 52, 86).

Factors influencing dimorphism. Nutritionalstudies have been concerned with the factors re-sponsible for the conversion and maintenance ofthe Y form. Salvin (86) grew the Y form in asemisolid peptone medium and found the follow-ing conditions favoring Y-form development:optimal temperature of 37 C, 0.1 to 0.3% con-centration of agar, pH of 8.2, and 60 to 80% CO2tension. He noted that the absence of agar, anacid environment, and a high concentration ofglucose results in a conversion to the M form.Bullen (11) was able to obtain the Y form on apeptone medium incubated at 37 C in an at-mosphere of 15% CO2. He concluded that a cer-tain concentration of CO2 is essential for Y-formdevelopment. Norden (71) stated that the Yform is obtained on complex organic media byincreasing the CO2 tension. Mariat and Drouhet(51, 52) grew the Y form in a liquid glucose-caseinhydrolysate-inorganic salts medium containingthiamine and biotin. A temperature of 37 C, a pHof 7.1, and the addition of biotin to the basal me-dium were found to favor Y-form development;mechanical agitation or increased CO2 tensionwas found to be essential for Y-form growth.

Agents demonstrating beneficial or deleterious

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effects on growth. The nonionic surface-activeagents which are stimulatory for C. neoformansare also stimulatory for the M form of S. schenckii(53-55). Thiourea (22) and p-hydroxymethylbenzoic acid (108) were found to be fungistaticfor the M form of this fungus.

Assimilation patterns. Archibald and Reiss (1)found that the M form grows not at all or poorlywith aspartic acid, p-aminobenzoic acid, lysine,or histidine as the sole source of nitrogen, butgrows well with 19 other amino acids studied.

Mariat and Drouhet (51, 52) performed an ex-tensive comparison of the effect of various nitro-gen sources as the sole source of nitrogen (inor-ganic salts, casein hydrolysate, 22 individualamino acids) on growth in shake, quiescent liquid,and solid cultures. They found that, regardless ofthe nitrogen source, the M form develops inquiescent liquid and solid cultures, and developsin shake cultures in the presence of ammoniumsulfate, ammonium nitrate, aspartic acid, as-paragine, serine, and the sulfur-containing aminoacids. They found that the Y form develops inshake cultures in the presence of casein hydroly-sate, casein hydrolysate plus amino acids, andthe following individual amino acids: alanine,arginine, glutamic acid, glycine, isoleucine, leu-cine, norleucine, norvaline, ornithine, phenylala-nine, threonine, and valine. When the investiga-tors increased the CO2 tension of the culture en-vironment, the Y form developed in still or shakeliquid cultures and solid cultures. They notedthat, without increased CO2 tension, only liquidshake cultures supplied with specific amino acidsallow Y-form development. Mariat and Drouhetconcluded that the amino acids, through decar-boxylation, serve as a source of CO2 required forY-form development, in place of atmosphericC02, and that the accumulation of CO2 is prob-ably favored by shake cultures.

NUTRITION OF THE DEMATIACEOUS FUNGI

The dematiaceous fungi which have undergonenutritional studies include the fungi of chromo-blastomycosis and related dematiaceous sapro-phytes and pathogens. Five species of dematia-ceous fungi are now recognized as causing chronmo-blastomycosis. These are Phialophora verrucosa,Cladosporium carrionii, Fonsecaea pedrosi, F.compacta, and F. dermatitidis. Representatives ofdematiaceous fungi which serve as agents ofmycetoma include Phialophora jeanselmei andCladosporium gougerotii. Saprophytic dematia-ceous fungi include Cladosporium sphaero-spermum, C. elatum, Phialophora obscura, P. ri-chardsiae, and Pullularia pullulans.

Vitamin-requirement studies indicate thatthiamine is required by F. pedrosi, F. verrucosa

(2, 14), F. compacta, and P. jeanselmei (2). Aspecies identified by Area Leao and Cury (2) asCladosporium wernecki requires no vitamin forgrowth. In recent studies, Silva (99) found that aB-vitamin supplement is essential for growth ofstrains of P. richardsiae and P. obscura, stimulatesgrowth of strains of F. pedrosi, F. compacta, F.dermatitidis, C. carrionii, C. gougerotii, C. sphaero-spermum, C. elatum, P. verrucosa, and P. jean-selmei, and has no effect on the growth of strainsof P. pullulans nor on other strains of P. jean-selmei and C. gougerotti.

Silva (99) found organic nitrogen to be essen-tial for the growth of a strain of P. jeanselmei, tostimulate the growth of strains of P. verrucosaand P. obscura, and to have no effect on growth ofany other strains studied. Those strains not re-quiring vitamin supplement or organic nitrogencan be grown on a GA-SM.

Silva (99) observed that alterations in the basalmedium are able to influence the type of sporula.tion. By increasing the concentration of inor-ganic nitrogen, substituting ammonium chloridefor sodium nitrate, or adding yeast extract, shewas able to increase the pseudo-Acrotheca typeof sporulation. No substance was found that con-sistently stimulated either the Cladosporium orPhialophora type of sporulation.Montemayor (62) reported, an optimal tem-

perature of 20 to 25 C for saprophytic cladosporia,of 30 C for agents of mycetoma, and of 37 C foragents of chromoblastomycosis. Silva (98, 99) re-ported the optimal temperature in the last-named group to range between 25 and 35 C. Sheconcluded that the faster growth rate at 25 C ofsaprophytic cladosporia than that of the fungi ofchromoblastomycosis does not provide a reliablecriterion for differentiation of these two groups.Furthermore, she observed that the absence ofgrowth at 30 C is a good sign of a saprophyte,but growth at this temperature is not found to bean exclusive characteristic of pathogenic clado-sporia.

Cycloheximide is known to inhibit saprophyticfungi, but Silva (99) found that, with the excep-tion of one isolate of P. obscura, all saprophyticisolates grow well on this antibiotic. She con-cluded that cycloheximide is not suitable forseparating a saprophyte from a pathogen.The factors responsible for the conversion to

the parasitic form of the fungi of chromoblasto-mycosis are unknown. Serial streaking of fungion Francis glucose-cystine-blood-agar (97) andCystine-Heart-Hemoglobin agar (99) incubatedat 37 C induces partial conversion to the para-sitic form. Complete suppression of the filamentshas been obtained only with P. jeanselnmei and C.gougerotii (99). Partial conversion also has been

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obtained in the chick embryo chorioallantoicmembrane and in the pupae of silkworms (97).Montemayor (62) reported the following car-

bon and nitrogen assimilation patterns: the agentsof chromoblastomycosis assimilate glucose, malt-ose, sucrose, galactose, and asparagine, but notlactose, urea, ammonium sulfate, or potassiumnitrate; the agents of mycetoma assimilate glu-cose, maltose, sucrose, galactose, urea, asparagine,ammonium sulfate, and potassium nitrate,but not lactose; the saprophytic cladosporiaassimilate all the above-mentioned substrates.Silva (98) reported that the agents of chromo-blastomycosis assimilate glucose, fructose, xylose,arabinose, sucrose, maltose, and starch, but notlactose or cellulose.

NUTRITION OF ALLESCHERIA (M1ONOSPORIUM)A few studies have been performed on one of

the causative agents of maduromycosis, Alle-scheria boydii (Monosporium apiospermum). Ithas been cultured on a glucose-asparagine-inor-ganic salts medium containing biotin (2, 21,106). Villela and Cury (106) reported a pH of 4to 5 and room temperature as optimal conditions,with no growth occurring at 37 C. They noticedthat nicotinic acid and casein hydrolysate en-hance growth, that cysteine and glutathionehave no effect on growth, and that pyridoxinedepresses growth. In their study of compoundsknown to replace biotin requirements, they foundthat pimelic acid, aspartic acid, oleic acid,and sorbitan monooleate are not effective. How-ever, they found that the analogues desthiobiotinand o-heterobiotin do partially replace biotin, butdo not show an additive response when added tobiotin. Wolf (108) observed that p-hydroxy-methyl benzoic acid has a fungistatic effect onthis fungus.

CONCLUSIONSData in Table 1 show that there is wide varia-

tion in the results obtained from vitamin-re-quirement studies among different strains of thesame species. This is true in Blastomyces, Histo-plasma, Sporotrichum, and some agents of chro-moblastomycosis. The requirements of Crypto-coccus, Candida, Coccidioides, and Allescheriaappear to be well substantiated. Cryptococcusrequires thiamine, Candida and Allescheria re-quire biotin, and Coccidioides has no requirement.Few definite statements can be made concern-

ing differences between the Y and M forms withrespect to their vitamin requirements. The Yform of H. capsulatum definitely requires a Bvitamin, but the M form probably requires novitamin. The Y form of S. schenckii is stimulatedby biotin, but biotin has no effect on the M form.

Definitive requirements for these organismswill be obtained only after repeated experimenta-tion by various investigators using a large num-ber of strains. An all-purpose basal medium con-taining biotin and thiamine would probably sup-port the growth of the majority, if not all, of thestrains of pathogenic fungi which have been cul-tivated on artificial media to date.The majority of the fungi will grow on a basal

medium supplied with inorganic ammonium salts.There are some important exceptions. The Y formof H. capsulatum requires a sulfhydryl compound.The Y form of S. schenckii requires organic nitro-gen, as does the filamentous form of A. boydii andP. jeanselmei. A difference is noted between theY and M forms of dimorphic fungi in regard tonitrogen requirements, with the Y form demon-strating a more exacting requirement, requiringorganic nitrogen. In most cases where organicnitrogen is not required, it is found to be stimula-tory.At present, little is known about the nutritional

requirements of the parasitic form of the agentsof chromoblastomycosis. With this exception,synthetic basal media have been developed whichwill support the growth of all the fungi includedin this review. An all-purpose basal medium in-corporating the same nitrogen source would beof only partial value, since some organisms re-quire sulfhydryl compounds and others havedemonstrated either inhibited or retarded growthwhen -SH-containing compounds are incorpo-rated in the medium.On the basis of evidence obtained to date, di-

morphism in C. albicans is controlled throughdisulfide - sulfhydryl maintenance. Sulfhydrylmaintenance is required for Y-form development,whereas the disulfide form favors production ofthe filamentous form. Dimorphism in Blastomycesis probably a function only of the temperature ofincubation. Temperature is a critical factor con-trolling conversion in C. immitis, H. capsulatum,and S. schenckii, but other factors, including di-sulfide-sulfhydryl maintenance, CO2 tension,presence of B vitamins, and tonicity of the growthmedium, influence dimorphism in these organismsas well. Little is known concerning the factorsthat control dimorphism in the agents of chromo-blastomycosis.

Physical and chemical modifications of the en-vironment and basal medium have demonstrateda beneficial effect on growth (Table 2). Besidesalterations in the pH of the medium and tem-perature of incubation, changes in the atmos-phere of incubation produce beneficial effects.Aeration with CO2 or mechanical agitation(shake cultures) is beneficial for Blastomyces, C.immitis, H. capsulatum, and S. schenckii. Shaking

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TABLE 1. Summary of nutritional requirements of pathogenic fungi as reported in the literature

Organism Vitamin requirements Nitrogen requirements Basal medium

Candida albicans.............

Cryptococcus neoformans......

Blastomyces dermatitidis......

B. brasiliensis................

Coccidioides immitis..........

Histoplasma capsulatum......

Biotin (numer-ous) *

Thiamine (stimu-latory; numer-ous)

Pantothenate (3)*

Thiamine (nu-merous)

Yeast formNo requirement

(22)Biotin (6)

Mold formNo requirement

(7)Biotin (6)

Yeast formNo requirement

(11)Mold formNo requirement

(2)Thiamine (2)Thiamine (stimu-

latory; 1)

Parasitic formNo requirement(numerous)

Filamentous formNo requirement

(numerous)

Yeast formBiotin (9)Thiamine (8)Thiotic acid (1)Mold formNo requirements

(24)Thiamine (2)Biotin (1)Pantothenate (1)Niacin (stimula-

tory; 1)Biotin (stimula-

tory; 1)

Inorganic ammoniumsalts


Amino acids(stimulatory)

Yeast formInorganic ammonium

saltsOrganic nitrogen

(stimulatory)Mold formInorganic ammonium


(stipulatory)

Yeast formInorganic ammonium


(stimulatory)Mold formInorganic ammonium


(stimulatory)

Parasitic formAmmonium acetateFilamentous formInorganic ammonium

salts

Yeast formSulfhydryl compoundsAspartic acid andglutamic acid(stimulatory)

Mold formInorganic ammonium

salts

Glucose

Ammonium-inorganicsalts

Biotin

Glucose


Thiamine

Glucose


GlucoseAmmonium-inorganic

salts

Parasitic formGlucoseAmmonium acetateInorganic saltsFilamentous formGlucoseAmmonium-inorganic

salts

Yeast formGlucoseCysteineInorganic saltsBiotin or thiamineMold formGlucoseAmmonium-inorganic

salts

(continued)

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TABLE 1-Continued

Organism Vitamin requirements Nitrogen requirements Basal medium

Fonsecaea pedrosoi............

F. compacta.................

F. dermatitidis...............

Cladosporium carrionii.

C. gougerotii................

C. sphaerospermum...........

C. elatum....................

Phialophora jeanselmei.

P. richardsiae................

P. verrucosa.................

P. obscura...................

Pullularia pullulans..........

Yeast formThiamine (2)Biotin (stimula-

tory; 2)Mold formThiamine (4)No requirement

(1)

Mold form (allspecies)

Thiamine (2)B vitamins (stim-

ulatory; 2)Thiamine (1)B vitamins (stim-

ulatory; 2)B vitamins (stim-


ulatory; 2)No requirement

(1)B vitamins (stim-



ulatory; 1)Thiamine (3)No requirement

(3)B vitamins (stim-

ulatory; 1)B vitamins re-

quired (1)Thiamine (2)B vitamins (stim-

ulatory; 2)B vitamins re-quired (1)

No requirement(1)

Yeast formOrganic nitrogenMold formInorganic ammonium


(stimulatory)

Asparagine

Mold form (allspecies)








Organic nitrogen re-quired


Organic nitrogen(stimulatory)

Organic nitrogen(stimulatory)


* Number of strains demonstrating stated requirement.

Yeast formGlucoseCasein hydrolysateInorganic saltsThiamineMold formGlucoseAmmonium-inorganic

salts

GlucoseAsparagineInorganic saltsBiotin

Mold form (all species)

GlucoseAmmonium-inorganic

salts(B-vitamin supplementand organic nitrogenfor deficient strains)

of the medium is reported as essential for the pro-duction of spherules of C. immitis and for the de-velopment of the Y form in S. schenckii. Thebeneficial effect in the latter two fungi is due tothe increased CO2 tension. Either CO2 or 02, or

both, may stimulate growth of the other fungi. It

should be remembered that other experimentshave shown that increased CO2 tension has noeffect on the growth of H. capsulatum and mayinhibit spherulation in C. immitis. The role of02 and CO2 tension in the growth of fungi re-

quires clarification.

Sporotrichum schenckii.......

Allescheria boydii ............ Biotin (5)

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TABLE 2. Summary of factors influencing dimorphism of pathogenic fungi, and physical andchemical agents affecting growth of pathogenic fungi, as reported in the literature

Organism Factors influencing Agents demonstrating a Agents demonstrating adimorphism beneficial effect on growth deleterious effect on growth

Candida albicans.............

Cryptococcus neoformans......

Blastomyces dermatitidis......I...

B. brasiliensis................

Coccidioides immitis..........

Disulfide-sulfhy-dryl main-tenance

TemperatureCO2 tension

Temperature

TemperatureTonicity of mediaSulfhydryl com-pounds

Yeast formFermentable carbo-hydrate

Presence of -SHcompounds

Increased 02 tension

Filamentous formAbsence of -SH com-pounds

Decreased 02 tensionAssimilable carbohy-

dratesPolysaccharidesPhosphorusPotassiumBiotinStarvation mediaLiquid media

Nonionic surface-activeagents

Yeast formSemisolid media (agar)Tween 80Aeration (shake cul-

tures)

Mold formNonionic surface-

active agents

Yeast formAeration (shake cul-

tures)

Parasitic formPyridoxineSodium, calcium ionsBicarbonateSulfhydryl compoundsMetabolic inhibitorsHemoglobinOleic, linoleic acidsTween 80, Tamol NMetabolic or bubbledC02

p-Hydroxymethylbenzoic acid

ThioureaHydrogen sulfideSodium acetateAscorbic acid


Thiourea

Yeast formTween 80Oxaloacetic acidSulfur-containingcompounds

Tyrosine

Mold formp-Hydroxymethylbenzoic acid

Yeast formTween 80Sulfur-containingcompounds

Mold formp-Aminobenzoic acid

Parasitic formSupplemental glucoseSulfur-containingcompounds

Shake cultures (aera-tion)Sulfhydryl inhibitors

Filamentous formp-Hydroxymethylbenzoic acid

Thiourea

(continued)

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TABLE 2.-Continued

Factors influencing Agents demonstrating a Agents demonstrating aOrganism dimorphism beneficial effect on growth deleterious effect on growth

TemperatureB vitaminsSulfhydryl com-pounds

TemperatureCO2 tension

Cladosporium species.........IUnknown

Yeast formAlbuminStarchAgar (semisolid media)Silica gelCO2 tensionAeration (shake cul-

tures)


active agents

Yeast formBiotinShake cultures (aera-

tion)Amino acidsCO2 tensionAgar (semisolid media)pH


active agents

Nicotinic acidCasein hydrolysate

Parasitic formCystine heart-hemo-globin agar

Francis cystine-blood-agar

Yeast formPotassium dihydrogesphosphate

Oleic acidAgar


ThioureaDiethylthiourea


Thiourea


Pyridoxine

Difficulty has been encountered in obtainingY-form growth in liquid media, but good growthis obtained as long as the basal requirementsare fulfilled. Physical factors will enhance thegrowth. The addition of agar or silica gel to a

semisolid medium is favorable to Y-form growthof H. capsulatum and S. schenckii. Luxuriantgrowth of Blastomyces is obtained in a basalliquid medium incubated with mechanical agita-tion. Mechanical agitation or increased CO2 ten-sion enhance Y-form development in S. schenckiiand H. capsulatum in liquid media.

Various surface-active agents such as Tween80 and Tamol N, and fatty acids such as oleicacid and linoleic acid, enhance growth. This hasbeen attributed to the ability of these agents toallow the nutrient material of the culture mediumto come in more intimate contact with the cell, toincrease the permeability of the cell, and to facili-tate faster utilization of the nutrients, with con-

sequent stimulation of growth. They have alsobeen described as serving as detoxifying agents.The role of albumin, starch, and agar in enhanc-ing growth is attributed to the fact that theseagents also detoxify the environment. But itshould be emphasized that there are instanceswhere Tween 80, agar, and oleic acid demonstratedeleterious effects on growth.

Table 2 shows that the majority of agentsdemonstrating a deleterious effect on growth are

reducing agents or sulfur-containing compounds.The inhibitory effect of the reducing agents maybe brought about by the lowering of the oxida-tion-reduction potential to the point wheregrowth is retarded or cannot be initiated. Thiswould explain the action of cysteine, thioglyco-late, glutathione, and ascorbic acid. The sulfurradical as found in methionine, cystine, and thio-urea may block the metabolism of the organismby inactivating enzyme systems through oxida-

Histoplasma capsulatum.

Sporotrichum schenckii.......

Allescheria boydii............I

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tion of sulfhydryl-active sites or by functioningas a chelating agent.

Assimilation patterns at present are too sketchyto make many conclusions. With respect to car-bon assimilation, these fungi are capable of as-similating all of the hexoses. Lactose is usuallynot available, whereas the assimilation of otherdisaccharides, namely, maltose, sucrose, tre-halose, and cellobiose, is variable. Variable re-sults are also noticed with regard to other oligo-saccharides, the pentoses, and the polyvalentalcohols. Carbohydrate derivatives assimilatedby one or more species include gluconic acidlactone, glyceraldehyde, pyruvic acid, and lacticacid. Krebs cycle intermediates assimilated in-clude a-ketoglutaric acid, succinic acid, fumaricacid, and oxaloacetic acid. Various amino acids,amino acid derivatives, and fatty acids are alsocapable of being used as the sole carbon source.

Inorganic nitrogen compounds assimilated asthe sole source of nitrogen include only am-monium salts. Nitrates, nitrites, and hydroxyl-amine are not available. Many of the amino acidsand amino acid derivatives are assimilated. Someof the more complex organic compounds assimi-lated as sole sources of nitrogen include citrulline,ornithine, asparagine, glutamine, glucosamine,glutathione, glycylglycine, f3-alanine, urea, andacetamide.The nutritional behavior of ascosporogenous

yeasts and the dermatophytes is used in theirclassification. Eventually, it may be possible toclassify systemic and subcutaneous fungi accord-ing to their nutritional characteristics. Beforethis is possible, definitive vitamin requirementsand more complete assimilation patterns must beobtained.

LITERATURE CITED

1. ARCHIBALD, R. M., AND F. REISS. 1950. Somebiochemical implications from a study ofgrowth of pathogenic fungi on media con-taining single amino acids. Ann. N.Y.Acad. Sci. 50:1388-1404.

2. ARtA LEiO, A. E. DE, AND A. CURY. 1950.Deficiencias vitaminicas de cogumelos pa-togenicos. Mycopathol. Mycol. Appl.5:65-90.

3. BAKER, E. E., AND E. M. MRAK. 1941.Spherule formation in culture by Coc-cidioides immitis (Rixford and Gilchrist,1896). Am. J. Trop. Med. Hyg. 21:589-596.

4. BAKER, E. E., AND C. E. SMITH. 1942. Utiliza-tion of carbon and nitrogen compounds byCoccidioides immitis (Rixford and Gil-christ). J. Infect. Diseases 70:51-53.

5. BENHAM, R. W. 1955. The genus Cryptococcus:The present status and criteria for theidentification of species. Trans. N.Y. Acad.Sci. 17:418-429.

6. BENHAM, R. W. 1956. The genus Cryptococcus.Bacteriol. Rev. 20:189-202.

7. BERGEIM, O., A. H. HANSZEN, L. PINCUSSEN,AND E. WEISS. 1941. Relation of volatilefatty acids and hydrogen sulfide to theintestinal flora. J. Infect. Diseases 69:155-166.

8. BIOCCA, E., AND C. S. LACAZ. 1945. Acdo"in vitro" do Acido para-amino-benz6icosdbre o Paracoccidioides brasiliensis e oActinomyces brasiliensis. Arquiv. Biol.(Sao Paulo) 29:151-152.

9. BONA, J. J., AND L. R. HEDRICK. 1952. Vita-min requirements for members of the yeastgenus Candida. Bacteriol. Proc., p. 135.

10. BRESLAU, A. M., AND M. Y. KUBOTA. 1964.Continuous in vitro cultivation of spher-ules of Coccidioides immitis. J. Bacteriol.87:468-472.

11. BULLEN, J. J. 1949. The yeast-like form ofCryptococcus farciminosus (Rivolta): (His-toplasma farciminosum). J. Pathol. Bac-teriol. 61:117-120.

12. BURKE, R. C. 1951. In vitro cultivation of theparasitic phase of Coccidioides immitis.Proc. Soc. Exptl. Biol. Med. 76:332-335.

13. BURKHOLDER, P. R. 1943. Vitamin deficien-cies in yeasts. Am. J. Botany 30:206-211.

14. BURKHOLDER, P. R., AND D. MOYER. 1943.Vitamin deficiencies of fifty yeasts andmolds. Bull. Torrey Botan. Club 70:372-377.

15. CAMPBELL, C. C. 1947. Reverting Histo-plasma capsulatum to the yeast phase. J.Bacteriol. 54:263-264.

16. CONANT, N. F., AND R. A. VOGEL. 1954.The parasitic growth phase of Coccidioidesimmitis in culture. Mycologia 46:157-160.

17. CONVERSE, J. L. 1955. Growth of sperules ofCoccidioides immitis in a chemically de-fined medium. Proc. Soc. Exptl. Biol.Med. 90:709-711.

18. CONVERSE, J. L. 1956. Effect of physico-chemical environment on spherulation ofCoccidioides immitis in a chemically de-fined medium. J. Bacteriol. 72:784-792.

19. CONVERSE, J. L. 1957. Effect of surface activeagents on endosporulation of Coccidioidesimmitis in a chemically defined medium.J. Bacteriol. 74:106-107.

20. CONVERSE, J. L., AND A. R. BESEMER. 1959.Nutrition of the parasitic phase of Coc-cidioides immitis in a chemically definedliquid medium. J. Bacteriol. 78:231-239.

21. CURY, A. 1951. Biotin-an essential growthfactor for the filamentous pathogenic fun-gus Allescheria boydii Shear. Mycopathol.Mycol. Appl. 5:125-127.

22. DANOWSKI, T. S., AND M. TAGER. 1948.Thiourea and the inhibition of growth offungi. J. Infect. Diseases 82:119-125.

23. DROUHET, E., AND M. M. COUTEAU. 1954.Sur la determination des Candida. Etudedes caractbres morphologiques et physio-

421VOL. 29, 1965


mbr.asm

.org/D

ownloaded from


BACTERIOL. REV.

logiques de 78 souches isol6es de preleve-ments pathologiques. Ann. Inst. Pasteur86 :602-617.

24. DROUHET, E., AND M. M. VIEU. 1957. Fac-teurs vitaminiques de croissance desCandida. Ann. Inst. Pasteur 92:825-831.

25. FALCONE, G., AND W. J. NICKERSON. 1959.Enzymatic reactions involved in cellulardivision of microorganisms, p. 65-70. InW. J. Nickerson [ed.], Biochemistry of mor-phogenesis. Pergamon Press, London.

26. FIRESTONE, B. Y., AND S. A. KoSER. 1960.Growth promoting effect of some biotinanalogues for Candida albicans. J. Bac-teriol. 79:674-676.

27. FOLEY, G. E., AND L. L. UZMAN. 1952. Studieson the biology of Cryptococcus. J. Infect.Diseases 90:38-43.

28. GILARDI, G. L., AND N. C. LAFFER. 1962.Nutritional studies of the yeast phase ofBlastomyces dermatitidis and B. brasilien-sis. J. Bacteriol. 83:219-227.

29. HALLIDAY, W. J., AND E. McCoy. 1955. Bio-tin as a growth requirement for Blastomycesdermatitidis. J. Bacteriol. 70:464-468.

30. JOHNSON, S. A. M., M. G. GUZMON, AND C. T.AGURLERA. 1954. Candida (Monilia) albi-cans. Effect of amino acids, glucose, pH,chlortetracycline (aureomycin), dibasicsodium and calcium phosphates, and anae-robic and aerobic conditions on its growth.Arch. Dermatol. 70:49-60.

31. KAO, C. J., AND J. SCHWARZ. 1957. The isola-tion of Cryptococcus neoformans frompigeon nests. Am. J. Clin. Pathol. 27:652-663.

32. KAPICA, L., AND F. BLANK. 1957. Growth ofCandida albicans on keratin as sole sourceof nitrogen. Dermatologica (Basel) 115:81-105.

33. KARNAKY, K. J. 1946. Hydrogen ion concen-tration (pH) of Monilia albicans infectionand treatment. Southern Med. J. 39:731-734.

34. KOSER, S. A., E. HODGES, I. TRIBBY, AND J.T. STUEDELL. 1960. Growth of lactobacilliin association with Candida albicans. J.Infect. Diseases 106:60-68.

35. KURUNG, J. M., AND D. YEGIAN. 1954. Me-dium for maintenance and conversion ofHistoplasma capsulatum to yeastlike phase.Am. J. Clin. Pathol. 24:505-508.

36. LACK, A. R. 1938. Spherule formation andendosporulation of the fungus Coccidioidesin vitro. Proc. Soc. Exptl. Biol. Med. 38:907-909.

37. LARSH, H. W., A. HINTON, AND S. L. SILBERG.1956. Conversion and maintenance of His-toplasma capsulatum in tissue culture. Proc.Soc. Exptl. Biol. Med. 93:612-615.

38. LEVINE, H. B., J. M. COBB, AND C. E. SMITH.1960. Immunity to coccidioidiomycosis in-duced in mice by purified spherule, arthro-

spore, and mycelial vaccines. Trans. N.Y.Acad. Sci. 22:436-449.

39. LEVINE, H. B., J. M. COBB, AND C. E. SMITH.1961. Immunogenicity of spherule-endo-spore vaccines of Coccidioides immitisfor mice. J. Immunol. 87:218-227.

40. LEVINE, S., AND Z. J. ORDAL. 1946. Factorsinfluencing the morphology of Blastomycesdermatitidis. J. Bacteriol. 52:687-694.

41. LITTMAN, M. L. 1958. Capsule synthesis byCryptococcus neoformans. Trans. N.Y.Acad. Sci. 20:623-648.

42. LITTMAN, M. L., AND T. MIWATANI. 1963.Effect of water soluble vitamins and theiranalogues on the growth of Candida albi-cans. I. Biotin, pyridoxamine, pyridoxine,and fluorinated pyrimidines. Mycopathol.Mycol. Appl. 21:82-108.

43. LOCHHEAD, A. G., AND G. B. LANDERKIN.1942. Growth factor requirements for os-mophilic yeasts. J. Bacteriol. 43:18.

44. LODDER, J., AND N. J. W. KREGER-VAN RIJ.1952. The yeasts. A taxonomic study.Interscience Publishers, Inc., New York.

45. LONES, G. W., AND C. L. PEACOCK. 1960.Role of carbon dioxide in the dimorphismof Coccidioides immitis. J. Bacteriol.79:308-309.

46. LONES, G. W., AND C. L. PEACOCK. 1960.Studies of the growth and metabolism ofCoccidioides immitis. Ann. N.Y. Acad.Sci. 89:102-108.

47. LONES, G. W., AND C. PEACOCK. 1964. Metab-olism of mannitol by Coccidioides immitis.J. Bacteriol. 87:1114-1117.

48. LUBARSKY, R., AND 0. A. PLUNKETT. 1955.In vitro production of the spherule phaseof Coccidioides immitis. J. Bacteriol. 70:182-186.

49. LURIE, H. I. 1950. Pathogenic sporotricha;their carbohydrate reactions. Mycologia42:624-641.

50. MANKIEWICZ, E. 1954. Mycobacterium tuber-culosis and Candida albicans: A study ofgrowth-promoting factors. Can. J. Micro-biol. 1:85-89.

51. MARIAT, F., AND E. DROUHET. 1952. Etudedes facteurs determinant le developpe-ment de la phase levure de Sporotrichumschenckii. Ann. Inst. Pasteur 83:506-515.

52. MARIAT, F., AND E. DROUHET. 1953. Actionde la biotine sur la croissance de Sporo-trichum schenckii. Ann. Inst. Pasteur 84:659-662.

53. MARWIN, R. M. 1956. Surface-active cultureadditives for growth of pathogenic. humanfungi. Bacteriol. Proc., p. 55.

54. MARWIN, R. M. 1959. Survey of surface-active culture additives for growth ofpathogenic human fungi. Mycologia 51:61-68.

55. MARWIN, R. M. 1962. Stimulatory effect ofselected surface-active agents on the

422 GILARDI


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.org/D

ownloaded from



growth of pathogenic human fungi. Myco-pathol. Mycol. Appl. 18:259-263.

56. MCCLARY, D. 0. 1952. Factors affecting themorphology of Candida albicans. Ann..Missouri Botan. Garden 39:137-164.

57. MCATEIGH, I., AND E. BELL. 1951. The aminoacid and vitamin requirements of Candidaalbicans Y475 and Mycoderma vini Y-939.Bull. Torrey Botan. Club 78:134-144.

58. MCVICKAR, D. L. 1948. The enhanced growthof Histoplasma capsulatum in peptonemedia detoxified by agar, plasma, andcharcoal. Bacteriol. Proc., p. 65-66.

59. MCA\ICKAR, D. L. 1951. Factors importantfor the growth of Histoplasma capsulatumin the yeast cell phase on peptone media.I. Blood and blood derivatives. J. Bac-teriol. 62:137-143.

60. MIYASHITA, S., T. MIWATANI, AND T. Fu-JINO. 1958. Studies on the nutrition ofCandida. I. Vitamin requirements of strainof Candida. Biken's J. 1:45-49.

61. MIYASHITA, S., T. MIWATANI, AND T. Fu-JINO. 1958. Studies on the nutrition ofCandida. II. Effect of amino acids on thegrowth of Candida albicans. Biken's J. 1:50-60.

62. MONTEMAYOR, L. DE. 1949. Estudio de laspropiedades biologicas de varias cepas dehongos pat6genos causantes de la cromo-micosis, y de especias vecinas saprofitas ypat6genas. Mycopathol. Mycol. Appl.4:379-383.

63. MORRIS, E. 0. 1958. Yeast growth, p. 251-321. In A. H. Cook [ed.], The chemistryand biology of yeasts. Academic Press,Inc., New York.

64. NICKERSON, W. J. 1948. Enzymatic controlof cell division in microorganisms. Nature162:241-245.

65. NICKERSON, W. J. 1963. Symposium on bio-chemical bases of morphogenesis in fungi.IV. Molecular bases of form in yeasts. Bac-teriol. Rev. 27:305-324.

66. NICKERSON, W. J., AND G. A. EDWARDS.1949. Studies on the physiological bases ofmorphogenesis in fungi. I. The respiratorymetabolism of dimorphic pathogenic fungi.J. Gen. Physiol. 33:41-55.

67. NICKERSON, W. J., AND Z. MANKOWSKI.1953. Role of nutrition in the maintenanceof the yeast-shape in Candida. Am. J.Botany 40:584-592.

68. NICKERSON, W. J., AND Z. MANKOWSKI.1953. A polysaccharide medium of knowncomposition favoring chlamydospore for-mation in Candida albicans. J. Infect.Diseases 92:20-25.

69. NICKERSON, W. J., W. A. TABER, AND G.FALCONE. 1956. Physiological bases of mor-phogenesis in fungi. 5. Effect of selefliteand tellurite on cellular division of yeast-like fungi. Can. J. Microbiol. 2:575-584.

70. NICKERSON, W. J., AND N. J. W. KREGER-VAN Rij. 1949. The effect of sulfhydrylcompounds, penicillin, and cobalt on thecell division mechanism of yeasts. Bio-chim. Biophys. Acta 3:461-475.

71. NORDEN, A. 1951. Sporotrichosis; clinicaland laboratory features and a serologicstudy in experimental animals and humans.Acta Pathol. Microbiol. Scand. Suppl.89:3-119.

72. NORTHEY, W. T., AND L. D. BROOKS. 1962.Studies on Coccidioides immitis. I. A sim-ple medium for in vitro spherulation. J.Bacteriol. 84:742-746.

73. PECK, S. M., AND H. ROSENFELD. 1938. Theeffects of hydrogen ion concentration,fatty acids, and vitamin C on the growthof fungi. J. Invest. Dermatol. 1:237-265.

74. PECK, S. M., H. ROSENFELD, AND W. BIER-MAN. 1939. Role of sweat as a fungicide.Arch. Dermatol. 39:126-148.

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

76. PINE, L. 1955. Studies on the growth of Histo-plasma capsulatum. II. Growth of the yeastphase on agar media. J. Bacteriol. 70:375-381.

77. PINE, L. 1957. Studies on the growth of Histo-plasma capsulatum. III. Effect of thiamineand other vitamins on the growth of theyeast and mycelial phases of Histoplasmacapsulatum. J. Bacteriol. 74:239-245.

78. REID, J. D. 1949. The influence of the vitaminB complex on the growth of Torulopsis(Cryptococcus) neoformans on a syntheticmedium. J. Bacteriol. 58:777-782.

79. REID, J. D., M. M. JONES, AND E. B. CAR-TER. 1953. A simple, clear medium fordemonstration of chlamydospores of Can-dida albicans. Am. J. Clin. Pathol. 23:938-941.

80. ROBBINS, W. J., AND N'. KAVANAGH. 1942.Vitamin deficiencies of the filamentousfungi. Botan. Rev. 8:411-471.

81. ROESSLER, W. G., E. J. HERBST, W. G.MCCULLOUGH, R. C. MILLS, AND C. R.BREWER. 1946. Studies with Coccidioidesimmitis. I. Submerged growth in liquidmediums. J. Infect. Diseases 79:12-22.

82. ROWLEY, D. A., AND M. HUBER. 1955. Patho-genesis of experimental histoplasmosis inmice. I. Measurement of infecting dosagesof the yeast phase of Histoplasma capsu-latum. J. Infect. Diseases 96:174-183.

83. ROWLEY, D. A., AND L. PINE. 1955. Somenutritional factors influencing growth ofyeast cells of Histoplasma capsulatum tomycelial colonies. J. Bacteriol. 69:695-700.

84. SALVIN, S. B. 1947. Cultural studies of the

VOL. 29, 1965 423


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.org/D

ownloaded from


BACTERIOL. REV.

yeastlike phase of Histoplasma capsulatumDarling. J. Bacteriol. 54:30.

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

86. SALVIN, S. B. 1947. Multiple budding inSporotrichum schenckii Matruchot. J.Invest. Dermatol. 9:315-320.

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

88. SALVIN, S. B. 1949. Phase-determining factorsin Blastomyces dermatitidis. Mycologia41:311-319.

89. SALVIN, S. B. 1950. Growth of the yeastlikephase of Histoplasma capsulatum in a fluidmedium. J. Bacteriol. 59:312-313.

90. SCHEFF, G. J. 1945. Biochemical and immuno-logical properties of Histoplasma capsula-tum No. 650. Yale J. Biol. Med. 18:41-54.

91. SCHERR, G. H. 1956. The influence of tem-perature and -SH groups on the growthof dimorphic pathogenic fungi. Bacteriol.Proc., p. 85.

92. SCHERR, G. H. 1957. Studies on the dimor-phism of Histoplasma capsulatum. I. Theroles of -SH groups and incubation tem-perature. Exptl. Cell Res. 12:92-102.

93. SCHERR, G. H., AND R. H. WEAVER. 1953.The dimorphism phenomenon in yeast.Bacteriol. Rev. 17:51-85.

94. SCHMIDT, E. G., J. A. ALVAREZ-DE CHOU-DENS, N. F. McELVAIN, J. BEARDSLEY,AND S. A. A. TAWAB. 1950. A microbio-logical study of Cryptococcus neoformans.Arch. Biochem. Biophys. 26:15-24.

95. SCHOPFER, W. H. 1944. Les facteurs de crois-sance pour quelques espbces de Candida etde Torulopsis. Compt. Rend. Soc. Phys.Hist. Nat. Geneva 61:147-152.

96. SEELIGER, H. P. R. 1955. Ein Neves Mediumzur Pseudomycelbildung von Candidaalbicans. Z. Hyg. Infektionskrankh. 141:488-491.

97. SILVA, M. 1957. The parasitic phase of thefungi of chromoblastomycosis: develop-ment of sclerotic cells in vitro and in vivo.Mycologia 49:318-331.

98. SILVA, M. 1958. The saprophytic phase of thefungi of chromoblastomycosis: effect ofnutrients and temperature upon growthand morphology. Trans. N.Y. Acad. Sci.21:46-57.

99. SILVA, M. 1960. Growth characteristics of thefungi of chromoblastomycosis. Ann. N.Y.Acad. Sci. 89:17-29.

100. SKINNER, C. E. 1947. The yeastlike fungi:Candida and Brettanomyces. Bacteriol.Rev. 11:227-274.

101. SKINNER, C. E., AND D. W. FLETCHER. 1960.A review of the genus Candida. Bacteriol.Rev. 24:397-416.

102. STEWART, R. A., AND K. F. MEYER. 1938.Studies in the metabolism of Coccidioidesimmitis (Stiles). J. Infect. Diseases 63:196-205.

103. STUDT, B. 1941. Morphological and physio-logical studies of a strain of Candida albi-cans associated with vaginitis. Am. J.Botany 28:509-515.

104. TAGER, M., H. B. HALES, AND T. S. DANOW-SKI. 1949. Studies on the suppression offungus growth by thiourea. III. The ef-fects of protein fractions, amino acids, andthiol compounds. J. Infect. Diseases 84:284-289.

105. UZMAN, L. L., H. ROSEN, AND G. E. FOLEY.1956. Studies on the biology of Cryptococ-cus. VI. Amino acid composition of thesomatic proteins of Cryptococcus neofor-mans. J. Infect. Diseases 98:208-210.

106. VILLELA, G. G., AND A. CURY. 1950. Studieson the vitamin nutrition of Allescheriaboydii Shear. J. Bacteriol 59:1-11.

107. WARD, J. M. 1958. Biochemical systems in-volved in differentiation of the fungi.Proc. Intern. Congr. Biochem., 4th,Vienna 6:33-64.

108. WOLF, F. T. 1950. Inhibition of pathogenicfungi in vitro by para-hydroxymethylbenzoate. Mycopathol. Mycol. Appl. 5:117-119.

109. ZARAFONETIS, C. J. D. 1952. Dubos' mediumwith albumin for yeast phase growth ofHistoplasma capsulatum. Am. J. Clin.Pathol. 22:911-912.

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