JOURNAL OF BACTERIOLOGY, Dec., 1966Copyright @ 1966 American Society for Microbiology

Ultraviolet Microscopy of Candida albicansEDWARD BALISH AND GEORGE SVIHLA

Division ofBiological and Medical Research, Argonne National Laboratory, Argonne, Illinois

Received for publication 25 July 1966

ABSTRACTBALISH, EDWARD (Argonne National Laboratory, Argonne, Ill.), AND GEORGE

SVIHLA. Ultraviolet microscopy of Candida albicans. J. Bacteriol. 92:1812-1820.1966.-Yeast and mycelial strains of Candida albicans were grown in medium sup-

plemented with sulfur amino acids in an effort to determine factors that control themorphology and pathogenicity of the organism. Ultraviolet microscopy revealeda greater concentration of S-adenosylmethionine in the vacuoles of the mycelialphase than in those of yeast phases. Supplementation with amino acids greatly in-creased the concentration of S-adenosylmethionine in the mycelial phase, and madethese cells more sensitive to the lytic action of snail gut enzymes than two yeast phasestrains. This indicates a difference in cell wall structure that may be related to thepathogenicity of the mycelial phase.

Ultraviolet microscopy has been used to showthat Candida utilis and Saccharomyces cerevisiaecan accumulate S-adenosylmethionine in theirvacuoles, when the yeasts are cultured in a

medium supplemented with methionine and re-lated amino acid (18, 19).The S-adenosylmethionine, which is stored in

the vacuole, is not readily catabolized upon pro-longed incubation in the absence of any sulfursupplementation. However, the sulfonium com-

pound can be transferred from the vacuole of themother cell into the vacuoles of buds (19). Itshould also be noted that growth in the presenceof methionine appears to alter the cell walls ofyeasts. Svihla, Schlenk, and Dainko (20) haveshown that the cell walls of yeast, cultured withmethionine supplementation, are more susceptibleto the action of snail gut enzyme than yeasts cul-tured without added methionine.Depending on the culture medium employed,

pathogenic cultures of C. albicans can grow eitheras budding yeastlike organisms, or in a myce-lial phase with blastospores and thick-walledchlamydospores. The capacity to transform froma yeastlike to a mycelial form of growth is thoughtto be an important factor in the pathogenicity ofC. albicans (1, 2, 8, 9, 22). Although other factorsare involved, it is evident that the sulfur aminoacids, methionine and cysteine, play a role incontrolling the cellular form of C. albicans (6, 11,21).The purpose of this investigation was to ascer-

tain whether growth in a medium supplementedwith methionine, methylmethionine, and homo-

cysteine had any influence on the morphology ofyeast and mycelial strains of C. albicans. Becauseof its existence in both cellular forms, C. albicansoffered a unique opportunity to look for differencesin the capacity to produce and accumulate S-adenosylmethionine, and an opportunity to in-vestigate cell wall structure changes caused by thesulfur amino acids.

MATERIALS AND METHODS

Organisms anid culture medium. We used threestrains of C. albicanis, ATCC 10231, 10259, and 10261.Strain ATCC 10259 is a filamentous mutant derivedfrom 10261 by MacKinnon (10). Stock cultures of allthree strains were maintained on Sabouraud DextroseAgar (Difco) slants at 23 C. Cultures were transferredto new slants every 2 weeks. Large batches of cellswere cultured in a medium which contained the fol-lowingper liter: KH2PO4, 10g; K2HPO4,5 g; (NH4)2SO4,2 g; trisodium citrate, 1.0 g; MgCl2-6H20, 0.1 g;MnSO4-7H20, 0.1 g; CaCl2, 0.1 g; ZnSO4.7H20, 0.1 g;biotin, 5,ug; glucose (sterilized separately), 15 g. Me-thionine, methylmethionine, and homocysteine (con-centration, 5 ,umoles/ml) were added to the abovemedium to stimulate the production of S-adenosyl-methionine (19). A medium containing the latter sup-plements will be referred to below as "supplementedmedium." L-Homocysteine was prepared from L-

homocysteine thiolactone by dissolving the lactonein 0.3 M NaOH for 7 min; an equal volume of 0.3 M

KH2PO4 was then added for neutralization.Culture of organisms. Cells grown for experimental

use were first cultured on slants of Sabouraud Dex-trose Agar for 18 hr at 30 C. From the slants, the cul-tures were transferred to 125-ml Erlenmeyer flasksthat contained 20 ml of the medium described above.

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The flask cultures were then incubated on a rotaryshaker for 24 hr at 30 C. At the end of this period, thecells were concentrated by centrifugation and placedin 100 ml of fresh medium (with or without organicsulfur supplements) in 500-ml flasks, culture wasthen continued for 48 hr at 30 C with agitation.

Assay of S-adenosylmethionine. The isolation andassay of S-adenosylmethionine was accomplished byextraction of the cells with cold 1.5 N perchloric acid(PCA) and by subsequent chromatography on Dowex50, H+ resin, as described by Schlenk, Dainko, andStanford (15).The 6 N H2SO4 eluates from the Dowex resin

columns showed an ultraviolet (UV) absorption spec-trum which was the same as that given by referenceS-adenosylmethionine (16). The absorption peak ob-served with the UV-absorbing material in the 6 NH2SO4 eluate shifted from 256 to 260 mit when theeluate was made alkaline with NaOH. This shift istypical of S-adenosylmethionine and is correlated withthe formation of adenine, ribose, and methionine fromS-adenosylmethionine under alkaline conditions (13).

UV-absorbing material was precipitated from 6 NH2SO4 eluate with phosphotungstic acid (20%) andpurified according to the method of Schlenk et al. (15).The purified UV-absorbing material was chromato-graphed on paper along with reference S-adenosyl-methionine. Butyl alcohol-acetic acid-water (60:25:15, v/v) and ethyl alcohol-acetic acid-water (64:1:35,v/v) were the solvent systems employed. The RF val-ues of the purified UV-absorbing material and refer-ence S-adenosylmethionine were the same in both sol-vent systems. Both substances migrated to the samespot when subjected to high-voltage electrophoresis for2 hr at 2,500 v on Whatman 2 mm filter paper in aformic acid-acetic acid-water buffer (6:24:170, v/v)ofpH 2. Heating the purified material in 0.1 N NaOHresulted in the formation of adenine and methionine.Heating under near neutral conditions resulted in theformation of methylthioadenosine and homoserine.The breakdown products of S-adenosylmethionineunder alkaline and neutral conditions have been de-scribed by Parks and Schlenk (13). The breakdownproducts were identified by the use of paper chroma-tography and electrophoresis with known compounds.It was concluded that the compound isolated fromC. albicans was S-adenosylmethione.

Preparation of spheroplasts. The digestive juice ob-tained from the gut of the snail Helix pomatia wasused as a source of enzymes to digest the cell walls ofthe three strains of C. albicans [Suc d'Helix pomatia,Industrie Biologique Frangaise, Gennevilliers (Seine),France]. When mannitol was used as an osmoticstabilizer, the juice was diluted with 2 volumes of 1 Mmannitol. When KCI was employed as an osmoticstabilizer, distilled water was used to dilute the diges-tive juice. For conversion of cells to spheroplasts, 1 mlof the diluted enzyme (cleared of debris by centrifuga-tion) was added to 4 ml of a 5% suspension of cellsin the desired concentration of osmotic stabilizer(1 M mannitol, and dilutions of KCl that resulted infinal concentrations of 0.4, 0.5, 0.6, 0.65, 0.75, and0.9 M). Digestion was allowed to proceed at 23 C with

occasional agitation. Progress of the digestion was ob-served by phase and UV microscopy.

Treatment of C. albicans with ribonuclease. Theribonuclease preparation was of commercial origin(Armour Pharmaceutical Co., Chicago, Ill.). The pro-cedures outlined by Schlenk and Dainko (14) wereused to determine the effects of ribonuclease on C.albicans.UV microscopy. Fresh preparations of cells, which

had been washed with and mounted in distilled water,were photographed at 295 and 265 mjs. Spheroplastswere examined in isosmotic medium. The quartz coverslips were blotted with sufficient pressure to insurethat cells would remain inactive during the 3-sec expo-sure at 295 m,u and the 7-sec exposure at 265 m,u.Melted paraffin was used to seal the cover slips to thequartz slides.The equipment consisted of monochromator,

microscope, and 35-mm camera. A small gratingmonochromator (Bausch & Lomb, Inc., 250 mm) witha high-intensity mercury arc of the U-shaped Hanoviatype and an achromatic condensing lens furnishedmonochromatic energy. To facilitate rapid selectionof pre-set wavelengths, the monochromator wasequipped with a servosystem. A touch of a switchyielded a choice of four wavelengths; 546 mM, forfocusing; 295 mMA, a region of little absorption, butuseful to indicate nonspecific absorption; 275 my, aregion of some specific absorption, useful when ab-sorption at 265 mA is too great; and 265 m,, a regionof high UV absorption by several compounds ofinterest. Other wavelengths may be easily preselectedby setting potentiometers to the desired wavelengths.The use of spectral lines permitted the monochromatorslits to be opened to the width of the lines to obtainmaximal energy with good spectral purity.

Zeiss Ultrafluar optics (objective 100X/1.25 na,condenser, and projective KIO: 1) were used for imageformation. Images were recorded on Kodak SpectrumAnalysis No. 1 film in a 35-mm camera (Leica, withnonscratching Leica cassettes) equipped with a reflexfocusing assembly and bellows. Exposure and filmprocessing were adjusted to the energy available ateach wavelength to give negatives with a backgrounddensity of about 1.0, and gamma of 0.9.

RESULTSUltraviolet-absorbing material, demonstrated

to be S-adenosylmethionine, was found in thethree strains of C. albicans studied (Table 1). Cellsgrown in medium supplemented with homo-cysteine, methionine, and methylmethionine con-tained much more of the sulfonium compoundthan those grown in nonsupplemented medium.Of unusual interest was the amount of adenosyl-methionine found in strain 10259 when culturedunder nonsupplemented conditions. It was almostas much as that obtained in early experimentswith C. utilis when methionine was used asa sulfur supplement (18).Growth in the presence of the sulfur supple-

ments was not as good as that observed in non-

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TABLE 1. S-adenosylmethionine in Candida albicans

Amt &umoles) ofS-adenosylmethionine/g

(dry wt)Supplement of sulfur in medium (dry_wt _

ATCC ATCC ATCC10231 10261 10259

None ...................... 3.0 2.5 25.0L-Methionine, L-homocys-

teine, S-methyl-L-methio-ninea ..................... 25.0 25.1 75.0

a A 5-,umole amount of each per milliliter.

TABLE 2. Growth of Candida albicans

Amt of cells (g, wetwt)/100 ml of mediumafter 48 hr at 30 C

Supplement of sulfur in mediumr

ATCC ATCC ATCC10231 10261 10259

None ...................... 5.0 5.2 2.5L-Methionine, L - homocys-

teine, S-methyl-L-methio-ninea . . 4.0 4.2 1.5

a A 5-,umole amount of each per ml.

supplemented medium (Table 2). The poorestgrowth was shown by the mycelial mutant. Insupplemented medium, only one-half as muchgrowth occurred as growth in a medium free fromsulfur supplement. The accumulation of S-adenosylmethionine in C. albicans was the samewhether the cells were incubated at 30 or 37 C.

Ultraviolet microscopy revealed that the S-adenosylmethionine was located in the vacuoles.Yeast-phase strains ATCC 10231 (Fig. la, b; Fig.2a, b) and 10261 (Fig. lc, d; Fig. 2c, d) resembledC. utilis when cultured under similar conditions;the absorption of 265 m,u UV energy by thevacuoles of cells grown in supplemented mediumwas greater than in those grown in nonsupple-mented medium. The mycelial mutant, ATCC10259 (Fig. le, f; Fig. 2e, f), showed S-adenosyl-methionine in the vacuoles of both mycelial andyeast phases in nonsupplemented as well as

supplemented medium. However, as can be seen

from the photographs and Table 1, the amount ofS-adenosylmethionine present in cells fromsupplemented medium was much greater than incells from nonsupplemented medium.Soon after receipt of strain 10261 from the

American Type Culture Collection, cells grownin supplemented medium were found to haveproduced filaments (Fig. 3a, b), whereas thosegrown in the nonsupplemented medium main-tained their typical yeast phase. Repetition of the

experiment after the stock had been maintainedin our laboratory for several months failed to givethe same results.

In an effort to learn whether cells of the myce-lial mutant could be depleted of S-adenosyl-methionine, the cells with S-adenosylmethioninewere incubated in a nitrogen-deficient medium.The latter medium was the same as that describedin Materials and Methods, except the (NH4)2SO4was omitted. Visual comparison of UV photo-micrographs showed a decline in the UV absorp-tion of the vacuoles as culture of the cellscontinued. Column chromatography of PCA ex-tracts from cells removed from the nitrogen-deficient medium at 0, 24, 48, and 72 hr alsoshowed a decrease in the amount of the sulfoniumcompound present in the cells (Fig. 4).

In an attempt at a simple correlation of thetwo methods of analysis, optical densities of thephotomicrographic negatives were measured andplotted (Fig. 4). Unfortunately, only densitiesgreater than 0.65 fell on the portion of the charac-teristic curve where proportionality of absorbancyand density exists. Nevertheless, the correlation isinteresting.

Spheroplasts. When cells of the three strains thathad been grown in supplemented and non-supplemented medium were digested with snailgut enzyme, the cells that had been grown insupplemented medium were more sensitive to theenzyme than were nonsupplemented cells. How-ever, there were great differences in sensitivity.Strain 10261 was quite resistant. Only a few cellswere affected after exposure to the enzyme for 24hr (Fig. 3h, i). With strain 10259, about 50% ofthe supplemented cells were affected in 4 hr (Fig.3g). Strain 10231 was intermediate in response tosnail gut enzyme. Strain 10259 was again of spe-cial interest because of its high S-adenosyl-methionine content in the nonsupplemented state.Strain 10259 was much more sensitive to the snailgut enzyme than yeast cells that contained less ofthe sulfonium compound, even though the yeastcells were cultured in supplemented medium.Although cell walls were affected by the enzyme

to varying extents, typical intact spheroplasts werenot readily produced. Of the few intact sphero-plasts that were observed, all appeared to derivefrom a single class of strain 10259 cells, namely,the blastospores (Fig. 3e, f). Intact spheroplastsdid not emerge through a hole in the cell wall(Fig. 3h, i) as described by Eddy and Williamson(4), Svihla et al. (20), or Garcia-Mendoza andVillanueva (7). Usually, intact vacuoles, or por-tions of vacuoles, were observed to emerge (Fig.3c, d, g). The bulk of the cytoplasm remainedwithin the damaged cell wall. With strain 10259,intact vacuoles were observed in 1 M mannitol, as

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ULTRAVIOLET MICROSCOPY OF C. ALBICANS

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FIG. 1. UV photomicrographs of Candida albicans grown in nonsupplemented medium. Frames a, c, and e weremade at 265 mu, and b, d, andf, at 295 mp. The a-b pair are ofATCC 10231; c-d, ofATCC 10261; e-f, ofATCC10259. The darker vacuoles reveal the presence of S-adenosydmethionine.

well as in 0.6, 0,65, 0.75, and 0.9 M KCI, in whichthey appeared to be stable, although they werenot observed for long periods of time, as werethose of C. utilis (20). Apparently, vacuoles varyin osmotic properties. Those that remained intacthappened to be isosmotic with the surroundingmedium. Rounded masses of cyptoplasm wereobserved that might be mistaken for spheroplasts,

especially by phase microscopy. However, thesewere assumed to be masses of cytoplasm re-maining after rupture of the cytoplasmic mem-brane and escape of the vacuole. The masses ofcytoplasm were much smaller than intact cells orintact spheroplasts, and were easily distinguishedfrom intact spheroplasts by UV microscopy (Fig.3c, d).

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FIG. 2. UV photomicrographs of Candida albicans grown in a medium supplemented to induce the accumulationofS-adenosylmethionine in the vacuole. UV wavelengths are as in Fig. 1. The dark vacuoles in a, c, and e revealthe location and relative concentration of the S-adenosylmethionine.

In general, the pattern of enzymatic digestion ofthe cell wall of those cells that did produce sphero-plasts appeared to be constant. The distal halfof the wall was affected, whereas the proximalportion usually remained intact and in contactwith its supporting cell (Fig. 3c, f). The cup-shaped fragments of cell wall that resulted wereproduced by the fracture of a cell wall at itsequator. In this case, the gathering of the proto-

plast (to become a spheroplast) helped separatethe two halves of the cell wall. Otherwise, budscars, or points of attachment between cells, weremore sensitive to the enzyme than were otherportions of the cell wall.Large intact spheroplasts, derived from fila-

mentous cells, were rare (Fig. 3g).Action of ribonuclease on C. albicans. In an

effort to explore further some of the differences

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FIG. 3. UV photomicrographs of Candida albicans grown in supplemented medium. Paired photographs made at265 and 295 m,u, except g, made at 265 mlu. Photos a and b illustrate hyphal growth obtained with ATCC 10261in an early experiment, details in text. Photos c and d, e and f, and g illustrate spheroplast formation from cells ofATCC 10259 that were treated with snail gut enzyme; sp = intact spheroplast, v = portion offree vacuole, 1=lipid body, cy = cytoplasmic mass remaining after breakage of cytoplasmic membrane and loss of vacuole. Photosh and i, a single spheroplast in the process offormation among many cells ofATCC 10261.

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BALISH AND SVIHLA

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TIME (hours)

FIG. 4. Decline in S-adenosylmethioninie content ofthe vacuoles and cells of a filamentous mutant ofCandida albicans (ATCC 10259) after transfer into anonsupplemented medium or a nitrogen-deficient me-dium. Cells were cultured in the indicated medium for48 hr and then transferred into the new medium for72 hr. AM-R = supplemented into nonsupplemented,AM-N = supplemented into nitrogen-deficient, M-N =methionine only supplement into nitrogen-deficient,R-N = nonsupplemented into nitrogen-deficient.

between the yeast and mycelial phases of C.albicans, all three strains were subjected to ribo-nuclease treatment in distilled water. Schlenk andDainko (14) have reported that yeast cells arerapidly killed and lose UV-absorbing materialwhen they are exposed to ribonuclease in distilledwater.

Ribonuclease caused the release of UV-absorb-ing components from the yeast strains of C.albicans (Fig. 5). Over 50% of the UV-absorbingmaterial was released after 20 min. That some ofthis material remained within the cell was evidentby extraction of an equivalent amount of cellswith boiling 1.5 N PCA. Doubling the concentra-tion of ribonuclease still did not remove all of theUV-absorbing constituents from the cells (Fig. 5).It was further demonstrated (Table 3) that theyeast and filamentous strains of C. albicans bothreleased UV-absorbing material when treated withribonuclease in distilled water.

Figure 6 shows that the action of ribonucleaseresulted in the rapid loss of viability with the two

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10 20 30 40 50 60TIME (minutes)

I"I FIG. 5. Release ofmaterial absorbing at 260 mp from72 Candida albicans (ATCC 10231 and 10261) after treat-

ment with ribonuclease in distilled water.

TABLE 3. Release of ultraviolet-absorbing materialfrom Candida albicans treated with

ribonuclease

Optical density at 260 miA ofmaterial released after

treatment withStrain Timea

Ribo- PerchloricWater nuclease acid

in water (1.5 N)

min

ATCC 10231 30 0.02 0.65 0.8260 0.03 0.67 0.85

ATCC 10261 30 0.02 0.54 0.8560 0.05 0.62 0.90

ATCC 10259 30 0.02 0.38 0.6160 0.05 0.39 0.63

a Amount of time after start of treatment.

yeast phase strains (10231 and 10261) of C.albicans. Strain 10259 was not used for quantita-tion of cell viability because of its mycelialmorphology. However, after a 30-min treatmentwith ribonuclease, few colonies of strain 10259developed when the ribonuclease-treated cellswere streaked over Sabouraud Dextrose Agarplates. Numerous colonies of the filamentous mu-tant developed from control cells not treated withribonuclease. Thus, the killing effect probablyoccurred with 10259 cells as well as with theyeast-phase strains 10231 and 10261.

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FIG. 6. Effect of ribonuclease on the viability ofyeastlike strains of Candida albicans.

It was also observed that no significant releaseof protein occurred during ribonuclease treatmentof yeast and mycelial strains. When 0.05 M phos-phate buffer (pH 5.8) was used instead of distilledwater, no release of UV-absorbing material wasnoted with any of the three strains of C. albicans.Schlenk and Dainko (14) showed a similar effectof buffer on ribonuclease activity with C. utilisand S. cerevisiae.

DISCUSSIONC. albicans is a dimorphic organism which is

capable of causing serious and even fatal infec-tions in animals and man. Although mycelialmutants of C. albicans have been reported to beless pathogenic for animals than yeast-phase cells(5, 10, 12), it is evident that the mycelial phase ofC. albicans predominates in infected tissue. Thereis increasing evidence that the capacity to formmycelia is related to pathogenicity (8, 22), andthat the presence of filamentous forms in stoolsis indicative of an alimentary-tract infection byC. albicans (1, 2, 9). Upon injection into mice,yeast-phase cells that are unable to formfilaments appear to be phagocytized and destroyed(8, 22).The present data show that the mycelial mutant

(ATCC 10259) had a much greater capacity toform and store S-adenosylmethionine in itsvacuoles than did the two yeast-phase strains. Itwas also evident that the mycelial mutant mayutilize the stored S-adenosylmethionine whenincubated in a nitrogen-deficient medium. Someutilization (25 %) of the S-adenosylmethioninestored in the vacuole of C. utilis was observed by

Svihla and Schlenk (18, 19) upon incubation ina sulfur-deficient medium for 24 hr.The capacity to form and store S-adenosyl-

methionine may be involved in the pathogenicityof C. albicans. The accumulation and storage ofan essential amino acid such as methionine, in theform of S-adenosylmethionine, may deplete hosttissues of a required nutrient, and hence mayweaken host capabilities to resist infection. Stor-age and utilization of S-adenosylmethionine bythe mycelial phase of C. albicans, along with theapparent difficulty of phagocytes in destroyingmycelial forms, may play a role in allowing theorganism to subsist for a longer period of timein host tissues, thus facilitating the transitionfrom a saprophytic to a parasitic existence.

It is evident from the data that there are dif-ferences in the cell wall structure of yeast and ofmycelial forms of C. albicans. The mycelial formswere found to be more susceptible to the lyticaction of snail gut enzyme than were the yeast-phase cells. Growth in a supplemented mediumrendered the yeast cells more susceptible to theaction of the enzyme than cells grown in mediumwithout supplementation. Svihla et al. (20) haveshown that S. cerevisiae and C. utilis are mostsusceptible to the lytic action of snail gut enzymewhen their vacuoles contain large amounts ofS-adenosylmethionine. Likewise, the most sensi-tive C. albicans cells were those of the mycelialmutant which had been grown in supplementedmedium and had accumulated large amounts ofS-adenosylmethionine in the vacuole.Another factor that may be of importance in

the pathogenicity of C. albicans is the structure ofthe cytoplasmic membrane. The failure to obtainsignificant yields of intact spheroplasts with C.albicans, under conditions favorable for otherorganisms, seems related to the lack of preserva-tion of the integrity of the cytoplasmic membraneor to its lack of formation in those cells in whichthe cell wall was attacked by snail enzyme. Eddyand Williamson (4) have shown diagrammaticallyhow the protoplast of S. carlsbergensis separatesfrom the cell wall and is released. Svihla, Dainko,and Schlenk (20) showed photographs of theseparation and release of a protoplast of C. utilis.The same phenomenon was observed with yeast-phase cells of C. albicans (Fig. 3h), but was seenonly in rare instances with the mycelial form (Fig.3c to g). Instead of intact spheroplasts being re-leased, intact vacuoles were released (Fig. 3c, g).This is an indication that the cytoplasmic mem-brane was damaged, as observed in earlier irradia-tion studies of spheroplasts of C. utilis (20).Apparently, the action of the snail gut enzyme onthe cell wall also affected the cytoplasmic mem-

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BALISH AND SVIHLA

brane of the C. albicans mycelial mutant. Thus,differences in the cell wall structure of yeast andmycelial forms of C. albicans were manifest bytheir differences in susceptibility to the snail gutenzyme. Just how these changes in cell wall struc-ure are brought about is not understood.

Svihla and Schlenk (18) demonstrated that thepresence of sulfur amino acids in a growthmedium can inhibit the growth of S. cerevisiaeand C. utilis. Supplementation of the growthmedium with sulfur amino acids also inhibitedthe growth of C. albicans. However, the supple-mentation did not appear to alter the morphologyof yeast and mycelial strains, except for oneexperiment in which hyphal forms were observedin supplemented medium with the yeast-phasestrain 10261. The latter cells resembled those de-scribed by Young (22) which were formed afterintraperitoneal injection of yeast-phase cells intomice.The rapid killing effect and the relase of UV-

absorbing material from all these strains of C.albicans by ribonuclease are also of interest, sincethey indicate that all three strains have cytoplas-mic membranes which are similar in their suscep-tibility to ribonuclease. However, the fact that thesnail gut enzyme had a greater effect on the cyto-plasmic membrane of the mycelial strain than onthe yeast strain may indicate differences in thisstructure as well. It should also be pointed outthat the UV-absorbing material is not released ifthe experiment is carried out in phosphate buffer.From the results of our experiments with ribo-nuclease, it is probable that the pore sizes of thecell wall of all three strains of C. albicans are atleast as large as those Schlenk and Dainko (14)reported for C. utilis and for S. cerevisiae on thebasis of their determination of the size of theribonuclease monomer (3).

ACKNOWLEDGMENTThis investigation was supported by the U.S.

Atomic Energy Commission.

LITERATURE CITED1. BALISH, E., AND A. W. PHILLIPS, 1966. Growth,

morphogenesis, and virulence of Candida albi-cans after oral inoculation in the germ-free andconventional chick. J. Bacteriol. 91:1736-1743.

2. BALISH, E., AND A. W. PHILLIPS. 1966. Growthand virulence of Candida albicans after oralinoculation in the chick with a monoflora ofeither Escherichia coli or Streptococcus faecalis.J. Bacteriol. 91:1744-1749.

3. CARLISLE, C. H., AND H. SCOULOUDI. 1951. Thecrystal structure of ribonuclease. Proc. Roy.Soc. (London) Ser. A 207:496-526.

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