JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 445-455Copyright © 1976 American Society for Microbiology

Vol. 128, No. 1Printed in U.S.A.

Light Microscopy of Basidia, Basidiospores, and Nuclei inSpores and Hyphae of Filobasidiella neoformans

(Cryptococcus neoformans)KEITH H. ERKE

Department of Medical Research and Development, William Beaumont Army Medical Center, El Paso,Texas 79920

Received for publication 14 July 1976

Three hypha-forming strains of Cryptococcus neoformans were induced toform basidia and basidiospores. Light microscopy showed that basidia formed atthe ends of terminal hyphal cells and were able to produce from a few to manybasidiospores. The morphology of the sexual structures indicated that thesestrains belonged to the recently described perfect state of C. neoformans, Filo-basidiella neoformans. The average dimensions of the basidiospores were 1.9,um in width by 2.7 am in length. Giemsa staining revealed that dikaryotic cellswere formed in all three strains. Only one strain had both terminal andsubterminal dikaryons, indicating functional clamp connections, whereas thetwo remaining strains had dikaryons restricted to the terminal cells. Basidio-spores of two strains were mononucleate, and yeast cell clones derived fromsingle basidiospores of these two strains were able to complete the sexual lifecycle, thus indicating their primary homothallic nature.

In 1975 Kwon-Chung (7) described the sexualstage of Cryptococcus neoformans. She gavethis yeast the perfect name of Filobasidiellaneoformans and placed it in the Filobasidi-aceae of the Ustilaginales. In the present studythree hypha-forming strains of C. neoformanswere found to form basidia and basidiospores. Alight-microscopy study of these structures coin-cided with the observations ofKwon-Chung (7),thus confirming that they had a perfect stagebelonging to the species F. neoformans. How-ever, a sexual stage developed in each of thethree individual strains without the necessityof mixing strains. Basidiospores were isolatedby micromanipulation to observe their germi-nation and to determine if they could completethe sexual life cycle. Finally, Giemsa stainingwas used to determine the nuclear patterns inhyphal filaments and basidiospores.

MATERIALS AND METHODSOrganisms and media. The source of C. neofor-

mans hyphal strains Coward (9.5), Stanford (9.26),and C-145, all originally isolated from human casesof cryptococcosis and pathogenic for mice (13-15; T.G. Mitchell, Ph.D. thesis, Tulane Univ., New Or-leans, La., 1971), was reported previously (4). Allstrains were maintained on Sabouraud dextroseagar at room temperature (22 to 24°C). Other mediaused in this study were Staib's nigerseed-creatinineagar (16), V-8 juice agar prepared from a filtratediluted 1:2 (10), and minimal medium agar minus

thiamine (MM-thi). Minimal medium (MM), ini-tial pH 5.7, has the following ingredients per liter ofdistilled water: glucose, 10 g; (NH4)2SO4, 5 g;KH2PO4, 1 g; MgSO4 * 7H20, 0.5 g; CaCl2*H2O, 0.1 g;NaCl, 0.1 g; thiamine-HCl, 0.5 mg; agar, 20 g; and 5ml of a trace element solution (containing the fol-lowing amounts in 100 ml: CuSO4 5H20, 0.8 mg;MnSO4 H20, 8 mg; FeCl3 6H20, 4 mg; KI, 2 mg;Na2MoO4 * 2H20, 4 mg; H3BO3, 10 mg; andZnSO4 * 7H20, 8 mg).Growth and sporulation. Hyphal growth was in-

duced from stock cultures by making four point inoc-ulations on V-8 or MM - thi agar and placing glasscover slips (22 mm) over the yeast cells. The plateswere sealed with masking tape, placed in plasticbags, and incubated at room temperature (22 to240C). Optimum conditions for sporulation varied foreach strain. Sporulation was best for the Stanfordstrain when cover slips were removed from a 40- to70-day V-8 culture and squares containing the hy-phal growth were cut out and transferred to thesurface of fresh V-8 plates. For the Coward strain,squares containing hyphal growth of a 35- to 50-dayV-8 culture were placed on the surface of freshMM - thi agar. Strain C-145 gave optimum sporula-tion with hyphal cultures maintained serially onMM - thi agar. This sporulation occurred after coverslips were removed from 6 to 10 days of growthwithout transfer to fresh medium.

Micromanipulation. A deFonbrune micromanip-ulator, equipped with microneedles fashioned free-hand (2) from 1-mm capillary tubes, was used toseparate basidiospores for observing germinationand obtaining monosporous yeast cell clones. Anumber of spores sticking to the microneedle from

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an inidividual basidium were transported to agar

blocks. The basidiospores were teased apart, andsingle spores adhering to the microneedle were car-

ried to other blocks and positioned near glass beadsso that they could be easily relocated (4). The agar

blocks were then placed on the surface of Sabourauddextrose agar plates for growth and microscopicobservation.Giemsa staining of nuclei. Small blocks of agar

containing hyphal growth were placed on a glassslide with a few drops of egg white. Another slidewas used to press the agar, and the slides were

drawn apart to give a thin preparation. To obtainstained terminal cells, hyphal growth was sand-wiched between dialysis tubing and glass cover slipsin the following manner. Dialysis tubing squares (22mm) were sterilized, placed on agar medium, anddried. Small blocks of agar containing hyphae were

inoculated onto the center ofthe tubing and overlaidwith 25-mm cover slips. After growth had occurred,both tubing and cover glass were removed from theagar and inverted, and the dialysis tubing was im-mediately peeled away, leaving hyphal cells adher-ing in place to the cover slip. To prepare basidio-spores for staining, areas with abundant sporulationon the surface of agar blocks were touched to coverslips having thin smears of egg white. After drying,all preparations were fixed, hydrolyzed, and stainedwith Giemsa (1). All photomicroscopy and measure-

ments of basidia and basidiospores were carried outwith a Zeiss photomicroscope equipped with an eye-

piece micrometer.

RESULTSThe hyphal growth of all three strains could

be maintained serially on MM - thi agar undercover slips. After removing the cover slips,yeast growth was limited on the agar surfacewhen grown on this medium. When cover slipswere removed from hyphal growth in MM agarthe surface area was overgrown with yeastcells. The surface area of similar cultures inMM - thi agar became covered with short,thick, white aerial hyphae and with noticeablyless yeast growth.Although some sporulation of all three

strains has been seen under a variety of differ-ent conditions on both MM - thi and V-8 agar,the conditions described in Materials and Meth-ods gave repeatable and the most abundantproduction ofbasidia and basidiospores for eachofthe three strains. The Stanford strain showedthe best sporulation of all three strains. Fourdays after cover slips had been removed andhyphal growth had been transferred to fresh V-8 agar, much of the surface area was coveredwith white aerial hyphae (Fig. 1) and hadabundant sporulation. Basidia and basidio-spores of the Coward strain appeared within 2days after V-8 agar blocks had been transferredto MM-thi agar and were most abundant on

the periphery of the hyphal colony. Strain C-145 sporulated in 2 to 3 days after removal ofcover glasses from a 6- to 10-day culture inMM - thi agar. After removal of the coverslips, each strain was also capable of producingyeast cells from short branches or from sessilepositions along the hyphae.The observations made on the sexual struc-

tures concurred with those made by Kwon-Chung (7). Figures 2, 3, and 4 show basidia andbasidiospores of the Stanford, Coward, and C-145 strains, respectively. Each basidium devel-oped as a swelling at the end of a terminalhyphal cell and was capable ofproducing from afew to many basidiospores. Spores appeared onbasidia in chains (Fig. 2 and 3) or in clusters(Fig. 4). Basidiospores were formed in the ma-jority of cases from four separate points on abasidium and resulted in four chains on mostbasidia. Basidia with more than four chainshave never been observed, and in only occa-sional instances have one, two, or three chainsbeen seen. In many cases where long chainswere formed, the ends were fused together, ap-parently giving more support to the individualchains (Fig. 5). All sporulation of the Stanfordand Coward strains and most sporulation of C-145 occurred on aerial hyphae above the surfaceof the agar. In some cases, C-145 had basidiawith small clusters ofbasidiospores that formedbeneath the surface of the agar.The enlarged ends of the basidia were gener-

ally cylindrical or clavate in shape (Fig. 6),with the dimensions of the inflated area for theStanford and Coward strains ranging in widthfrom 4.7 to 7.7 ,m and in length from 6.6 to 15.3,um. Basidiospores were formed successively onthe basidium, with the older spores beingpushed outward. Sterigmata were not present.Basidiospores were either oval, round, cylindri-cal, or teardrop in shape, with the distal endfrom the basidium being rounded and the proxi-mal end generally truncate (Fig. 7). Fifty-sevenspores from the Stanford and Coward strainsvaried in width from 1.3 to 2.2 ,m (average, 1.9,um) and in length from 2.1 to 3.4 ,m (average,2.7 ,um). Giemsa-stained preparations of Stan-ford and Coward basidiospores revealed thepresence of a single nucleus in each spore (Fig.8). Only rarely were spores seen with two orthree nuclei, and these spores were consider-ably larger than spores with a single nucleus.The first step in the germination process was

a swelling of the basidiospore. It becamerounded and enlarged approximately to the sizeof a C. neoformans yeast cell. The next stepresembled typical yeast budding with the for-mation of a daughter cell. The first daughter

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BASIDIA AND BASIDIOSPORES OF C. NEOFORMANS

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FIG. 1. Colony of C. neoformans Stanford strain producing basidia and basidiospores among the areas ofwhite aerial hyphae. Bar, 1 cm.

FIG. 2. Basidium (arrow) of the Stanford strain with four chains of basidiospores (only three chains arepartially in focus). Other basidia and/or basidiospores are seen in the surrounding area. Bar, 30 i.tm.

447VOL. 128, 1976

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448 ERKE

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4 111 - _FIG. 3. Basidium (arrow) of the Coward strain with four chains of basidiospores. Bar, 16 ,um.FIG. 4. Basidium (arrow) ofstrain C-145 bearing clusters of basidiospores. Two basidia directly to the left

have similar spore clusters. Bar, 20 ,um.

cell very often grew until it equaled the size ofthe mother cell before either cell initiated newbudding. Thereafter, new cells arose by a bud-ding process from either mother cell, daughter

cell, or both. The germination sequence is illus-trated in steps A through E in Fig. 11.Table 1 indicates the ability of 25 yeast cell

clones derived from single spores of the Stan-

J. BACTERIOL.

BASIDIA AND BASIDIOSPORES OF C. NEOFORMANS

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6 4i- ^FIG. 5. Four basidiospore chains with ends (arrow) fused together; Stanford strain. Bar, 30 ,um.FIG. 6. Tease preparation of Coward hyphae showing basidium (arrow) with four basidiospores still

attached. A clamp connection can be seen four bar lengths to the right of the basidiospores. Phase contrast.Bar, 12 ,um.

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450 ERKE

A 5

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FIG. 7. Basidiospores ofStanford strain micromanipulated to agar surface. Phase contrast. Bar, 6 ,um.FIG. 8. Giemsa stain illustrating nuclei ofStanford strain basidiospores. Spores are scattered throughout

field with clusters at end of arrows. Larger yeast cells can also be seen in the field. Bar, 10 ,um.

ford and Coward strains to form hyphae, basi-dia, and basidiospores when induced to sporu-late under the same conditions as the parentalstrains. All of the single-spore cultures were

able to form basidia and basidiospores, al-though three cultures originating from sporesof a single basidium of the Coward strainshowed markedly reduced sporulation when

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BASIDIA AND BASIDIOSPORES OF C. NEOFORMANS 451

TABLE 1. Hyphal growth pattern ofStanford and Coward strain single-spore isolates and their ability to formbasidia, basidiospores, and brown pigment

Source of single-spore Brown pigment onStrain clones Staib's nigerseed- Hyphal growth pat- Basidia and ba-

.> ~~~~creatinineagartensdoprSBasidium Spore

Stanford strain 1 1 + Normal + +2 1 + Normal + +3 1 + Normal + +

2 + Normal + +3 + Normal + +

4 1 + Normal + +2 + Normal + +3 + Normal + +4 + Normal + +

5 1 + Normal + +2 + Normal + +3 + Normal + +4 + Normal + +5 + Normal + +

Coward strain 1 1 + Normal + +2 + Normal + +

2 1 + Normal + +2 + Stunted +3 + Normal + +4 + Stunted +5 + Normal + +6 + Stunted +

3 1 + Normal + +2 + Normal + +

4 1 + Normal + +a +, Basidia and basidiospores present but very few as compared to the parental strain; + +, basidia and

basidiospores formed as well or better than the parental strain.

compared with that of the parental strain.These three isolates also showed reducedgrowth in both hyphal and yeast cultures, thesquare area of growth reaching only about one-halfthe size of other single-spore isolates. All ofthe cultures formed a brown pigment whengrown on Staib's nigerseed-creatinine agar.Giemsa staining revealed the presence of di-

karyotic cells in all three hyphal strains grownon V-8 agar; however, dikaryons in the Stan-ford and C-145 hyphae were restricted to termi-nal cells. Figure 9 shows the predominent ter-minal cell and first subterminal cell nuclearpattern for the Stanford strain. From 70 to 90%of the terminal cells were dikaryotic. The firstsubterminal cell was monokaryotic, and a veryhigh percentage of the clamps, whether be-tween the terminal and first subterminal cellsor farther down the hyphae, contained nuclei.The majority of the remaining terminal cellswere monokaryotic, with occasional anucleateor trinucleate cells. The nuclear pattern forstrain C-145 was similar to that of the Stanfordstrain but with fewer dikaryotic terminal cells(25 to 30%). The number of dikaryons in theCoward hyphae was difficult to estimate sincethey varied significantly from one preparation

to another. Dikaryons tended to occuir in sec-tors that generally had large numbers of suchcells. In the Coward strain dikaryons appearedin both terminal and first subterminal cells(Fig. 10), and only a few nuclei were seen in theclamp connections. The majority of the remain-ing cells were monokaryotic. Clamp connec-tions were found at the septa of adjacent cells inall three strains regardless of whether the cellswere monokaryotic or dikaryotic. Figure 11summarizes the sexual and asexual life cyclesof C. neoformans based on the information thatis presently known.

DISCUSSIONThe limited yeast growth on the surface of

MM-thi agar when cover slips were removedmade it possible to observe more abundantsporulation on the hyphae ofthe Coward and C-145 strains. The ability to maintain serial hy-phae cultures of all three strains in MM-thiagar under cover slips was most likely due tothe less than absolute requirement ofC. neofor-mans for thiamine (9).The capacity of the Stanford and Coward

strain single-spore clones to complete the lifecycle coupled with the mononuclear condition of

VOL. 128, 1976

452 ERKE

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FIG. 9. Three Giemsa-stained hyphal filaments of the Stanford strain illustrating the dikaryotic terminalcells (large arrow), nuclei trapped in clamp structures (between arrows), and monokaryotic first subterminalcells (small arrow). Bar, 40 a.m.

FIG. 10. Giemsa-stained sector ofCoward strain hyphae showing several dikaryotic cells. Arrows indicatehyphal strands having both terminal and first subterminal dikaryons. Bar, 40 t,m.

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BASIDIA AND BASIDIOSPORES OF C. NEOFORMANS

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FIG. 11. Life cycle of C. neoformans illustrating both asexual and sexual phases. (A-E) Sequence repre-sents swelling ofa basidiospore and subsequent budding. (F) Asexual yeast cell cycle. (G) Brackets indicate asyet unknown alternatives to initiate hyphal formation by either germination of individual yeast cells orpreviously conjugated yeast cells. (H) Development of hyphae having clamp connections and dikaryotic cells.(I) Budding yeast cells formed along hyphal strands. (J) Basidia and basidiospores formed at ends ofterminal hyphal cells.

the basidiospores strongly suggest primaryhomothallism (as defined by Whitehouse [18])for these two strains of C. neoformans. Therestill remains the possibility that spore nucleiwere diploid and formed from haploid nuclei ofopposite mating type. The small size and theconsiderable number of spores that many basi-dia were capable of forming, together with thefact that sporulation generally occurred on eachbasidium at four separate points, arguesagainst diploidy. Kwon-Chung (7) reported herstrains to be heterothallic but did not specifythe type of heterothallism, bipolar or tetra-polar. The presence of both homothallic andheterothallic forms in C. neoformans wouldseem to be akin to the situation for the wood-rotting basidiomycete Sistotrema brinkmannii,which consists of an aggregate of biologicalforms having homothallic, bipolar heterothal-lic, and tetrapolar heterothallic patterns of sex-uality (17). However, it seems safe to say at thispoint that more C. neoformans strains would

have to be studied and genetic analysis carriedout with several strains before any conclusionscould be reached. Such matings and subsequentgenetic analysis could perhaps elucidate themating types present in the heterothallicforms. Additionally, such studies may shedlight on whether the homothallic behavior ofthe strains reported in this study are due tomutations at mating loci, as is the case forhomothallic behavior that sometimes occurs inheterothallic species (Schizophyllum communeand Coprinus lagopus; 12).Among the three hyphal strains, only the

Coward strain had both terminal and subter-minal dikaryotic cells, indicating the presenceof fused clamp connections. A recent electronmicroscopic study by Kwon-Chung and Popkin(8) revealed the presence of fused clamp connec-tions in the hyphae of two compatible strains ofC. neoformans. The restriction of dikaryons tothe terminal cells of the Stanford and C-145strains and the many nuclei trapped in the

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clamps showed that the final fusion of the hookcells to subterminal cells did not occur. Evi-dence for the failure of clamp fusions in thehyphae of the Stanford strain has been demon-strated in an earlier electron microscopic study(3). Clamps that fail to fuse have been termedpseudoclamps and have unequivocally beenshown to be characteristic of common B mat-ings among tetrapolar heterothallic species ofthe higher basidiomycetes, including S. com-mune and Coprinus species (12). What factorsare responsible for such nuclear behavior inthese apparently homothallic strains of C. neo-formans remain unknown. The restriction ofdikaryons to terminal cells did not inhibit theability to fruit, since the Stanford strain formedthe most abundant basidia and basidiosporesamong the three strains. Kwon-Chung (7) indi-cated that the sexual stage was initiated byconjugation of two yeast cells. In the presentstudy it was not determined at what point di-karyons were established among the strains ofC. neoformans. As was indicated in step G ofFig. 11, it was unclear whether the fusion ofyeast cells was a prerequisite for hyphal forma-tion and dikaryotization or whether othermechanisms cause yeast cells to form hyphaethat can subsequently anastamose to establishdikaryons.The formation of basidia and basidiospores

among hyphal strains of C. neoformansprompts interesting speculation on three ques-tions that might have ecological and epidemio-logical significance for this pathogenic yeast.First, although only a relatively few laboratorystrains have demonstrated ability to form hy-phae and therefore may have sporulating po-tential, it remains unknown as to what extentthis stage might exist in the natural environ-ment of C. neoformans. Second, if such a stagedoes exist in nature, what role do the basidio-spores play in the dispersal of this organism inthe environment? Powell et al. (11) have shownthat a small percentage of C. neoformans air-bome particles from pigeon excreta exist withdiameters of less than 5.5 Am. Both basidio-spores and small yeast cells (9) have dimen-sions less than this, although the basidiosporeswould seem to be better designed aerodynami-cally than yeast cells to become airborne. Fi-nally, what possible role might the basidio-spores play as infective agents? In order for C.neoformans to become deposited in the lungalveoli, Powell et al. (11) and Farhi et al. (5)have postulated that the infective particle of C.neoformans exists in a small form, less than 5.0,um or approximately 3.0 Am, respectively. Gu-tierrez et al. (6) have recently reported a granu-lomata of the lung due to a small form of C.

neoformans. Measurements of the nonbuddingorganisms in the lung averaged 2.0 by 2.6 ,um,which approximates the average size (1.9 by 2.7Am) of basidiospores formed by the Stanfordand Coward strains. Whether such an infectioncould result from exposure to basidiospores re-mains an interesting but unanswered question.

ACKNOWLEDGMENTSI gratefully acknowledge the assistance of Ralph Banez,

Chief Technologist of the Department of Medical Researchand Development, during the course of this study and thehelp of Shirley Patton in preparing the media. I am alsoindebted to the personnel of the Medical Illustration andPhoto Lab for their support with the photography. Specialthanks are due to Leon J. Krzemien and Forrest W. Lyonfor their helpful advice on photomicroscopy and for thephotography leading to Fig. 1, and to Paulino Magallanesfor preparing the final draft of Fig. 11. Thanks are alsoextended to Leonard Davis of Medical Research and Devel-opment and Gordon Robertstad of the Department of Bio-logical Sciences, University of Texas at El Paso, for theircritical reading of the manuscript.

LITERATURE CITED

1. Banno, I. 1967. Studies on the sexuality of Rhodoto-rula. J. Gen. Appl. Microbiol. 13:167-196.

2. Chambers, R. W., and M. J. Kopac. 1950. Micrurgicaltechnique for the study of cellular phenomena, p.492-543. In R. McC. Jones (ed.), McClungs handbookof microscopical technique, 3rd ed. Harper (Hoeber),New York.

3. Cutler, J. E., and K. H. Erke. 1971. Ultrastructuralcharacteristics ofCoccidioides immitis, a morphologi-cal variant of Cryptococcus neoformans and Podosy-pha ravenelii. J. Bacteriol. 105:438-444.

4. Erke, K. H., and J. D. Schneidau, Jr. 1973. Relation-ship of some Cryptococcus neoformans hypha-formingstrains to standard strains and to other species ofyeasts as determined by deoxyribonucleic acid basesand homologies. Infect. Immun. 7:941-948.

5. Farhi, F., G. S. Bulmer, and J. R. Tacker. 1970. Crypto-coccus neoformans. IV. The not-so-encapsulatedyeast. Infect. Immun. 1:526-531.

6. Gutierrez, F., Y. S. Fu, and H. I. Lurie. 1975. Crypto-coccosis histologically resembling histoplasmosis.Arch. Pathol. 99:347-352.

7. Kwon-Chung, K. J. 1975. A new genus, Filobasidiella,the perfect state ofCryptococcus neoformans. Mycolo-gia 67:1197-1200.

8. Kwon-Chung, K. J., and T. J. Popkin. 1976. Ultrastruc-ture of septal complex in Filobasidiella neoformans(Cryptococcus neoformans). J. Bacteriol. 126:524-528.

9. Phaff, H. J., and J. W. Fell. 1970. Genus 3. Cryptococ-cus Kutzing emend. Phaffet Spencer, p. 1088-1147. InJ. Lodder (ed.), The yeasts. A taxonomic study.North-Holland Publishing Co., Amsterdam.

10. Pitt, J. I., and M. W. Miller. 1968. Sporulation in Can-dida pulcherrima, Candida reukaufii and Clamydo-zyma species: their relationships with Metschniko-wia. Mycologia 60:663-685.

11. Powell, K. E., B. A. Dahl, R. J. Weeks, and F. E. Tosh.1972. Airborne Cryptococcus neoformans: particlesfrom pigeon excreta compatible with alveolar deposi-tion. J. Infect. Dis. 125:412-415.

12. Raper, J. 1966. Genetics of sexuality in higher fungi.The Ronald Press Co., New York.

13. Shadomy, H. J. 1970. Clamp connections in two strainsof Cryptococcus neoformans, p. 67-72. In D. Ahearn(ed.), Recent trends in yeast research, vol. I, Spec-trum: monograph series in arts and sciences. Georgia

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State University, Atlanta.14. Shadomy, H. J., and H. I. Lurie. 1971. Histopathologi-

cal observations in experimental cryptococcosiscaused by a hypha-producing strain of Cryptococcusneoformans (Coward strain) in mice. Sabouraudia9:6-9.

15. Shadomy, H. J., and J. Utz. 1966. Preliminary studieson a hypha-forming mutant of Cryptoeoccus neofor-mans. Mycologia 58:383-390.

16. Staib, F. 1963. Membranfiltration und Negersaat (Gui-

zotia abyssinica)-Nahrboden fur den Cryptococcusneoformans-Nachweis (Braunfarbeffekt). Z. Hyg. In-fektionskr. 149:329-335.

17. Ullrich, R. C., and J. R. Raper. 1975. Primary homo-thallism-relation to heterothallism in the regula-tion of sexual morphogenesis in Sistotrema. Genetics80:311-321.

18. Whitehouse, H. L. K. 1949. Heterothallism and sex inthe fungi. Biol. Rev. Cambridge Philos. Soc. 24:411-447.

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