Inorganic ions. The addition of various levels

Transcription

1 NUTRITION OF THE PARASITIC PHASE OF COCCIDIOIDES IMMITIS IN A CHEMICALLY DEFINED LIQUID MEDIUM JOHN L. CONVERSE AND ARTHUR R. BESEMER U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland Received for publication February 2, 1959 In previous reports (Converse, 1955, 1956, 1957) a chemically defined medium was described which supported the growth of the parasitic phase (spherule) of the dimorphic fungus Coccidioides immitis. Certain physicochemical factors were shown to influence the growth of spherules in this medium. The work to be described here is an extension of the investigation of the nutritional and environmental factors which affect the spherulation in vitro of C. tmmitis. MATERIALS AND METHODS C. immitis strain Mll was used in this investigation. Experimental cultures were incubated for 72 hr at 34 C on a reciprocating shaker (operating through a 412 in stroke at 96 excursions per min) in a liquid basal medium composed of ammonium acetate, M; glucose, M; KH2PO4, M; K2HPO4, M; MgSO4-7H20, M; and ZnSO4-7H20, 1.24 X 10-5 M, with a final ph of 6.3 after autoclaving. Fifty ml of medium in 250-ml Erlenmeyer flasks were inoculated with 1 X 107 arthrospores. The flasks were closed with cotton-packed modified thistle tubes inserted through rubber stoppers. The inoculum was prepared by transferring arthrospores from a stock agar slant to a liquid synthetic medium (Basal no. 2 of Roessler et al., 1946; with 4 ppm Zn+ as ZnSO4) and incubating for 14 days under similar conditions. This culture was stored at 5 C and discarded after 1 month. Spherule yields were determined by microscopic examination of wet mounts stained with lacto-phenol-blue. The degree of spherulation and total growth was estimated visually for each culture and assigned an arbitrary number on a scale ranging from 0 to 8. Endospore cultures were filtered by gravity through 6 layers of surgical gauze to remove trace amounts of hyphae. The endospore yields were measured by viable counts, using the pourplate technique with a plating medium composed of peptone, 1 per cent; glucose, 2 per cent; yeast autolysate, (yeast-75, Vico Products Company) 0.1 per cent; and agar, 2 per cent; adjusted to a final ph of 6.9 to 7.2. Plates were incubated for 48 hr at 34 C, and an additional 16 hr at 25 C, before counting. Gas mixtures used for the determination of the oxygen and carbon dioxide requirements were prepared according to Umbreit et al. (1957), using commercial gases of known purity. Analyses of the composition and utilization of gas mixtures during growth were performed with the Orsat gas analyzer. The growth containers for these experiments were 1-L glass reagent bottles closed with rubber stoppers through which two 13-gauge hypodermic needles, one 2 in and the other 6 in in length, had been inserted. The growth containers were flushed with the experimental gas mixtures after which both needles were sealed by attaching short lengths of rubber tubing clamped at the free end. RESULTS The following factors were examined to determine their effect on growth and spherulation in the basal medium. Concentration of total solids in the medium. It was previously reported that varying the total solids of the basal medium from 3.36 to 0.34 per cent resulted in a progressive increase in spherule yield, paralleled by a decrease in total growth of the cultures (Converse, 1956). Findings in the present investigation indicated that concentrations of the nitrogen source (0.016 M) and of the carbon source (0.022 M) were critical for maximal spherulation. Inorganic ions. The addition of various levels of sodium chloride, calcium chloride, and sodium bicarbonate, singly and in combination to the basal medium, resulted in increased spherulation. Maximal spherulation was obtained with the combined use of 2.4 X 104 M sodium chloride, 231

2 232 CONVERSE AND BESEMER [VOL. 78 TABLE 1 Effect of gaseous environment on growth and spherulation of Coccidioides immitisa Gas Mixture 02 C02 N2 Visual Growthb Spherule Yieldb No ~% % Oc Oc Od Air control Oc oc Air control a Mean values of 4 cultures. b 0 to 8 = increasing degrees of growth or spherulation. c Abortive spherules (no endospores). d Hyphal growth only. 2 X 10-5 M calcium chloride, and 1.4 X 104 M sodium bicarbonate. The addition of manganese sulfate, at various concentrations and in combination with the above three compounds, did not affect spherulation. Oxygen and carbon dioxide. Bullen (1949) was able to grow the yeastlike phase of the dimorphic pathogenic fungi, Histoplasma capsulatum, Blastomyces dermatitidis, and Sporotrichum schenckii, on peptone agar by controlling the carbon dioxide content of the atmosphere above the cultures. More recently, Lubarsky and Plunkett (1955) demonstrated the favorable effects of increased amounts of carbon dioxide on the growth of the parasitic phase of C. immitis in the presence of serum and chick embryo extract. In addition to the preceding observations, the increased spherulation of C. immitis, obtained upon the addition of sodium bicarbonate to the defined basal medium, and the effect of aeration of cultures during growth suggested that carbon dioxide might be required for the development of the parasitic phase in defined medium. Preliminary experiments, using 1-L growth containers with 50 ml of the basal medium per container revealed equivalent growth in both sealed and unsealed air controls. Growth also occurred in atmospheres of 100 per cent oxygen or nitrogen, but not in 100 per cent carbon dioxide. It was determined later that the commercial nitrogen used in these experiments was contaminated with 0.5 per cent oxygen. The requirement for oxygen was established by incubating cultures in sealed containers under atmospheres containing the varied concentrations of oxygen and nitrogen as shown in table 1. Optimal growth was obtained with 5 and 10 per cent oxygen and optimal spherulation with 20 per cent; however, only 1 to 2 per cent of the oxygen was consumed in each container during the 72-hr growth period. The optimal carbon dioxide level for spherulation was determined in a similar manner. The oxygen level was kept constant at 20 per cent, and the carbon dioxide concentration varied as indicated in table 1. The highest level of growth was obtained with atmospheres containing 20 per cent carbon dioxide or less. Spherulation, which was inhibited at a CO2 concentration greater than 10 per cent, also occurred in the absence of added C02. It was noted further that the initial ph of the medium decreased when increased concentrations of carbon dioxide were used to flush the containers. These experiments were repeated under conditions of controlled hydrogen ion concentration with similar results, thus indicating that alterations in growth and spherulation resulted from the presence of the carbon dioxide and not from variations in the initial ph of the media. Fatty acids. Observations in this laboratory and those reported by Conant and Vogle (1954) have shown that Tween 80, an ester of oleic acid, stimulated in vitro the conversion of the saprophytic to the parasitic phase of C. immitis thus suggesting that fatty acids may play a role in the phasic differentiation of C. immitis. The effects of various saturated and unsaturated fatty acids, with carbon chain lengths ranging from 6 to 26 added to the basal medium, were examined. Stearic and palmitic acids at 0.1 and 0.01 per cent levels, and oleic and myristic acids at the per cent level stimulated growth. A marked stimulation of spherule formation was noted in cultures containing oleic and linoleic

3 1959] NUTRITION OF C. IMMITIS 233 Effect of sulfur TABLE 2 compounds on growth and spherulation of Coccidioides immitis Compound Conc Visual Growth* Spherule Yield* Morphology H Glutathione , 1,1, 1 0, 0, 0, 0 Abortive spherulest , 3, 5, 5 4, 4, 6, 5 Spherules 30-50, in diameter , 3, 5, 5 8, 5, 8, 5 Spherules I in diameter Cysteine , 2, 3, 3 0, 0, 0, 0 Abortive spherules Iu in diameter , 3, 4, 4 0, 0, 0, 0 Abortive spherules 50-80,M in diameter , 4, 4, 4 0, 0, 0, 0 Abortive spherules u in diameter Methionine , 1, 3, 3 0, 0, 0, , 3, 3, 3 0, 0, 0, , 3, 4, 3 0, 0, 0, 0 Abortive spherules I in diameter Sodium thio , 1, 1, 1 0, 0, 0, 0 glycolate , 3, 3, 3 0, 0, 0, ,4,4,4 0,0,0,0 None (control) - 4 4, 4, 4 4, 4, 4, 4 Spherules A in diameter * Values of quadruplicate cultures. t Abortive spherules: immature (no endospoores in evidence). TABLE 3 Effect of metabolic inhibitors on growth and spherulation of Coccidioides immitis Inhibitor Con. ~ce S heru- Spherule Diameter Inhibitor Conc : w F)ation M NaF * 5( CuS t * 3-5 Sulfaguani dine LiCl None (control) * Ninety-eight per cent of the growth in these cultures consisted of spherules. t Abortive spherules: immature (no endospores in evidence). acids at the per cent level. The addition of stearic, palmitic, cerotic, or arachidic acids resulted in the formation of very large spherules, the majority of which were abortive (maturation of endospores incomplete). Lauric acid was toxic for C. immitis in concentrations as low as per cent. Sulfur compounds. Investigations on dimorphism in various pathogenic fungi (Salvin, 1949b) indicated that sulfides or sulfhydryl compounds were essential for the growth of the yeast phase of H. capsulatum in a chemically defined medium. The growth and spherulation of C. immitis were investigated in basal medium supplemented with the sulfur compounds that are listed in table 2 Spherulation was stimulated by M glutathione. This stimulation was further shown by the increase in the size of the spherules (50 to 80 A in diameter). The addition of cysteine at 0.01 to 0.03 M levels resulted in the formation of very large abortive spherules (20 to 80,u in diameter) that failed to mature at 72 hr incubation. Previous experiments with cysteine indicated that incubation periods of 9 days were frequently required for maturation of spherules. The addition of methionine or sodium thioglycolate, at the concentrations tested, inhibited spherulation. I Metabolic inhibitors. Various metabolic inhibitors were tested to determine whether

4 234 CONVERSE AND BESEMER [VOL. 78 a4.. ::.:. A C Downloaded from Figure 1. Morphology of Coccidioides immitis strain M-11, grown under various conditions: a, Arthrospore inoculum; b, chain of spherules grown in basal spherule medium plus 2.5 X 10-' M CUS04; c, endospore culture grown in basal spherule medium plus 0.05 per cent Tamol "N"; d, spherules grown in basal spherule medium plus 0.05 per cent Tamol "N"; e, spherules and endospores grown in basal spherule medium plus m sodium fluoride; and f, spherules grown in basal spherule medium plus 0.05 per cent Tamol "N" and m sodium fluoride. (Magnification X323; reduced 45 per cent in reproduction here.) spherule formation would be influenced by altering the physiological activity. It was postulated that the addition of specific metabolic inhibitors to basal medium might influence the phasic development of the cultures (table 3). Marked stimulation (98 per cent spherules) of spherulation with little effect on growth resulted upon the addition of sodium fluoride (8 X 103 M) or copper sulfate (2.5 X 105 M) to the basal medium. A marked difference in spherule morphology also was noted with these two compounds. The addition of sodium fluoride resulted in an increase in spherule diameter to 50 to 80, (figure le). Moreover, the spherule size decreased proportionately with a decrease in sodium fluoride concentration. Approximately 50 per cent of the spherules grown in the presence of sodium fluoride had ruptured, releasing the endospores as large clumps. On the other hand increasing the concentrations of copper sulfate proportionately decreased the spherule size (figure lb) to that of normal arthrospores (3 to 5,u in diameter). These spherules occurred in chains of 6 to 14 units, and contained fully developed endospores. Sulfaguanidine and lithium chloride also stimulated spherulation but to a small degree. Other inhibitors (sodium fluoroacetate, sodium benzoate, sodium propionate, or sodium azide) either failed to exhibit significant effects, or inhibited growth and spherulation of the orgamsm. The indications of the importance of sulfhydryl groups in phasic development of H. capsulatum and the stimulation of spherule formation on January 31, 2019 by guest

5 1959] NUTRITION OF C. IMMITIS 235 TABLE 4 Inhibition of spherulation of Coccidioides immitis by sulfhydryl inhibitors and reversal of inhibition by glutathione Growth Inhibition of Spherulation Inhibitor Conc With With With inhibitor With inhibitor inhibitor and gluta- inhibitor and glutathione* thione* p-chloromercuribenzoate 2.8 X X X X Sodium iodoacetate 5.4 X X X X None (control) * Glutathione, 5 X 10-3 M, used with p-chloromercuribenzoate; 1 X 10-2 M used with iodoacetate. in C. immitis by glutathione reported above, suggested an examination of the effects of sulfhydryl inhibition on morphology. The addition of p-chloromercuribenzoate to the basal medium in levels of 2.8 X 10-8 M to 2.8 X 10-6 M (table 4) inhibited spherulation by 20 to 80 per cent, respectively, without inhibiting growth (mycelium). This inhibition was reduced to 0 to 20 per cent by the addition of 5 X 103 M glutathione. Iodoacetate in 5.4 X 10-5 and 5.4 X 10-4 M concentrations resulted in 30 and 50 per cent inhibition of spherulation (without inhibition of growth), respectively, which was completely reversed by the addition of 1 X 10-2 M glutathione. Surface active compounds. Representative types of anionic, cationic, and nonionic surface active agents were investigated. As previously reported (Converse, 1957), Tamol "N" was most effective in stimulating growth, spherulation, and rupture of the spherule walls, with subsequent dispersion of endospores. Continued experiments have indicated that the level of Tamol "N" which was most effective in increasing endospore yield did not result in the highest conversion of arthrospores to spherules. In an attempt to increase the endospore yield, Tamol "N" (1.0 and 0.1 per cent concentrations) was tested in chemically defined media with the total solids content varying from 3.36 to 0.34 per cent as indicated in figure 2. After 72 hr incubation total viability (mycelium and endospore) of the cultures was determined. The cultures then were filtered through gauze and viable counts of the resulting suspensions determined. Microscopic examinations of these suspensions showed approximately 100 per cent endospores (figure lc). In every case, the higher level of Tamol "N" resulted in a higher endospore count. The lower concentration of Tamol "N" generally resulted in a higher percentage of endospores. The highest endospore yield (290 x 105 endospores per ml) was obtained in the medium containing 1.34 per cent total solids and 1.0 per cent Tamol "N," with 52 per cent conversion to the parasitic growth phase. On the other hand, 100 per cent conversion to the parasitic growth phase was obtained in media having 0.67 and 0.34 per cent total solids, but with lower endospore yields (180 and 116 x 105 per ml, respectively). A combination of Tamol "N" (0.05 per cent) and sodium fluoride (0.008 M) in the medium stimulated spherule formation but inhibited spherule rupture, resulting in suspensions of intact spherules (figure lf). Furthermore, the total growth was consistently higher with the combination of compounds than with either compound alone. The anionic surface active compounds, Triton X-200 and Triton X-301 (the sodium salts of

6 236 CONVERSE AND BESEMER [vol PERCENT TAMOL "N' ( ) I. 1.1I00 10J PE0.67 C S0.34 ) PER CENT SOLIDS ( ) Figure 2. Effect of Tamol "N" concentration and total solids content of the medium on growth level and morphology of Coccidioides immitis at 72 hr. Open bar = total count (mycelium and endospores); solid bar = endospore count; numbers above solid bar = endospore count as per cent of total; and dotted bar = no determination made. alkyl aryl polyether sulfonate, and alkyl aryl polyether sulfate, respectively), Rohm and Haas Company, stimulated spherulation slightly at 0.1 to per cent concentrations. A modified phthalic glycerol alkyd resin (B-1956, Atlas Powder Company) possessed the dispersive characteristics of Tamol "N" in the 0.01 to per cent range, but failed to stimulate growth or spherulation. Tween 80 (a nonionic polyoxyethylene sorbitan monooleate, Atlas Powder Company) in 0.05 per cent concentration resulted in cultures containing 98 per cent spherules (50 to 80,u in diameter). No effect on the rupture of the spherule walls or dispersion of endospores was noted with the latter compound. Abortive spherules. The occurrence of partially developed or immature spherule-like bodies which lack endospores and are too large to be called arthrospores or chlamydospores have been referred to as "abortive" spherules (Baker and Braude, 1956). These abortive spherules have been noted in this laboratory on numerous occasions, (figure 3a, b). Observations on spherule development indicate that endospores may be formed by a gathering and thickening of the cytoplasm around the periphery of the wall of the developing spherule until the spherule is filled with endospores. Interruption of this maturation process results in large thick-walled spherule-like structures (figure 3a). Figure 3b shows the degeneration or shrinking of the cytoplasm away from the spherule wall, following an interruption of the maturation process. On the assumption that this interruption might result from a nutritional deficiency, cultures containing abortive spherules (figure 3b) were supplemented with additional glucose and ammonium acetate during the growth period and observed microscopically after further incubation. It was noted that the normal development continued after supplementation of the cultures (figure 3c), followed by maturation 48 hr later (figure 3d), with spherule rupture and the discharge of endospores into the medium. The abortive spherules in unsupplemented control cultures did not change in appearance.

7 1959]. :'., Mn :.' NUTRITION OF C. IMMITIS 237..;d --.. r w$e >8 i.i S *:2 ^.:. E f.:.;. g.' :..' ^.'.E.:...: if % s.u... L. '... ^- '. i., ::: o * ;8 id IF t:x b. X ry ::w'{ zb ':... S f 0.': omw.^.d. *' ' t.f X :,.S B:... : ''''- '. Figure 3. Effect of supplementary feeding of degenerating, immature (abortive) spherules during incubation. a, Normal spherule development after 48 hr incubation; b, degenerated spherules after 5 days incubation (48 hr past the normal 72 hr maturation time). Note shrinkage of cytoplasm away from the spherule wall; c, the same culture 24 hr after supplementary feeding (after 6 days incubation). The process of maturation has resumed; and d, the same culture 72 hr after feeding (after 8 days incubation). Maturation is complete. Spherules ruptured, endospores released into the medium. (Magnification; 476X; reduced 45 per cent in reproduction here.) DISCUSSION Some of the physiological conditions involved in the conversion of one growth phase to the other in dimorphic fungi have been defined but the basic mechanisms of this phenomenon in C. immitis still remain unknown. A decrease in total solids content appears to be one of the most important factors favoring the parasitic phase in defined medium. None of the other factors stimulating the parasitic growth phase have been effective in other than diluted medium. Whether this factor is due to changes in osmotic pressure, or to change in rate of metabolism, remains to be determined. It has been demonstrated (Converse, 1956) that if spherulation does not occur within 48 to 72 hr after inoculation, conversion will not take place. &0. SQ... g}$ F :'..j f o. Levine and Ordal (1946) and Salvin (1949a) found that the conversion in vitro of the mycelial to the yeast phase (or parasitic form) in Blastomyces braziliensis and B. dermatitidis was independent of nutritional factors, and was governed by temperature alone. The yeast phase developed at 37 C and reverted to the mycelial phase at 25 C. This conversion was attributed by Nickerson (1951) to the failure of the cells grown at 25 C to maintain the yeast-phase cellular division rate, thus resulting in cells morphologically different in length and size. Although increased temperature induced spherulation in C. immitis (Converse, 1956), it was not the only factor influencing phasic development in this organism. Salvin (1949b) also reported that conversion of the mycelial phase to the yeast phase of H.

8 238 CONVERSE AND BESEMER [VOL. 78 capsulatum in chemically defined medium was not entirely temperature dependent, but required the presence of a sulfide or sulfhydryl group in a small organic molecule. Nickerson and Romano (1952), working with an abnormal strain of Candida albicans which had lost the ability to maintain itself in the yeastlike growth phase, were able to convert the filamentous phase to the yeast phase by growing it in the presence of cysteine or other sources of -SH groups. This also restored the pathogenicity of this strain. Furthermore, Nickerson and Van Rij (1949), using a nitroprusside staining reaction, were able to demonstrate a marked increase in intracellular foci of sulfhydryl groups in the yeast cells over that found in filamentous cells. Even though C. immitis will spherulate in a defined medium in the absence of added sulfhydryl groups, the addition of glutathione markedly increased this conversion to the parasitic phase. The observation that spherulation was inhibited by sulfhydryl inhibitors and that this inhibition was reversed by the addition of glutathione strongly suggests that the same or similar metabolic processes are operative within this organism. The stimulatory action of surface active agents may be manifold. The effect of Tamol "N" on the rupture of the spherule wall in 72 hr may result either from accelerated growth and maturation or from a change in the permeability of the spherule wall. The stimulation of growth and spherulation of C. immitis by fatty acids may be related to the physical activity of surface active compounds. A number of the surface active compounds, such as the "Tweens" and "Spans," are esters of fatty acids. On the other hand, fatty acids may play a role as growth factors or as detoxifying agents in the metabolism of the organism (Neiman, 1954). The reports of Conant (1941), Bullen (1949), Nord6n (1951), Lubarsky and Plunkett (1955), and Larsh et al. (1956) indicated that the growth of the parasitic phase of practically all of the dimorphic pathogenic fungi cultivated in complex media could be obtained by the addition of sodium bicarbonate or by altering the carbon dioxide tension of the atmosphere. The results in this report show that added CO2 is not required for spherulation in defined media. It cannot be stated at this time whether conversion of the saprophytic to the parasitic growth phase is due to a single factor or to multiple factors, but the data presented herein strongly suggests that the tonicity of the growth environment and the presence of sulfhydryl groups may be the most critical factors in formation of spherules by C. immitis. ACKNOWLEDGMENTS The authors wish to express appreciation to Dr. N. D. Gary for comments and suggestions during the preparation of this manuscript and to Mr. George A. Evans for preparing the photomicrographs. SUMMARY Some of the nutritional requirements for iinproved spherulation of Coccidioides immitis in a chemically defined medium have been investigated. Optimal spherule formation occurred in atmospheres containing 20 per cent oxygen. In defined medium with low total solids content, a requirement for added carbon dioxide was not demonstrated. Stimulation of spherulation resulted from the addition of (a) sodium and calcium ions, (b) sodium bicarbonate, (c) the sulfhydryl compound glutathione, (d) the fatty acids, oleic and linoleic, and (e) the action of such metabolic inhibitors as sodium fluoride, copper sulfate, sulfaguanidine, and lithium chloride. The sulfhydryl inhibitors, p-chloromercuribenzoate, and iodoacetate inhibited spherulation and this inhibition was reversed by the addition of glutathione. The anionic surface active compound, Tamol "N," was more effective in stimulating growth, spherulation, spherule rupture, and dispersal of endospores than any other surface active compound tested. A combination of Tamol "N" and sodium fluoride resulted in practically pure suspensions of intact spherules. REFERENCES BAKER, 0. AND BRAUDE, A. I A study of stimuli leading to the production of spherules in coccidioidomycosis. J. Lab. Clin. Med., 47, BULLEN, J. J The yeast like form of Cryptococcus farciminosus (Rivolta): (Histoplasma farcimonosum). J. Pathol. Bacteriol., 61, CONANT, N. F A cultural study of the life-cycle of Histoplasma capsulatum Darling J. Bacteriol., 41,

9 1959] NUTRITION OF C. IMMITIS 239 CONANT, N. F. AND VOGEL, R. A The parasitic growth phase of Coccidioides immitis in culture. Mycologia, 46, CONVERSE, J. L Growth of spherules of Coccidioides immitis in a chemically defined liquid medium. Proc. Soc. Exptl. Biol. Med., 90, CONVERSE, J. L The effect of physicochemical environment on spherulation of Coccidioides immitis in a chemically defined medium. J. Bacteriol., 72, CONVERSE, J. L Effect of surface active agents on endosporulation of Coccidioides immitis in a chemically defined medium. J. Bacteriol., 74, LARSH, H. W., HINTON, A., AND SELBERG, S. L Conversion and maintenance of Histoplasma capsulatum in tissue culture. Proc. Soc. Exptl. Biol. Med., 93, LEVINE, S. AND ORDAL, Z. J Factors influencing the morphology of Blastomyces dermatitidis. J. Bacteriol., 52, LUBARSKY, R. AND PLUNKETT, 0. A In vitro production of the spherule phase of Coccidioides immitis. J. Bacteriol., 70, NEIMAN, C Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriol. Revs., 18, NICKERSON, W. J Physiological basis of morphogenesis in animal disease fungi. Trans. N. Y. Acad. Sci., 13, NICKERSON, W. J. AND ROMANO, A. H Enzymatic reduction of cysteine by coenzyme I. Science, 115, NICKERSON, W. J. AND VAN RIJ, N. J. W The effect of sulfhydryl compounds, penicillin, and cobalt on the cell division mechanism of yeasts. Biochem. Biophys. Acta, 3, NORDEN, A Sporotrichosis; clinical and laboratory features and a serological study in experimental animals and humans. Acta. Pathol. Microbiol. Scand. Suppl., 89, ROESSLER, W. G., HERBST, E. J., MCCULLOUGH, W. G., MILLS, R. C., AND BREWER, C. R Studies with Coccidioides immitis. I. Submerged growth in liquid mediums. J. Infectious Diseases, 79, SALVIN, S. B. 1949a Phase-determining factors in Blastomyces dermatitidis. Mycologia, 41, SALVIN, S. B. 1949b Cysteine and related compounds in the growth of the yeast-like phase of Histoplasma capsulatum. J. Infectious Diseases, 84, UMBREIT, W. W., BURRIS, R. H., AND STAUFFER. J. F Manometric techniques, revised ed. Burgess Publishing Co., Minneapolis. Downloaded from on January 31, 2019 by guest