INFECTION AND IMMUNITY, Mar. 1976, p. 830-835 Copyright © 1976 American Society for Microbiology
Vol. 13, No. 3 Printed in U.S.A.
Germination of Candida albicans Induced by Proline NINA DABROWA, SHARON S. S. TAXER, AND DEXTER H. HOWARD* Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, California 90024
Received for publication 30 September 1975
Blastospores of Candida albicans germinated in a proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glutamate, aspartate, and pyruvate families also fostered germination but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of Lproline inhibited proline-stimulated germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts. The blastospores of Candida albicans germinate when incubated at 37 C in serum or plasma from humans and other animals (6, 8, 19), and this fact has been developed into a useful diagnostic test for identification of yeast isolates from clinical materials (3, 5, 15, 20, 23). Landau et al. (12) established that tissue culture medium 199 (TC 199) supports germ tube production by C. albicans nearly as well as does serum. Although there is no evidence that the ingredients of serum which fostered germination are the same as those in medium 199, it was obviously tempting to work with a chemically defined medium to discover which component(s) supported germination. Therefore, Dabrowa studied the ability of each amino acid ingredient of TC 199 to support germ tube production in C. albicans, and eventually reported that proline was virtually the only amino acid to stimulate good germination (N. Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52). This fact was also established by Nancy Pickering working independently in another laboratory (unpublished observation). Recently Land et al. (10) reported that proline and several other amino acids, chiefly of the glutamate, aspartate, and pyruvate families, stimulate germination of C. albicans. Furthermore, these workers have advanced the hypothesis that morphogenesis of the mycelial phase of growth in C. albicans is correlated with repression of mitochondrial activity (10, 11). This suggestion integrates a large number of fragmentary observations on incitants of filamentation in C. albicans (11). The purpose of this report is to publish data
originally presented in abstract form (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) which are confirmed by the results of Land et al. (10), to present new data which show that isolates vary in their response to different amino acids, and to indicate that a simple amino acid-buffer medium cannot be substituted for serum or TC 199 in a diagnostic test to assess germ tube production by clinical isolates of yeasts. (This paper is taken in part from a thesis submitted by S.S.S.T. to the University of California, Los Angeles, in partial fulfillment of the requirements for the M.S. degree.) MATERIALS ANI) METHODS Fungi. Two stock cultures of C. albicans were employed. Strain H was from the culture collection of the Division of Dermatology, Department of Medicine, UCLA, which was used in previous studies (Dabrowa, Proc. V. Cong. Int. Soc. Hum. Anim. Mycol., 1971, p. 51-52; 2); and strain 304 was from the culture collection of the Department of Microbiology and Immunology, UCLA, which was used by Pickering in her unpublished experiments alluded to above. Additional clinical isolates of C. albicans were obtained from the Mycology Division of the Clinical Laboratories, UCLA Hospital. All of these isolates formed germ tubes in human serum and conformed to accepted criteria for the identification of C. albicans (22). Media. Glucose-peptone agar (GPA) contained 2% glucose (Difco Laboratories, Detroit, Mich.), 1% neopeptone (Difco), and 2% agar (Difco). Glucose-peptone broth (GPB) had the same recipe as GPA except for the omission of agar. A glucose-ammonium sulfate-salts medium (CM), originally devised by Janson and Nickerson (9), consisted of: KH2PO4, 3 g;
830
GERMINATION OF C. ALBICANS
VOL. 13, 1976 (NH4)2SO4, 3 g; CaCl, 0.25 g; MgSO4-7H2O, 0.25 g; ZnSO4 7H2O, 5 mg; Fe (NH4)2(S04)2-6H2O, 15 mg; glucose, 20 g; biotin (Nutritional Biochemicals Corp., Cleveland, Ohio), 5 mg; water, 1 liter. TC 199 (Hyland Laboratories, Los Angeles, Calif.) was used as the positive control medium in most experiments. Amino acids and analogues were obtained from Calbiochem (Los Angeles, Calif.), and were dissolved at a concentration of 0.01 M in 0.033 M phosphate buffer (PB) containing 10 ,g of biotin/liter. Preparation of inocula. Twenty milliliters of CM or GPB contained in 150-ml flasks was inoculated with growth from a 24-h GPA culture of C. albicans. The flasks were incubated at 37 C on a gyratory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) for 24 h. The blastospores were harvested by centrifugation, washed three times with sterile distilled water, and suspended in PB (pH 7.2). Cells were counted in a Neubauer hemocytometer and the desired densities were prepared in PB. Germination tests. Tubes containing 0.5 ml of PB with individual amino acids or TC 199 were inoculated with 0.05 ml of a suspension of blastospores of C. albicans in PB. The tubes were incubated at 37 C for 3 h without agitation. All experimental situations were run in duplicate and each experiment was repeated at least three times. After 3 h of incubation, the contents of the tubes was agitated and a drop was emulsified with a drop of 0.05% trypan blue in lactophenol. A cover slip was added and the percentage of germ tubes was assessed. The results from duplicate tubes of the same experimental situation were averaged. Procedures which vary slightly from those detailed in this section will be explained with the results. RESULTS
Preliminary observation. Blastospores of C. albicans germinate when incubated at 37 C in TC 199 (12). This medium was used as the positive control in most experiments. Accordingly, a few preliminary studies were performed to determine the optimum conditions for germination of C. albicans in TC 199. The results of such studies will be presented in this section but will not be documented in detail. Germination was not appreciably altered by the pH of TC 199 over the range pH 6.0 to 8.0. For convenience, therefore, TC 199 and the PB were used at pH 7.0 to 7.2. Three hours was chosen as the termination time of the experiments because very few additional germ tubes appeared after that time under the conditions used. A series of experiments established that 0.01 M was the optimum concentration for the amino acids in the biotin-PB medium. The density of the inoculum was important. The highest percentage of germination was observed at the lowest cell concentration (1 x 10i cells/ml). Very dense populations of yeasts (1 x 10" cells/ml) did not germinate well. A final
831
concentration of 1 x 107 cells/ml was chosen because a high percentage of germination (70 to 90%) occurred at this density and because it provided a sufficient number of cells to facilitate rapid assay of results. The medium in which yeast cells are grown before preparation for use in experiments was critical. Although this point was not exhaustively explored, it was obvious that the percentage of germination of C. albicans in TC 199 was consistently higher when yeast cells were harvested from GPB than it was when they were harvested from CM. Stock cultures may undergo changes over periods of time which alter their response to various stimuli of germination. Strain H, which was used in the studies reported by Dabrowa (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52), did not give as high a percentage of germination in TC 199 or various amino acids as it had when first studied. Another strain, 304, which had behaved consistently over the years was, therefore, used in establishing the conditions described in this section. The strain designation or isolate number will be noted in the results reported in the remaining portions of this paper. Germination of C. albicans in amino acids. The percentage of yeast cells which germinated by 3 h in PB containing various amino acids is recorded in Table 1. The amino acids chosen for the table were those that fostered the highest percentages of germination in these particular experiments. Sixteen other amino acids supported either no germination or very low percentages. The results in Table 1, obtained with strain 304, correspond closely to those reported earlier in abstract form by Dabrowa with strain H (Dabrowa, Proc. V. Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) and to those of Land et al. (10). Proline consistently supported the highest percentage of germination. Inhibitors of germination. Various proline TABLE 1. Germination of Candida albicans, 304, incubated in various amino acids in PB-biotin medium Amino acid (0.01 M)
Percentage of germination
L-Arginine L-Alanine L-Glutamine L-Glutamic acid L-Aspartic acid L-Proline
18.0 12.0 20.0 44.0 20.0 86.0
PB
0 97.0
TC 199
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DABROWA, TAXER, AND HOWARD
analogues and other potential inhibitors were studied for their ability to ameliorate or abolish proline-stimulated germination of C. albicans. The substances were added at several different concentrations to PB containing 0.01 M proline and the percentage of germination was recorded after 3 h at 37 C. The results are recorded in Table 2. At a concentration of 0.01 M all the potential inhibitors chosen reduced the amount of germination fostered by proline. The inhibitory effect diminished or disappeared as the concentration of the inhibitor was decreased (Table 2). It was consistently noted that levels of glucose from 0.001 to 0.28 M markedly diminished or abolished the proline stimulation of germination. Concentrations of phosphate from 0.1 to 0.5 M and of ammonium ion from 0.0002 to 0.2 M also depressed germination by C. albicans in the proline medium. Variation in response to proline stimulation by various stock cultures and clinical isolates. Dabrowa found that only three of the eight stock cultures maintained in her collection formed significant numbers of germ tubes in the proline-PB medium (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 5152). All eight cultures germinated promptly in serum or in TC 199. Working with another set of stock cultures of C. albicans, Pickering (unpublished observation) found five out of seven cultures germinated in proline. Again all seven cultures germinated in serum. Fifteen clinical isolates of C. albicans were collected and tested for their ability to germiTABLE 2. Inhibition ofproline-stimulated germination of Candida albicans (strain H)a Percentage of germination' Inhibitor
-
L-Cysteine D-Proline 4-Thiazolidinecarboxylic acid L-Azetidine-2 carboxylic acid
10-2 M
10-3 M
10-4 M
12 12 32
35 50
50 61 55
20
47
52
55 Controld 55 55 a Data originally reported by Dabrowa at the General Meeting of the International Society for Human and Animal Mycology, Paris, August, 1971. t Inhibitors alone (0.01 M) did not stimulate ger-
mination of C. albicans. X Percentage of yeast cells with germ tubes after 3 h at 37 C in proline-PB medium with the inhibitor at indicated concentrations. d Control is biotin-PB medium with 0.01 M L-
proline.
INFECT. IMMUN.
nate in buffer with 22 individual amino acids (Table 3). Proline was the only amino acid which consistently fostered the germination of a large percentage of isolates. One isolate, 2100, gave a very low percentage of germination in proline. Four isolates (2218, 1725A, 2087, 2086) were stimulated to germinate by proline alone, whereas isolate 2065A was stimulated to some degree by a rather large number of different amino acids. The other isolates showed different patterns of germ tube stimulation but, in keeping with the results of Land et al. (10), amino acids entering metabolism by conversion to glutamate were more often effective than other groups. A large number of clinical isolates were collected to assess the usefulness of a PB-proline medium substituted for serum or TC 199 in a germ tube test. The conditions of a rapid screening test like that proposed by Dolan and Ihrke (3) were used in the survey. Such a screening test cannot include the carefully controlled adjustment of inocula used to obtain the data shown in Table 3. Under these less wellcontrolled circumstances, 40% of several hundred clinical isolates, which germinated in human serum, did not form germ tubes in the proline-PB medium. Furthermore, the percentage of germination among the 60% that did germinate was frequently quite low.
DISCUSSION C. albicans reproduces by budding when grown on most routine laboratory media (13, 26). Under certain conditions, however, blastospores of this fungus germinate and thereby initiate a mycelial form of growth (12, 13, 14, 21). Many investigators have set about to describe environmental factors that control this expression of dimorphism. In some instances there are apparent contradictions among reported results (10, 16, 18). One possible source of confusion is the failure to recognize that when authors speak of "filamentation" by C. albicans some are referring to pseudohyphal formation (16) whereas others (19, 23) mean germ tube formation, and still others signify a combination of both germination and pseudohyphal formation with the term (10). Blastospore germination in C. albicans has characteristics that have led some to refer to it as pseudo-germ tube formation (15), and the subsequent growth of germ tubes involves pseudohyphal formation and budding. Nevertheless, referring to both pseudohyphal formation and germ tube formation as "filamentation" might lead to some confusion because it is clear that proline fosters germination (10) but
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833
TABLE 3. Germination of clinical isolates of Candida albicans in amino acids Amino acids (0.02 M)
L-Lysine HCI L-Valine DL-Serine DL-Methionine L-ISOleucine Glycine L-Cysteine HCI L-Arginine L-Phenylalanine L-Histidine DL-Threonine L-Alanine
a
Germinationa of clinical isolates: _ 2282 2286 1956 1725A 2087 2065A 1968 2100 9292 2086 734 2059 1792 1966 1580C
L-Glutamine L-Leucine L-Tryptophan L-Glutamic acid L-Aspartic acid L-Cystine L-Tyrosine L-Ornithine HCI L-Proline OH-Proline
0 0 2 0 0 0 0 2 0 0 0 6 1 0 0 2 0 0 0 0 70 0
2 0 4 0 2 4 0 8 2 2 1 27 2 0 1 0 0 0 0 0 49 0
2 8 10 1 1 2 6 30 3 3 2 56 22 3 2 4 0 2 0 2 74 14
0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 24 0
PB TC 199
0 83
0 68
0 96
0 99
0 0 0 0 0 0 0
0 0 46 0
2 26 12 17 8 11 0 62 19 2 43 29 60 24 32 70 34 34 15 50 78 64
0 0 0 0 0 0 0 24 0 0 0 10 8 0 0 10 6 0 0 2 80 1
0 81
4 98
0 99
0 0 0 0 4 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 16 0
4 4 4 4 4 8 3 8 3 2 5 32 14 0 0 22 7 4 4 4 56 16
0 4 0 0 0 16 3 0 0 3 5 2 0 8 86 6
0 92
1 82
0 98
1
0 0 0 0 0 0
1 1 6 2 3 8 12
14 53 32
2 4 5 0 3 10 15 15 8 1 6 15 15 6 5 26 6 23 6 16 38 26
3 0 3 0 2 10 4 0 8 0 2 0 4 0 31 22 2 5 0 10 4 1 11 8 48 5 2 0 1 0 18 4 2 0 2 1 1 2 8 0 80 60 8 1
4 70
2 62
4 93
18 8 0 9 31 28 2 3 21 2 8 3
0 99
1 2 0 0 2 1 2 8 2 0 3 17 30 2 0 39 16 0 2 7 78 10
0 97
Results shown as percentage of germination under conditions of the test (see Materials and Methods).
does not stimulate pseudohyphal formation (16). However, even when one is careful to distinguish between the two obvious types of "filaments" expressed by C. albicans, there are still instances in which different results have been obtained by different workers under seemingly similar experimental circumstances. The one constant feature among these various results is strain variation. For example, Mardon et al. (17) studied a number of strains of C. albicans which were stimulated to germinate by L-alpha-amino-n-butyric acid, but we were not able to observe this effect with three stock cultures in one of our collections (unpublished observation). Evans et al. (4) have consistently observed germination on a glucose-peptone medium whereas we routinely use a similar medium for production of large yields of blastospores devoid of germ tubes (see Materials and Methods). There are other examples of such discrepancies and one can always rely on slight differences in media recipes or culture conditions to overcome these apparent contradictions. However, the results of the present study point up yet another important variable, i.e., strain variation, which may be important in determining the divergence of results among investigators. The data in Table 3 reveal a considera-
ble difference in response to stimulation by different amino acids among clinical isolates. This result helps to explain how an individual amino acid could be singled out as a promoter of germination in one instance, whereas a family of amino acids might be similarly recognized if a different isolate had been employed in the experiments. The data in this report establish that proline is prominent among amino acids for its ability to stimulate germ tube formation by blastospores of C. albicans. This fact was originally reported in abstract form by Dabrowa (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52), and was greatly extended by Land et al. (10, 11). This amino acid also stimulates germination of the sporangiospores of Rhizopus stolonifer and R. arrhizus (25) and the conidiospores of Aspergillus niger (28). Land et al. (10) report that the amino acids entering metabolism by conversion to glutamate are generally good stimulators of germination, and this fact was also noted in our results. Analogues of proline or the D-isomer markedly reduced germination of C. albicans in the proline-PB medium. These observations augment the conclusions of Land et al. (10) that normal proline catabolism is involved in its
834
DABROWA, TAXER, AND HOWARD
stimulation of germination. In addition, our results correspond to those of Weber and Ogawa (25) who observed that analogues of proline did not stimulate germination in R. arrhizus spores. In keeping with theories developed by Nickerson and his colleagues (for review see reference 1), addition of cysteine to the proline-containing medium prevented germination of the blastospores probably by fostering budding. However, Wain et al. (24) did not observe inhibition of germination of C. albicans in plasma containing 10-2 M cysteine, and the role of sulfur-containing amino acid in the morphogenesis of C. albicans is reopened by their contradictory findings. Dabrowa previously reported (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) that glucose suppressed germination in a proline-PB medium. This observation was repeated several times. However, Land et al. (10) have shown that high levels of glucose (.0.1 M) promote germination in a NH4Cl medium and that 0.01 M glucose does not suppress proline-stimulated germination. Moreover, TC 199, which supports germination of C. albicans nearly as well as serum or plasma, contains 0.006 M glucose, a level which suppressed proline-stimulated germination in our experiments (see Results). This contradiction is probably related to the phosphate concentration of the various media. Thus, Land et al. (11) relate the effects of glucose, as well as those of proline and other environmental factors, to a Crabtree-like response, i.e., repression of mitochondrial activity. Such repression is also related to phosphate concentrations (see reference 9 for a discussion of the literature on these relationships). For example, it has been shown in mammalian systems that the repressing effect of glucose on respiration is antagonized by 0.01 M phosphate in the medium (9). It thus seems possible that our failure to observe the same effects of glucose on germination as those reported by Land et al. (10) was related to the fact that our buffer medium (0.033 M) had four times as much phosphate as did theirs (0.008 M). Indeed, the latter workers noted negligible amounts of germination in their prolineglucose-biotin medium when it contained 0.1 and 0.01 M phosphate. The phosphate concentration in TC 199 is much lower (0.0008 M) than that used in our buffer or that of Land et al. (10). Thus either differences in phosphate concentration in the medium or differences in transport of phosphate by the strains used are likely explanations for the seemingly divergent results. In keeping with this suggestion is the observation by Widra that the glucose-to-inor-
INFECT. IMMUN.
ganic phospate ratio is crucial to pseudohyphal formation by C. albicans in Sabouraud dextrose agar (27). Land et al. have extended their original observations (10) and provided additional evidence that germination of C. albicans is correlated with a repression of mitochondrial activity (11). Any environmental circumstance fostering such repression is likely to stimulate germination. Accordingly, the observations of Land et al. (10, 11) have provided a means of correlating a great many different and, at times, apparent diverse observations. Under carefully controlled conditions it is possible to show that a large percentage of clinical isolates of C. albicans germinate in a proline-PB medium (see Table 3). However, a simple amino acid medium will not substitute for serum or TC 199 in a useful diagnostic test where such factors as cell concentration cannot be rigorously controlled (3). The obvious practical conclusion to be derived from our work is that the proline-PB medium is unsuitable for routine diagnostic work. ACKNOWLEDGMENTS We are indebted to M. Sakamoto and Jennie Pang for excellent technical assistance. We thank Evelyn Graham and Judith Rhodes for the supply of clinical isolates of C. albicans, and we acknowledge the fact that some of the work was performed in a laboratory at the Veterans Administration Hospital, Sawtelle, Calif., the use of which was temporarily extended to us by E. T. Wright, Chief of Staff. S.S.S.T. was supported by Public Health Service grant AI-07461-09 from the National Institute of Allergy and Infectious Diseases. Other aspects of the study were partially supported by Public Health Service grant AI-10478-03 from the National Institute of Allergy and Infectious Diseases.
ADDENDUM Recently, a paper was published by K. L. Lee, H. R. Buckley, and C. C. Campbell on germination of C. albicans in a liquid synthetic medium (Sabouraudia 13:148-153). LITERATURE CITED 1. Bartnicki-Garcia, S., and I. McMurrough. 1971. Biochemistry of morphogenesis in yeast, p. 441-491. In A. H. Rose and J. S. Harrison (ed.). The yeasts, vol. 2. Academic Press, Inc., New York. 2. Dabrowa, N., D. H. Howard, J. W. Landau, and Y. Shechter. 1970. Synthesis of nucleic acids and proteins in the dimorphic forms of Candida albicans. Sabouraudia 8:163-169. 3. Dolan, C. T., and D. M. Ihrke. 1971. Further studies of the germ-tube test for Candida albicans identification. Am. J. Clin. Pathol. 55:733-734. 4. Evans, E. G. V., F. C. Odds, M. D. Richardson, and K. T. Holland. 1975. Optimum conditions for incitation of filamentation in Candida albicans. Can. J. Microbiol. 21:338-342. 5. Fahlberg, W. J., C. D. Dukes, and R. K. Guthrie. 1957. Rapid classification of Candida (monilia) albicans. J. Invest. Dermatol. 29:111-118. 6. Hu, F., C. S. Livingood, P. Johnson, and C. M. Pom-
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8.
9. 10.
11.
12. 13. 14. 15.
16. 17.
erat. 1954. Tissue culture studies on human skin. Arch. Dermatol. 70:1-15. Janson, V. K., and W. J. Nickerson. 1970. Chemical composition of chlamydospores of Candida albicans. J. Bacteriol. 104:910-921. Johnson, S. A. M. 1954. Candida (monilia) albicans. Arch. Dermatol. 70:49-60. Koobs, D. H. 1972. Phosphate mediation of the Crabtree and Pasteur effects. Science 178:127-133. Land, G. A., W. C. McDonald, R. L. Stjernholm, and L. Friedman. 1975. Factors affecting filamentation in Candida albicans: relationship of the uptake and distribution of proline to morphogenesis. Infect. Immun. 11:1014-1023. Land, G. A., W. C. McDonald, R. L. Stjernholm, and L. Friedman. 1975. Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation. Infect. Immun. 12:119-127. Landau, J. W., N. Dabrowa, and V. D. Newcomer. 1965. The rapid formation in serum of filaments by Candida albicans. J. Invest. Dermatol. 44:171-179. Lodder, J., and N. J. W. Kreger-van Rij. 1952. The yeasts: a taxonomic study. North Holland Publishing Co., Amsterdam. McClary, D. 0. 1952. Factors affecting the morphology of Candida albicans. Ann. Mo. Bot. Gard. 39:137-164. Mackenzie, D. W. R. 1962. Serum tube identification of Candida albicans. J. Clin. Pathol. 15:563-565. Mardon, D., E. Balish, and A. W. Phillips. 1969. Control of dimorphism in a biochemical variant of Candida albicans. J. Bacteriol. 100:701-707. Mardon, D. N., S. K. Hurst, and E. Balish. 1971. Germtube production by Candida albicans in minimal liquid culture media. Can. J. Microbiol. 17:851-856.
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18. Nickerson, W. J., and C. W. Chung. 1953. Cellular division in a mutant yeast. Am. J. Bot. 41:114-120. 19. Reynolds, R., and A. Braude. 1956. The filament-inducing property of blood for Candida albicans: its nature and significance. Clin. Res. Proc. 4:40-47. 20. Ridley, M. F. 1960. A comparison of methods for identification of Candida albicans. Aust. J. Dermatol. 5:209-213. 21. Scherr, G. H., and R. H. Weaver. 1953. The dimorphism of yeasts. Bacteriol. Rev. 17:51-92. 22. Silva-Hutner, M., and B. H. Cooper. 1974. Medically important yeasts, p. 491-507. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 23. Taschdjian, C. L., J. J. Burchall, and P. J. Kozinn. 1960. Rapid identification ofCandida albicans by filamentation on serum and serum substitutes. J. Dis. Child. 99:212-215. 24. Wain, W. H., M. F. Price, and R. A. Cawson. 1975. A re-evaluation of the effect of cysteine on Candida albicans. Sabouraudia 12:74-82. 25. Weber, D. J., and J. M. Ogawa. 1965. The specificity of proline in the germination of spores of Rhizopus arrhizus. Phytopathology 55:262-266. 26. Wickerham, L. J., and L. F. Rettger. 1939. A taxonomic study of Monilia albicans with special emphasis on morphology and morphological variation. J. Trop. Med. Hyg. 42:174-177, 187-192, 204-216. 27. Widra, A. 1964. Phosphate directed Y-M variation in Candida albicans. Mycopathol. Mycol. Appl. 23:197202. 28. Yanagita, T. 1957. Biochemical aspects in the germination of conidiospores of Aspergillus niger. Arch. Microbiol. 26:329-344.
Vol. 13, No. 3 Printed in U.S.A.
Germination of Candida albicans Induced by Proline NINA DABROWA, SHARON S. S. TAXER, AND DEXTER H. HOWARD* Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, California 90024
Received for publication 30 September 1975
Blastospores of Candida albicans germinated in a proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glutamate, aspartate, and pyruvate families also fostered germination but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of Lproline inhibited proline-stimulated germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts. The blastospores of Candida albicans germinate when incubated at 37 C in serum or plasma from humans and other animals (6, 8, 19), and this fact has been developed into a useful diagnostic test for identification of yeast isolates from clinical materials (3, 5, 15, 20, 23). Landau et al. (12) established that tissue culture medium 199 (TC 199) supports germ tube production by C. albicans nearly as well as does serum. Although there is no evidence that the ingredients of serum which fostered germination are the same as those in medium 199, it was obviously tempting to work with a chemically defined medium to discover which component(s) supported germination. Therefore, Dabrowa studied the ability of each amino acid ingredient of TC 199 to support germ tube production in C. albicans, and eventually reported that proline was virtually the only amino acid to stimulate good germination (N. Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52). This fact was also established by Nancy Pickering working independently in another laboratory (unpublished observation). Recently Land et al. (10) reported that proline and several other amino acids, chiefly of the glutamate, aspartate, and pyruvate families, stimulate germination of C. albicans. Furthermore, these workers have advanced the hypothesis that morphogenesis of the mycelial phase of growth in C. albicans is correlated with repression of mitochondrial activity (10, 11). This suggestion integrates a large number of fragmentary observations on incitants of filamentation in C. albicans (11). The purpose of this report is to publish data
originally presented in abstract form (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) which are confirmed by the results of Land et al. (10), to present new data which show that isolates vary in their response to different amino acids, and to indicate that a simple amino acid-buffer medium cannot be substituted for serum or TC 199 in a diagnostic test to assess germ tube production by clinical isolates of yeasts. (This paper is taken in part from a thesis submitted by S.S.S.T. to the University of California, Los Angeles, in partial fulfillment of the requirements for the M.S. degree.) MATERIALS ANI) METHODS Fungi. Two stock cultures of C. albicans were employed. Strain H was from the culture collection of the Division of Dermatology, Department of Medicine, UCLA, which was used in previous studies (Dabrowa, Proc. V. Cong. Int. Soc. Hum. Anim. Mycol., 1971, p. 51-52; 2); and strain 304 was from the culture collection of the Department of Microbiology and Immunology, UCLA, which was used by Pickering in her unpublished experiments alluded to above. Additional clinical isolates of C. albicans were obtained from the Mycology Division of the Clinical Laboratories, UCLA Hospital. All of these isolates formed germ tubes in human serum and conformed to accepted criteria for the identification of C. albicans (22). Media. Glucose-peptone agar (GPA) contained 2% glucose (Difco Laboratories, Detroit, Mich.), 1% neopeptone (Difco), and 2% agar (Difco). Glucose-peptone broth (GPB) had the same recipe as GPA except for the omission of agar. A glucose-ammonium sulfate-salts medium (CM), originally devised by Janson and Nickerson (9), consisted of: KH2PO4, 3 g;
830
GERMINATION OF C. ALBICANS
VOL. 13, 1976 (NH4)2SO4, 3 g; CaCl, 0.25 g; MgSO4-7H2O, 0.25 g; ZnSO4 7H2O, 5 mg; Fe (NH4)2(S04)2-6H2O, 15 mg; glucose, 20 g; biotin (Nutritional Biochemicals Corp., Cleveland, Ohio), 5 mg; water, 1 liter. TC 199 (Hyland Laboratories, Los Angeles, Calif.) was used as the positive control medium in most experiments. Amino acids and analogues were obtained from Calbiochem (Los Angeles, Calif.), and were dissolved at a concentration of 0.01 M in 0.033 M phosphate buffer (PB) containing 10 ,g of biotin/liter. Preparation of inocula. Twenty milliliters of CM or GPB contained in 150-ml flasks was inoculated with growth from a 24-h GPA culture of C. albicans. The flasks were incubated at 37 C on a gyratory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) for 24 h. The blastospores were harvested by centrifugation, washed three times with sterile distilled water, and suspended in PB (pH 7.2). Cells were counted in a Neubauer hemocytometer and the desired densities were prepared in PB. Germination tests. Tubes containing 0.5 ml of PB with individual amino acids or TC 199 were inoculated with 0.05 ml of a suspension of blastospores of C. albicans in PB. The tubes were incubated at 37 C for 3 h without agitation. All experimental situations were run in duplicate and each experiment was repeated at least three times. After 3 h of incubation, the contents of the tubes was agitated and a drop was emulsified with a drop of 0.05% trypan blue in lactophenol. A cover slip was added and the percentage of germ tubes was assessed. The results from duplicate tubes of the same experimental situation were averaged. Procedures which vary slightly from those detailed in this section will be explained with the results. RESULTS
Preliminary observation. Blastospores of C. albicans germinate when incubated at 37 C in TC 199 (12). This medium was used as the positive control in most experiments. Accordingly, a few preliminary studies were performed to determine the optimum conditions for germination of C. albicans in TC 199. The results of such studies will be presented in this section but will not be documented in detail. Germination was not appreciably altered by the pH of TC 199 over the range pH 6.0 to 8.0. For convenience, therefore, TC 199 and the PB were used at pH 7.0 to 7.2. Three hours was chosen as the termination time of the experiments because very few additional germ tubes appeared after that time under the conditions used. A series of experiments established that 0.01 M was the optimum concentration for the amino acids in the biotin-PB medium. The density of the inoculum was important. The highest percentage of germination was observed at the lowest cell concentration (1 x 10i cells/ml). Very dense populations of yeasts (1 x 10" cells/ml) did not germinate well. A final
831
concentration of 1 x 107 cells/ml was chosen because a high percentage of germination (70 to 90%) occurred at this density and because it provided a sufficient number of cells to facilitate rapid assay of results. The medium in which yeast cells are grown before preparation for use in experiments was critical. Although this point was not exhaustively explored, it was obvious that the percentage of germination of C. albicans in TC 199 was consistently higher when yeast cells were harvested from GPB than it was when they were harvested from CM. Stock cultures may undergo changes over periods of time which alter their response to various stimuli of germination. Strain H, which was used in the studies reported by Dabrowa (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52), did not give as high a percentage of germination in TC 199 or various amino acids as it had when first studied. Another strain, 304, which had behaved consistently over the years was, therefore, used in establishing the conditions described in this section. The strain designation or isolate number will be noted in the results reported in the remaining portions of this paper. Germination of C. albicans in amino acids. The percentage of yeast cells which germinated by 3 h in PB containing various amino acids is recorded in Table 1. The amino acids chosen for the table were those that fostered the highest percentages of germination in these particular experiments. Sixteen other amino acids supported either no germination or very low percentages. The results in Table 1, obtained with strain 304, correspond closely to those reported earlier in abstract form by Dabrowa with strain H (Dabrowa, Proc. V. Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) and to those of Land et al. (10). Proline consistently supported the highest percentage of germination. Inhibitors of germination. Various proline TABLE 1. Germination of Candida albicans, 304, incubated in various amino acids in PB-biotin medium Amino acid (0.01 M)
Percentage of germination
L-Arginine L-Alanine L-Glutamine L-Glutamic acid L-Aspartic acid L-Proline
18.0 12.0 20.0 44.0 20.0 86.0
PB
0 97.0
TC 199
832
DABROWA, TAXER, AND HOWARD
analogues and other potential inhibitors were studied for their ability to ameliorate or abolish proline-stimulated germination of C. albicans. The substances were added at several different concentrations to PB containing 0.01 M proline and the percentage of germination was recorded after 3 h at 37 C. The results are recorded in Table 2. At a concentration of 0.01 M all the potential inhibitors chosen reduced the amount of germination fostered by proline. The inhibitory effect diminished or disappeared as the concentration of the inhibitor was decreased (Table 2). It was consistently noted that levels of glucose from 0.001 to 0.28 M markedly diminished or abolished the proline stimulation of germination. Concentrations of phosphate from 0.1 to 0.5 M and of ammonium ion from 0.0002 to 0.2 M also depressed germination by C. albicans in the proline medium. Variation in response to proline stimulation by various stock cultures and clinical isolates. Dabrowa found that only three of the eight stock cultures maintained in her collection formed significant numbers of germ tubes in the proline-PB medium (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 5152). All eight cultures germinated promptly in serum or in TC 199. Working with another set of stock cultures of C. albicans, Pickering (unpublished observation) found five out of seven cultures germinated in proline. Again all seven cultures germinated in serum. Fifteen clinical isolates of C. albicans were collected and tested for their ability to germiTABLE 2. Inhibition ofproline-stimulated germination of Candida albicans (strain H)a Percentage of germination' Inhibitor
-
L-Cysteine D-Proline 4-Thiazolidinecarboxylic acid L-Azetidine-2 carboxylic acid
10-2 M
10-3 M
10-4 M
12 12 32
35 50
50 61 55
20
47
52
55 Controld 55 55 a Data originally reported by Dabrowa at the General Meeting of the International Society for Human and Animal Mycology, Paris, August, 1971. t Inhibitors alone (0.01 M) did not stimulate ger-
mination of C. albicans. X Percentage of yeast cells with germ tubes after 3 h at 37 C in proline-PB medium with the inhibitor at indicated concentrations. d Control is biotin-PB medium with 0.01 M L-
proline.
INFECT. IMMUN.
nate in buffer with 22 individual amino acids (Table 3). Proline was the only amino acid which consistently fostered the germination of a large percentage of isolates. One isolate, 2100, gave a very low percentage of germination in proline. Four isolates (2218, 1725A, 2087, 2086) were stimulated to germinate by proline alone, whereas isolate 2065A was stimulated to some degree by a rather large number of different amino acids. The other isolates showed different patterns of germ tube stimulation but, in keeping with the results of Land et al. (10), amino acids entering metabolism by conversion to glutamate were more often effective than other groups. A large number of clinical isolates were collected to assess the usefulness of a PB-proline medium substituted for serum or TC 199 in a germ tube test. The conditions of a rapid screening test like that proposed by Dolan and Ihrke (3) were used in the survey. Such a screening test cannot include the carefully controlled adjustment of inocula used to obtain the data shown in Table 3. Under these less wellcontrolled circumstances, 40% of several hundred clinical isolates, which germinated in human serum, did not form germ tubes in the proline-PB medium. Furthermore, the percentage of germination among the 60% that did germinate was frequently quite low.
DISCUSSION C. albicans reproduces by budding when grown on most routine laboratory media (13, 26). Under certain conditions, however, blastospores of this fungus germinate and thereby initiate a mycelial form of growth (12, 13, 14, 21). Many investigators have set about to describe environmental factors that control this expression of dimorphism. In some instances there are apparent contradictions among reported results (10, 16, 18). One possible source of confusion is the failure to recognize that when authors speak of "filamentation" by C. albicans some are referring to pseudohyphal formation (16) whereas others (19, 23) mean germ tube formation, and still others signify a combination of both germination and pseudohyphal formation with the term (10). Blastospore germination in C. albicans has characteristics that have led some to refer to it as pseudo-germ tube formation (15), and the subsequent growth of germ tubes involves pseudohyphal formation and budding. Nevertheless, referring to both pseudohyphal formation and germ tube formation as "filamentation" might lead to some confusion because it is clear that proline fosters germination (10) but
GERMINATION OF C. ALBICANS
VOL. 13, 1976
833
TABLE 3. Germination of clinical isolates of Candida albicans in amino acids Amino acids (0.02 M)
L-Lysine HCI L-Valine DL-Serine DL-Methionine L-ISOleucine Glycine L-Cysteine HCI L-Arginine L-Phenylalanine L-Histidine DL-Threonine L-Alanine
a
Germinationa of clinical isolates: _ 2282 2286 1956 1725A 2087 2065A 1968 2100 9292 2086 734 2059 1792 1966 1580C
L-Glutamine L-Leucine L-Tryptophan L-Glutamic acid L-Aspartic acid L-Cystine L-Tyrosine L-Ornithine HCI L-Proline OH-Proline
0 0 2 0 0 0 0 2 0 0 0 6 1 0 0 2 0 0 0 0 70 0
2 0 4 0 2 4 0 8 2 2 1 27 2 0 1 0 0 0 0 0 49 0
2 8 10 1 1 2 6 30 3 3 2 56 22 3 2 4 0 2 0 2 74 14
0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 24 0
PB TC 199
0 83
0 68
0 96
0 99
0 0 0 0 0 0 0
0 0 46 0
2 26 12 17 8 11 0 62 19 2 43 29 60 24 32 70 34 34 15 50 78 64
0 0 0 0 0 0 0 24 0 0 0 10 8 0 0 10 6 0 0 2 80 1
0 81
4 98
0 99
0 0 0 0 4 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 16 0
4 4 4 4 4 8 3 8 3 2 5 32 14 0 0 22 7 4 4 4 56 16
0 4 0 0 0 16 3 0 0 3 5 2 0 8 86 6
0 92
1 82
0 98
1
0 0 0 0 0 0
1 1 6 2 3 8 12
14 53 32
2 4 5 0 3 10 15 15 8 1 6 15 15 6 5 26 6 23 6 16 38 26
3 0 3 0 2 10 4 0 8 0 2 0 4 0 31 22 2 5 0 10 4 1 11 8 48 5 2 0 1 0 18 4 2 0 2 1 1 2 8 0 80 60 8 1
4 70
2 62
4 93
18 8 0 9 31 28 2 3 21 2 8 3
0 99
1 2 0 0 2 1 2 8 2 0 3 17 30 2 0 39 16 0 2 7 78 10
0 97
Results shown as percentage of germination under conditions of the test (see Materials and Methods).
does not stimulate pseudohyphal formation (16). However, even when one is careful to distinguish between the two obvious types of "filaments" expressed by C. albicans, there are still instances in which different results have been obtained by different workers under seemingly similar experimental circumstances. The one constant feature among these various results is strain variation. For example, Mardon et al. (17) studied a number of strains of C. albicans which were stimulated to germinate by L-alpha-amino-n-butyric acid, but we were not able to observe this effect with three stock cultures in one of our collections (unpublished observation). Evans et al. (4) have consistently observed germination on a glucose-peptone medium whereas we routinely use a similar medium for production of large yields of blastospores devoid of germ tubes (see Materials and Methods). There are other examples of such discrepancies and one can always rely on slight differences in media recipes or culture conditions to overcome these apparent contradictions. However, the results of the present study point up yet another important variable, i.e., strain variation, which may be important in determining the divergence of results among investigators. The data in Table 3 reveal a considera-
ble difference in response to stimulation by different amino acids among clinical isolates. This result helps to explain how an individual amino acid could be singled out as a promoter of germination in one instance, whereas a family of amino acids might be similarly recognized if a different isolate had been employed in the experiments. The data in this report establish that proline is prominent among amino acids for its ability to stimulate germ tube formation by blastospores of C. albicans. This fact was originally reported in abstract form by Dabrowa (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52), and was greatly extended by Land et al. (10, 11). This amino acid also stimulates germination of the sporangiospores of Rhizopus stolonifer and R. arrhizus (25) and the conidiospores of Aspergillus niger (28). Land et al. (10) report that the amino acids entering metabolism by conversion to glutamate are generally good stimulators of germination, and this fact was also noted in our results. Analogues of proline or the D-isomer markedly reduced germination of C. albicans in the proline-PB medium. These observations augment the conclusions of Land et al. (10) that normal proline catabolism is involved in its
834
DABROWA, TAXER, AND HOWARD
stimulation of germination. In addition, our results correspond to those of Weber and Ogawa (25) who observed that analogues of proline did not stimulate germination in R. arrhizus spores. In keeping with theories developed by Nickerson and his colleagues (for review see reference 1), addition of cysteine to the proline-containing medium prevented germination of the blastospores probably by fostering budding. However, Wain et al. (24) did not observe inhibition of germination of C. albicans in plasma containing 10-2 M cysteine, and the role of sulfur-containing amino acid in the morphogenesis of C. albicans is reopened by their contradictory findings. Dabrowa previously reported (Dabrowa, Proc. V Cong. Int. Soc. Hum. Anim. Mycol. 1971, p. 51-52) that glucose suppressed germination in a proline-PB medium. This observation was repeated several times. However, Land et al. (10) have shown that high levels of glucose (.0.1 M) promote germination in a NH4Cl medium and that 0.01 M glucose does not suppress proline-stimulated germination. Moreover, TC 199, which supports germination of C. albicans nearly as well as serum or plasma, contains 0.006 M glucose, a level which suppressed proline-stimulated germination in our experiments (see Results). This contradiction is probably related to the phosphate concentration of the various media. Thus, Land et al. (11) relate the effects of glucose, as well as those of proline and other environmental factors, to a Crabtree-like response, i.e., repression of mitochondrial activity. Such repression is also related to phosphate concentrations (see reference 9 for a discussion of the literature on these relationships). For example, it has been shown in mammalian systems that the repressing effect of glucose on respiration is antagonized by 0.01 M phosphate in the medium (9). It thus seems possible that our failure to observe the same effects of glucose on germination as those reported by Land et al. (10) was related to the fact that our buffer medium (0.033 M) had four times as much phosphate as did theirs (0.008 M). Indeed, the latter workers noted negligible amounts of germination in their prolineglucose-biotin medium when it contained 0.1 and 0.01 M phosphate. The phosphate concentration in TC 199 is much lower (0.0008 M) than that used in our buffer or that of Land et al. (10). Thus either differences in phosphate concentration in the medium or differences in transport of phosphate by the strains used are likely explanations for the seemingly divergent results. In keeping with this suggestion is the observation by Widra that the glucose-to-inor-
INFECT. IMMUN.
ganic phospate ratio is crucial to pseudohyphal formation by C. albicans in Sabouraud dextrose agar (27). Land et al. have extended their original observations (10) and provided additional evidence that germination of C. albicans is correlated with a repression of mitochondrial activity (11). Any environmental circumstance fostering such repression is likely to stimulate germination. Accordingly, the observations of Land et al. (10, 11) have provided a means of correlating a great many different and, at times, apparent diverse observations. Under carefully controlled conditions it is possible to show that a large percentage of clinical isolates of C. albicans germinate in a proline-PB medium (see Table 3). However, a simple amino acid medium will not substitute for serum or TC 199 in a useful diagnostic test where such factors as cell concentration cannot be rigorously controlled (3). The obvious practical conclusion to be derived from our work is that the proline-PB medium is unsuitable for routine diagnostic work. ACKNOWLEDGMENTS We are indebted to M. Sakamoto and Jennie Pang for excellent technical assistance. We thank Evelyn Graham and Judith Rhodes for the supply of clinical isolates of C. albicans, and we acknowledge the fact that some of the work was performed in a laboratory at the Veterans Administration Hospital, Sawtelle, Calif., the use of which was temporarily extended to us by E. T. Wright, Chief of Staff. S.S.S.T. was supported by Public Health Service grant AI-07461-09 from the National Institute of Allergy and Infectious Diseases. Other aspects of the study were partially supported by Public Health Service grant AI-10478-03 from the National Institute of Allergy and Infectious Diseases.
ADDENDUM Recently, a paper was published by K. L. Lee, H. R. Buckley, and C. C. Campbell on germination of C. albicans in a liquid synthetic medium (Sabouraudia 13:148-153). LITERATURE CITED 1. Bartnicki-Garcia, S., and I. McMurrough. 1971. Biochemistry of morphogenesis in yeast, p. 441-491. In A. H. Rose and J. S. Harrison (ed.). The yeasts, vol. 2. Academic Press, Inc., New York. 2. Dabrowa, N., D. H. Howard, J. W. Landau, and Y. Shechter. 1970. Synthesis of nucleic acids and proteins in the dimorphic forms of Candida albicans. Sabouraudia 8:163-169. 3. Dolan, C. T., and D. M. Ihrke. 1971. Further studies of the germ-tube test for Candida albicans identification. Am. J. Clin. Pathol. 55:733-734. 4. Evans, E. G. V., F. C. Odds, M. D. Richardson, and K. T. Holland. 1975. Optimum conditions for incitation of filamentation in Candida albicans. Can. J. Microbiol. 21:338-342. 5. Fahlberg, W. J., C. D. Dukes, and R. K. Guthrie. 1957. Rapid classification of Candida (monilia) albicans. J. Invest. Dermatol. 29:111-118. 6. Hu, F., C. S. Livingood, P. Johnson, and C. M. Pom-
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8.
9. 10.
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12. 13. 14. 15.
16. 17.
erat. 1954. Tissue culture studies on human skin. Arch. Dermatol. 70:1-15. Janson, V. K., and W. J. Nickerson. 1970. Chemical composition of chlamydospores of Candida albicans. J. Bacteriol. 104:910-921. Johnson, S. A. M. 1954. Candida (monilia) albicans. Arch. Dermatol. 70:49-60. Koobs, D. H. 1972. Phosphate mediation of the Crabtree and Pasteur effects. Science 178:127-133. Land, G. A., W. C. McDonald, R. L. Stjernholm, and L. Friedman. 1975. Factors affecting filamentation in Candida albicans: relationship of the uptake and distribution of proline to morphogenesis. Infect. Immun. 11:1014-1023. Land, G. A., W. C. McDonald, R. L. Stjernholm, and L. Friedman. 1975. Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation. Infect. Immun. 12:119-127. Landau, J. W., N. Dabrowa, and V. D. Newcomer. 1965. The rapid formation in serum of filaments by Candida albicans. J. Invest. Dermatol. 44:171-179. Lodder, J., and N. J. W. Kreger-van Rij. 1952. The yeasts: a taxonomic study. North Holland Publishing Co., Amsterdam. McClary, D. 0. 1952. Factors affecting the morphology of Candida albicans. Ann. Mo. Bot. Gard. 39:137-164. Mackenzie, D. W. R. 1962. Serum tube identification of Candida albicans. J. Clin. Pathol. 15:563-565. Mardon, D., E. Balish, and A. W. Phillips. 1969. Control of dimorphism in a biochemical variant of Candida albicans. J. Bacteriol. 100:701-707. Mardon, D. N., S. K. Hurst, and E. Balish. 1971. Germtube production by Candida albicans in minimal liquid culture media. Can. J. Microbiol. 17:851-856.
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18. Nickerson, W. J., and C. W. Chung. 1953. Cellular division in a mutant yeast. Am. J. Bot. 41:114-120. 19. Reynolds, R., and A. Braude. 1956. The filament-inducing property of blood for Candida albicans: its nature and significance. Clin. Res. Proc. 4:40-47. 20. Ridley, M. F. 1960. A comparison of methods for identification of Candida albicans. Aust. J. Dermatol. 5:209-213. 21. Scherr, G. H., and R. H. Weaver. 1953. The dimorphism of yeasts. Bacteriol. Rev. 17:51-92. 22. Silva-Hutner, M., and B. H. Cooper. 1974. Medically important yeasts, p. 491-507. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 23. Taschdjian, C. L., J. J. Burchall, and P. J. Kozinn. 1960. Rapid identification ofCandida albicans by filamentation on serum and serum substitutes. J. Dis. Child. 99:212-215. 24. Wain, W. H., M. F. Price, and R. A. Cawson. 1975. A re-evaluation of the effect of cysteine on Candida albicans. Sabouraudia 12:74-82. 25. Weber, D. J., and J. M. Ogawa. 1965. The specificity of proline in the germination of spores of Rhizopus arrhizus. Phytopathology 55:262-266. 26. Wickerham, L. J., and L. F. Rettger. 1939. A taxonomic study of Monilia albicans with special emphasis on morphology and morphological variation. J. Trop. Med. Hyg. 42:174-177, 187-192, 204-216. 27. Widra, A. 1964. Phosphate directed Y-M variation in Candida albicans. Mycopathol. Mycol. Appl. 23:197202. 28. Yanagita, T. 1957. Biochemical aspects in the germination of conidiospores of Aspergillus niger. Arch. Microbiol. 26:329-344.
