Preliminary and Short Report

Transcription

1 Tuz JOURNAL OF INvRSrIOATIVE DERMATOLOOT Copyright 1965 by The Williams & Wilkins Co. Vol. 45, No. 3 Printed in U.S.A. Preliminary and Short Report ULTRASTRUCTTJRE OF BLASTOSPORES OF CANDIDA ALBICANS AFTER FERMANGANATE FIXATTON* L. F. MONTES, M.D., T. A. PATRICK, M.D., S. A. MARTIN AND M. SMITH, MA. Although several publications describe the ultrastrueture of yeast Cells (1 8) only a few refer briefly to Condido olbicons (9 12). In view of this and of the increasing importance of C. elbicens as a cause of both superficial and systemic disease (13), a study of its fine structure was considered worthwhile. MATERIAL AND METIIODS In agreement with the observations of Gale (10) that osmium tetroxide is not a very satisfactory fixative for C. elbicens, preference also has been given in our laboratory to the use of potassium permanganate (12 2 per cent) in aqueous unbuffered solutions. The fixation is carried out at 2 4 C. for a period of 50 minutes to one hour with favorable results (Fig. 1) which compare with those of Gale (10, 11) and Bakerspiegel (12). In more recent experiments, the use of lithium permanganate (LiMnO4), as suggested by Mollenhauer (14) and Afzelius (15), also resulted in good fixation of C. elbicens. Cells were scraped from the surface of the colooies after 24 to 96 hours of room temperature growth in Sabouraud's dextrose agar or in Mycological Agar (Difco). They were then fixed in unbuffered aqueous solutions of LiMnO4 (5 15 per cent) from three to four hours at room temperature. Dehydration was performed using graded concentrations of acetone, and embedding in a mixture of Epon and Araldite (16). Ultrathin sections were obtained with a Porter- Blum microtome. The sections were studied with an RCA EMU 3F electron microscope using an accelerating voltage of 50 kv. RESULTS AND DI5cUSSION Observations thus far have been performed on blastospores only. Our preliminary observations of Supported in part by a research grant from the Upjohn Company, Kalamazoo, Michigan, and Training Grant HE from the National Heart Institute, National Institutes of Health, Bethesda Maryland. Received for publication April 15, * From the Departments of Dermatology and Pathology, Baylor University College of Medicine, Houston, Texas. these cells as seen after potassium and lithium permanganate fixation are summarized in this report. The cell well was seen as a thick layer whose electrondensity was greater in the outer part in contact with the external medium (Figs. 1, 4, 5). Well-defined layers within the cell wall as described by Bakerspiegel (12) in both blastospores and chlamydospores were not observed. The plasme membrene frequently showed short intracytoplasmie invaginations (Fig. 5). These invaginations were particularly apparent in material fixed with LiMnO4. Mitechondria were well developed and numerous, varying in size and number from cell to cell. They had an external double membrane and cristae which often were parallel (Figs. 3, 4). A welldeveloped endople.smic reticulum made up of relatively straight double membranes was particularly evident in blastospores fixed with LiMnO4 (Fig. 4). Fixation in KMnO, also preserved these membranes although not as well as LiMnO,. In cells fixed with LiMnO4, the cytoplasmic ground substance was finely granular (Fig. 3). With KMnO4-fixed cells, this granularity was increased to an undesirable extent. For this reason we preferred LiMnO4 to KMnO4. This and other effects of the permanganates, such as the lack of preservation of ribesomes and the washed-out appearance of the nucleus, were recently discussed by Afzelius (17). As observed in various yeasts (5, 7, 8) one or several vecueles, sometimes of large Size, were seen in almost every cell (Figs. 1, 2, 4). These vacuoles had a limiting membrane. The uniformity of this membrane often was altered by intravacuular protrusions of the cytoplasm. The nucleus was encompassed by a doublelayered nuclear membrane (Fig. 5) with several nuclear pores (Fig. 1). These pores were about 700 A in diameter. Similar pores were described by Agar and Douglas (2) in 2. cereeisiee. Frequently, eleetrondense areas in the nueleoplasm were observed to alternate with other less dense regions. The adjacency of the nucleus to the cytoplasmic vacuole, already described in yeasts (2), also was observed in our material (Figs. 1, 2). In Some of our sections electrondense round bodies having approximately the size of Small mitochendria were seen in the cytoplasm. The identity of these bodies remains to be determined; however, they could represent lysosomes, since cytochemical experiments recently performed in our laboratory (18) have demonstrated eytoplas- 227

2 228 THE JOURNAL OF INVEST1GATIVE DERMATOLOGY.-Jcw Fro. 1. Blastospore from a 48-hour culture of C. albicans grown in Sabouraud's medium at room temperature. The cell wall (CW), a large vacuole (V), and mitoehondria (M) are shown. The nucleus (N) is well demarcated by an electrondcnse nuclear membrane with several pores (arrows). Fixed in 2 per cent KMnO4 for 50 minutes at 4 C. >< 27,800.

3 t 4 M V Fro. 2. Blastospores of C. albir,ans from a 24-hour culture incubated at room temperature (Myeological Agar). These cells show numerous mitoehoadria (M) and one or several vaeuoles (V) located close to the nuclei (N). Fixed in 15 per cent LiMnO4 for three and one-half hours at room temperature. X 9,100. FIG. 3. Well-developed mitoehondria showing parallel eristae. C. olbicons cultured in Sabouraud's dextrose agar at room temperature for 45 hours. The cytoplasmie ground substance appears finely granular. Fixation in 5 per cent LiMaO4 for three and one-half hours at room temperature. X 37,

4 230 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY m Fic. 4. Elongated blastospore of C. albicans. Culture incubated for 24 hours on Mycological Agar at room temperature. Mitochondria, the endoplasmic reticulum (ER), the cell wall (CW), the plasma membrane (PM) and a small vacuole (V) are shown. Same fixation as in Fig. 2. X 16,500.

5 IJLTRASTRUCTURE OF CANDIDA ALBICANS CW PMVK).I N 4 FIG. 5. The plasma membrane (PM) of C. olbicons frequently shows intracytoplasmic invaginations (5). The cell wall (CW) and the double layered nuclear membrane (NM) are shgwn. Culture incubated fgr 24 hgurs on Mycological Agar. Same fixation as in Fig. 2. X 32,100. mic granules with acid phgsphatase activity in Can clida albicans. Further experiments are presently being carried out to determine whether lithium permanganate fixation may be successfully applied to the electron microscupic study of tissues infected with C. albicans. Also it seems possible that this fixative nny be useful in ultrastructural studies of pathogenic yeast-like fungi. 5UMMARY The fine structure of blastospores of C. albicans growing at room temperature, and following fixation with potassium permanganate and lithium permanganate, is described. The characteristics of the various submicroscopic components of these cells are shown. The possibility of further application of lithium permanganate fixation for electron microscopic study of other mycological material is discussed. REFERENCES 1. Bartholomew, J. W. and Levin, H.: The structure of Saccharomyces carlsbergensis and Saccharomyces cerevisiae as determined by ultrathin sectioning methods and electron microscopy. J. Gen. Microbiol., 12: 473, Agar, H. D. and Douglas, H. C.: Studies on the cytological structure of yeast: electron microscopy of thin sections. J. Bact., 73: 365, Yotsuyanagi, Y.: Etudes Sur Le Chondriome de la Levure. J. ljltrastruct. Res., 7: 121, Hirano, T.: The fine structure of the nuclear apparatus in saccharomyces. J. ljltrastruct. Res., 7: 201, Thyagarajan, T. H., Conti, S. H. and Naylor, H. B.: Electron microscopy of Rhoclotorula Glutinis. J. Bact., 83: 381, Mundkur, B.: Electron microscopical studies of frozen-dried yeast. IV. Schizosaccharomyces, Nadsonia and Saccharomycedcs. Z. Naturforsch., 18: 1073, Hashimoto, T., Gerhardt, P., Conti, S. F. and Naylor, H. F.: Studies on the fine structure of microorganisms. V. Morphogenesis of nuclear and membrane structures during ascospore formation in yeast. J. Biophys. Biochem. Cytol., 7: 305, Vitols, E., North, H. J. and Linnane, A. W.: Studies on the oxidative metabolism of Sacchoromyces cerevisioe. I. Observations on the fine structure of the yeast cell. J. Biopbys. Biochem. Cytol., 9: 689, Adams, J. N., Painter, B. G. and Payne, W. J.: Effects of sodium caprylate on Candida Albicans. I: Influence of concentration on ultrastructure. J. Bact., 86: 548, Gale, G.: Cytology of C. Albicans as influenced by drugs acting on the cvtoplasmic membrane. J. Bact., 86: 151, Gale, G. H. and McClain, H. H.: Effect of

6 232 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY thiobenzoate on cytology of Gondido Albicons. J. Gen. Microbiol., 36: 297, Bakerspiegel, A.: Some observations on the cytology of Condido Albicons. J. Bact., 87: 228, Winner, H. I. and Hurley, R.: Candida Albicans. Boston, Little, Brown and Co., Mollenbauer, H. H.: Personal communication to the authors. 15. Afzelius, B. A.: Experiments with simple fixatives. J. Ultrastruct. Res., 9: 393, Mollenhauer, H. H.: Plastic embedding mixtures for use in electron microscopy. Stain Techn.,39: 111, Afzelius, B. A. and Harris, R. J. C.: Interpretation of Ultrastructure. New York, Academic Press, Inc., Montes, L. F.: Unpublished data.