ELECTRON MICROSCOPIC STUDIES ON THE CYTOLOGICAL STRUCTURE OF TRICHOPHYTON MENT AGROPHYTES

Abstract

A rapid progress made for ultrathin sectioning technique in electron microscopy has contributed much to the analysis of microbial cell structures. Recently, several papers (3,(7)(8)(9)17,18,23) have appeared on the subject of electron microscopic studies of certain dermatophytes, and the structural details of fungal cells have been exposed to some extent through electron microscopy of ultrathin sections. However, the published electron micrographs of the organism, especially those in its early stage, were not of sufficient quality to reveal the detail architectures of the fungus. Moreover, the findings of the cell structures hitherto obtained have not been always agreeable among workers, and many problems of the fine structures of the organism still remained to be studied.Since Watson (1958)(25,26) succeeded in enhancing the contrast and resolution in thin sections of biological materials, using stain technique with heavy metals, several modifications(4,10,16) of his method have been reported, and these electron-opaque stains have been widely applied to tissue sections for electron microscopy. Vitols, North and Linnane (1961)(24) first reported that uranyl nitrate treatment of the whole cells of Saccharomyces cerevisiae after permanganate fixation leads to better preservation of the membrane structures and also to an improvement in general contrast. This paper describes some of the fine structures of Trichophyton mentagrophytes as observed chiefly by staining the thin sections of fungus cells with various heavy metals. MATERIALS AND METHODSOrganism employed: A stock culture of Trichophyton mentagrophytes, strain J-812, maintained on a glucose peptone agar at 27 C, was used. This strain was obtained from Dr. T. Tsuchiya, Professor of Juntendo University School of Medicine.Culture media employed: The medium mostly used was a glucose (1 per cent) peptone (1 per cent) water adjusted to pH 5.6.Electron microscopy of thin sections : Hyphal pellets of the strain grown on the medium for 5 to 7 days at 27 C were harvested by centrifugation and washed with sterile veronal-acetate buffer solution several times. These pellets of about 1 to 2 mm in diameter were fixed in 1 per cent osmium tetroxide solution, or 2 per cent potassium permanganate buffered at pH 6.99 with veronal-acetate buffer, for 24 hr at 4 C.

CYTOLOGICAL STRUCTURE OF ASPERGILLUS NIGER BY ELECTRON MICROSCOPY

“…Shatkin and Tatum(l6) previously indicated that the outer region of the cell wall of the filamentous Ascomycete, Neurospora crassa, consisted of a fibriform meshwork of great density, and in the cell wall interior a network of fibers, finer and less dense than those on the outer region, was discernible in the area where the wall usually appeared to be structureless. A fibrillar architecture of the whole cell wall has been reported on S. cerevisiae by Houwink and Kreger(7), C. neoformans by Tsukahara (20), and, subsequently, on T. mentagrophytes by the present authors (22). The cell wall of these fungi was found to be composed of fibrous and rather electrondense structures running longitudinally to the cell body.…”

“…11, a rather electron-transparent area is visible in the nearly central portion of the septal wall, and intercellular streaming of the cytoplasmic materials can be seen through this area. These findings have already been pointed out in the thin sections of hyphae of N. crassa by Shatkin and Tatuma6), and, successively, in the septate hyphae of some other fnngi (17,22). The electrontransparent area, situated at the central part of the wall, appears to correspond to the " septal pore " previously demonstrated in some filamentous fungi.…”

“…Subsequently, potassium permanganate has also been demonstrated to be a reliable fixative for the electron microscopic studies of the fine structure of certain fungal cells, Saccharomyces cerevisiae (Hashimoto et al, 1958(4,5)), Cryptococcus neoformans (Tsukahara, 1963(20)), and Candida albicans (Tsukahara et al, 1964(21)). Permanganate treatment, however, was unsuccessful for the satisfactory fixation of hyphal cells of Trichophyton mentagrophytes (22). Potassium permanganate therefore is not always regarded as a favourable fixative for every fungal cell.…”

“…Many workers (1)(2)(3)7,10,(13)(14)(15)(20)(21)(22)24) have regarded the fungus cell wall as a considerably thick-walled organelle which consists possibly of several layers. Similar findings were also obtained when most sections of the used strain of A. niger were treated with uranyl nitrate after permanganate fixation.…”

“…However, under the given conditions the cytoplasmic membrane of the mold rarely represents such an unit composition as described above. Also, this membrane is electron-dense but rarely has any sinuate structure, whereas the plasma membrane of most species of fungi has been found to have numerous infoldings at its surface (2,14,16,(20)(21)(22)24). Previously, O'Hern et al (14) and Shatkin et al"6) postulated that the presence of the infoldings or invaginations in the cytoplasmic membrane of fungi might be closely related to the metabolic activity of the cells.…”