Dark-Field Electron Microscopy of Thin Sections of

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1 JOURNAL OF BACTERIOLOGY, Oct. 1974, p Copyright American Society for Microbiology Vol. 120, No. 1 Printed in U.S.A. Dark-Field Electron Microscopy of Thin Sections of Trichosporon cutaneum CLAES WEIBULL Department of Microbiology, University of Lund, S Lund, Sweden Received for publication 18 June 1974 Dark-field electron micrographs of aldehyde-fixed, unstained yeast-phase cells of Trichosporon cutaneum exhibit remarkable contrast and good resolution when the sections studied are less than 25 nm thick and conical illumination is used. High-resolution, dark-field electron microscopy of unsectioned, particulate matter, e.g., ribosomes, deoxyribonucleic acid, and messenger ribonucleic acid has been described (1, 2, 3). However, to my knowledge hardly any micrographs of sectioned material obtained with dark-field illumination have been published. In the present investigation cells of Trichosporon cutaneum (5) were fixed for 2 h at room temperature with 4% glutaraldehyde (7). Sections approximately 20 nm thick (6) were cut with a diamond knife, mounted on plastic micro grids (4), and studied with a Philips EM 300 electron microscope. An annular diaphragm was inserted into the second condenser (2). Thus a dark-field generated by conical illumination was obtained. Figure 1 shows a micrograph of the material studied. The yeast cell wall appears dark, indicating that it scatters few electrons. The mitochondria are clearly seen with cristae mitochondriales brighter than the surrounding stroma. Vacuoles containing electron-dense bodies, most probably consisting of polyphosphate, are also present. All structures appear, as could be expected, with reversed brightness in comparison with micrographs of similar specimens photographed with conventional illumination (7) (Fig. 2). However, the contrast in the micrographs obtained with dark-field illumination is much higher, although a less contrasty film (Kodak fine grain release positive) was used. Figure 3 illustrates the marked influence of the section thickness on contrast and resolution. Sections thinner than 20 nm could sometimes be cut, but a reduction of the section thickness below this value hardly improved the resolution. Such sections, moreover, very easily broke. Generally, the illumination had to be kept at a minimum to avoid beam damage to the specimen. Thus, the cell wall of the yeast cell often spearated slightly from the surrounding embedding material (Fig. 3b). Cells of a bacterium (Proteus mirabilis) and a fungus imperfectus (Fusarium roseum) were also studied, but less satisfactory results were obtained with these organisms than with the yeast cells. The skillful technical assistance of Ulla Wulf and Torbji5rn Magnusson is gratefully acknowledged. This investigation was financially supported by the Swedish Natural Science Research Council. 527

2 528 NOTES J. BACTERIOL. FIG. 1. A glutaraldehyde-fixed, unstained cell of T. cutaneum. Dark-field electron micrograph. Note cell wall (CW), mitochondria (M) with cristae mitochondriales, vacuole (V) with polyphosphate granules (P) and sites of lipid material extracted during the dehydration (L). Bar equals 0.5 Am.

3 VOL. 120,1974 NOTES 529 I L L..e...1 q ;.-.,. L. FiG. 2. A glutaraldehyde-fixed, unstained cell of T. cutaneum. Bright-field electron micrograph. Designations as in Fig. 1. Bar equals 0.5 jam.

4 530 NOTES J. BACTERIOL. Downloaded from ow I,{'~:.1 tt K on September 25, 2018 by guest W, 4E ~ ~ ~ ~~~~ FIG. 3. Mitochondria of T. cutaneum. Dark-field electron micrographs. (a) section thickness approximately 50 nm. (b) section thickness approximately 20 nm. Bar equals 0.1 im.

5 VOL. 120, 1974 NOTES 531 LITERATURE CITED 1. Brakenhoff, G. J., N. Nanninga, and J. Pieters Relative mass determination of darkfield electron micrographs with an application to ribosomes. J. Ultrastruct. Res. 41: Dubochet, A., M. Ducommun, M. Zollinger, and E. Kellenberger A new preparation method for darkfield electron microscopy of biomacromolecules. J. Ultrastruct. Res. 35: Dubochet, J., C. Morel, B. Lebleu, and M. Herzberg Structure of globin mrna and mrna-protein particles. Use of dark-field electron microscopy. Eur. J. Biochem. 36: Fukami, A., and K. Adachi A new method of preparation of a self-perforated micro plastic grid and its application. J. Electronmicros. 14: Neujahr, H. Y., and J. N. Varga Degradation of phenols by intact cells and cell-free preparations of Trichosporon cutaneum. Eur. J. Biochem. 13: Weibull, C Estimation of the thickness of films used in electron microscopy. Allg. Mikrobiol. 12: Weibull, C Electron microscope studies on aldehyde-fixed, unstained microbial cells. J. Ultrastruct. Res. 43: