THE USE OF DIMETHYLSULFOXIDE FOR FIXATION OF YEASTS FOR ELECTRON-MICROSCOPY

Conventional methods of chemical fixation are often inadequate for pre serving yeast ultrastructure. The thick cell wall severely limits pene tration of fixatives rendering poor detail of the cell wall, membranes , and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration o f chemicals and has been added to fixatives to improve cell preservati on. At high concentrations (5 to 50%), however, it affects ultrastruct ure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glab rata and Aspergillus fumigatus were fixed for 3 hr in 3% paraformaldeh yde, 1% glutaraldehyde, 1 mM MgCl2, 1 mM CaCl2, 0.1% DMSO in 0.1 M sod ium cacodylate buffer followed by 1% OsO4, 1% K2Cr2O7, 0.85% NaCl, 0.1 % DMSO in the same buffer. Thin epoxy sections were post-stained in ur anyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or al cian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner mannoprotein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significa ntly improved.