Improved method for ultrastructural preservation of Saccharomyces cerevisiae JHY-31-11D

Investigation in the National Institute of Environmental Health Sciences (N IEHS) laboratories of the possibility that the JHY-31-11D strain of Sacchar omyces cerevisiae might provide a simple model for studying apoptosis was n ecessarily preceded by developing an electron microscopic protocol for supe rior preservation of ultrastructural detail. Since the biochemical composit ion of the thick cell wall of this particular strain of yeast presumably im peded penetration of routine fixatives, we developed a new combinational pr ocedure in which a mixture of 2% paraformaldehyde, 2.6% glutaraldehyde, and 3% sucrose functioned as postfixative following the more traditional prima ry fixative, potassium permanganate (KMnO4), used at 4% concentration. Uran yl acetate(1%) was subsequently applied as Fixative and en bloc stain. All steps in our 4-day procedure were performed by suspending the cells in the reagents following light centrifugation in a tabletop microfuge, thus elimi nating often troublesome agarose embedding and assuring consistent reproduc ibility of the method. No chemical treatments that might have altered wall structure and morphology were used. Our superior fixation results indicate the apparent importance of the concentration of the permanganate, the use o f a double-aldehyde postfixative, and the order of the application of the f ixatives.