Improved Fixation and Embedding Methods for Electron Microscopy of Alternaria alternata Spores

Attempts were made to improve fixation and embedding methods for electron microscopy of Alternaria alternata spores. Among 16 methods tested, the ultrastructural images were greatly improved by the use of an en bloc alkaline bismuth stain or an osmium tetroxide-potassium ferrocyanide (reduced osmium) fixative. The cellular membranes were highly contrasted even in unstained sections obtained from en bloc alkaline bismuthstained spores after prefixation in aldehyde and postfixation in potassium permanganate. The use of the reduced osmium, followed by en bloc uranyl acetate staining, resulted in fine and enhanced staining of cellular membranes after primary fixation of spores in aldehyde. An en bloc alkaline bismuth also selectively reacted with the cell wall and plasma membrane in aldehyde and osmium tetroxide-fixed spores. This indicates that the stain is useful for observation of cell wall and plasma membrane. Spores fixed with the reduced osmium always exhibited a poor infiltration of Spurr resin. This was considerably improved by placing the reduced osmium-fixed spores in a resin/propylene oxide mixture at 60C for 3hr, rinsing in propylene oxide, and performing the infiltration-process with prolonged exposure of the spores to resin. (Received July 17, 1989)