HOT FIXATION OF FUNGAL SPORES FOR TRANSMISSION ELECTRON-MICROSCOPY - APPLICATION TO THICK-WALLED SPORES OF THE SMUT FUNGUS ENTORRHIZA

Thick walled fungal spores are often difficult to prepare for transmis sion electron microscopy. The fixative and embedding medium usually in completely penetrates the interior of the spore, causing the inner wal l layers and protoplast to become dislodged during thin sectioning. Ou r experiments explored the use of hot fixation as a means of improving preservation in thick walled spores of Entorrhiza. The retention of s tructure in material stored after long periods in the dried and chemic ally preserved states was also assessed to determine the validity of s uch material for use in taxonomic or morphological work. Spores from h erbarium specimens over a 100 years old, host gall tissue containing s pores preserved in formalin-aceto-alcohol (FAA) and glutaraldehyde for 20 years, and freshly collected spores were examined. Spores prepared by freeze-fracturing followed by conventional chemical fixation and e mbedding for transmission electron microscopy were used as controls. F ixation was at temperatures of 20-degrees-C, 40-degrees, 60-degrees an d 80-degrees. The duration of fixation was 1, 2, 4, 6 and 8 h. Results indicate that hot fixation can improve preservation: spores retained more of the inner wall layers and protoplast compared with material fi xed at ambient temperature (20-degrees). However, fixation of the prot oplast was inferior to controls, as lipid bodies were marred by compre ssion caused during sectioning. The optimal temperature of hot fixatio n was found to vary and depend on the material examined. Fresh spores were the most difficult to fix and required a temperature of up to 80- degrees for 8 h, and even then preservation remained imperfect. Dried spores, and those previously stored in fixative for long periods, were reasonably well preserved after 4 h fixation at 40-degrees. The layer ing and other ultrastructural features of the wall in spores stored fo r many years corresponded to that of controls; such material can there fore be used with confidence in structural or taxonomic studies. Mater ial stored in FAA for 20 years was the least well fixed of the long te rm dried or chemically preserved material. Prolonged storage in fixati ve apparently lowers the permeability of the wall, thereby allowing so me extraction of lipid matrix and for the lipid bodies therefore to be cut without compression. Storage in FAA mostly caused lipid bodies to fuse. With storage in glutaraldehyde lipid bodies collapsed slightly.