Improvement of the specimen fixation method before t-butyl alcohol freeze-drying for SEM observation of the hymenium of Pleurotus ostreatus

Abstract

To observe biological specimens using scanning electron microscope (SEM), generally, specimens are subjected to fixation, dehydration, and drying to avoid morphological deformation due to the vacuum condition. t-Butyl alcohol freeze-drying is one of the methods for drying specimens, and it is simple and convenient when compared to other methods, such as critical point drying. However, the occurrence of gross shrinkage and cracks on the cell surface is a critical problem when the hymenophore of <i>Pleurotus ostreatus</i> is treated by conventional double fixation with glutaraldehyde and osmium tetroxide (OsO4) followed by t-butyl alcohol freeze-drying. The present study revealed that when the hymenophore was treated by triple fixation with OsO4-tanic acid-OsO4 before t-butyl alcohol freeze-drying, shrinkage and cracks were substantially suppressed, and it was possible to observe the ultrastructure of the cell surface. The intracellular structure of the specimens treated by the triple fixation method was also observed using transmission electron microscope. The significance of the triple fixation method is discussed from both extracellular and intracellular morphological aspects.