Culturing and Embedding of Filamentous Fungi for Electron Micrography in the Plane of the Filaments

Abstract

One-day-old mycelia of Volvariella volvacea, grown on water-permeable cellophane membrane applied to an agar medium, were stripped from the medium and fixed 1 hr in 3% glutaraldehyde followed by 2 hr in 1% OsO4. The specimens were washed in water, dehydrated with acetone and embedded in Vestopal W. The flat end of the precast tissue carrier was placed on the top (tissue side) of the specimen in a polyethylene dish. The preparation was first allowed to harden 12 hr at 50 C. After hardening was completed at 60 C., the block was removed, the cellophane stripped off, and the face of the block trimmed for sectioning. Since the mycelium had grown as a thin flat layer, the face of the block required little or no trimming, and sections in the plane of the mycelial filaments were readily obtained.