Use of Freeze Substitution for TEM Studies of Fungal Spores

Abstract Since its initial use to fix fungal hyphae (1), plunge freezing followed by freeze substitution has become the “gold standard” for TEM studies of fungal hyphae and spores. In this presentation we discuss results we have obtained using plunge freezing and freeze substitution to fix various types of spores produced by plant pathogenic fungi. Examples include basidiospores and aeciospores of rust fungi, teliospores of rust and smut fungi and conidia of a variety of ascomycetes and deuteromycetes. Generally speaking, plunge freezing followed by freeze substitution usually yields best results when spore diameter does not exceed 10-15 μm. However, in a number of cases we (2) have obtained good to excellent results with much larger spores. A key to good freezing appears to be direct exposure of spores to liquid propane during plunging. We have had best results by placing spores on small pieces of dialysis membrane and then plunging these membranes. In most cases the spores remain attached to membranes throughout the freezing, substitution, infiltration and embedment procedures.