Safe Specimen Preparation for Electron Microscopy of Pathogenic Fungi by Freeze-substitution after Glutaraldehyde Fixation

A safe method is described for observing ultrastructure of highly infectious fungi by ultrathin sectioning electron microscopy. The fungal cells were first chemically fixed by glutaraldehyde to kill them. They were then rapidly frozen by propane slush in liquid nitrogen and freeze-substituted in acetone containing 2% osmium tetroxide. This method gave clear cell images with high resolution in a natural state, close to the image obtained by rapidly frozen freeze-substituted specimen of living cells. Although we have demonstrated the utility of this method using Exophiala dermatitidis and Cryptococcus neoformans, it could also be used for observing highly infectious fungi such as Coccidioides immitis.