Vapor fixation of intractable fungal cells for simple and versatile scanning electron microscopy

Imaging of exudation of conidia from fruiting bodies by scanning electron microscopy is challenging because of the difficulty in achieving specimen fixation that enables conidia to be immobilized from fruiting bodies. Conidia in chains borne on conidiophores are readily dissipated on coming in contact with liquid fixatives before fixation becomes effective. Two phialide-forming fungi including Aspergillus and Penicillium species, and a pycnidium-forming fungus Fusicoccum species were subjected to a vapor fixation procedure for scanning electron microscopy rather than conventional immersion fixation procedures. The intractable fungi were exposed to the vapor of 2% glutaraldehyde and 2% osmium tetroxide each for at least 2 h, subsequently followed by gold coating. Through the vapor fixation, excellent retention of conidial chains in phialides was achieved in this study. It appeared that conidial masses from pycnidia were usually held together in globose to oval drops. The simple vapor fixation procedure for preserving delicate fungal cells could be widely used for routine applications to facilitate morphological characterization of diverse plant pathogenic fungi as they are in ex planta ecological niches during the saprophytic phase as well as in host parts during the parasitic phase.