FREEZE-DRYING OF FUNGAL HYPHAE

Methods of long-term preservation in which metabolism is inhibited during storage are preferable to maintenance on agar slants. The last mentioned method is laborious because regular transfers onto fresh media are required. In addition, repeatedly subcultured strains may undergo mutations. Current methods for long-term preservation are freeze-drying and preservation at temperatures below 130 C. Cryopreservation methods give high survival rates and are universally applicable. However, some disadvantages are encountered. Methods using nitrogen are rather expensive. Regular supply of liquid nitrogen is not always guaranteed. The dependence of ultra-deep freezers on electricity may be problematic in developing countries. Organisms must be dispatched under frozen conditions or activated before transport. Freeze-drying is a good alternative. Ampoules can be stored easily in dense packing without any special requirements. Cultures need not be revived on agar slants prior to dispatch. The product is light, inactive and dry, enabling easy distribution by mail. However, so far only conidia or spores can be freeze-dried successfully. Most attempts to revitalize dehydrated hyphae have failed, except for some successes reported for vesicular arbuscular mycorrhizal fungi (Tommerup, 1988), Claviceps (Pertot et al., 1977) and some Basidiomycetes (Bazzigher, 1962). Previous studies have shown that the survival rate of frozen fungal cells increases considerably when cells are cooled at a rate of1 C/min (Mazur et al., 1972; Hwang et al., 1976; Grout and Morris, 1987). Several authors have stressed the beneficial effect of trehalose (Crowe et al., 1984, 1990; van Laere, 1990; Wiemken, 1990). Addition of peptides to the resuscitation medium is aimed to improve recovery (MacLeod and Calcott, 1976). After optimization of these parameters, hyphae of a test series of Ascomycetes and Basidiomycetes were freeze-dried. Trehalose was added to the growth medium rather than to the protectant to allow absorption by the cell. Absorption would result in protection of cellular membranes as well as membranes of organelles.