Preservation of Saprolegniaceous Fungi for Light Microscopy

Abstract

Saprolegniaceous fungi are generally preserved for light microscopy by immersing hyphae in Amann's solution, or less commonly in formalin-acetic-alcohol (FAA) (Seymour, 1970), and mounting specimens on glass slides (Johnson, 1956). Material prepared in this manner is inadequate since the slides cannot be sealed permanently. More importantly, the morphological characteristics of the fungi become distorted (Figs. 1, 2). Thus, key diagnostic features (e.g., antheridial cell shape, mode of antheridial branch attachment, and oogonial wall ornamentation) often cannot be recognized in such preparations. The difficulty in preserving saprolegniaceous fungi has hindered the establish? ment of permanent reference collections of these fungi. Type specimens of most genera have been lost and preserved material, where it exists, is all but useless for taxonomic work. Collections of living cultures (Hawksworth, 1974; Stevens, 1974) are limited and incomplete. Furthermore, living cultures are subject to genetic change (Goos and Pollack, 1965) and can be lost easily as a result of contamination or improper maintenance; they cannot substitute for preserved specimens which provide a permanent record ofthe morphological features pres? ent in an organism at a given time and under specific environmental conditions. A technique has been developed which eliminates most ofthe distortion normally associated with saprolegniaceous fungi preserved by traditional methods. Fungi prepared with this technique can be permanently mounted on glass slides and are suitable for taxonomic studies at the light microscope level. Isolates of Achlya treleaseana (Humphrey) Kauffman, Brevilegnia bispora Couch, and Dictyuchus pseudodictyon Coker et Braxton were grown on split, sterilized hemp seed in glass Petri plates containing sterilized, charcoal-filtered, distilled water. Colonies exhibiting the desired structural features were submerged individually in 1% glutaraldehyde in 0.1 m sodium cacodylate buffer (pH 7.2). After 30 seconds, each colony was transferred and held for 10 minutes in a plate containing 0.1 m sodium cacodylate buffer. This was followed by two 10 minute rinses in distilled water. Colonies were stored in distilled water prior to staining and slide preparation. Permanent slide mount preparations (modified from the method described by Kohlmeyer and Kohlmeyer, 1972) were made by placing a square coverglass (No. 1, 25 mm) in the center of a clean glass slide (76 x 25 x 1 mm). The coverglass and slide were temporarily sealed together by applying a small drop of water at the interface. Portions of mycelium were cut from an intact colony with micro-