A Modified, Short Protocol for Preparation of Bryophytes for Scanning Electron Microscopy

Abstract

Live bryological specimens can be prepared for scanning electron microscopy in under one hour without prior fixation by dehydrating with 2,2-di- methoxypropane followed by critical point drying. Unfixed specimens exhibited little distortion, thus eliminating the requirement for prolonged and/or expensive fixation that may cause collapse of cell walls or other artifacts. Critical point drying of specimens prior to examination by scanning electron micros- copy (SEM) is now a generally accepted procedure and its utility in bryology was dem- onstrated by Magill, Seabury and Mueller in 1974. Muller and Jacks (1975) proposed the use of 2,2-dimethoxypropane (DMP) for the rapid preparation of biological specimens for transmission electron microscopy. This procedure was adapted by Johnson et al. (1976) to specimen preparation for SEM. This adaptation required fixation in glutaraldehyde and osmium, prior to dehydration in 2,2-dimethoxypropane and critical point drying (CPD). The essential element of the procedure is dehydration by DMP, which upon exposure to water instantaneously hydrolyzes to form acetone and methanol. Johnson et al. (1976) suggested that fixation of specimens may be unnecessary. In an effort to simplify and shorten fixation and dehydration schedules, the effects of DMP dehydration on fixed and unfixed material were tested using leafy and thalloid liv- erworts and mosses. The protocol reported here yielded acceptable specimens for SEM