An Ice-Solvent Method of Drying Frozen Tissue for Plant Cytology

Publisher Website
Google Scholar
Abstract
A freeze-dry method where cold absolute ethanol is used as a dehydrating agent in place of vacuum dehydration has been applied to various plant materials with good cytological results. The method involves: (a) freezing rapidly small pieces of tissue 1 cubic mm or less in partly frozen isopentane cooled with liquid nitrogen, (b) transferring quickly to vials of cold absolute ethanol at -41° to -45°C, and (c) holding within this temperature range for 3 days to dissolve the ice. A simply constructed cryostat is used to maintain the vials of absolute alcohol and tissue at the cold temperature. This consists of a semi-frozen constant temperature bath of either 65% ethanol or pure diethyl oxalate in a tightly covered beaker which fits within a large dewar flask half filled with dry ice. The bath is arranged so that it will be on top of and in contact with the dry ice but properly insulated to prevent freezing completely. The resulting dried tissue is very unstable in either water or hot absolute ethanol; therefore, to prevent loss of cytological detail during further processing, the tissue must be treated to render the proteins insoluble. Either (a) replace the cold absolute ethanol in the tissue vials with cold (approx. -40°C) 75% ethanol, warm slowly to 60°C, and hold for 1 hour, or (b) replace with cold acidulated 95% ethanol (100 ml. of 95% ethanol + 0.30 ml. of glacial acetic acid), warm to room temperature, and hold for 30 minutes. Following either treatment the tissues are dehydrated to absolute alcohol and embedded in paraffin by the usual technics. Sections are attached to slides by flattening over warm water and drying. When epidermis from onion bulbs was used as a basis of comparison of fixed and living material with the phase-contrast microscope, the mitochondria, plastids, and other fine structures in fixed preparations appear to be nearly identical with the living. Fat droplets disappear. With larger tissues such as onion root tips, thin freehand sections must be prepared before freezing to obtain good cytological results. The application of the method to cytochemical studies is discussed and in many ways it seems to be as useful as the freeze vacuum-dry method.