The preservation of unfixed cytological detail by dehydration with “Inert” agents

Abstract

Remarkable cytoplasmic detail can be preserved without chemical fixation if living tissues are properly dehydrated with increasing concentrations of inert, water-soluble, permeable, organic substances. Success is thought to depend upon adequate stabilization and consequent immobilization of the macromolecular systems before physiologically damaging the cytomembranes, particularly the plasma membranes. Thus, the most critical period is midway in dehydration when the rate and kind of exchange appears to be of great significance. Once immobilization is achieved, the final stages of dehydration can be completed in a variety of ways without importantly altering the end result. The simplest successful procedure is to use ethylene glycol as the dehydrating agent and to transfer the tissue directly from this into prepolymerized hydroxypropyl methacrylate. Glycerol and glucose syrup can be substituted for the first stage of dehydration, and then tissue is transferred to ethylene glycol to complete dehydration and allow subsequent infiltration with the hydroxypropyl methacrylate embedding mixture. If other embedments are desired, Cellosolve can serve as an intermediate solvent. Dimethyl sulfoxide and urea have not so far worked well as primary dehydrating agents. Methanol has been useless. Postdehydration fixation with anhydrous crotonaldehyde adds little or nothing to the final image. Treatment with osmium tetroxide dissolved in anhydrous Cellosolve in general degrades the final pattern. Fixation with glutaraldehyde and/or osmium tetroxide after partial dehydration is possible. The described method of tissue preparation, which may be termed “inert dehydration,” is basically a physical method, presumably leaving proteinaceous systems preserved essentially in their native states. Hopefully, the technique will find histochemical, immunological, and autoradiographic applications. The general method of dehydration is easily performed, and quantities of tissue can be processed reliably. Thus tissue can be stored for long periods in glycol or glycerol, and subsequently be further processed for biochemical or morphological study.