The Use of Potassium Permanganate as an Electron-Dense Stain for Sections of Tissue Embedded in Epoxy Resin

Potassium permanganate has become an established fixative in electron microscopy (Luft, 1956) and has special value in the study of cell membranes. During an investigation of methods of increasing the contrast in the electron microscope of tissues embedded in epoxy resin, t a solution of potassium permanganate was used to treat the sections, using a technique similar to that of Watson (1958). Potassium permanganate was found to produce a marked increase in contrast, but this was usually accompanied by a granular precipitate of electron-opaque material on the surface of the section. It was found that a weak solution of citric acid could be used to remove this precipitate without seriously reducing the contrast of the section. The following method is suggested as a starting point for further improvement. Staining Solution.-Dissolve 1 gram of potassium permanganate crystals in 100 ml. of distilled water. Do not filter. Allow to stand at least 24 hours before use. Staining Procedure.-1. Fill a small vessel with staining solution removed with a pipette from beneath the surface film in the stock bottle. 2. Sweep the surface of the staining solution clean with the edge of a microscope slide coated with paraffin wax. 3. Float thin sections (without removal of the epoxy resin) mounted on collodion-coated copper grids, section side down, on the surface of the staining solution immediately after the surface has been cleaned. Stain for 15 minutes to 2 hours. 4. Rinse in running distilled water for a few seconds. 5. Immerse and agitate in a solution of one drop of 5 per cent citric acid in 1 ml. distilled water for 30 seconds to 1 minute. 6. Rinse in distilled water. 7. Dry by placing grids section side up on filter paper. Even with the precautions used in this procedure , precipitation may occur, but it does not usually limit the usefulness of the section. The time of staining required varies with fixation (both Dalton's and Palade's fixative have given good results), the degree of contrast desired, the type of epoxy resin, and other unknown factors, and must be determined by trial. Potassium permanganate appears to stain nearly all of the known components of tissues when used in this manner, and, therefore, the resulting micrographs lack the clarity of micrographs of tissues fixed in permanganate solutions in which cell membranes are selectively stained. The citric acid has a tendency to cause tissue damage, for …