Permanganate—A New Fixative for Electron Microscopy

During the early phases of tissue sectioning for electron microscopy, virtually all of the standard cytological fixatives were tried. Of these, osmium tetroxide appeared to give the best preservation, and this was further improved by buffering the fixative solution at a slightly alkaline pH (Palade (1)). A variant developed by Dalton (2) contains Os04 in a chromate-dichromate system. Formalin has been used for special purposes for electron microscopy, and a fixative without OsO4 has been described by Low (3), consisting of a chromic acid-formaldehyde mixture. Potassium permanganate, or more specifically the permanganate ion, has occasionally been used as a fixative or stain in light microscopy (4). It provides remarkable preservation of many cell components at the electron microscope level, as the following plates illustrate. The tissue fine structure looks similar to that following osmium treatment, thus rendering less likely the criticism that electron microscopists are merely seeing artifacts of osmium fixation. Besides preserving fine structure, perman-ganate fixation enhances tissue density and contrast. This feature is of interest because of the low atomic weight of manganese (54.9) in comparison to the high atomic weight of osmium (190.8). It would seem that explanations of tissue contrast must consider other factors besides the atomic weight or number of the fixative components. Permanganate appears to fix membrane systems first, (within 15 to 30 minutes), allowing the remaining cellular components either to leach out during dehydration, or to be volatilized in the electron beam as reported by Morgan et al. (5). Longer fixation (1 to 12 hours) retains other structures such as nuclear chromatin, the nucleo-lus, and the matrix of mitochondria. One hour fixation retains enough nuclear material to give a moderate Feulgen reaction in sections 2# thick. Sodium permanganate appears to offer no advantage over the potassium salt, nor does the addition of OsO4 to the per-manganate. The actual details of the method are as follows: A stock solution of reagent grade potassium permanganate (KMnO~) is made up in distilled water to 1.2 per cent w/v and stored in the refrigerator. It is best to keep the stock solution in a well filled, glass-stoppered bottle since KMnO4 solutions are slowly reduced on contact with air. This solution replaces the 2 per cent Os04 stock solution used to prepare the usual 1 per cent buffered OsO4 (Palade (1)). For use, equal volumes of the permanganate stock solution are mixed with the veronal-acetate buffer, giving a …