Sodium Permanganate Fixation for Electron Microscopy

Heightened interest in the cytology of certain free-living flatworms has been evident in the literature throughout the past year (5, 6, 10-12). The variety of problems dealt with by various investigators reflects the abundance of curious cellular specialization found in this group of animals. Moreover, the suitability of the Turbellarians for the study of regenerative processes is well recognized. Difficulties encountered in achieving satisfactory preservation of fine structure detail in Dugesia tigrina, a fresh-water Turbellarian, have led to a re-examination of several of the parameters involved in standard methods of preparation. During the course of these investigations, several of the more commonly used fixing agents have been tried with varied success. These include osmium tetroxide (9), chromeosmium (2), buffered formalin, and potassium permanganate (7). Of these fixing agents, osmium tetroxide has yielded acceptable results, while potassium permanganate fixation has shown especially good preservation of all membranes except the cytoplasmic membrane. Sodium permanganate as a fixative for electron microscopy has been previously mentioned, but without recommendation (7). A summary of the reasoning leading to its present use may be of interest. It is noted that careful maintenance of a delicate balance among sodium, potassium, calcium, and to some extent magnesium ions in the immediate environment of a living cell is necessary to the maintenance of cell function and structure. Disturbance of this ion balance can seriously disrupt a cell, though the tonicity of the solution is kept constant. The use of 1 per cent sodium permanganate as a fixing agent augments the concentration of sodium ion in the fixative by a value (51 mM/1 . ) well below the sodium ion concentrations generally reported for the body fluids of most organisms. Consequently, the ion balance of the fixative can be readily adjusted to approximate the ion balance of the cell's normal environment. When 1 per cent potassium permanganate is used as a fixative, however, the concentration of potassium ion contributed by the fixing agent alone (63 mM/1 . ) exceeds the values generally reported for potassium ion concentration in the body fluids of most organisms, This discrepancy in ion balance between the fixing fluid and the cell's normal environment might interfere with opt imum fixation of the cell, perhaps more markedly in some tissues than in others. The rationale employed in this investigation assumes that an approximation of the physiological extracellular conditions of a tissue is advantageous in formulating a successful fixing fluid. Such an assumption can only gain validity through a meticulous and exhaustive analysis of the prerequisites and mechanisms of satisfactory tissue preservation. Fixation is indeed so inadequately understood that the cytologist has little basis for choosing the extracellular environment over the intracellular one as a guide for formulating his fixative. Even the degree to which a fixing fluid need be "physiological" is still in question. Certainly a general evaluation of the role of ion balance in fixation cannot be drawn from the above-mentioned empirical approach to a particular problem. Yet, the results of this investigation would seem to merit careful consideration.