DIVISION OF MICROBIOLOGY: PROBLEMS OF FIXATION AND STAINING IN MICROBIAL CYTOLOGY*

Abstract

In securing preparations of meaningful cytological delicacy, particularly of the smaller microorganisms, the avoidance of artifacts is inevitably a more exacting requirement than is frequently the c a s e with large, well-differentiated tissue cells. T h e problems encountered in preserving structure in microorganisms in a form closely approximating that prevailing in t h e living cell are severe in morphological, and more so in cytochemical studies at the levels of both the light and electron microscopes. T h e s e difficulties are attributable largely to the two special properties of microbial cells which, among others, distinguish them from higher cell typesdiminutive cell sizes and relative paucity of total concentration of cell componentsand are a reflection of their place in evolution. They a r e a reminder of the c a r e that must b e exercised in interpreting microbial cytology and in seeking analogies with structural components of advanced cell types. In the smaller microorganisms, limitations in light microscopical resolution preclude accurate descriptions, counts, or behavioral observation of intracellular constituents, particularly in studies of their nuclei. Low concentrations of such components as DNA, enzymes, and sometimes of polysaccharides in wall layers, are liable to prevent spatially accurate localizations with t h e same facility, positiveness or ease of microscopical visualization possible with most cytochemical reactions performed on higher cell types. T h e literature of microbial cytology is replete with statements as to t h e difficulties experienced in obtajning satisfactory preparations for definitive descriptions of detail in internal morphology or chemical identification of cellular components.The sharply divergent and polemical views arising from technical and observational problems are a measure of t h e uncertainties in interpreting material studied by t h e usual methods of fixation and staining.An extensiveanalysis of the various technical factors that have yielded discrepant cytological conclusions h a s been made in a recent critical review (Mundkur, 1962). For t h e purpose of t h i s paper, therefore, I confine attention to only a few selected examples of problems encountered by t h e microbial cytologist; and, by a comparison of photomicrographs and electronmicrographs of conventionally processed material with that obtained by freezing-drying and specific staining, to indicate the potential of improved techniques. *This paper, illustrated with slides.