A comparison of methods for morphological studies of cultured cells.

Abstract

A number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouin's solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouin's solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope.