Structure of HeLa cells after treatment with glycerol as revealed by freeze-etching and electron microscopic methods

Abstract

HeLa cells were incubated for 2 h with a 30% glycerol concentration. Ultrathin sections of the glycerinated cells, chemically fixed with a conventional fixative isotonic to blood plasma, showed considerable changes in the cell's fine structure. These changes did not occur to the same degree in freeze-etched cells. The alteration-marked disruptions of cellular organelles—interruptions of membranes and extraction of the ground plasm—were related to the level of the glycerol concentration. Cellular damages interpreted as arising from osmotic shock were not observed when glycerol was added to the fixative corresponding to the degree of the cellular glycerol inhibition. Thus the osmolality of the fixative was adapted to the changed osmolality of the cells impregnated with glycerol. The best preservation was seen when cells were incubated with a slowly increasing concentration of glycerol that reached 30% at the end of 2 h. The only changes in the fine structure of these cells were a slight increase in the electron density of the cytoplasm of approx. 15% of the cells, which also displayed slightly wrinkled nuclei. These results are discussed with regard to the consequences for freeze-conservation of biological material, especially tissue culture cells. Freeze-etching revealed the three-dimensional structure of cells with their different compartments. All cell organelles known from conventional ultrathin section techniques were also seen in freeze-etched specimens. Hitherto unknown from conventional electron microscopic techniques, however, are the particles on the plasma membrane and membranes of the ergastoplasm, the nuclear envelope, the vacuoles, and the Golgi cisternae. The character of the particles on the outer side of the plasma membrane is discussed.