Electron microscopic study of microorganisms subjected to freezing and drying: Cinematographic observations of yeast and coli cells

Abstract The processes of sublimation of extra- and intracellular ice and the changing cellular morphology of microorganisms during freezing and drying were recorded by special cinematographic equipment incorporated in an electron microscope equipped for low temperature work. Yeast and coli cells suspended in distilled water or 5 per cent glucose or 1 per cent gelatin solution were sprayed on carbon-collodion films and rapidly frozen by immersion in liquid nitrogen. On completion of the freezing process they were transferred to the specimen chamber of a specially equipped electron microscope. Cinematographic recording revealed the following: under low magnification the frozen droplets were observed to have diameters of 20 to 50 μ. These were scattered throughout the field and were opaque to the electron beam. As sublimation proceeded the cells embedded in the droplets gradually became visible and of recognizable contour. After the ice surrounding the cells disappeared, the transfer of intracellular water, frozen or unfrozen, occurred from within the intact cells. Sublimation of water from damaged cells took place simultaneously with sublimation of extracellular ice. Yeast cells retaining their original size and shape during sublimation of the surrounding extracellular ice gradually lost their opacity, attributed to intracellular ice crystals, and a porous structure within the cell became evident. During and after the sublimation of extracellular ice, morphological changes including swelling, cell contraction, and the development of small irregularities in the cell surface were observed. These changes are believed due to the irradiation with the electron beam. In contrast to yeast cells, most coli cells underwent shrinking, probably because the absence of intracellular freezing permitted the water transfer to the extracellular environment. After drying, coli cells exhibited no remarkable change in morphology. After drying, a residual network of glucose or gelatin was observed among the cells suspended in those media before freezing. The changes in cellular morphology seen in the aqueous suspensions were not observed during and after drying of the cells suspended in these media.