Growth of ice crystals in frozen specimens

The formation of ice crystals, which might be the possible artefact in cryo-techniques for electron microscopy, was examined during the rewarming process of rapidly frozen erythrocytes. Intracellular ice formation, which is usually found in cells suspended in saline by rapid freezing, was inhibited by the addition of 30% glycerol. When such glycerinated cells, having no ice crystals at liquid nitrogen temperature, were rewarmed to higher temperatures above ? 80° C, recrystallization of ice occurred. Ice particles became visible within the cells even at ? 80°C and grew larger with a temperature rise. From the results obtained in the morphological and physiological investigations, it also became evident that the recrystallization of ice appeared prior to the increase in haemolysis during the rewarming process.