Vitrification and Crystallization of Organic Cells at Low Temperatures

Abstract

I. Theoretical considerations.—The conditions which determine whether a homogeneous liquid will solidify into the crystalline or into the vitreous state are discussed and a brief account of the rôle of the speed of crystallization, V, and that of grain formation, N, is given. These concepts are applied to the causes of the death due to crystallization of nondehydrated organic cells, and the physical conditions of cooling are developed under which such organisms can be solidified with a minimum of crystallization, assuring to this extent the preservation of their potential life. II. Technical considerations.—Methods for obtaining cooling rates up to 10⁴ deg. sec.⁻¹ applicable to organic cells in a highly dispersed state are developed. As subject for the experiments, pure strains of yeast (Saccharomyces cerevisiae) were chosen on account of the high sensitivity to low temperatures. As death criterion, the permeability of the cells to a standard aqueous solution of methylene blue was chosen, the death rate was thus determined microphotographically or with a counting chamber. Different techniques of exposure favoring and preventing crystallization are developed. III. Experimental results.—The rate of cooling of suspensions of living cells in aqueous media was varied from 1 to 10⁴ deg. sec.⁻¹ and thus conditions favoring either crystallization or vitrification of the cell plasma were produced. The death rate at an exposure of −185°C was thus varied from approximately 75 percent to approximately 3 percent. The duration of exposure at this temperature was varied from 5′ to 6000′; no appreciable influence of the time of exposure was found. The temperature to which the cells were cooled was varied from −50°C to −252°C and it was found that between −185°C and −252°C the temperature did not affect the death rate. Above app. −150°C an increase of the death rate was found, and a dependence upon the time of exposure, since the speed of crystallization begins to be appreciable. Repeated exposures of the same cells under conditions not especially favoring vitrification were studied and the integral death rate, δ_n (death rate after nth exposure) was investigated with respect to a constancy of the death rate (δ) at each freezing. It appears that the death rate for repeated freezings is not controlled by simple probability relations, but that other effects enter, such as weakening of cells by previous exposures and the selection of cold‐resistant cells by survival.