Survival of Escherichia coli from freeze–thaw damage: a theoretical and practical study

Abstract

The effect of cooling and warming rates on the cryosurvival of chemostat populations of Escherichia coli was investigated. Survival curves were obtained which were similar in shape to those obtained by others for other cell types, with saline always a more lethal environment than distilled water.For organisms frozen in either distilled water or saline, as the cooling rate was increased survival increased to a maximum at 6 C/min, decreased to a minimum at 100 C/min, and then continued to increase as the cooling rate was increased to ultrarapid rates (about 6000 C/min). At these ultrarapid rates of cooling but not at lower rates, increasing the rate of warming enhanced survival; slow warming rates were detrimental to survival and eliminated the increase in survival associated with these cooling rates. Solution of a theoretical equation predicted that supercooling of cell water, and thus the likelihood of internal freezing, should increase as the cooling rate was increased from 3.5 to 350 C/min. This increase in probability of internal freezing was paralleled by a decrease in viability of cells as the cooling rate was increased from 6 to 100 C/min.The effectiveness of three protectants was investigated over the whole cooling rate range. Glycerol or sucrose added to suspensions of cells in distilled water protected over the whole range; but at lower cooling rates (less than 6 C/min) they did so with an altered stoichiometry. Sucrose protected organisms frozen in saline and Tween 80, a non-ionic detergent, protected organisms frozen either in saline or distilled water from rapid or ultrarapid cooling only. The evidence supports a two-factor hypothesis to explain cryoinjury.