ABSTRACT

Survival at liquid nitrogen temperature requires both the prevention of intracellular freezing by rapid cooling and the protection from harmful effects by slow cooling. A simple method of immediate immersion in liquid nitrogen generally results in a very poor survival rate except for a few organisms, which are probably able to lose intracellular water so rapidly as to prevent intracellular freezing and have a great tolerance to freezing. A wide-applicable technique is a two-step cooling method, which consists of a slow cooling step to an intermediate sub-zero temperature and a subsequent rapid cooling step to liquid nitrogen temperature. The stresses caused by slow cooling cease to increase at the end of the first step, and intracellular ice-crystallization during the rapid cooling is inhibited by the cell-content concentration that occurs during the first step. If cells are adequately dehydrated, residual intracellular liquid water transforms to a glassy state or crystals that are small enough to be innocuous. Resultingly, cells survive cooling to liquid nitrogen temperature.