SUMMARY: Steadily growing organisms were largely killed by slow freezing in buffer or by freeze drying. 100% survival was obtained after dropping bacteria suspended in 10% aqueous glycerol into liquid nitrogen and thawing. Ten per cent solutions of diethylene glycol, -erythritol, glucose, sucrose or polyethylene glycol (MW = 10,000) protected equally well; the last three substances did not penetrate the cell cytoplasm. The most lethal medium was dilute NaCl; broth, water or a dilute salt mixture were moderately lethal. These findings are incompatible with the view that the lethal effects of freezing are connected with osmotic shock or that protection from freezing damage requires penetration of the protective agent or osmotic dehydration of the cytoplasm. Cells frozen and thawed, even with a protective agent, showed a lowered rate of glycerol oxidation and a higher death rate when starved at the optimal temperature and pH value for growth. The storage life of frozen organisms at -20° depended on the protective agent used; only glycerol permitted extended storage.


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