The effects of various salts on the viabifity of T4 and T4 Bo phages during freezing and thawing have been studied. The effectiveness of different salts in causing inactivation or protection of phage during freeze—thawing was of the same order as has been observed for their effects on the conformational stability of macromolecules. There were two important contributory factors causing inactivation of phage during freeze—thawing: (i) concentration of solutes during freezing, and (ii) lowering the temperature, without change in salt concentration.
The viability of phage following storage at subzero temperatures was dependent on the storage temperature, the species of ions in the suspending medium and the initial salt concentration. Viability was greatest when phage had been initially suspended in dilute solutions.
Survival of T4 Bo phage, following cooling to subzero temperatures and rewarming, was greater in D2O solutions than in H2O solutions.
It was found that 20% glycerol completely protected against inactivation of T4 Bo phage, suspended in 3·5 molal NaBr, which were cooled to subzero temperatures and rewarmed in the absence of ice. Without added glycerol the viability of T4 Bo phage suspended in 3·5 molal NaBr, which were cooled to −20° C and rewarmed was less than 1%. This protective effect of glycerol is in contradiction to the accepted views of its mode of action in prevention of freezing injury.
This work has been supported in part by grants from the Medical Research Council and the Office of Naval Research under contract F 61052–68-C-0041.