Studies on the conditions required for optimum recovery of Tetrahymena pyriformis strain S (Phenoset A) after freezing to and thawing from -196 C.

The toxicity of several cryoprotective agents was tested at room temperature (23 C) against Tetrahymena pyriformis strain S (Phenoset A) at different stages of growth cycle. Polyvinylpyrrolidone (PVP-40) at 10 per cent (v/v) concentration was without effect at any stage in the growth cycle, while 1.2 M glycerol immobilized the cells which were disrupted very shortly afterwards. The toxicity of 0.25 M glucose was largely independent of the position of the cells in the growth cycle, but the toxicity of 0.25 M sucrose and 1.4 M dimethylsulfoxide (DMSO) was most marked in late log- and stATIONARY-PHASE CELLS. After log-phase cells had been equilibrated with 1.4 M DMSO for 1 hr, the number of cells surviving cooling at defined rates from 0.45 to 12 C/min decreased as the final temperature decreased from -30 to -60 C. A temperature of -53 C was found to be the optimum from which cells cooled at a given rate could be cooled rapidly to -196 C. Nevertheless, when cells were cooled at defined rates to -35, -45, or -53 C and then rapidly to -196 c the optimum rate of cooling to these temperatures was found to be 1 C/min. The optimum rate of cooling to -60 C prior to plunging into liquid nitrogen was found to be 2.7 C/min.