Effect of Storage Temperature, Equilibration Time, and Buffers on Survival of Tritrichomonas foetus in the Presence of Glycerol at Freezing Temperatures *

Tritrichomonas foetus survives when frozen in the presence of 1 M glycerol after growth in cysteine‐peptone‐liver‐maltose (CPLM) medium. In the present study, survival upon storage was better in a chest‐type freezer at a constant −21°C than in an upright freezer from which were recorded cyclic temperature fluctuations between −23 and −25° or in a dry‐ice chest with a nominal temperature of −72°. By the use of special thermocouples inside the freezing tubes, it was found that the temperature fluctuated between −22 and −24° in the chest‐type freezer, between −19 and −30° in the upright freezer, but rising as high as ‐2° when the door was opened and samples were removed, and markedly in the dry‐ice chest as the dry ice melted and was replaced, sometimes rising to as high as −27°. The poorer survival in the latter 2 freezers was considered due to temperature fluctuation. When equilibration with glycerol was carried out at room temperature, survival upon subsequent freezing was better following rapid equilibration (glycerol added all at once; equilibration time, 1 hour) than following slow equilibration (1/6 of the final amount of glycerol added each hour for 6 hr.; equilibration time, 7 hr.). Survival was extremely poor following either rapid or slow equilibration in the refrigerator (4°). Since T. foetus can grow indefinitely at 37° in the presence of 10% glycerol and since it is protected by glycerol when frozen, there is a critical zone near 4° in which glycerol appears to be toxic. Buffering the storage medium to pH's 6.3 to 7.1 with glycyl‐glycine increased survival upon freezing, but buffering to the same pH's with triethanolamine had no significant effect upon survival, and buffering to pH's 7.1 to 7.5 with phosphates decreased survival upon freezing.