Cryopreservation of Resting Cysts of the Freshwater Ciliate Meseres corlissi by Conventional Two-Step Methods and One-step Vitrification Protocols

Trophic cells of the freshwater ciliate Meseres corlissi CCAP 1647/1 proved to be recalcitrant to all the cryopreservation methods tested; however, resting cysts of this strain were amenable to both conventional two-step cryopreservation and ultra-rapid vitrification methods. Conventional controlled rate cooling and Mr. Frosty cooling methods, employing 5% dimethylsulphoxide (DMSO) as a cryoprotectant were effective in preserving cysts in either liquid medium or soil suspensions. Alternative cryopreservation methods involving dehydration of soil/cyst suspensions, in the absence of any colligitative cryoprotectant, and rapid cooling over liquid nitrogen or plunge freezing were effective. The level of residual moisture was the critical factor with no survival observed in any samples with >35% residual moisture, and high levels of survival (>50%) in samples with <14% residual moisture. Trophic cells obtained from cryopreserved cysts appeared healthy and did not differ obviously from the controls with respect to morphology, movement, division and encystment/excystment reactions. In a test culture derived from material which had been cryopreserved by the rapid cooling method a maximum growth rate of 1.32 d^-1 (at 22°C) was determined, a value which agrees well with earlier observations on the original strain.