Cryoprotectants for Crithidia fasciculata Stored at ‐20 C, with Notes on Trypanosoma gambiense and T. conorhini

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Abstract
SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log‐phase or end‐of‐log‐phase cultures were frozen for 24–48 hr at ∼ ‐20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2′‐dioxyethanol (diethylene glycol); 1,2,4‐butanetriol; 1,4‐cyclohexanediol; dimethylsulfoxide; propylene glycol; and N‐acetylethanolamine. Several sugars were active, e.g., D‐arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood‐enriched media + 2‐2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3‐week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.