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

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.