Cryopreservation of Pathogenic African Trypanosomes In Situ: Metacyclic and Bloodstream Forms

SYNOPSIS. Of several methods developed for cryopreservation of Trypanosoma vivax, Trypanosoma congolense , and Trypanosoma brucei metacyclic forms in tsetse fly organs, as well as bloodstream forms in host blood, one proved the most satisfactory. In this method, infected fly proboscises and salivary glands were placed in glass capillary tubes containing fetal calf serum with 8% (v/v) glycerol as the cryoprotectant. The method for bloodstream forms involved the addition of glycerol directly to infected blood, which was then dispensed into capillary tubes. Next the tubes were placed in paper containers inside a glass test tube with a 5 mm thick plasticine jacket. The insulated assembly was suspended in the liquid nitrogen vapor phase in an LR-35 (Union Carbide) refrigerator for 45 min. Under these conditions, the cooling rate was 2 C/min. The frozen samples were transferred to permanent storage. The viability and infectivity of the preserved organisms were found to be satisfactory upon testing, and no antigenic changes were observed. Laboratory and field applications of the method are discussed.