Evaluation of the Viability and Pathogenicity of Hemoflagellates after Freezing and Storage

As a method of maintaining hemoflagellates in the laboratory, 11 strains of culture forms of Trypanosoma cruzi and T. duttoni, 3 species of culture forms of Leishmania, and blood and tissue forms of T. cruzi, T. lewisi, and L. donovani were successfully frozen in the presence of glycerol and stored at -64 C for 6 to 18 months. At 18 months of storage three strains were still alive shortly after mechanical failure of the freezer. Many investigators have reported that various protozoa can survive and thawing. Gaylord (1908) found that Trypanosoma gambiense resisted the temperature of liquid air for 20 min, but was killed after 40 min. Flagellates, both blood and culture forms, have been frozen and stored for variable periods of time (Weinman and McAllister, 1947; Levaditi, 1952; Most et al., 1964). However, large numbers of the organisms were killed during freezing. Since the discovery that glycerol and some related substances can protect cells from the injurious effects of and thawing (Polge et al., 1949), many species of organisms have been successfully frozen by this method. Wide variations among different species in their resistance to have been reported and different methods of have to be used. Blood and tissue stages of hemoflagellates have been successfully preserved by in the presence of glycerol and have survived storage for long periods of time at -70 C (Reusse, 1956; Polge and Soltys, 1957; Walker and Ashwood-Smith, 1961; Cunningham et al., 1963a, b; Gordon, 1961). Application of these methods to hemoflagellates in vitro has been more limited. Since the routine maintenance of cultures of hemoflagellates is both costly and time-consuming, an attempt was made to put to practical use the methods developed for and storing both trypanosomes and leishmania cultures. Following the slow freezing method of Polge and Soltys (1957), 15 different strains of hemoflagellates were frozen, stored for various lengths of time, thawed, and checked for survival, viability, and pathogenicity. In addition, blood and tissue stages of some of the organisms were also frozen. An evaluation of this method is presented. MATERIALS AND METHODS Strains: The following species and strains of Trypanosoma and Leishmania hemoflagellates were preserved by freezing: