Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates

Callow L. L. and Farrant J. 1973. Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates. International Journal for Parasitology, 3: 77–88. An investigation of the optimal conditions for the preservation of Leishmania tropica var major by freezing was undertaken because it was important to obtain a high yield when the thawed organisms were cultured. As a prerequisite for comparing different conditions, assay methods were devised. One method was based on the reduced growth of promastigotes in diphasic medium that was found to follow inoculation of relatively few organisms. The other employed serial ten-fold dilutions of suspensions of organisms, and the inoculation of medium at each dilution stage. Viability was related to the time taken for flagellates to be found in the medium. A 1·0 m solution of glycerol in the flagellate suspension inhibited growth when diphasic medium was inoculated. This effect was removed by separating the organisms from the glycerol before inoculation, or by diluting the suspension approximately one hundred-fold. A similar inhibition was not observed for dimethylsulphoxide (DMSO). Glycerol (1·0 m), DMSO (1·5 m), polyvinylpyrrolidone (10% w/v) and sucrose (0·3 m) were not obviously detrimental to the organisms. Normal growth in diphasic medium resulted when these additives were removed after being in contact with organisms for 5 h in an ice bath. In freezing experiments, flagellates survived freezing and thawing while they were still in contact with their nutrient medium, and also after they had been separated, washed and resuspended in isotonic saline with 10 mm of glucose. The survival rate was much greater when either 1·5 m DMSO or 1·0 m glycerol was added. These additives were compared at one rate only, $\text{0·3°C}\text{min}$, and DMSO gave better protection. With 1·5 m DMSO, maximal survival was obtained at a cooling rate of $\text{1·9°C}\text{min}$. Relatively high rates of cooling, that is, over $\text{400°C}\text{min}$ were detrimental to the organisms.