Growth of Isolated Amastigotes of Trypanosoma cruzi in Cell-Free Medium1

Amastigotes of Trypanosoma cruzi were purified from overlays of infected Vero cell cultures by centrifugation over a discontinuous gradient of metrizamide. Pure amastigote preparations were usually recovered from the pellet under the layer of specific gravity 1.086. The isolated amastigotes grew in cell-free ML-15HA medium. Growth rate for the different strains of T. cruzi were in the order Y > Tulahuan > CL. The generation time of amastigotes in ML-15HA medium was 16.8, 18.0, and 26.4 h for the Y, Tulahuen, and CL strains, respectively, in the presence of 5% CO₂) and 16.8, 31.2, and 36.4 h, respectively, in the absence of CO₂. Intracellular amastigotes did not differ ultrastructurally from amastigotes from either the density-gradient fractionation or culture in cell-free medium.