Abstract
A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G1 cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.
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