Trypanosoma cruzi: obtaining extracellular amastigotes and studying their development under different culture conditions

This work describes a protocol to obtain pure populations of extracellular amastigotes of Trypanosoma cruzi. The amastigote stage was obtained by means of temperature changes and human plasma added to the culture medium. Epimastigotes (clon BraC15C2) were first grown in F69 medium at 27 degrees C during 96 h and then at 36.5 degrees C. After three subcultures of 96 h each at the latter temperature a subsequent incubation in the presence of 5% human plasma, was needed to obtain a population of amastigotes that could be maintained indefinitely in the F69 or F29 media. This amastigote population was similar morphologically to that obtained through other methods. The kinetic of growth depended on the culture medium used (F29 or Brain-Heart Infusion, BHI). When culture was incubated at 27 degrees C in both media, the pre-exponential and logarithmic phases of growth were observed at 72-96 h and 24-48 h respectively. The change in stage from amastigote to epimastigote dependent whether amastigote were subcultured or not. The growth of amastigotes in BHI medium at 36.5 degrees C did not occurred. The growth of amastigotes was similar to those observed at 27 degrees C when F29 medium was used although the transformation to epimastigotes did not take place at this temperature. A population over 99% of amastigotes were maintained at 36.5 degrees C indefinitely by means of subcultures in F29 medium.