An axenic culture system for the transformation of bloodstream forms to procyclic forms of Trypanosoma brucei brucei in vitro.

ABSTRACT

An axenic culture system for continuous cultivation of bloodstream forms of Trypanosoma brucei GUT at 3.1 and subsequent transformation of bloodstream forms to procyclic forms is described. Bloodstream forms were continuously grown at 37 degrees C in Iscove's modification of Dulbecco's medium (M-DMEM, with bovine serum albumin, transferrin and soybean lecithin supplemented with 100 microM hypoxanthine, 30 microns thymidine, 40 microM adenosine, 1mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% (v/v) heat-inactivated fetal bovine serum. In this system, 2-mercaptoethanol (2-ME) was essential and in the absence of 2-ME, 100 microM L-cysteine and 10 microM bathocuproine sulfonate could not be substituted for 2-ME. This culture system was useful for long-term culture of bloodstream forms of this clone. Axenic cultivation of bloodstream forms at 27 degrees C resulted in transformation to procyclic forms within 5 days in the same medium supplemented with 5 mM L-proline, 8 micrograms/ml hemin and 4 micrograms/ml hematin, respectively and, instead of FBS, 20% (v/v) hemoglobin-poor fraction of fetal bovine serum.