Leishmania donovani: A Chemically Defined Medium Suitable for Cultivation and Cloning of Promastigotes and Transformation of Amastigotes to Promastigotes

A chemically defined medium using commercially available α-MEM supplemented with HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 × 10⁷/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients’bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16–20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.