Development of a modified MTT assay for screening antimonial resistant field isolates of Indian visceral leishmaniasis

Abstract

The semi-automated MTT colorimetric assay has previously been applied on Leishmania promastigotes based on the ability of viable parasites to reduce the tetrazolium salt to an insoluble formazan product. As promastigotes are non-adherent, application of the MTT assay in its original form has a major drawback of a high and variable background absorbance due to incomplete removal of phenol red, a component of most media. We have accordingly optimised a modified MTT assay wherein the absorbance linearity was maintained for cells ranging from 1 × 10⁴ to 1 × 10⁷ being 0.04 ± 0.003-2.38 ± 0.04. In contrast, the original MTT assay had a narrower linearity range of 1 × 10⁶-1 × 10⁷ cells, absorbances being 0.05 ± 0.005-1.54 ± 0.005. The modified MTT assay was effectively applied to study growth kinetics and identification of antimonial resistant field isolates. Considering the growing problem of antimonial unresponsiveness in the Indian subcontinent, this modified MTT assay is a useful tool for Leishmania research.