A new photometric assay with bromocresol purple for testing in vitro antitrichomonal activity in aerobic environment

Abstract

A new colorimetric assay relying on the acidic metabolism of Trichomonas vaginalis was developed for in vitro screening of various compounds against axenically grown trichomonads. Parasites from continuous culture were exposed to series of drug dilutions in a microtiter plate. After an incubation period of 48 h at 37 degrees C, the pH indicator of the medium had changed its colour in non-inhibited cultures due to the production of lactate and acetate. Inhibited cultures showed no colour changes. The use of bromocresol purple, a pH indicator, was suitable for several reasons: it is not toxic at the concentration used in the assay; the absorbance of bromocresol purple at 405 nm showed a linear correlation with both the pH of the medium and the viability of the trichomonads observed microscopically; plates could easily be read by eye or using an enzyme-linked immunosorbant assay (ELISA) reader. By comparison of the decreases in absorbance in test cultures with those in control cultures, IC50 values could be determined. Thus, IC50 of metronidazole was calculated at 25.5 +/- 2.3 mumol/l (n = 5) with the bromocresol purple assay and about 25 mumol/l after microscope observation. Minimal lethal concentrations (MLCs) could be read by eye. This test is now routinely used for anti-trichomonas evaluation of various chemical compounds and natural products.