After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 μl) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. The data were evaluated in Excel. For each test concentration, the percent growth inhibition was calculated in comparison with an untreated control and benznidazole at 10 μg/mL was included as positive control.