THE USE OF A WATER-SOLUBLE FORMAZAN COMPLEX TO QUANTITATE THE CELL NUMBER AND MITOCHONDRIAL-FUNCTION OF LEISHMANIA-MAJOR PROMASTIGOTES

One of the methods to quantitate Leishmania major promastigotes (LmP) has been to utilize the formation of a formazan dye, which in turn is produced via conversion of an artificial substrate, (4,5-dimethylthiaz ol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method has one maj or drawback in that the formazan complex precipitates inside the paras ites and has to be extracted by denaturants before measurements can be performed. By using a new synthetic substrate, 3-(4,5-dimethylthiazol - arboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS), the extrac tion procedure is eliminated as the formazan-like dye is released spon taneously into the medium, making it possible to perform several measu rements on the same parasite culture without disturbing or killing the parasites. The measurements were shown to reflect the numbers of para sites as confirmed via comparative experiments using radioactive thymi dine uptake and cell counting, respectively. The method is simple, fas t, and highly reproducible and is suitable for drug screening, identif ication of drug-resistant isolates, and growth-kinetics studies. It is therefore contemplated that the MTS method will be a general and usef ul technique in this field of parasitology.