Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTI' [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the M'IT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colonyforming units was demonstrated for all of the species investigated, indicating that the M" assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts. The ability to reduce tetrazolium salts varies among mycoplasma species and is used as a means of characterization. This ability is usually determined by using the test described by Aluotto et al. (2). In this stucty we investigated the suitability of the MTT [3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide] assay for demonstrating tetrazolium reduction by mycoplasmas. The MTT assay is a colorimetric test that was originally developed to determine the survival and growth of eucaryotic cells in proliferation and cytotoxicity tests (8) and was later adapted for monitoring the growth of bone marrow cells in microtiter plate cultures (7). Mycoplasmas. The mycoplasma species that we used for this study are listed in Table 1. All of the mycoplasmas were cultivated in modified Friis medium (5) under aerobic conditions (5% COz) and anaerobic conditions (anaerobic jars; Oxoid) at 37°C. The numbers of colony-forming units were determined by using the method of Albers and Fletscher (1). M'IT assay. Mycoplasmas (100-pl volumes) in the logarithmic growth phase (lo8 to lo9 CFU/ml) were diluted 1:2 with modified Friis medium in 96-well microtiter plates. A 10-pl portion of an MTT solution (5 mg of MTI' per ml of phosphate-buffered saline) was added to each well, including two wells that contained only medium (100 pI) (controls). All tests were run in triplicate and preparations were incubated for 4 h at 37°C in an atmosphere containing 5% COz. For the spectrophotometric evaluation, 100 pI of a solution containing 20 g of sodium dodecyl sulfate, 50 ml of dimethylformamide, and 50 ml of distilled water (adjusted to pH 4.7) was added to each well, and the plates were incubated for 15 h (overnight) at 37°C to dissolve the formazan crystals. The plates were then read spectrophotometrically with a model SLT 400AT photometer at 540 nm. The results were evaluated by using a modification of a computer program that was written to evaluate enzyme-linked immunosorbent assay data. Briefly, we performed an analysis of variance, a lack of fit test, and a linear regression analysis of the linear part of