Adaptation of the methyl thiazole tetrazolium (MTT) reduction assay to measure cell viability in Vibrio spp.

Abstract

Pathogenic vibrios of farmed shrimp have acquired resistance to antibiotics commonly used in aquaculture, leading to a constant search for new, effective products to control them. In vitro methods currently used to evaluate possible solutions are expensive, tedious, and time-consuming. In contrast, the methyl thiazole tetrazolium (MTT) reduction assay, widely used in mammalian cell lines to measure the cytotoxic effect of drugs, is inexpensive and reproducible. The objective of this study was to standardize an in vitro method, based on the reduction of the MTT compound, to measure cell viability and to evaluate the anti-Vibrio effect of bioproducts. The reduction capacity of MTT was evaluated by the formation of formazan crystals in Vibrio harveyi, V. campbellii, V. parahaemolyticus and V. vulnificus. Subsequently, the method was validated using essential oil of Origanum vulgare (EOOv) at different concentrations to obtain a dose-response effect. Oxytetracycline was used as a positive control at microbicidal doses. All vibrios were able to reduce the MTT to formazan and the effect of EOOv on the ability to reduce MTT depended on the Vibrio strains. The antibiotic affected the viability of the four vibrios at all concentrations evaluated and is understandable due to its microbicidal effect. In conclusion, the MTT reduction test was successfully adapted to measure viability and constitutes a suitable alternative to evaluate antimicrobial products quickly and accurately.