A rapid and simple spectroscopic method for the determination of yeast cell viability using methylene blue

Determination of cell viability is important in various microbiological studies. The microscopic method, counting dead cells stained by methylene blue (MB), has often been used for the determination of viability, although it is not efficient for the measurement of a large number of samples. Alternatively, some spectroscopic methods have been proposed to avoid tedious cell counting. One of these proposed methods detects the decrease in MB absorbance in the supernatant of cell suspension, because dead cells incorporate MB more efficiently than viable cells. However, at present, this spectroscopic method is rarely used due to its low throughput. Therefore, we devised a small-scale, rapid and simple method by improving several points as follows. (1) The peak wavelength of MB absorbance, 665 nm, was used to detect MB efficiently at the microtube scale. (2) The composition of the MB solution was improved by adding trisodium citrate. (3) The reaction time was shortened. And (4) the concentration ranges of both MB and cells, with which absorbance is linearly related to cell viability, were determined. The improved method enabled us to evaluate the dose-dependent toxicities of alcohols, antifungal/antimalarial quinacrine, and UV-C irradiation. The results were compatible with those of conventional microscopic counting and colony formation. The method would be applicable to automated determination and to various organisms such as bacteria and filamentous fungi which are difficult to be counted microscopically.