NONDESTRUCTIVE AND CONTINUOUS SPECTROPHOTOMETRIC MEASUREMENT OF CELL RESPIRATION USING A TETRAZOLIUM-FORMAZAN MICROEMULSION

Triton X-100 was incorporated into a tetrazolium dye reduction assay u sing ,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). Triton X-100 combines with the insoluble formazan product to form a h omogeneous dispersion, or microemulsion, which allows reliable spectro photometric measurement. The combination of this nonionic detergent an d dye allows respiration to be measured nondestructively and thus cont inuously. The assay combines MTT with phenazine methosulfate, the appr opriate cell growth substrate, and Triton X-100. Addition of the cells results in the conversion of MTT to the colored formazan. This assay was tested using cultures of bacteria and yeast. Various factors such as MTT concentration, cell number, and concentration of Triton X-100 w ere optimized. A comparison was then made between viable cell counts o btained by the plate count method and MTT-formazan production measured spectrophotometrically. A linear relationship was demonstrated betwee n cell number and MTT-formazan production. The advantage of this metho d is that the stabilized MTT-formazan microemulsion eliminates the nee d for organic solvent extraction steps and also inhibits further cellu lar reduction of the formazan to its colorless end-product. Because th e cells are not destroyed, as is the case with other tetrazolium metho ds using organic solvents, cell viability can be assessed rapidly. Som e bacteria can be assayed in only 30 min as compared to 6-12 h for oth er traditional tetrazolium methods.