Rp. Stowe et al., NONDESTRUCTIVE AND CONTINUOUS SPECTROPHOTOMETRIC MEASUREMENT OF CELL RESPIRATION USING A TETRAZOLIUM-FORMAZAN MICROEMULSION, Journal of microbiological methods, 22(3), 1995, pp. 283-292
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.