Abstract
Monoformazan (MF) and diformazan (DF) are the reduction products of nitroblue tetrazolium (Nitro BT), commonly used for the histochemical detection of dehydrogenase activity. We designed a microphotometric image-analysis system, and used it to measure the separate amounts of MF and DF in a single Paramecium caudatum. We stained this cell in the presence of succinate and 1-methoxy phenazine methosulfate at 30°C, and took the observed density images (ODI) at 530nm and 650nm every 3min for 15 min. We used the component-separation method to reconstruct the individual absorption images of MF and DF from a pair of ODIs of the single cell at each reaction time. The estimation of MF and DF quantities in the cell are based on their total absorbance at each reaction time, and molar extinction coefficients at 530nm and 650nm, respectively. Our study indicates the difference in the spatial distribution and also appreciable kinetic differences in the formation between MF and DF, and provides a comparison between the kinetic data from the separate measurements and that taken at 590nm, which is near the isobestic wavelength of the two formazans (585 nm).
