Aspects of tetrazolium salt reduction relevant to quantitative histochemistry

Summary Uncertainty about the nature of the reduction products of ditetrazolium salts may have limited their use in quantitative histochemistry. Our studies have shown that under appropriate conditions pure Nitro-BT reduces through a red intermediate substance to a stable blue diformazan. Nitrobenzene was found to be a satisfactory solvent for this diformazan. The monotetrazolium INT may also be reduced to a formazan through an intermediate phase. The amounts of definitive formazan produced from both monotetrazolium and ditetrazolium salts may be influenced by the solubility of their intermediate reduction compounds in the systems in which reduction is occurring. The yield of definitive diformazan from Nitro-BT after chemical reduction, and after enzymatic reduction in liver homogenate and sections of a mock tissue, was not in linear proportion to the strength of reducing conditions; however, the yields of formazan from the monotetrazoliums INT and MTT were linear. This finding suggests that in quantitative histochemistry it is essential to calibrate reactions involving ditetrazolium reduction.This work was supported by a grant from the National Health and Medical Research Council of Australia.