Some aspects of the use of tetrazolium salts in quantitative histochemistry

The reduction of tetrazolium salts to highly coloured insoluble formazans is a reaction which has been widely exploited in histochemistry. This presentation considers three important aspects of the use of these compounds in quantitative studies, namely purity, intermediate reduction products, and calibration and measurement. Thin layer chromatography of tetrazolium salts from different suppliers has shown that standards of purity can vary widely, and that some samples are seriously contaminated. Studies with chromatographically pure compounds have demonstrated that the red diffuse colour seen in some reactions with di-tetrazolium salts is due to a half-reduced reduction intermediate. This "half-formazan" is part of the total activity, and cannot be disregarded in quantitative work. An understanding of the chemical nature of formazan end-products in tissue sections has made it possible to calibrate these reactions for microdensitometry.