Ultracytochemistry for Detection of O2- in Polymorphonuclear Leukocytes Using the Tetrazolium Method: Comparison of Various Tetrazolium Salts

The reduction of nitroblue tetrazolium (NBT) produces diformazan which shows localization of superoxide anion (O2-) in polymorphonuclear leukocytes (PMNs). The detection of O2- in PMN is performed exclusively by using a microscopic NBT test, while an ultracytochemical method in NBT has seldom been employed. The present study compared various tetrazolium salts, including nitroblue tetrazolium (NBT), tetranitroblue tetrazolium (TNBT) and 2- (2′-benzothiazolyl) -5-styryl-3- (4′-phthalhydrazidyl) tetrazolium chloride (BSPT), by detecting O2- using a histochemical and an ultracytochemical method. Peripheral rabbit leukocytes which ingested opsonized zymosan were prepared for detection of O2-. The reacted leukocytes in each tetrazolium salt contained a reaction medium which was examined by light and electron microscopy. Each reaction of the tetrazolium salts took place in the phagosomal membrane. The color of formazan in the TNBT was black rather than dark purplish-blue, and darker than that of NBT. On the other hand, the reaction color of BSPT was purplish-red. The reaction formazans of NBT and TNBT were coarse and electron-dense by ultracytochemistry. Some of these diformazans were not associated with the membrane, while the formazan of BSPT demonstrated fine deposits and was electron-dense. These reaction products corresponded well with the membrane. These results suggest that BSPT could be employed for detection of O2- not only in the histochemical method but also in ultracytochemistry and can be used as a new method.