Ultracytochemistry for detection of O-2(-) in polymorphonuclear leukocytesusing the tetrazolium method: Comparison of various tetrazolium salts

The reduction of nitroblue tetrazolium (NBT) produces diformazan which show s localization of superoxide anion (O-2(-)) in polymorphonuclear leukocytes (PMNs). The detection of O-2(-) 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, includ ing nitroblue tetrazolium (NBT), tetranitroblue tetrazolium (TNBT) and 2-(2 '-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BS PT), by detecting O-2(-) using a histochemical and an ultracytochemical met hod. Peripheral rabbit leukocytes which ingested opsonized zymosan were pre pared for detection of O-2(-) The reacted leukocytes in each tetrazolium sa lt contained a reaction medium which was examined by light and electron mic roscopy. Each reaction of the tetrazolium salts took place in the phagosoma l membrane. The color of formazan in the TNBT was black rather than dark pu rplish-blue, and darker than that of NBT. On the other hand, the reaction c olor 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 demonstra ted fine deposits and was electron-dense. These reaction products correspon ded well with the membrane. These results suggest that BSPT could be employ ed for detection of O-2(-) not only in the histochemical method but also in ultracytochemistry and can be used as a new method.