NONOSMIOPHILIC TETRAZOLIUM SALTS THAT YIELD OSMIOPHILIC, LIPOPHOBIC FORMAZANS FOR ULTRASTRUCTURAL LOCALIZATION OF DEHYDROGENASE ACTIVITY

Abstract

Monotetrazolium salts were designed and prepared which incorporate a phthalhydrazide moiety to make them lipophobic and a benzothiazole moiety to make them react with osmium tetroxide after they are reduced to the corresponding formazans. The tetrazolium salts themselves do not react with osmium tetroxide under the conditions used for the cytochemical demonstration of enzyme activities. Although all of the new formazans, when dissolved in dimethylformamide, were reoxidized to the tetrazolium salts by osmium tetroxide, they were not reoxidized by osmium tetroxide when they were dissolved in tetrahydrofuran or precipitated by reduction in tissue, but gave dark complexes. One of the tetrazolium salts, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT), was readily reduced by succinic dehydrogenase activity, gave a formazan which produced a dark osmium complex relatively rapidly and gave good localization of succinic dehydrogenase activity on the membranes of mitochondria of rat myocardial cells. Although this tetrazolium salt (BSPT) was not photoreduced by fresh cells of Elodea to stain chloroplasts as seen with nitroblue tetrazolium or distyryl nitroblue tetrazolium, the 5-p-nitrophenyl derivative of BSPT was photoreduced by chloroplasts. The preparation of BSPT and its use in demonstrating succinic dehydrogenase activity ultracytochemically is given here. The preparation and ultracytochemical use of the 5-p-nitrophenyl derivative in chloroplasts will be published later.