ELECTRON MICROSCOPIC DEMONSTRATION OF DEHYDROGENASE ACTIVITY WITH A NEW OSMIOPHILIC DITETRAZOLIUM SALT (TC-NBT)

Dissatisfaction with the degree of contrast produced by currently available tetrazolium salts in electron microscopy of the dehydrogenases stimulated us to design and prepare an osmiophilic ditetrazolium salt (TC-NBT)3 containing thiocarbamyl groups in innocuous positions of the nitro-BT molecule. The new reagent has the same favorable properties as nitro-BT for light microscopy and, in addition, is convertible to osmium black when tissue sections containing the diformazan are exposed to osmium tetroxide vapor. The stained tissue sections withstand dehydration and embedding in Araldite. Fresh and formalin-fixed rat heart, cut in thin slices without freezing, were stained for SDH and NADH2-D activity. In fresh heart, both dehydrogenases produced dense and thickened cristae in mitochondria that were active. Heterogeneity of mitochondria ris à vis dehydrogenase activity was confirmed. In fresh preparations stained mitochondria were contracted with narrowing of intercristal spaces as compared to inactive mitochondria. This was not the case in formalin-fixed heart. The dehydrogenase activity of individual mitochondria appeared to be an all-or-none phenomenon in these preliminary studies and the diformazan deposits were quite uniformly distributed along both surfaces of the cristae with encroachment on intracristal spaces in heavily stained preparations. The outer limiting membranes of the active mitochondria did not have diformazan deposits in contrast to the heavily stained cristae. Unreactive mitochondria revealed the usual contrast seen in control sections exposed to osmium tetroxide or in deep parts of the blocks to which TC-NBT did not penetrate.