and the Dajac Laboratories of the Chemical Division of the Borden Company.Acknowledgement is due Mrs. Soveig Varnauskas for technical assistance, and Mr Harold Thomas for photomicrography. Research Fellow of the Rockefeller Institute for Medical Research. * One exception to this observation was noted. Apparently the high concentration of lipid within the adrenal cortex and its characteristic steroid composition favors the formation of crystal clumps. This phenomenon was not observed in tissue sections of the central nervous system. 422 MARVIN M. NACHLAS ET AL. bation solution was needed, 10 ml. of the buffered substrate reagent were added to 10 ml. of an aqueous solution containing 10 mg. of Nitro BT.* During this study, three additional solutions were prepared to compare the activating effect of certain ions upon succinic dehydrogenase activity. These were calcium chloride (0.03 M), * This reagent may be purchased from the Dajac Laboratories, Chemical Divuion,

A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion.

A simultaneous coupling azo dye method for the histochemical demonstration of yglutamyl transpeptidase activity using the new substrate 'y-glutamyl-4-methoxy-2-naphthylamide has been described. The method appears superior to previously reported methods for-y-glutamyl transpeptidase activity and can easily be modified for the electron microscopic localization of the enzyme by bridging osmium to the copper chelate of the azo dye via thiocarbohydrazide. The optimum conditions for the histochemical reaction were developed and the distribution of enzymatic activity in the tissues of the rat is described for light microscopy and with rat pancreas for electron microscopy. The electron-opaque deposits were seen in the endoplasmic reticulum in the vicinity of the zymogen granules 1 This investigation was supported by Research