THE VARIABLE OF pH IN THE BROMINDOXYL ACETATE METHOD FOR THE DEMONSTRATION OF ESTERASE

Abstract

In the new incubation medium, 1.5 mg of 5-bromoindoxyl acetate was dis3olved om 0.1 cc of absolute ethanol and to this was added 0.3 cc of hexazonium pararosanilin (Barka, T. and Anderson, P. J., J. Histochem. Cytochem. 10: 241, 1962) buffered to pH 6.1 with 9.6 cc of 0.1 M tris (hydroxymethyl) aminomethane/HCl buffer. Another solution was prepared as above to which was added 3 mg/cc of sodium fluoride, a reversible inhibitor of cytoplasmic esterase. Neither of these solutions contained potassium ferror-ferricyanide. Incubation time was ten minutes. With this experimental design, the enzymatically liberated 5-bromoindoxyl is immediately coupled with the pararosanilin before any diffusion of product or its oxidation to 5,5''-dibromoindigo has an opportunity to occur. This eliminates the need for an oxidant in the incubation medium and the variables attending its use (Schnitka and Seligman, J. Histochem. Cytochem. 9: 514, 1961). The results indicated a significant amount of cytoplasmic reaction and discrete red droplets. The cytoplasmic reaction was inhibited by the addition of sodium fluoride to the incubation medium.