206 Microscopic Histochemistry 



within the collodion membrane itself and can be washed off 

 together with the latter by acohol-ether before clearing the 

 section. 



The hydrolysis of T weens is susceptible to the effects of 

 activators and inhibitors of esterase-lipase, such as bile salts, 

 quinine, arsanilate, etc.^^^ 



The sensitivity of the Tween method (determined by 

 Coujard's technique) is ±7 fjM of acid liberated in 24 

 hours and per gram of active structure. Stearates are hy- 

 drolyzed at about one-third of the rate of laurates. 



B. The azo dye technique.— 



The first azo dye method for esterase was published by 

 Nachlas and Seligman in 1949;^^^ it was based on the enzy- 

 matic hydrolysis of /5-naphthyl acetate in the presence of a 

 diazonium salt. Their method is not entirely satisfactory be- 

 cause of diffusion artifacts which make fine localization im- 

 possible,^^^ although for gross orientation it produces usable 

 results. By replacing ^-naphthyl acetate with the corre- 

 sponding ester of a-naphthol or of naphthol AS and by se- 

 lecting the right pH and temperature, it is possible to avoid 

 diffusion artifacts completely and to obtain exceedingly 

 sharp pictures on a cytological scale. 



1) The a-naphthol technique— Although the optimal ac- 

 tivity of esterase in case of phenolic substrates is around pH 

 6.5, it is advisable to run the incubation at a pH of ±8 be- 

 cause of the much prompter azo-coupling of a-naphthol at 

 this pH. The loss in enzymatic activity at this nonoptimal 

 pH is a decided advantage, especially in the case of highly 

 active tissues, such as liver, intestine, pancreas, etc., which 

 often liberate naphthol at such a fast rate that the supply of 

 diazonium salt cannot keep pace with it ( see p. 146 ) . Even 

 at pH 8, diffusion may be quite disturbing. It is readily rec- 

 ognizable, even grossly, by the development of off-shades in 

 the more active tissues; ff it does occur, the incubating mix- 

 ture must be refrigerated and/or stirred. 



114. Gomori, G.: ibid., 67:4, 1948. 



115. Gomori, G.: J. Lab. & CHn. Med., 35:802, 1950. 



