142 Microscopic Histochemistry 



In other cases, including all hydrolytic enzymes, the prod- 

 uct of enzymatic activity is a colorless and soluble substance 

 (ions, phenolic compounds) and must be precipitated in a 

 second step by a suitable reagent, added to the incubating 

 mixture. If correct localization and high sensitivity are to be 

 achieved, precipitation must be very prompt (of the veloc- 

 ity of ionic reactions ) in order to bind the reaction product 

 as fast as it is liberated, and the precipitate must be exceed- 

 ingly insoluble. The less perfectly these conditions are satis- 

 fied, the less sensitive the method and the poorer the local- 

 ization of activity will be. 



These important principles will require a somewhat more 

 detailed consideration. 



No substance is totally insoluble. If an "insoluble" com- 

 pound is made from its components in a solution, it will pre- 

 cipitate only if and when its solubility (or, in the case of a 

 salt, solubihty product) is exceeded. Constancy of results, 

 maximum sensitivity and correct localization in a histochemi- 

 cal experiment can be attained only if the velocity of precipi- 

 tation is almost infinite and the precipitate is practically in- 

 soluble. These two aspects do not necessarily run parallel; 

 even in the case of highly insoluble precipitates a transient 

 phase of supersaturation or colloidal state may occur. For- 

 tunately, this is very rarely observed in practice. In the opti- 

 mal case all the reaction product v^ll be precipitated imme- 

 diately and at the exact site where it is formed. Otherwise, 

 some of it will diflFuse away from the primary sites of forma- 

 tion before it can be precipitated and raise the concentration 

 in the ambient fluid. In unfavorable cases very little or even 

 none will precipitate locally, in spite of high enzymatic activ- 

 ity; practically all the reaction product will be washed away 

 and contribute to the saturation of the medium, first only 

 around centers of high activity and later everywhere. The 

 difference between the rates of hydrolysis and of loss by dif- 

 fusion must have a minimum absolute value, proportional to 

 the solubility of the precipitate, for a local precipitation to 



