GENERAL TECHNICAL CONSIDERATIONS 31 



and distilled water should each have an addition of 0.5 milli- 

 liter of sodium veronal per 100 milliliters of solution. 



The method just described localizes the phosphate end of the 

 ester. A method published by Menten, Junge, and Green in 

 1944 and Danielli in 1946 localizes the alcoholic end of the 

 molecule as an insoluble diazo dye. In this method the incuba- 

 tion medium contains a phenol phosphate and a diazonium hy- 

 droxide. Phenol phosphates do not react with diazonium hy- 

 droxides. But, in the presence of phosphatase, the ester is split, 

 giving rise to phenol, which reacts very rapidly with the dia- 

 zonium hydroxide. By a correct choice of phenol and dia- 

 zonium hydroxide, it can be arranged that the reaction product 

 of the phenol and diazonium hydroxide is very insoluble in 

 water and is therefore precipitated in the section in the vicinity 

 of the enzyme. 



The medium which is conveniently used for these experiments 

 consists of a phenol phosphate solution in sodium veronal, con- 

 taining 0.1 gram of phenol phosphate suspended or dissolved 

 in 50 milliliters of 2 percent sodium veronal solution. To this 

 is added, just before use, 25 milliliters of diazonium hydroxide 

 solution containing 0.2 percent of diazonium hydroxide. The 

 diazonium hydroxide solutions may be prepared by the methods 

 given by Saunders (1936). The diazonium hydroxide may be 

 kept for a period of hours in the hydrochloric acid solution in 

 which it is prepared, or often for more prolonged periods as a 

 stabilized compound in solid form. Just before use it is brought 

 to pH 9.2-9.3 by the use of thymol blue as an indicator, and 

 then mixed with phenol phosphate solution and filtered, keeping 

 the temperature between 6 and 8° C. throughout the operation. 

 After nitration the slides may be placed in the medium. Many 

 diazonium hydroxides decompose rather rapidly under these 

 conditions, and it may be necessary to change the incubation 

 medium every 20 minutes to obtain optimal results. 



Some workers have had difficulty in using this technique, 

 through paying insufficient attention to the adjustment of the 

 pH of the diazonium hydroxide solution. The diazonium hy- 

 droxide reacts with the indicator thymol blue, which is itself 

 a phenol, to give another indicator substance which has a color 

 change very similar to that of thymol blue, but with which the 



