ALKALINE PHOSPHATASE 79 



same procedure but oniitting the treatment with substrate. Another 

 procedure for use with bone has been given by Bourne (1943) in- 

 volving the addition of 0.01% sodium alizarin sulfonate to Gomori's 

 substrate medium. The calcium phosphate produced is automatically- 

 stained red by the alizarin dye. 



The use of magnesium ions to activate the phosphatase was intro- 

 duced by Kabat and Furth (1941) and is employed in the revised 

 method of Gomori ( 1946c) . 



It is of interest to call attention to the different approach to the 

 staining technique for alkaline phosphatase that was brought for- 

 ward by Menten, Junge, and Green (1944). These investigators em- 

 ployed a reaction of the organic, rather than the phosphate, moiety 

 of the substrate to precipitate a reddish-purple dye at loci of phos- 

 phatase action. Employing calcium /j-naphthol phosphate as the sub- 

 strate, |3-naphthol liberated by the enzyme was made to react at 

 once with diazotized a-naphthylamine present in the substrate solu- 

 tion. While this procedure can undoubtedly be applied in many in- 

 stances, it would appear to offer no advantage over the Gomori 

 method, and, as Menten et al. readily admit, in its present form the 

 test is intricate and probably not well suited to routine use. Never- 

 theless, Yin (1945) employed this method for plant tissues in order 

 to avoid interference by preformed phosphates. Since the preformed 

 phosphates can be removed with citrate buffer (page 78) it would 

 appear that interference from this source need not be made a deter- 

 mining factor in the choice of a method. 



Gomori Revised Method for Alkaline Phosphatase 



SPECIAL REAGENTS 



0% Acetylcellulose (Eastman's No. 4644) in acetone. Optional. 



1-2% Cobalt Acetate, Chloride, or Nitrate. 



Ammonium Sulfide Solution. A few drops of yellow ammonium 



sulfide soln. to a Coplin jar of distilled water. 

 Substrate Medium, pH 9.4. (Will keep in refrigerator for months.) 



Combine 25 ml. 2% sodium glycerophosphate, 25 ml. 2% sodium 



barbital, 50 ml. distilled water, 5 ml. 2% calcium chloride, 2 ml. 



2% magnesium sulfate, and a few drops of chloroform. 



PROCEDURE 



1. Place slices of fresh tissue, under 2 mm. thick, in chilled abso- 



