Enzymes 145 



nonoptimal conditions of incubation, such as too low pH 

 values or Ca ion concentration. As the pH and Ca ion con- 

 centration are raised, the localization becomes progressively 

 sharper, and the stained areas shrink concentrically. Actually, 

 under optimal conditions (p. 184) and an incubation time 

 not exceeding IM hours, the diffusion artifacts described by 

 Danielli^ and by Jacoby and Martin^- ^ cannot be repro- 

 duced.^^ This observation, together with the facts that (1) 

 artificial gradual supersaturation of the substrate mixture or 

 the diffuse impregnation of the section with enzyme produce 

 pictures entirely different form those obtained by the regular 

 method and (2) the localization of activity is the same with 

 the calcium-cobalt and the azo dye methods, does not sup- 

 port the theory of the importance of false secondary localiza- 

 tions. Furthermore, the thesis that phosphatase is distributed 

 evenly (or at random) among all cells or even within one 

 cell body cannot be accepted. Comparison with quantitative 

 Coujard slides clearly shows that in many (if not most) cases 

 the enzyme is concentrated in very small, discontinuous 

 spots, the activity of which may exceed the average value 

 for the whole tissue by a factor of the order of 100. Activities 

 as high as 700 Bodansky units per gram of active structure 

 (as against the average value of 1 unit, assumed by the au- 

 thors) have been observed.^^ On the basis of the facts men- 

 tioned, it is safe to assume that the highest intensity of the 

 histochemical reaction coincides with centers of enzymatic 

 activity. Further experiments will have to decide whether 

 the diffusion artifacts persisting in spite of optimal condi- 

 tions are within or beyond the resolving power of high-power 

 dry objectives. 



It must be mentioned here that diffusion of the enzyme 

 itself has been blamed for false localizations.^^' ^^' ^^ Diffusion 



14. Gomori, G.: Unpublished. 



15. Gomori, G.: Exper. Cell Research, 1:33, 1950. 



16. Danielli, J. F.: Nature, 165:762, 1950. 



17. Hebert, S.: Arch, de biol, 61:235, 1950; Yokoyama, H. O., Stowell, 

 R. E., and Tsuboi, K. K.: J. Nat. Cancer Inst., 10:1367, 1950; Yokoyama, 

 H. O., Stowell, R. E., and Mathews, R. M.: Anat. Rec, 109:139, 1951. 



