Enzymes 151 



purplish, or blue formazans, insoluble in water and soluble 

 in fats; and (3) tellurites (first introduced into bacteriologi- 

 cal technique by Klett),^* reduced to insoluble black ele- 

 mentary tellurium. 



The methylene blue technique is not recommended be- 

 cause (a) the negative image it gives allows only a very 

 poor locahzation and (b) the method is cumbersome and 

 must be carried out under strictly anaerobic conditions to 

 prevent the reoxidation of the leuco-base by atmospheric 

 oxygen. The tetrazolium method is the most sensitive of the 

 three and aflFords an excellent localization, except for an 

 occasional secondary staining of fat droplets by formazan, a 

 complication which should be borne in mind to avoid mis- 

 interpretation of the pictures. It should also be mentioned 

 that some of the commercial batches of tetrazoHum com- 

 pounds may contain oxidizing substances (probably lead 

 tetra-acetate ) as an impurity and that these interfere with 

 the reaction. The formation of dye should be quite notice- 

 able after about 5-10 minutes of incubation if good active 

 material is used (e.g., rat kidney); if not, the reagent must 

 be recrystallized by dissolving it in a small volume of ab- 

 solute alcohol and precipitating it with 4-5 volumes of ether. 

 The tellurite method is considerably less sensitive, but it 

 gives nice, sharp pictures. 



Even without the use of any substrate, positive reactions 

 will be obtained in most cases on account of the presence of 

 various endogenous substrates in the tissues. Although such 

 reactions of an undefinable substrate specificity are usually 

 weak, the substrate being exhausted rapidly, one should try 

 to avoid them by rinsing the sections before incubation. For 

 all practical purposes, reactions obtained in rinsed sections 

 will be specific for the dehydrogenases of the substrate 

 supplied. 



Maintaining the right pH under incubation is important. 

 The optimum for the enzymatic activity is pH 7.3-7.6; at 



24. Klett, A.: Ztschr. f. Hyg., 33:137, 1900. 



