192 Microscopic Histochemistry 



stained by lead impregnation ( Mallory'^^ ) . Nonenzymatic 

 staining of axons by lead can easily be mistaken for an en- 

 zymatic reaction. "^^ Control sections with either inactivated 

 ( by Lugol's solution or by boiling water ) tissue or with active 

 tissue incubated in the presence of 0.005 M fluoride must be 

 used to detect nonenzymatic impregnation; it may be ex- 

 tremely difficult or impossible to determine whether the pic- 

 ture obtained is not a composite of both enzymatic and 

 nonenzymatic staining. 



5. Nonoptimal composition of the incubating mixture. 

 The effect of the composition of the substrate mixture on 

 the constancy of the results has been studied by Gomori, 

 who found that the ratio between the concentrations of buf- 

 fer and substrate is an important factor. An unduly high 

 concentration of buffer will greatly reduce the sensitivity of 

 the method, thereby giving rise to "all or none" phenomena. 

 The original formula is definitely nonoptimal, and there can 

 be no doubt that some of the failures obtained with it are 

 due to this fact. 



6. Undertermined factors. This group includes the queer- 

 est types of failure, unexplainable by any of the causes enu- 

 merated. To mention only a few examples: sections cut from 

 a single block stain uniformly one day but utterly refuse to 

 stain the next day, although the incubating mixture is pre- 

 pared in exactly the same way both times; out of a ribbon 

 of several serial sections, mounted on one slide, one or two 

 stain exceedingly poorly while the majority show an excellent 

 reaction. 



Fixation of thin blocks in cold acetone, rapid embedding 

 at a temperature not exceeding 56° C, and the use of re- 

 cently cut sections and of the correct substrate mixture will 

 produce good results in a vast majority of instances; however, 

 occasional unexplainable failures cannot be eliminated com- 

 pletely. 



74. Mallory, F. B.: Am. J. Path., 12:569, 1936. 



75. Bartelmez, G. W., and Bensley, S. H.: Science, 106:639, 1947; 

 Heinzen, B.: Anat. Rec, 98:193, 1947; Lassek, A. M.: Stain Technol., 

 22:133, 1947. 



