CHAPTER IV 



CONTROLS TO PROVE VALIDITY 

 OF TECHNIQUE 



In a number of cases the reaction used is so highly specific 

 that no control is necessary; a positive reaction indicates the 

 presence of the substance searched for with absolute cer- 

 tainty (for instance, the Prussian blue reaction for Fe"^ + + ). 

 In other cases, however, "blanks" must be run, as in ana- 

 lytical chemistry or biochemistry, to avoid confusing the 

 genuine reaction with other similar reactions of a nonspecific 

 nature. These blank runs are especially important in the 

 identification of enzymes. 



The two main methods for the verification of the speci- 

 ficity of enzymatic reactions are ( 1 ) the omission of essential 

 ingredients (e.g., the substrate or Ca ions in the technique 

 for alkaline phosphatase) and (2) the use of inactivators or 

 inhibitors, such as excessive heat, strong acids, oxidants, 

 fluoride, eserine, etc., depending on the nature of the enzyme 

 investigated. Whatever reaction persists after such treatment 

 cannot be due to enzymatic activity. 



One of the difiiculties of identification in histochemistry is 

 the impracticability of applying reactions to purified sub- 

 stances. The compounds investigated almost invariably occur 

 in association with, and often adsorbed on, other compounds. 

 The presence of these may profoundly modify the typical 

 reactions, solubility, color, and other properties of the com- 

 pound investigated as listed in textbooks of analytical chem- 

 istry. In addition, fixatives may cause such significant 

 changes in the reactive groups as to make them unrecogniz- 

 able by the accepted identifying reactions. These are the 

 main reasons why test-tube reactions, even if they otherwise 



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