Organic Substances 83 



fixatives the optimum duration of hydrolysis is much less 

 critical (10-30 minutes ).^^ 



SchifF's reagent itself may cause some hydrolysis on long 

 exposure. ^^^ This is especially important in the case of stain- 

 ing unhydrolyzed control sections because of the possibility 

 of obtaining false positive reactions. 



The following procedure is recommended: 



Method 



Hydrolyze tissue sections in N HCl preheated to 58°-62°C. 

 for 8-10 minutes. Wash them under the tap and stain in 

 Schiff's reagent as described in the section on polysaccha- 

 rides (p. 59). Naturally, omit counterstaining with hema- 

 toxylin; a yellow or green acid counterstain (picric acid, 

 orange G, or light green) is permissible and may actually 

 be advantageous. 



3. The acid moiety. There are a few methods published for 

 the specific identification of phosphoric acid in the nucleo- 

 tides. ^^^ The reliability of these methods, especially as far as 

 correct localization is concerned, is questionable (see under 

 "Phosphorus" ) . 



Other methods demonstrate simply the presence of acidic 

 groups of any kind. The use of indicators cannot be depended 

 upon to yield accurate information because the color con- 

 trasts are not sharp enough. 



The only methods to be used extensively are based on 

 basophilia, the afiinity for basic dyes, which is a property 

 of any substance possessing acidic groups. To obtain con- 

 firmatory evidence for nucleotides as the cause of basophilia, 

 specific extraction procedures must be resorted to in many 

 cases because of the presence of basophilic substances other 

 than nucleic acids (mucopolysaccharides) in the tissues. 



The basic dyes most often used are methyl green, meth- 



111. Serra, J. A.: Bol. Soc. Broteriana, 17:203, 1943. 



112. Lilienfeld, L., and Monti, A.: Ztschr. f. wissensch. Mikr., 9:332, 

 1892; Serra, J. A., and Queiroz Lopes, A.: Port, acta bioL, 1:111, 1945. 



