method for staining chromosomes. To demonstrate the presence of sub- 

 stances such as proteins, nucleic acids, carbohydrates, and Hpids in a 

 particular cell structure requires the use of specific cytochemical tests 

 employing chromogenic agents which are selectively bound to one or 

 more molecular groupings of the substance under study. This specificity 

 can be improved in many cases by differential digestion with enzymes to 

 eliminate interfering substances. 



The methods found most reliable for the routine detection of proteins 

 in tissues are (1) the Millon reaction for tyrosine, and (2) the diazo- 

 nium reaction for histidine, tyrosine, and tryptophan. Both of these 

 reactions involve the use of a colorless chromogenic agent which, when 

 applied to fixed tissues under appropriate conditions, gives a colored 

 product by combining with a specific group (s) of the protein. When 

 tissues are exposed to Millon's reagent (a solution of mercuric nitrite 

 and nitrate in a mixture of nitric and nitrous acids) a red precipitate is 

 obtained due to the presence of the phenolic ( — ^ ^ — OH) group 



of tyrosine. In the diazonium reaction, the chromogenic reagent is a 

 diazonium hydroxide which reacts with the phenolic group of tyrosine, 

 the indole group of tryptophan, and the imidazole group of histidine to 

 produce a complex which is colored. 



The cytochemical determination of specific amino acids is limited by 

 the relatively few techniques available for their identification and the 

 difficulty of applying them to fixed materials. The methods most often 

 used with fixed tissues are (1) the Sakagiichi reaction for arginine, and 

 (2) the mercurial method for protein sulfhydryl groups. The latter tech- 

 nique involves the treatment of tissues with the red-colored mercurial, 

 Mercury Orange, which combines with — SH groups as follows: 



R— SH -f ClHg <^ \— N=N 



/ 



OH 



R— SHg <^^ \— N=N— / \ + HCl 



OH 



The — SH content of cell components can also be measured quantita- 

 tively by photometric analysis of tissues stained by this technique 

 (Bennett and Watts, 1958). 



SURVEY OF CYTOLOGICAL TECHNIQUES / 219 



