Organic Substances 127 



(over 24 hours) a number of additional structures will be 

 stained; an early and intense blackening of eosinophilic and 

 neutrophilic granules is especially conspicious.^^' ^^ After 48 

 hours or more, practically the entire slide may turn solid 

 black. If a darkish background develops on account of over- 

 staining, differentiate slide as described under the Ag tech- 

 nique for glycogen and mucin (p. 64). 



According to Burtner and Lillie,^^ the argentaflBn reaction 

 can be greatly accelerated by performing it at 60° C. 



2) The azo-coupling reaction.— 



Method 



Dissolve about 50 mg. of either Red B Salt or Black B Salt 

 or Garnet GBC Salt (chemical constitutions, p. 171 ) in about 

 10 ml. of cold water; add a few drops of a saturated solution 

 of borax and pour the more or less turbid solution on the 

 slide. Leave it on for 30-60 seconds; wash slide under the 

 tap, counterstain lightly with hematoxylin, dehydrate, and 

 mount. EC granules stain deep orange (Red B Salt), rusty 

 red-brown (Black B Salt), or red (Garnet GBC Salt). The 

 background is light yellow. 



In order to obtain darker shades, Lison^^ recommends the 

 use of tetrazotized diamines, in the hope that only one diazo 

 group will couple with the tissue, and the second one can be 

 coupled with naphthol. In this way intense purple or bluish 

 shades could be produced. The method does work in prac- 

 tice, but the background becomes stained so heavily that the 

 net gain in contrast is negligible. The two-step modification 

 of Clara^^ (using unilaterally diazotized diamines and diaz- 

 otizing the other side after coupling with the tissue has 

 taken place ) is not better than Lison's original method. 



30. Cordier, R.: Arch, de biol., 36:427, 1926. 



31. Burtner, H. J., and Lillie, R. D.: Stain Technol., 24:225, 1949. 



32. Clara, M., and Canal, F.: Ztschr. f. Zellforsch. u. mikr. Anat., 15:801, 

 1932; Clara, M.: Ztschr. f. wissensch. Mikr., 51:316, 1934. 



