Hematoxyliji Staining Procedures 135 



comments: 



The shorter staining schedule produces bkie-black nuclei and the 

 overnight staining a truer black. The latter is recommended for 

 mitochondria. Picric acid usually perfects a more sharply diflferenti- 

 ated nucleus than destaining with iron alum. Prolonged destaining 

 in the latter case can leave a tan or yellow tinge in the cytoplasm. 

 Yellow color left in the tissue after picric acid is due to insufficient 

 washing following the destaining. Striations of muscle and some 

 protozoan structures are better differentiated by iron alum. After 

 either method thorough washing is essential or the sections will con- 

 tinue to destain slowly and fade after a time. (Picric acid destaining, 

 Tuan, 1930) 



The destaining agents act both as an acid and an oxidizer, reacting 

 in two ways: (I) as an acid they extract stain faster from the cytoplasm 

 than from the nucleus; and (2) as an oxidizer they bleach the stain 

 uniformly. If an acid is used alone, the staining of the nuclei can be 

 favored, and if the oxidizer is used alone, the staining of the cyto- 

 plasm can be enhanced, 



Hutner (1934) recommends for nuclei: 



1. Mordant in 4% iron alum: I hour. 



2. Stain hematoxylin: 1 hour. 



3. Destain in saturated aqueous picric acid. 



4. Wash and blue by adding 1-2 drops ammonia to 70% alcohol. 



Hutner recommends for cytoplasm: 



1. Mordant in iron alum: 30 minutes. 



2. Stain in hematoxylin: 30 minutes. 



3. Destain in freshly made 95% alcohol 2 parts, Merk's superoxol 

 (30%, H,.Oo) 1 part. 



4. Rinse in 1 change 70% alcohol, 10 minutes. 



For brevity, intermediate steps were omitted in the above. Oxidizer and 

 acid can be combined to approximate iron alum destaining effects: 



0.25% HCl in 95% alcohol, 2 parts; Superoxol, 1 part. 



Wash in 70% alcohol and blue. 



Iron Hematoxylin groat's single solution (1949) 

 fixation: any general fixative, preferably containing HgCl2. 



solutions: 



Hematoxylin, page 126. 

 Counterstains, page 129. 



