HEMOGLOBIN, BILE PIGMENTS AND ACIDS 63 



Dunn Method for Hemoglobin 



SPECIAL REAGENTS 



Cyanol Stock Solution. Dissolve 1 g. cyanol {National Aniline 

 Division, Allied Chemical and Dye Corp.) in 100 ml. distilled 

 water, add 10 g. pure zinc powder, and 2 ml. glacial acetic acid. 

 Bring mixtm^e to a boil and the blue color will soon fade out. The 

 soln. is stable for several weeks. 



Cyanol Working Solution. Just before use filter 10 ml. of the stock 

 soln., add 2 ml. glacial acetic acid and 1 ml. commercial 3% 

 hydrogen peroxide. 



PROCEDURE 



1. Prepare frozen or paraffin sections of tissue fixed in 4% 

 formaldehyde buffered to pH 7.0. 



2. Bring sections to water and stain in cyanol working solution 

 3-5 min. 



3. Rinse in water and counterstain in safranin (1:1000 in 1% 

 acetic acid) 1 min. 



4. Wash in water, dehydrate, clear, and mount in Clarite. 



Result. Hemoglobin stains dark blue to bluish-gray; nuclei, 

 red; and cytoplasm, light pink. 



BILE PIGMENTS AND ACIDS 



The well-known Gmelin test has been adapted to the microscopic 

 detection of bile pigments by simply adding a drop of nitric acid 

 containing some nitrous acid to the sample on a slide. A positive 

 test is indicated by the appearance of a green color changing to red 

 and finally to blue. Stein's test ( 1935) , given below, is probably more 

 satisfactory. Bile salts and acids may be precipitated by barium and 

 the precipitate stained with acid fuchsin according to the technique 

 of Forsgren ( 1928) . 



Stein Test for Bile Pigments 



SPECIAL REAGENTS 



Iodine Reagent. 2 or 3 vol. Lugol solution (6 g. potassium iodide 

 and 4 g. iodine dissolved in 100 ml. distilled water) plus 1 vol. 

 tincture of iodine. 



5% Sodium Hyposidfite. 



