40 MICROSCOPIC TECHNIQUES 



with 450 ml. of 95% alcohol and heat on a water bath to simmer- 

 ing. Stir occasionally and then filter while hot. Transfer to a 

 stoppered bottle and place in a refrigerator overnight. Filter while 

 cold, and add distilled water dropwise from a burette, while stir- 

 ring, to reduce the alcohol concentration to 80%. Allow to stand 

 24 hr. ; filter and keep stoppered. 



Acetic-Carbol-Siidan III Reagent. To a given volume of Sudan III 

 stock soln., slowly add 5% phenol dropwise from a burette, stir- 

 ring after each addition, to bring the alcohol concentration to 60% 

 {e.g., add 2 ml. phenol soln. to 6 ml. stock soln.). Let stand for 

 several hr. keeping the bottle stoppered. Then, in the same drop- 

 wise manner, add glacial acetic acid in the proportion of 2.5 drops 

 per ml. carbol-Sudan soln. After standing for 24 hr. in a stoppered 

 bottle, filter the reagent. Do not use when the soln. is more than 

 several days old. 



5% Glacial Acetic Acid in 50% Alcohol. 



PROCEDURE 



1. Transfer formalin-fixed frozen sections to 50% alcohol for 

 1 min. 



2. Place in the acetic-carbol-Sudan III reagent for 1.5 hr. or 

 longer; keep vessel stoppered. 



3. Differentiate in the acetic-alcohol soln. for 10-60 sec. and 

 wash in distilled water for 1 min. In some cases it may be well to 

 dilute the acetic-alcohol with more 50% alcohol. 



4. A counterstain of recently filtered Delafield hematoxylin 

 diluted with 2 vol. distilled water may be applied for 15 min., 

 followed by differentiation in 0.5% hydrochloric acid until reddish 

 (10-20 sec), and treatment with very dilute ammonium hydroxide 

 for 5 min. to develop the blue color. 



5. Wash in distilled water and mount in glycerin jelly. 



Result. The lipid will be stained red. 



Telford Govan Technique for Sudan Dye Staining 

 in Aqueous Media 



SPECIAL REAGENTS 



Sudan Dye Suspension. Add a saturated soln. of a Sudan dye in 

 acetone dropwise from a capillary pipette to 1% gelatine soln. 



