256 ^ Cytoplasmic Elements (chap. 18) 



procedure: 



1. Frozen sections, 15 microns, into water. 



2. Place in pure propylene or ethylene glycol: 3-5 minutes, 2 changes. 

 Agitate. 



3. Stain, 2 changes: 5-7 minutes each, agitate occasionally. 



4. Differentiate in glycol and water (85:15): 3-5 minutes, agitate. 



5. \Vash in distilled water: 3-5 minutes. 



6. Counterstain in hematoxylin if desired. 



7. Mount in glycerol jelly. _ 



results: 



fat — Sudan IV, orange to red; Sudan black B, blue-black to black 



comments: 



Chiffelle and Putt recommend glycols as a perfect solvent for a fat 

 stain because it does not extract lipids. 



Ziigibe et al. (1938, 1959) suggest Carbowax 400 as a solvent for Oil 

 red O and Sudan IV. 



Gomori (1952) questions the use of glycols because they are sol- 

 vents for so many water-insoluble substances, and he prefers triethyl 

 phosphate. It has a low volatility and is harmless to lipids. His method 

 follows. 



procedure: 



1. Frozen sections are rinsed in water and transferred into 50% al- 

 cohol: few minutes. 



2. Stain in a saturated, filtered solution of any of the fat dyes in 60% 

 triethylphosphate, 5-20 minutes. 



3. Differentiate in 50% alcohol: 1 minute. 



4. Counterstain in hematoxylin or any preferred stain. 



5. Mount in glycerol jelly. 



Osmic Acid (mallory, 1944) 

 fixation: 10% formalin, no alcohol. 



solution: 

 Osmic acid: 



osmic acid (osmium tetraoxide) 1.0 gm. ampule 



distilled water lOO.O ml. 



With file, score a circle around ampule and drop it into bottle Avith 

 the distilled water. Several sharp shakes will break the ampule and 



