2C)6 Cytoplasmic Elements (chap. 1(S) 



Osmic Method (newcomer, 1940) 



fixation: Zirkle's solution: 48 hours, 



potassium dichromate 1.25 gm. 



ammonium dichromate 1.25 gm. 



copper sulfate 1 .0 gm. 



distilled water 100.0 ml. 



procedure: 



1. Wash tissue blocks 8 hours to overnight. 



2. Impregnate in 2% osmic acid (2 gm./lOO ml. water): 4-6 days. 

 Change solution on alternate days. 



3. Wash for 8 hours or overnight. 



4. Dehydrate, clear, and embed. Use benzene, not xylene, for clear- 

 ing. 



5. Cut 5-micron sections, moiuit on slides. 



6. Deparaffinize and hydrate slides to water. 



7. Bleach in 1% potassium permanganate (1 gm./lOO ml. water): 5 

 minutes. 



8. Rinse in distilled water. 



9. Treat with 3% oxalic acid (3 gm./lOO ml. water): 2-3 minutes. 



10. Wash in running water: 15 minutes. 



11. Dehydrate, clear, and mount. 



results: 



mitochondria — black 



comments: 



Newcomer used his method on plant cells. A counterstain such as 

 acid fuchsin may be added. 



Saccharides (Carbohydrates) 



Carbohydrates appear as four types of substances: (1) simple polysac- 

 charides (includes glycogen); (2) mucoid substances (mucopolysaccha- 

 rides, mucoproteids, glycoproteins); (3) glycolipids; and (4) nucleic 

 acids. In addition to the use of certain specific stains, aldehyde reactions 

 are a common means of demonstrating carbohydrates. The aldehyde 

 groups must first be liberated by some chemical agent, either oxidized 

 (periodic or chromic acid), or hydrolyzed (dilute hydrochloric acid). 

 Then the specific reagent for aldehydes can be applied. See the Feitlgen 

 and periodic acid-Schiff reactions, page 292. 



