Nerve Cells and Fibers 201 



longer the tissue is left in silver nitrate, the greater the tendency 

 for everything to stain. This time will have to be determined by 

 trial and error. 



4. Wash in distilled water: 20 minutes to 1 hour. This is a critical 

 step. The amoimt of silver Avashed from the tissue depends on 

 whether the Avater is changed often or whether the tissue is shaken 

 in the water. Change every 10 minutes or use a large volume of 

 water and shake every 10 minutes. Equal staining of the periphery 

 as well, as central parts of the tissue determines correct washing. 

 A light periphery indicates too much washing. 



5. Reduce in 4% aqueous pyrogallol solution: 4 hotirs. 



6. Dehydrate, clear, embed, and section. 



7. Moimt on slides, deparaflmize, and clear. Add mountant and cover 

 glass. 



results: 



ner\ e cells — yellow to brown 



neurofibrils — brown to black 



axis cylinders of myelineated fibers — yellow to brown 



axis cylinders of nonmyelinated fibers — black 



comments: 



If the preparation tends to be too light, reduce the time in pyridine 

 and washing. 



If the preparation is too dark, omit the pyridine and wash 48 hours 

 after fixation, or reduce the concentration of the silver nitrate solu- 

 tion and wash longer between impregnation and reduction. 



Bielschowsky Method (modified bv davenport el al., 1934) B 



fixation: 10% formalin: 2 days. For embryos add 0.5% of trichlor- 

 acetic acid to 10% formalin. 



SOLUTION : 



Ammoniated silver: 



Add 5.0 ml. of concentrated ammonia to 40.0 ml. of 2% sodiinn hy- 

 droxide (aqueous). Mix well. Add slowly from a burette or pipette 

 with shaking 8.5% aqueous silver nitrate luitil an opalescence re- 

 mains in the sohuion (about 40.0 ml.). Add 0.5-1.0 ml. of ammonia. 

 Dilute with about 5 parts of distilled water. (Diliuion is not critical.) 



procedure: 



1. After fixation, wash for I hour. 



2. Transfer to 50%, aqueous pyridine: 1-2 days. 



J 



