318 Microorganisms (chap. 20) 



While stirring add: 



formalin 4.0 ml. 



glycerol 5.0 ml. 



When thoroughly mixed add, drop by drop, with constant stirring: 



equal parts of gum mastic saturated in absolute 



alcohol and absolute alcohol 8.0 ml. 



pyridine 2.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Remove any formalin pigment in 2% ammonium hydroxide (2 

 ml./98 ml. water): few minutes. 



3. Transfer to 80% alcohol: 2-3 minutes. 



4. Wash in distilled water: 5 minutes. 



5. Treat with uranium nitrate 2-3% (2-3 gm./lOO ml. water), pre- 

 viously warmed to 60°C: 10 minutes. 



6. Wash in distilled water: 1-2 minutes. 



7. Transfer to 95% alcohol: 1-2 minutes. 



8. Treat with dilute gum mastic: 5 minutes. 



9. Wash in distilled water, 3-4 changes, initil rinse is clear. 



10. Impregnate with silver nitrate, 2% (2 gm./lOO ml. water), pre- 

 viously warmed to 60°C: 30-40 minutes. 



11. Warm developing solution to 60°C. Dip slide up and down in 

 solution initil sections turn light tan or pale brown. 



12. Rinse in 95% alcohol, then in distilled water. 



13. Treat with silver nitrate, 2% (2 gm./lOO ml. water): 1-2 minutes. 



14. Wash in distilled water: 1-2 minutes. 



15. Dehydrate, clear and mount. 



results: 



spirochetes — black 

 background — yellow 



comments: 



Use thin sections, 4-5 microns. 



The uranium nitrate prevents the impregnation of nerve fibers 

 and reticulum. 



Warthin-Starry Silver Method (kerr, 1938; faulkner and 

 LiLLiE, 1945A; BRmcES and luna, 1957) 



fixation: 10% formalin. 

 Notes of caution: 



All glassware must be cleaned with potassium dichromate-sulfuric 

 acid. 



