294 Fe^ilgeu and PAS Technics (chap. 19) 



solutions: 



N hydrochloric acid, see page 408. 

 Schiff's reagent (Lillie, 1951 B): 



basic fuchsin, C.I. 42500 0.5-1.0 gm. 



distilled water 85.0 ml. 



sodium metabisulfite, NaaSoOj 1.9 gm. 



N HCl 15.0 ml. 



Place in bottle with approximately 50-60 ml. of free air space. Shake 

 at intervals for at least 2 hours or overnio^ht. Add 200 ms^. activated 

 charcoal: 2 minutes; shake occasionally. Filter. If solution is not 

 water-white the charcoal is old. Try a fresh batch and refilter. Store 

 Schiff reagent in a bottle with a minimum of air space above the solu- 

 tion and keep in refrigerator. This wdll decrease loss of SO2 {Eljtman, 

 1959). 



Bleaching solution (sulfurous acid): 



N HCl (page 408) 5.0 ml. 



potassium or sodium bisulfite, 10% aqueous 



K2S0O5 or NaoSsOs 5.0 ml. 



distilled water 100.0 ml. 



or: 



HCl, concentrated 1.0 ml. 



potassium or sodium bisulfite 0.4 gm. 



distilled water 100.0 ml. 



For best residts, make up bleach fresh each time. 



Fast Green: 



fast green FCF, C.I. 42053 0.05 gm. 



95% ethyl alcohol 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water; remove HgCl2. (Leav- 

 ing slides in 95% alcohol overnight will remove lipids Avhich 

 might cause a plasmal reaction.) 



2. Rinse at room temperature in N HCl: 2 minutes. 



3. Hydrolyze at 50°C in N HCl: 20 minutes or 5 minutes at 60°C. 



4. Rinse at room temperature in N HCl; rinse in distilled water. 



5. Stain in Schiff's reagent: 2 hours in dark. 



6. Drain and transfer quickly into bleaching solution, 3 changes: 

 1.5-2 minutes in each. 



7. Wash in rimning water: 10-15 miniites, 



