STAINING TECHNIQUES 5_jq 



a. Fixation, 8-10 minutes. 



b. Tap water wash, 10-15 minutes. 



c. Distilled water, 2 minutes. 



d. Acid fuchsin-methyl green mixture, 25-30 minutes. 



e. Direct transfer to 95% alcohol until green stain ceases 

 to leave smears in clouds. 



f. Rapid dehydration in absolute alcohol. 



g. Clear, two 2-5 minute changes in toluene and mount. 

 Results: Chromosomes - dark green 



Centrosome - red 

 Nucleolus - pale green 

 Cytoplasm - pink 



3. Pyronin Y- methyl green. 

 Prepare the staining solution: 



Methyl green 0.50 gm. 



Pyronin Y 0.05 gm. 



Alcohol 5.00 ml. 



Glycerine 10.00 ml. 



0.5% carbolized H2O to 100 ml. 

 Follow the schedule given above for acid fuchsin-methyl 

 green, using this stain mixture 15-20 minutes. 

 Results: Chromosomes - dark green 



Centrosome - pale green 



Nucleolus - pale green 



Cytoplasm - pink 



4. Safranin - fast green. 



a. Fixation, 8-10 minutes. 



b. Tap water wash, 10-15 minutes. 



c. Distilled water, 2 minutes. 



d. Stain in 1% aqueous safranin 0, 1/2-1 hour. 



e. Distilled water, rinse. 



f. 50% alcohol, 2 minutes. 



g. Fast green in 95% alcohol (enough fast green to 

 color the alcohol faintly), 3-5 minutes. 



h. Absolute alcohol, rinse. 



i. Clear, two 2-5 minute changes in toluene, mount. 



Results: Chromosomes - red 



Centrosome - red 



Nucleolus - pale green 



Cytoplasm - pale green 



5. Crystal violet-anilin blue. This stain combination used after 

 the I and KI mordant proved an excellent one for showing the paired 

 chromosomes. 



a. Fixation, 8-10 minutes. 



b. Tap water wash, 10-15 minutes. 



c. Pass through 50%, 70% alcohols, 2 minutes each. 



