STAINING, PRACTICAL AND THEORETICAL 



C. Giemsa stain . . . . . . i ml. 



Distilled water (buffered to pH y-o) 20 ml. 



D. Acetic acid o-o8% aqueous 



Technique: 



1 . Fix material in Solution A (above) from twelve to forty-eight 

 hours. 



2. Dehydrate in the alcohols and clear as usual; embed in 

 paraffin wax and cut sections not exceeding 5^ in thickness. 



3. Fix sections to slides and remove wax with xylol. 



4. Wash well with two changes of pure methyl alcohol. 



5. Stain sections with a measured volume of Jenner stain, which 

 should be freshly filtered. 



6. Cover the shdes with a Petri dish lid lined with two or three 

 sheets of moistened filter paper (this is to prevent the evapor- 

 ation of the alcohol and the consequent formation of a precipitate 

 on the sections), and allow the stain to act for three minutes. 



7. Add a volume of distilled water (buffered to pH 7-0), equal to 

 that of the stain, to the slides, which should now be gently rocked 

 to ensure thorough mixing of the stain and water. 



8. Allow this diluted stain to act for one minute. 



9. Pour off excess stain; then without washing, immerse the 

 slides in a stoppered staining jar containing diluted Giemsa 

 stain (Solution C above) and leave the stain to act for forty-five 

 minutes. 



10. Rinse and differentiate in Solution D. 



1 1 . Rinse thoroughly in distilled water. 



12. Dehydrate quickly in 95% alcohol, followed by two changes 

 of absolute alcohol. 



13. Clear in xylol and mount in Cristalite. 



Results: 



Erythrocytes are stained orange. Cytoplasm of lymphocytes 

 and blastocytes are blue. Nuclei: deep blue to violet. Mast cell 

 granules : violet to violet-red. 



Reference: Pappenheim, A. (1912). 



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