STAINING, PRACTICAL AND THEORETICAL 



Technique : 



1. Fix smears immediately, while still wet, in solution A for at 

 least five to fifteen minutes. 



Note: The smears may remain in the fixative indefinitely at room 

 temperature. 



2. Rinse for about ten seconds in 80% alcohol. 



3. Transfer to 70% alcohol for about ten seconds. 



4. Transfer to 50% alcohol for about ten seconds. 



5. Rinse in distilled water for about ten seconds. 



6. Immerse in solution B (1% acetic acid) for ten seconds to 

 one minute. 



Note: This is to prevent rapid fading of fluorescence. 



7. Rinse in distilled water for ten to fifteen seconds. 



8. Stain in the buffered acridine orange (solution H) for 

 three minutes or a little longer. 



9. Immerse directly into phosphate buffer (solution F or G) 

 for at least one minute to remove excess dye. 



Note: The smears may be left in the buffer solution for several hours 

 in cases where it is desired to re-examine them later, or where prepara- 

 tions are processed in batches and screened successively. 



10. Differentiate for one to two minutes, or longer if found 

 necessary, with solution J (i.e. M/io calcium chloride), controlling 

 by examination under the microscope while the smear is still 

 wet, until the nuclei, especially of granulocytes, fluoresce in 

 bright translucent green. 



Note: The calcium chloride solution promotes differentiation between 

 RNA and DNA. 



11. Rinse with phosphate buffer (solution F or G). 



12. Mount with a coverslip placed over a drop of the buffer 

 solution, and examine with the fluorescence micrsocope. 



Note: Permanent preparations can be made by mounting in Uvak 

 (Edward Gurr), which is an aqueous non-fluorescent mountant. 



Results : 



Normal cells from the tracheal, bronchial and bronchiolar 

 epithelium (celiated columnar cells, goblet cells, and basal cells), 

 and pulmonary macrophages (alveolar cells) show brown to dull 

 reddish-brown cytoplasm. Squamous cells from the oesophageal 



98 



