INTRODUCTION:-

Consistency and reliability are most important in cytological interpretation. Cytologists rely heavily on the quality and appearance of the stain. The Papanicolaou stain is recommended for the staining of alcohol fixed cytology slides. Romanowsky stains may also be used for wet fixed slides, but are primarily applied to air-dried smears.
Special stains are used as per requirements: Modified Ziehl Neelson (for acid fast bacilli), Gram staining (Bacteria), Mucicarmine (mucins), PAS (for glycogen, fungal wall, lipofuscin, etc), Oil red O (lipids), Perl’s Prussian blue (iron), modified Fouchet’s test (bilirubin), etc. Recently, immunocytochemistry is also being increasingly used in cytology specimens. These special stains and immunocytochemistry will be discussed along with respective sections in
histopathology as the principles and methods remain the same.

 STAINING IN CYTOLOGY:-

The universal stain for cytological preparations is the Papanicolaou stain. Harris’hematoxylin is the optimum nuclear stain and the combination of OG6 and EA50 give the subtle range of green, blue and pink hues to the cell cytoplasm.

Papanicolaou stain:-

Papanicolaou formula:-

1. Harris’ hematoxylin:-

Hematoxylin                               5g 
Ethanol                                        50ml
Potassium alum                        100g
Distilled water (50°C)               1000 ml
Mercuric oxide                           2-5g
Glacial acetic acid                     40ml

2. Orange G 6:-

Orange G (10% aqueous)                50ml 
Alcohol                                               950ml
Phosphotungstic acid                     0-15g

3. EA 50:-

0.04 M light green SF                      10ml 
0.3M eosin Y                                     20ml 
Phosphotungstic acid                     2g 
Alcohol                                              750ml
Methanol                                           250ml
Glacial acetic acid                           20ml
Filter all stains before use

Original Papanicolaou staining method:-

 1. 96% ethyl alcohol 15 seconds                                                                                                                                                     2. 70% ethyl alcohol 15 seconds                                                                                                                                                     3. 50% ethyl alcohol 15 seconds                                                                                                                                                     4. Distilled water 15 seconds                                                                                                                                                             5. Harris hematoxylin 6 minutes                                                                                                                                                       6. Distilled water 10 dips                                                                                                                                                                   7. Hydrochloric acid 0.5% solution, 1-2 quick dips                                                                                                                       8. Distilled water 15 seconds                                                                                                                                                           9. Few dips in 0.1% ammoniated water. The smear turns to blue.                                                                                         10. 50% ethyl alcohol 15 seconds
 11. 70% ethyl alcohol 15 seconds
 12. 96% ethyl alcohol 15 seconds
 13. OG-6 (orange) 2 minutes
 14. 96% ethyl alcohol 10 dips
 15. 96% ethyl alcohol 10 dips
 16. EA 50 eosin yellowish 3 minutes
 17. 96% ethyl alcohol (10 dips)
 18. 100% ethyl alcohol (10 dips)
 19. Xylene (10 dips)
 20. Mount: in DPX using coverslip

Results:-

The nuclei should appear blue/black
The cytoplasm (non-keratinising squamous cells) – blue/green
Keratinising cells- pink/orange

Precautions:-

1. Use stains only after filtering them
2. Change stains frequently
3. Check staining under microscope for good quality control