Papanicolaou Stain (Pap Stain) Protocol


Papanicolaou Stain Reagents


Description: Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic staining histological technique developed by George Papanikolaou, the father of cytopathology. Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells. Pap staining is a very reliable technique. As such, it is used for cervical cancer screening in gynecology. The entire procedure is known as Pap smear.


The classic form of Pap stain involves five dyes in three solutions:

  • A nuclear stain, haematoxylin, is used to stain cell nuclei.
  • First OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
  • Second EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dyes, eg. EA-50, EA-65.
    • Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.
    • Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, however it produces visually different results and is not considered satisfactory by some.
    • Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.

When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin patterns are well visible, the cells from borderline lesions are easier to interpret, the photomicrographs are better, and the stained cells are pretty. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.

On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.


Fixation: Fix smear according to standard procedure. For example, 95% alcohol or 100% methanol.


Procedure 1 (Standard Method):

  1. 95% Ethanol 15 minutes (fixation)

  2. Rinse in tap water

  3. Harris or Gill Hematoxylin 1-3 minutes (Time vary with selection of hematoxylin solution)

  4. Rinse in tap water or Scott's tap water

  5. 95% Ethanol 10 dips

  6. OG-6 stain for 1.5 minutes.

  7. 95% Ethanol 10 dips

  8. EA-50, or Modified EA-50, or EA-65 stain for 2.5 minutes.

  9. 95% Ethanol 10 dips, 2 changes

  10. 100% Ethanol 1 minute

  11. Clear in 2 changes of xylene, 2 minutes each

  12. Mount with permanent mounting medium

Procedure 2 (Modified Pap Procedure):

  1. 95% Ethanol 15 minutes (fixation)

  2. Distilled water 10 dips, 2 changes

  3. Gill Hematoxylin 2 minutes

  4. Distilled water 10 dips

  5. Scott's tap water 1 minute

  6. Distilled water 10 dips, 2 changes

  7. 95% Ethanol 10 dips, 2 changes

  8. OG-6 stain for 1-2 minutes

  9. 95% Ethanol 10 dips, 3 changes

  10. EA-50 or EA-65 stain for 6-10 minutes

  11. 95% Ethanol 20-30 dips, 3 changes

  12. Absolute ethanol 10 dips

  13. Clear in xylene

  14. Mount with permanent mounting medium

Procedure 3 (Rapid Economic, Acetic Acid, Papanicolaou Stain Method):

  1. 1% acetic acid 10 dips

  2. Harris’s Haematoxylin, preheated 60˚ C 10 dips

  3. Tap water 10dips

  4. 1% acetic acid 10 dips

  5. OG-6 10dips

  6. 1%acetic acid 10 dips

  7. EA-50 10 dips

  8. 1% acetic acid 10 dips

  9. Methanol 10 dips

  10. Xylene 10 dips

Blotting was done after each step. Mount by D.P.X




Rapid Economic, Acetic Acid, Papanicolaou Stain Method

Ultrafast Papanicolaou stain. An alternative preparation for fine needle aspiration cytology

Pap Staining from Histonet