Pap Stain Procedure in Cytology

Pap stain procedure in cytology post we have briefly explained about Pap stain method’s principle, requirements, pap staining procedure, and result with label.

Pap Stain Procedure in Cytology

Pap staining procedure is a multichromatic staining histological technique developed by George Papanikolaou, the father of cytopathology in 1942.

It is a stain with a polychromatic structure that uses multiple dyes to stain different parts of cells selectively. It is a histological and staining method used to distinguish cells in a smear preparation. The most commonly used method of screening for cervical cancer.

Many specimens can be used to make the Pap smear based on the type of infection being screened, such as urine, sputum cerebrospinal fluid and abdomen tissue, tumour biopsy synovial fluid, fine needle aspirates, the pleural fluids.


The stain employs basic and acidic dyes so that the base dye stain cells with acidic components while the dyes that are acidic stain the essential components of the cells. This is based on the ionic charges of the components of the cell with the principle of attraction and repulsion of the ions and the dyes. Five dyes are employed in three solutions to serve as the principal reagents for staining.

Hematoxylin: It is a natural dye that stains the nuclear cells blue. The dye can attach to the DNA sulfate groups due to its high affinity to nuclear chromatins.

Orange Green 6: It is an acidic counterstain that stains the cytoplasm of mature keratinized cells. The components of the target stain orange in varying intensities of the dye.

Eosin Azure: This is the 2nd counterstain, a mixture of eosin Y and light green SF and Bismarck brown. Eosin Y stains cells of the mature squamous cells, red blood cells, nucleoli, and cilia pink.


Harris’ hematoxylin

1. Hematoxylin: 2.5g

2. Ethanol: 25ml

3. Potassium alum: 50g

4. Distilled water (50°C): 500ml

5. Mercuric oxide: 1-3g

6. Glacial acetic acid: 20ml

Orange G 6

1. Orange G (10% aqueous): 25ml

2. Alcohol: 475ml

3. Phosphotungstic acid: 0.8g

EA 50

1. 0.04 M light green SF: 5ml

2. 0.3M eosin Y: 10ml

3. Phosphotungstic acid: 1g

4. Alcohol: 365ml

5. Methanol: 125ml

6. Glacial acetic acid: 10ml


1. Standard Method

  1. 95% Ethanol 15 minutes (fixation)
  2. Rinse in tap water
  3. Harris or Gill Hematoxylin 1-3 minutes
  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

2. Modified 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.

3. Rapid 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


Staining dyes will stain different components of the cell with different colours and intensities as follows:

Pap Stain Procedure in Cytology

Image Source: Wikipedia.

Nuclei: Blue

Acidophilic cells: Red

Basophilic cells: Blue Green

Erythrocytes: Orange-red to dark pink

Keratin: Orange-red

Superficial cells: Pink

Intermediate and Parabasal Cells: Blue-Green

Eosinophil: Orange Red

Metaplastic cells: May contain both blue/green and pink

Candida: Red

Trichomonas: Grey-green


1. Used in the Pap smear (or pap staining procedure).

2. Pap staining procedure used for screening for cervical cancer.

3. Examination of myeloma cancer cells of the liver.

4. Assessment and characterization of benign tumours.


1. Pap staining procedure is only a screening test that must be followed up with more specialized tests. 

2. Pap staining procedure has very low sensitivity with limited accuracy.

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