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Papanicolaou’s Alcoholic Trichrome for Exfoliated Cells

Papanicolaou's Alcoholic Trichrome

for Exfoliated Cells


This is the Papanicolaou technique commonly used to screen for cervical cancer.


  • Ehrlich’s or Harris’ alum hematoxylin or equivalent
  • Solution A (OG6)
    Orange G5g
    Phosphotungstic acid0.15g
    Ethanol, 95%1L
  • Solution B
    EA 25EA 36EA 50EA 65
    Light green SF2.2g2.25g0.45g1.125g
    Bismarck brown0.6g0.5g0.5g0.5g
    Eosin Y2.2g2.25g2.25g2.25g
    Phosphotungstic acid1.7g2g2g2g
    Lithium carbonate5mg5mg5mg5mg
    Ethanol, 95%1L1L1L1L


For each variant:

  1. Dissolve the bismarck brown in 100 mL ethanol, the light green in 450 mL ethanol, and the eosin Y in another 450 mL ethanol.
  2. Combine the three solutions.
  3. Add the acid and lithium carbonate.
  4. Filter.

Tissue Sample

Ethanol-ether fixed smears of exfoliated epithelial cells.


  1. Bring smears to water via graded ethanols.
  2. Stain nuclei with alum hematoxylin for 2 minutes.
  3. Blue.
  4. Rinse with 95% ethanol.
  5. Place into solution A for 1 minute.
  6. Rinse with 95% ethanol.
  7. Place into solution B for 2 minutes.
  8. Rinse well 95% ethanol.
  9. Dehydrate rapidly with ethanol.
  10. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cells  –  shades of orange, pink or green


  • Although the method calls for smears fixed in an equal parts mixture of ethanol and diethyl ether, smears fixed in 95% or absolute ethanol are completely satisfactory.
  • Lithium carbonate saturates in water at 1% at room temperature, so 0.5 mL contains 5 mg.
  • EA 65 is recommended for specimens with large amounts of mucus.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.


  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Papanicolaou, (1942)
    Science, v.95, pp.438
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
  3. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.