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 G 5 g
Phosphotungstic acid 0.15 g
Ethanol, 95% 1 L
Solution B   EA 25   EA 36   EA 50   EA 65
Light green SF 2.2 g 2.25 g 0.45 g 1.125 g
Bismarck brown 0.6 g 0.5 g 0.5 g 0.5 g
Eosin Y 2.2 g 2.25 g 2.25 g 2.25 g
Phosphotungstic acid 1.7 g 2 g 2 g 2 g
Lithium carbonate 5 mg 5 mg 5 mg 5 mg
Ethanol, 95% 1 L 1 L 1 L 1 L
For each variant: –
Dissolve the bismarck brown in 100 mL ethanol, the light green in 450 mL ethanol,
and the eosin Y in another 450 mL ethanol. Then combine the three solutions.
Add the acid and lithium carbonate. 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


  1. 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.
  2. Lithium carbonate saturates in water at 1% at room temperature, so 0.5 mL contains 5 mg.
  3. EA 65 is recommended for specimens with large amounts of mucus.


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

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.

Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.




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