Papanicolaou's Alcoholic Trichrome
for Exfoliated Cells
This is the Papanicolaou technique commonly used to screen for cervical cancer.
Materials
- Ehrlich’s or Harris’ alum hematoxylin or equivalent
- Solution A (OG6)
Material Amount Orange G 5 g Phosphotungstic acid 0.15 g Ethanol, 95% 1 L - Solution B
Material Variant 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
Preparation
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.
- Combine the three solutions.
- Add the acid and lithium carbonate.
- Filter.
Tissue Sample
Ethanol-ether fixed smears of exfoliated epithelial cells.
Protocol
- Bring smears to water via graded ethanols.
- Stain nuclei with alum hematoxylin for 2 minutes.
- Blue.
- Rinse with 95% ethanol.
- Place into solution A for 1 minute.
- Rinse with 95% ethanol.
- Place into solution B for 2 minutes.
- Rinse well 95% ethanol.
- Dehydrate rapidly with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Cells – shades of orange, pink or green
Notes
- 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.
References
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
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.
