Papanicolaou's
Alcoholic Trichrome for Exfoliated Cells

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

Solutions

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

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. Then combine the three solutions.

Add the acid and lithium carbonate. Filter.

Tissue sample
Ethanol-ether fixed smears of exfoliated epithelial cells.

Method

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

Notes

1. Although the technique calls for ethanol-ether fixed smears, material fixed with 95% or absolute ethanol is 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.

 

Reference

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.