Boone & Drijver's
Alcoholic Trichrome for Exfoliated Cells

Solutions

Mayer's hemalum or equivalent

Solution A
Orange G	1	g
Phosphotungstic acid	0.075	g
Ethanol, absolute	250	mL
Distilled water	250	mL

Combine the ethanol and water.

Add the orange G and phosphotungstic acid.

Filter. The pH should be between 2.0 and 2.8.

Solution B
Eosin Y	0.9	g
Light green SF	0.35	g
Phosphotungstic acid	1.5	g
Ethanol, 100%	250	mL
Distilled water	250	mL
Lithium carbonate	–

Combine the ethanol and water.

Dissolve the dyes, then the acid.

Stirring continuously, adjust to pH4.6 with lithium carbonate powder.

Filter.

Tissue sample
Ethanol 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 with running tap water.
4. Rinse with 50% ethanol, 15 dips.
5. Place into solution A for 1 minute.
6. Rinse with 50% ethanol, two changes of 15 dips each.
7. Place into solution B for 2 minutes.
8. Rinse with 50% ethanol, 15 dips.
9. Dehydrate with absolute ethanol, two changes.
10. Clear with t-butanol and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Cytoplasm  –  pink or blue-green
• Keratinized cells  –  orange
• Erythrocytes  –  pink to orange

 

Reference
Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.
  Citing:–
    Boone, M. E., and Drijver, J. S., (1986)
    Routine cytological staining. Theoretical background and practice.
    Elsevier, New York.