Protocol
Boone & Drijver's Alcoholic Trichrome
for Exfoliated Cells
Materials
- Mayer’s hemalum or equivalent
- Solution A
Material Amount 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
Material Amount 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 as needed
Tissue Sample
Ethanol fixed smears of exfoliated epithelial cells.
Protocol
- Bring smears to water via graded ethanols.
- Stain nuclei with alum hematoxylin for 2 minutes.
- Blue with running tap water.
- Rinse with 50% ethanol, 15 dips.
- Place into solution A for 1 minute.
- Rinse with 50% ethanol, two changes of 15 dips each.
- Place into solution B for 2 minutes.
- Rinse with 50% ethanol, 15 dips.
- Dehydrate with absolute ethanol, two changes.
- 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
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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.