Papanicolaou Stain: The Pap
Cellular Detail for Cytological Evaluation and Cancer Detection
Author: Kelsey Goldman, HTL (ASCP)
Last Update: 16 March 2025
Copyright: 2025, HistologyOutlines.com
Cite this Page! -> Goldman, Kelsey. “Papanicolaou Stain.” HistologyOutlines.Com, 16 March 2025. Accessed (today).
Purpose
- Often used in cervical and vaginal smears to look for abnormal cells (Disbrey, 1970).
- A cytology protocol; uses smears not tissue.
Control Tissues
- Internal control in smear
Staining Pattern
- Nuclei: blue
- Acidophilic cells: red to orange
- Basophilic cells: green
- Blood vessels: orange (Humanson & Humason, 1979)
Biochemistry Theory
Troubleshooting
- Cells need to be fixed immediately, or they will not be usable. Most cell protocols recommend a 15-minute 95% alcohol wash (Humanson & Humason, 1979).
- Do not aggressively wash. The cells are not firmly fixed to the slide and may wash off (Humanson & Humason, 1979).
- Be careful not to lose cells in the reagents in a way that they could cross-contaminate other slides(Humanson & Humason, 1979).
Additional References
- Disbrey, B. (1970). Histological Laboratory Methods. E.& S. Livingstone.
- Humanson, G. L., & Humason, G. L. (1979). Animal tissue techniques (4. ed). Freeman.
Sample Protocol
Humason’s Protocol
This protocol is for FFPE slides.
Reagents:
- Harris hematoxylin
- Orange G
- Orange G 0.5% 100 mL
- Phosphotungstic Acid 0.015 g
- Eosin Azure 36
- Light green SF yellowish 0.5% 45 mL
- Bismark Brown 0.5% 10 mL
- Eosin Y 0.5% 45 mL
- Phosphotungstic acid 0.2 g
- Lithium carbonate 1 drop
Method:
- Deparaffinize and bring slides back to water.
- Stain in a 1:1 mixture of Harris hematoxylin and water for 8 minutes.
- Wash in lightly running tap water for 3-4 minutes.
- Differentiate nuclei in 0.5% hydrochloric acid in 70% alcohol until the nuclei are sharp and the background is pale blue when viewed under the microscope.
- Wash in running tap water for 5 minutes. Wash until the nuclei are blue.
- Wash in deionized water.
- Pass slides through 70%, 80%, and 95% alcohol.
- Stain in Orange G 6 for 1-2 minutes.
- Rinse in three changes of 95% alcohol.
- Stain in Eosin Azure 36 for 2-3 minutes.
- Pass again through three changes of 95% alcohol.
- Dehydrate through 100% alcohol, clear in xylene, and coverslip. Adapted from (Humanson & Humason, 1979)