The Papanicolaou method is commonly used for routine cytological staining. Papanicolaou Stain was initially used only to assess estrogen levels in vaginal epithelium and detect pathogens such as Candida and Trichomonas in the genital tract. The combination of Orange G6 with EA36 or EA50 stains the cytoplasm in distinct bright green, blue, and pink colors. Currently, most laboratories use ready‑to‑use staining solutions; therefore, the optimal conditions for each modified solution should be observed. The final cytoplasmic staining should be transparent and distinct, and nuclear chromatin should be easily identifiable. The modified Papanicolaou staining solution contains various ions and exhibits polychromatic staining properties, resulting in bright, transparent cytoplasm and clear nuclear membrane, nucleolus, and chromatin structures.
Sperm Morphology Staining Solution (Papanicolaou Method). Proteins with different isoelectric points in sperm and cells carry different charges at the same pH and can selectively bind corresponding dyes. The nucleus consists of acidic substances and has a strong affinity for basic dyes, whereas the cytoplasm contains basic substances and has a high affinity for acidic dyes. This product uses a modified EA50 staining solution specifically optimized for sperm cytoplasmic staining, and a non‑toxic modified hematoxylin staining solution for nuclear staining. It is especially suitable for sperm staining and can also be used for cytological samples such as pleural effusion, ascites, and sputum. This reagent is for research use only and not intended for clinical diagnostic or other purposes.
Materials Required:
Fixative solution (e.g., 95% ethanol‑ether fixative), graded ethanol, bluing reagent
Protocol (For Reference Only):
1. Fix cell smears in equal parts of 95% ethanol‑glacial acetic acid fixative or 95% ethanol for 10–15 min.
2. Immerse in 80% ethanol for 1 min.
3. Immerse in 70% ethanol for 1 min.
4. Immerse in 50% ethanol for 1 min.
5. Immerse or rinse in distilled or tap water for 1 min.
6. Stain with hematoxylin stain for 3–5 min, then rinse with tap water for 2 min.
7. Differentiate in differentiation solution for approximately 4–10 s, then rinse with tap water for 2 min.
8. Bluing in running water for 4 min, then rinse with tap water for 2 min.
9. Dehydrate in 50% ethanol for 2 min.
10. Dehydrate in 70% ethanol for 2 min.
11. Dehydrate in 80% ethanol for 2 min.
12. Dehydrate in 90% ethanol for 2 min.
13. Stain with Orange G6 stain for 2 min.
14. Rinse in 95% ethanol (I) and (II) for 2 min each.
15. Stain with modified EA50 stain for 5 min.
16. Dehydrate in 95% ethanol (I) and (II) for 1 min each.
17. Dehydrate in absolute ethanol (I) and (II) for 1 min each.
18. Clear in xylene or dewaxing clearing agent, and mount with neutral resin.
Staining Results:
Nuclei: blue-purple or black
Cytoplasm of non-keratinized cells: pale blue or pale green
Cytoplasm of keratinized cells: pink or orange-red
Precautions:
1. This reagent has been filtered. If the staining result is unsatisfactory, it may be filtered again, and the staining solution should be replaced regularly.
2. For your safety and health, please wear a lab coat and disposable gloves during operation.
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