Papanicolaou staining 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. When Orange G6 is combined with EA36 or EA50, the cytoplasm can be stained in distinct colors including green, blue, and pink. At present, most laboratories use ready‑to‑use staining solutions, so the optimal conditions for each modified staining solution should be noted. The final cytoplasmic staining should be transparent and visible, and nuclear chromatin should be easily distinguishable. Currently, modified Papanicolaou staining solutions contain various ions and exhibit polychromatic staining efficiency. After staining, the cytoplasm is bright and transparent, and the structures of the nuclear membrane, nucleolus, and chromatin are clear. The main nuclear stain is Harris hematoxylin solution, and the main cytoplasmic stains are EA36 solution and EA50 solution. Papanicolaou Stain is used for exfoliated cell specimens. Cell nuclei appear blue or black, and the cytoplasm of keratinized squamous cells appears pink or orange‑red.
Papanicolaou Stain Solution (Papanicolaou EA50) uses EA50 solution for cytoplasmic staining and non‑toxic modified hematoxylin solution for nuclear staining. It is suitable not only for gynecological cytological smears such as screening for cervical cancer and precancerous lesions, but also for non‑gynecological cytological samples including pleural effusion, ascites, and sputum.
Materials Required:
1. Fixative solution (e.g., 95% ethanol‑glacial acetic acid fixative)
2. Graded ethanol series
3. Microscope
4. Hydrochloric acid ethanol differentiation solution
Protocol (For Reference Only):
1. Fix cell smears in 95% ethanol‑glacial acetic acid fixative for 10–15 min.
2. Immerse in 95% ethanol for 1 min.
3. Immerse in 80% ethanol for 1 min.
4. Immerse in 70% ethanol for 1 min.
5. Immerse or rinse in distilled or tap water.
6. Stain in hematoxylin stain.
7. Rinse in tap water.
8. Differentiate in hydrochloric acid ethanol differentiation solution or aqueous hydrochloric acid solution.
9. Rinse in tap water.
10. Blue in bluing reagent.
11. Rinse in tap water.
12. Dehydrate in 70% ethanol for 2 min.
13. Dehydrate in 80% ethanol for 2 min.
14. Dehydrate in 95% ethanol (I) and (II) for 2 min each.
15. Stain in Orange G6 stain.
16. Rinse in 95% ethanol (I) and (II) for 2 min each.
17. Stain in EA50 stain for 3–5 min.
18. Dehydrate in 95% ethanol (I) and (II) for 1 min each.
19. Dehydrate in absolute ethanol (I) and (II) for 1 min each.
20. Clear in xylene and mount with neutral balsam.
Staining Results:
Nuclei: bluish-purple or black
Cytoplasm of non-keratinized cells: pale blue or pale green
Cytoplasm of keratinized cells: pink or orange-red
Precautions:
1. All staining solutions must be filtered and replaced regularly.
2. For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F0434308