Which stain is used as the first counterstain in Pap smear?

• A. Harris hematoxylin
• B. EA 50 stain
• C. OG 6 stain
• D. Papanicolaou

Answer: (C)

Pap staining builds color contrast by first staining nuclei and then applying two cytoplasmic counterstains. After the nuclei are colored with a blue-black hematoxylin, the first counterstain used is orange G (OG-6). This step gives the cytoplasm an orange hue, helping to highlight differences between cell types and improve morphological contrast, especially for identifying squamous versus glandular cells. A second counterstain with EA-50 (a mix of eosin Y and light green) adds additional color differentiation to further refine cytoplasmic staining. Harris hematoxylin is the nuclear stain, not a counterstain, and EA-50 is the second counterstain. Papanicolaou refers to the overall stain method, not a specific counterstain.