ASCP Hematoxylin and Eosin Staining 1 — Questions and Answers
Question 1: What cellular component is primarily stained by hematoxylin in an H&E stain?
- Cytoplasm
- Collagen
- Nuclei (DNA/RNA) (Correct answer)
- Lipids
Correct answer: Nuclei (DNA/RNA)
Hematoxylin is a basic dye that binds to negatively charged nucleic acids in the nucleus, staining it blue-purple.
Question 2: What color does eosin stain the cytoplasm and collagen in a routine H&E preparation?
- Blue
- Purple
- Pink/red (Correct answer)
- Green
Correct answer: Pink/red
Eosin is an acidic dye that stains positively charged proteins in the cytoplasm and extracellular matrix pink to red.
Question 3: What is the purpose of the mordant in the hematoxylin staining process?
- To remove excess hematoxylin
- To link the hematoxylin dye to tissue components (Correct answer)
- To differentiate background from nuclei
- To prevent fading
Correct answer: To link the hematoxylin dye to tissue components
A mordant (typically alum) acts as a bridge between the dye and tissue, forming a stable dye-mordant-tissue complex.
Question 4: What is the 'bluing' step in H&E staining intended to accomplish?
- Increase eosin intensity
- Convert soluble hematoxylin to an insoluble blue-black complex by raising pH (Correct answer)
- Remove excess eosin
- Deparaffinize sections
Correct answer: Convert soluble hematoxylin to an insoluble blue-black complex by raising pH
Exposure to a slightly alkaline solution (Scott's water or ammonia water) shifts the hematoxylin complex from red-brown to a permanent blue-black.
Question 5: What is the purpose of the differentiation step using acid-alcohol in H&E staining?
- To remove eosin background
- To selectively remove excess hematoxylin from non-nuclear elements (Correct answer)
- To increase nuclear stain intensity
- To deparaffinize tissue
Correct answer: To selectively remove excess hematoxylin from non-nuclear elements
Acid-alcohol removes excess hematoxylin from cytoplasm and connective tissue, leaving stain only in the nuclei.
Question 6: Before H&E staining, paraffin sections must be deparaffinized using which reagent?
- Ethanol
- Xylene (Correct answer)
- Acetone
- Water
Correct answer: Xylene
Xylene dissolves paraffin wax before the section is rehydrated through graded alcohols for aqueous staining.
ASCP Hematoxylin and Eosin Staining 1
What cellular component is primarily stained by hematoxylin in an H&E stain?