Papanicolaou Staining (Pap stain) 50 FAQs And 30 MCQs:
Papanicolaou Staining (Pap stain) 50 FAQs:
What is Papanicolaou (Pap) staining?
It is a polychromatic staining technique used in cytopathology to differentiate cells in smear preparations for cancer screening and diagnosis.
Who developed the Pap stain?
George Papanicolaou in 1942.
What is the primary use of Pap staining?
Mainly for cervical cancer screening (Pap smear), but also for other bodily fluids and secretions.
What types of specimens can be used for Pap staining?
Cervical smears, sputum, urine, CSF, pleural fluid, synovial fluid, fine needle aspirates, tumor biopsies, etc.
Why is Pap staining considered reliable?
It provides clear differentiation of cell structures (nuclei, cytoplasm, keratin) and helps detect malignant and precancerous cells.
What are the main dyes used in Pap staining?
Hematoxylin (nuclear stain), Orange G-6 (keratin stain), and EA (Eosin Azure) counterstain (cytoplasm stain).
What does hematoxylin stain in Pap staining?
It stains cell nuclei blue/black.
What is the purpose of Orange G-6 in Pap staining?
It stains keratin and glycogen yellow/orange.
What does EA (Eosin Azure) stain?
Eosin Y stains superficial cells, RBCs, and cilia pink, while Light Green SF stains intermediate and parabasal cells blue-green.
Why is Bismarck Brown sometimes omitted?
It does not stain anything and is often excluded from modern formulations.
What is the difference between EA-50 and EA-65?
EA-50 is commonly used for gynecological smears, while EA-65 is used for non-gynecological samples.
What is the role of phosphotungstic acid in Pap staining?
It acts as a mordant, helping dyes bind to cellular components.
How long should a smear be fixed in 95% ethanol?
15 minutes (can be stored for up to 3 days if needed).
What is Scott’s tap water used for?
It enhances nuclear staining after hematoxylin.
What is the purpose of xylene in the procedure?
It clears the sample before mounting.
What is the standard Pap staining procedure?
Fixation → Hematoxylin → Orange G-6 → EA counterstain → Dehydration → Clearing → Mounting.
How long is hematoxylin applied in the standard method?
1-3 minutes (varies with type).
What is the modified Pap staining procedure?
Uses distilled water rinses and longer EA staining (6-10 minutes).
What is the rapid acetic acid Pap staining method?
A quick method using 1% acetic acid and 10-dip steps for faster results.
Why is blotting done in the rapid method?
To remove excess liquid between steps for quicker drying.
What mounting medium is used for Pap-stained slides?
DPX (a permanent mounting medium).
Can prolonged fixation affect staining?
Yes, it may alter staining reactions if left beyond 3 days.
What happens if a smear dries before fixation?
It can cause artifacts and poor staining quality.
Why is dehydration important in Pap staining?
It removes water and prepares the sample for xylene clearing.
What is the difference between Harris’ and Gill’s hematoxylin?
Gill’s hematoxylin is preferred for cytology due to better nuclear clarity.
What color do nuclei appear after Pap staining?
Blue to black.
How do keratinized cells appear?
Orange-red or yellow.
What color are superficial squamous cells?
Pink to orange
How do intermediate and parabasal cells stain?
Blue-green or turquoise.
What color are red blood cells (RBCs) in Pap staining?
Orange-red to dark pink.
How do eosinophils appear?
Orange-red.
What does Candida look like in a Pap stain?
Red hyphae or yeast forms.
How are Trichomonas vaginalis identified?
Grey-green with pear-shaped appearance.
What does metaplastic cell staining look like?
Mixed green and pink cytoplasm.
Why do some cells stain both pink and green?
Due to variations in cytoplasmic composition (e.g., metaplastic cells).
What is the main clinical use of Pap staining?
Cervical cancer screening (Pap smear).
Can Pap staining detect infections?
Yes, it can identify Candida, Trichomonas, and bacterial vaginosis.
Is Pap staining used for non-gynecological samples?
Yes, for sputum, urine, CSF, and fine-needle aspirates.
Can Pap staining diagnose thyroid cancer?
Yes, in fine-needle aspiration cytology (FNAC).
How does Pap staining help in lung cancer diagnosis?
By examining sputum or bronchial washings for malignant cells.
Is Pap staining used for prostate cancer detection?
Yes, in prostatic secretion smears.
Can Pap staining detect gastric cancer?
Yes, in gastric washings or biopsy smears.
Why is Pap staining preferred for cervical smears?
It provides clear nuclear and cytoplasmic differentiation for detecting dysplasia.
What are the limitations of Pap staining?
It is screening, not diagnostic—requires biopsy confirmation for malignancy.
Can Pap staining replace histopathology?
No, it is complementary—tissue biopsy is needed for definitive diagnosis.
What causes poor nuclear staining in Pap smears?
Inadequate hematoxylin time or old reagents.
Why might cytoplasm appear too dark?
Over-staining with EA or insufficient rinsing.
What if the slide appears cloudy after mounting?
Incomplete dehydration or clearing in xylene.
Can alcohol substitutes be used in Pap staining?
No, 95% and 100% ethanol are critical for proper fixation and dehydration.
Why is Light Green SF sometimes replaced with Fast Green FCF?
Due to cost and availability, but it may alter staining results.
Papanicolaou Staining (Pap stain) 30 MCQs :
Who developed the Papanicolaou staining technique?
a) Louis Pasteur
b) George Papanicolaou
c) Robert Koch
d) Rudolf Virchow
2. What is the primary purpose of Pap staining?
a) To detect bacterial infections
b) To screen for cervical cancer
c) To identify fungal hyphae
d) To measure blood glucose levels
3. Which dye is used as a nuclear stain in Pap staining?
a) Eosin Y
b) Hematoxylin
c) Orange G-6
d) Bismarck Brown
4. What color do cell nuclei appear after Pap staining?
a) Pink
b) Blue/Black
c) Orange
d) Green
5. Which component does Orange G-6 primarily stain?
a) Nuclei
b) Keratin
c) Red blood cells
d) Cilia
6. What does EA (Eosin Azure) stain in Pap smears?
a) Only nuclei
b) Only cytoplasm
c) Both nuclei and cytoplasm
d) Only bacteria
7. Which of the following is NOT a specimen used for Pap staining?
a) Sputum
b) Urine
c) Blood plasma
d) Cerebrospinal fluid (CSF)
8. How long should a smear be fixed in 95% ethanol for Pap staining?
a) 5 seconds
b) 15 minutes
c) 1 hour
d) 24 hours
9. What is the role of xylene in Pap staining?
a) Staining nuclei
b) Clearing the slide
c) Fixing cells
d) Counterstaining
10. Which of these dyes is often omitted in modern Pap stain formulations?
a) Hematoxylin
b) Eosin Y
c) Bismarck Brown
d) Light Green SF
11. What color do superficial squamous cells appear after Pap staining?
a) Blue
b) Green
c) Pink/Orange
d) Black
12. Which stain is used for non-gynecological specimens like sputum?
a) EA-36
b) EA-50
c) EA-65
d) OG-6
13. What is the purpose of Scott’s tap water in Pap staining?
a) To remove excess stain
b) To enhance nuclear staining
c) To dehydrate the slide
d) To fix cells
14. How are Candida species identified in a Pap smear?
a) Blue nuclei
b) Red hyphae/yeast
c) Green cytoplasm
d) Orange keratin
15. What is the final step in Pap staining before microscopy?
a) Fixation
b) Mounting with DPX
c) Rinsing in water
d) Staining with eosin
16. Which of the following is a rapid Pap staining method?
a) Using 1% acetic acid
b) Prolonged hematoxylin staining
c) Skipping dehydration
d) Omitting xylene
17. What does poor nuclear staining indicate in Pap smears?
a) Over-staining with eosin
b) Inadequate hematoxylin
c) Excess xylene
d) Too much Orange G-6
18. Which dye is responsible for staining RBCs orange-red?
a) Hematoxylin
b) Eosin Y
c) Light Green SF
d) Bismarck Brown
19. What is the advantage of Pap staining over simple H&E?
a) Cheaper
b) Faster
c) Better cytoplasmic transparency
d) Doesn’t require fixation
20. What is the most critical step before Pap staining?
a) Immediate fixation
b) Heating the slide
c) Adding a coverslip
d) Using distilled water
21. Which component is NOT part of EA (Eosin Azure)?
a) Eosin Y
b) Light Green SF
c) Orange G-6
d) Bismarck Brown
22. What happens if a Pap smear dries before fixation?
a) Better staining
b) Artifacts and poor results
c) No effect
d) Faster processing
23. Which of these is a counterstain in Pap staining?
a) Harris hematoxylin
b) OG-6
c) Phloxine
d) Methylene blue
24. What is the purpose of phosphotungstic acid in EA?
a) To act as a mordant
b) To dehydrate cells
c) To fix nuclei
d) To clear the slide
25. What color do intermediate squamous cells stain?
a) Pink
b) Blue-Green
c) Orange
d) Black
26. Which of these is NOT a use of Pap staining?
a) Cervical cancer screening
b) Diagnosing diabetes
c) Detecting Trichomonas
d) Lung cancer cytology
27. What is the most common fixative for Pap smears?
a) Formalin
b) 95% Ethanol
c) Methanol
d) Acetone
28. What is the role of glacial acetic acid in Pap staining?
a) To enhance nuclear staining
b) To dehydrate cells
c) To act as a clearing agent
d) To fix RBCs
29. How are metaplastic cells identified in Pap staining?
a) Pure blue cytoplasm
b) Mixed pink and green
c) Only red nuclei
d) Black keratin
30. What is the main limitation of Pap staining?
a) It is only for research
b) It cannot detect infections
c) It is a screening, not diagnostic test
d) It doesn’t work on blood samples
