Hematoxylin and Eosin (H&E) Staining 100 FAQs and 60 MCQs
Fematoxylin and Eosin (H&E) Staining 100 FAQs:
What is the primary purpose of H&E staining in histology?
To recognize tissue morphology and pathological changes, especially in cancer diagnosis.
Why is H&E the most widely used histological stain?
Simplicity, clarity in demonstrating diverse tissue structures, and compatibility with most fixatives.
What structures does hematoxylin stain, and what color do they appear?
Nuclei (blue-black) via binding to nucleic acids.
What structures does eosin stain, and what colors are produced?
Cytoplasm, connective tissue (pink/red) by interacting with proteins.
How do acidic and basic dyes interact with tissue components?
Eosin (acidic, anionic) binds cationic structures; hematoxylin (basic, cationic with mordant) binds anionic sites.
What is the difference between progressive and regressive staining?
Progressive stains nuclei directly; regressive over-stains and differentiates.
What are the advantages of automated H&E staining over manual methods?
Consistency, speed, reduced manual error.
Why is differentiation necessary in H&E staining?
Removes excess dye to sharpen nuclear detail.
How does fixation affect H&E staining results?
Acidic fixatives (e.g., unbuffered formalin) impair nuclear staining; chromate fixatives enhance PTAH.
What are common artifacts in H&E-stained sections?
Precipitates, uneven staining, fading.
What is hematoxylin, and where is it derived from?
Extracted from Hematoxylon campechianum heartwood
Why is hematoxylin not a stain by itself?
Requires oxidation to hematein for staining.
What is hematein, and how is it produced from hematoxylin?
Hematein: Oxidized hematoxylin; the active dye component.
What role do mordants play in hematoxylin staining?
Mordant role: Binds hematein to tissue (e.g., aluminum, iron).
Name the types of mordants used in hematoxylin solutions.
Mordant types: Aluminum (alum), iron, tungsten, molybdenum, lead.
What are the differences between alum and iron hematoxylins?
Alum vs. iron hematoxylins: Alum for nuclei; iron for connective tissue/muscle.
How does natural oxidation (“ripening”) of hematoxylin work?
Natural oxidation: Slow ripening (months) via light/air exposure (e.g., Ehrlich’s).
How does chemical oxidation (e.g., sodium iodate) affect hematoxylin?
Chemical oxidation: Sodium iodate (Mayer’s) or mercuric oxide (Harris’s) for instant use.
What are the key components of Ehrlich’s hematoxylin?
Ehrlich’s components: Hematoxylin, alcohol, glycerin, acetic acid, alum.
Why is Harris’s hematoxylin preferred for cytology?
Harris’s in cytology: Precise nuclear detail, resistant to fading.
What is the role of acetic acid in Harris’s hematoxylin?
Acetic acid in Harris’s: Sharpens nuclear staining.
How does Gill’s hematoxylin differ from Mayer’s?
Gill’s vs. Mayer’s: Gill’s uses ethylene glycol for stability; Mayer’s is progressive.
Why does Carazzi’s hematoxylin work well for frozen sections?
Carazzi’s for frozen sections: Rapid staining (1–2 minutes) with potassium iodate.
What are the disadvantages of alum hematoxylins in acidic conditions?
Alum in acidic conditions: Mordant stripped, leading to poor nuclear staining.
How does Weigert’s hematoxylin resist acid decolorization?
Weigert’s acid resistance: Iron mordant creates strong bonds.
What structures are demonstrated using Heidenhain’s iron hematoxylin?
Heidenhain’s use: Mitochondria, muscle striations, chromatin.
How does Verhöeff’s hematoxylin stain elastin fibers?
Verhöeff’s elastin stain: Iron-iodine mordant binds elastin (black).
Why is celestine blue used with alum hematoxylin in some protocols?
Celestine blue: Enhances alum hematoxylin’s acid resistance (e.g., van Gieson).
How does lead hematoxylin demonstrate endocrine cell granules?
Lead hematoxylin: Targets endocrine granules via lead mordant.
What is eosin Y, and why is it the most common eosin variant?
Eosin Y: Water/alcohol-soluble, most common for consistent pink staining.
How does eosin interact with cytoplasmic proteins?
Eosin interaction: Binds cytoplasmic proteins via electrostatic attraction.
Why is thymol added to eosin solutions?
Thymol: Prevents fungal growth in aqueous solutions.
How does acetic acid enhance eosin staining?
Acetic acid: Sharpens eosin staining by lowering pH.
What causes over-differentiation of eosin, and how is it corrected?
Over-differentiation: Prolonged washing; correct by reducing dehydration time.
How does mercuric fixation affect eosin staining intensity?
Mercuric fixation: Increases eosin intensity due to tissue hardening.
What alternatives to eosin exist, and why are they less popular?
Eosin alternatives: Phloxine, Biebrich scarlet (less differentiation control).
How does the eosin-phloxine B combination improve tissue contrast?
Eosin-phloxine combo: Enhances contrast (muscle vs. collagen).
Why is sodium sulfate detrimental to eosin solutions?
Sodium sulfate: Causes granular precipitate, reducing dye efficacy.
What is the optimal eosin concentration for routine H&E staining?
Optimal eosin: 0.5–1% aqueous solution.
Outline the steps for routine H&E staining of paraffin sections.
Routine H&E steps: Dewax → hydrate → hematoxylin → differentiate → blue → eosin → dehydrate → mount.
How is rapid H&E staining performed for frozen sections?
Frozen sections: Fix in formalin (20 sec) → Carazzi’s hematoxylin (1 min) → eosin (10 sec).
What modifications are needed for H&E staining after acid decalcification?
Acid decalcification: Use resistant hematoxylin (e.g., Ehrlich’s) and extend staining time.
How does the Papanicolaou stain differ from routine H&E?
Papanicolaou vs. H&E: Uses Harris’s hematoxylin, OG 6 (orange G), and EA 50 (eosin-azure) for cytology.
What are OG 6 and EA 50 in the Papanicolaou protocol?
OG 6 & EA 50: OG 6 stains keratin; EA 50 differentiates cytoplasmic hues.
Why is Scott’s tap water substitute used during “blueing”?
Scott’s tap water: Alkaline solution accelerates blueing.
How does dehydration impact eosin staining intensity?
Dehydration impact: Removes unbound eosin; over-dehydration lightens stain.
What is the purpose of acid alcohol in H&E differentiation?
Acid alcohol: Differentiates hematoxylin by removing excess dye.
How are nuclei “blued” after hematoxylin staining?
Blueing: Alkaline solutions (e.g., tap water) convert hematein to blue-black.
Why are frozen sections stained for shorter durations?
Frozen section timing: Prevents ice crystal artifacts and over-staining.
Why might hematoxylin-stained nuclei appear muddy or faint?
Muddy nuclei: Acidic fixatives; use Ehrlich’s or celestine blue.
How do over-oxidized hematoxylin solutions affect staining?
Over-oxidized hematoxylin: Brown color; discard and prepare fresh.
What causes hematoxylin to stain glass slides or adhesive?
Glass staining: Aluminum mordant in Gill’s binds charged glass.
How can eosin over-staining be resolved?
Eosin over-staining: Rinse in 70% alcohol briefly.
Why do sections from acidic fixatives require special hematoxylins?
Acidic fixatives: Require acid-resistant hematoxylins (e.g., Ehrlich’s).
How does prolonged formalin fixation impact H&E results?
Prolonged formalin: Acidic pH impairs staining; neutralize with buffer.
What steps prevent fungal growth in eosin solutions?
Fungal prevention: Add thymol to eosin.
How do aging hematoxylin solutions affect staining times?
Aging hematoxylin: Increases staining time; filter before use.
Why might collagen appear indistinct in H&E sections?
Indistinct collagen: Poor differentiation; optimize eosin timing.
How is poor nuclear detail corrected in H&E staining?
Poor nuclear detail: Ensure proper differentiation and blueing.
What is the role of celestine blue in nuclear staining?
Celestine blue: Iron-based mordant for acid-resistant nuclear staining.
How does Heidenhain’s hematoxylin demonstrate mitochondria?
Heidenhain’s mitochondria: Differentiation removes excess dye, retaining organelle stain.
What is the Loyez hematoxylin technique used for?
Loyez hematoxylin: Myelin demonstration via iron mordant.
How does Verhöeff’s hematoxylin differ from other elastin stains?
Verhöeff’s vs. others: Uses iodine for elastin specificity.
Why is phosphotungstic acid hematoxylin (PTAH) used for muscle striations?
PTAH muscle striations: Binds fibrin/myosin with phosphotungstic acid.
How does the Thomas molybdenum hematoxylin stain collagen?
Thomas molybdenum: Stains collagen via phosphomolybdic acid.
What fixatives are incompatible with Mallory’s PTAH stain?
Incompatible with PTAH: Dichromate fixatives; use Zenker’s instead.
How does the Movat’s pentachrome incorporate hematoxylin?
Movat’s pentachrome: Combines hematoxylin with multiple dyes for connective tissue.
Why is Weigert-Pal hematoxylin used for myelin in block preparations?
Weigert-Pal myelin: Chromium/copper mordant in block preparations.
How does Solcia’s lead hematoxylin target endocrine cells?
Solcia’s lead hematoxylin: Targets endocrine granules (replaced by IHC).
How does H&E aid in cancer diagnosis?
Reveals nuclear abnormalities, cytoplasmic changes, tissue disruption, and mitotic activity, key for identifying tumors.
Why are nuclei blue and cytoplasm pink in H&E sections?
Nuclei: Hematoxylin binds nucleic acids (blue).
Cytoplasm: Eosin binds proteins (pink).What cytoplasmic features are highlighted by eosin?
Proteins, RBCs (bright red), collagen (pink), muscle fibers (deep pink).
How does H&E staining demonstrate fibrin or keratin?
Fibrin: Bright pink strands.
Keratin: Intense pink in keratinized cells.What structures are poorly visualized with H&E, requiring special stains?
Reticulin, microorganisms, specific granules, lipids.
How does H&E staining vary in connective tissue vs. epithelial cells?
Epithelial: Prominent nuclei (blue), variable cytoplasm (pink).
Connective: Collagen (pink), fibroblasts (small, dark nuclei).Why is Ehrlich’s hematoxylin recommended for cartilage staining?
Binds mucopolysaccharides in cartilage, providing clear staining in acidic conditions.
How does prolonged eosin staining improve photomicrography?
Enhances contrast and color intensity for clearer images.
What causes red blood cells to appear bright red in H&E?
Eosin binds hemoglobin, staining RBCs bright red.
How are mucins stained by hematoxylin in certain protocols?
Mucins bind hematoxylin via sulfated/carboxylated groups, appearing blue-purple.
How does the pH of tap water affect hematoxylin “blueing”?
pH in blueing: Alkaline pH (≥7) ensures proper hematein conversion.
Why do iron hematoxylins require microscopic control during differentiation?
Iron hematoxylin control: Differentiation requires microscopy to avoid over-removal.
What is the mechanism behind Heidenhain’s differentiation using iron alum?
Heidenhain’s mechanism: Iron alum removes excess dye selectively.
How does ethylene glycol stabilize Gill’s hematoxylin?
Ethylene glycol: Prevents precipitate formation in Gill’s.
Why are mercury-based hematoxylins being phased out?
Mercury phased out: Toxicity and environmental harm.
How does sodium iodate act as an oxidizing agent in Mayer’s hematoxylin?
Sodium iodate: Oxidizes hematoxylin in Mayer’s.
What is the role of chloral hydrate in Mayer’s hematoxylin formulation?
Chloral hydrate: Stabilizes Mayer’s hematoxylin.
How does post-chroming (dichromate treatment) enhance PTAH staining?
Post-chroming (PTAH): Enhances tissue affinity via dichromate.
Why does Verhöeff’s hematoxylin include Lugol’s iodine?
Lugol’s in Verhöeff’s: Enhances elastin binding.
How does the choice of mordant influence hematoxylin’s tissue affinity?
Mordant influence: Determines dye-tissue affinity (e.g., aluminum for nuclei).
Compare Delafield’s, Ehrlich’s, and Harris’s hematoxylins.
Delafield’s vs. Ehrlich’s vs. Harris’s: Delafield’s (natural), Ehrlich’s (acid-resistant), Harris’s (cytology).
Why is natural ripening of hematoxylin less common today?
Natural ripening decline: Time-consuming; replaced by chemical oxidation.
How have commercial H&E solutions impacted traditional methods?
Commercial impact: Standardized solutions reduce variability.
What historical alternatives to eosin were explored, and why failed?
Eosin alternatives: Phloxine (intense but less versatile).
How did Mallory’s early work influence modern connective tissue stains?
Mallory’s legacy: Pioneered connective tissue stains (e.g., PTAH).
Why is Weigert’s hematoxylin still used despite newer alternatives?
Weigert’s relevance: Resists acidic stains (e.g., van Gieson).
How did Gill’s modifications improve hematoxylin stability?
Gill’s stability: Ethylene glycol reduces oxidation/precipitation.
What are the environmental concerns with mercury-based hematoxylins?
Mercury concerns: Toxic to labs/environment.
How has immunohistochemistry replaced lead hematoxylins?
IHC replacement: Specific markers supersede lead hematoxylins.
What future advancements might replace or enhance H&E staining?
Future advancements: Digital pathology, AI-assisted analysis, novel dyes.
What is the role of phosphotungstic acid in Mallory’s PTAH stain?
PTAH role: Phosphotungstic acid mordants hematoxylin for muscle/fibrin (blue).
Hematoxylin and Eosin (H&E) Staining 60 MCQs:
- What is the primary purpose of H&E staining?
a) To identify microorganisms
b) To visualize tissue morphology and diagnose diseases ✔
c) To stain lipids
d) To highlight collagen fibers - Which dye in H&E stains nuclei blue-black?
a) Eosin
b) Hematoxylin ✔
c) Phloxine
d) Methyl blue - What color does eosin stain cytoplasm?
a) Blue
b) Pink/red ✔
c) Green
d) Yellow - What is the role of a mordant in hematoxylin staining?
a) To oxidize hematoxylin
b) To bind hematein to tissue ✔
c) To differentiate eosin
d) To dehydrate tissues
- Which hematoxylin is naturally oxidized and ideal for acidic tissues?
a) Harris’s
b) Ehrlich’s ✔
c) Gill’s
d) Mayer’s - What is the active dye component in hematoxylin staining?
a) Hematein ✔
b) Eosin
c) Mordant
d) Acetic acid - Which mordant is used in Weigert’s hematoxylin?
a) Aluminum
b) Iron ✔
c) Tungsten
d) Lead - What is the purpose of “blueing” in H&E staining?
a) To remove excess eosin
b) To convert hematein to blue-black ✔
c) To differentiate nuclei
d) To dehydrate tissues
- What is the most common eosin variant used in H&E?
a) Eosin B
b) Eosin Y ✔
c) Ethyl eosin
d) Phloxine - Why is thymol added to eosin solutions?
a) To enhance staining
b) To prevent fungal growth ✔
c) To differentiate tissues
d) To oxidize eosin - What causes red blood cells to appear bright red in H&E?
a) Hematoxylin binding
b) Eosin binding to hemoglobin ✔
c) Acid alcohol differentiation
d) Blueing
- Which stain is used to demonstrate elastin fibers?
a) Weigert’s hematoxylin
b) Verhöeff’s hematoxylin ✔
c) Mallory’s PTAH
d) Heidenhain’s hematoxylin - What is the role of phosphotungstic acid in Mallory’s PTAH?
a) To oxidize hematoxylin
b) To act as a mordant for hematoxylin ✔
c) To differentiate eosin
d) To dehydrate tissues - Why is celestine blue used with alum hematoxylin?
a) To enhance nuclear staining in acidic conditions ✔
b) To stain collagen fibers
c) To differentiate eosin
d) To oxidize hematoxylin - What is the primary disadvantage of alum hematoxylins?
a) They stain glass slides
b) They are sensitive to acidic conditions ✔
c) They fade quickly
d) They are toxic
- Which structure is NOT stained by eosin?
a) Cytoplasm
b) Collagen
c) Nuclei ✔
d) Red blood cells - What does H&E staining reveal in cancer diagnosis?
a) Lipid accumulation
b) Nuclear abnormalities and tissue architecture disruption ✔
c) Microorganisms
d) Reticulin fibers - Which hematoxylin is recommended for cartilage staining?
a) Harris’s
b) Ehrlich’s ✔
c) Gill’s
d) Mayer’s - What causes nuclei to appear blue in H&E staining?
a) Eosin binding
b) Hematoxylin binding to nucleic acids ✔
c) Acid alcohol differentiation
d) Blueing with tap water - Which fixative is incompatible with Mallory’s PTAH?
a) Formalin
b) Zenker’s ✔
c) Bouin’s
d) Carnoy’s
- Which hematoxylin is chemically oxidized using sodium iodate?
a) Ehrlich’s
b) Mayer’s ✔
c) Delafield’s
d) Weigert’s - What is the primary role of acetic acid in Harris’s hematoxylin?
a) Oxidize hematoxylin
b) Sharpen nuclear staining ✔
c) Prevent fungal growth
d) Differentiate eosin - Which hematoxylin is ideal for frozen sections due to rapid staining?
a) Ehrlich’s
b) Carazzi’s ✔
c) Gill’s
d) Cole’s - What differentiates Heidenhain’s iron hematoxylin from alum hematoxylins?
a) Requires microscopic control ✔
b) Stains collagen blue
c) Uses tungsten mordant
d) Does not need blueing
- Over-differentiation of eosin may result in:
a) Bright red RBCs
b) Loss of cytoplasmic detail ✔
c) Blue collagen
d) Faded nuclei - Which component is added to eosin to inhibit fungal growth?
a) Acetic acid
b) Thymol ✔
c) Sodium sulfate
d) Ethanol - Prolonged eosin staining is recommended for:
a) Highlighting nuclei
b) Enhancing photomicrography ✔
c) Staining lipids
d) Differentiating collagen
- Which stain demonstrates muscle striations and fibrin?
a) Verhöeff’s
b) Mallory’s PTAH ✔
c) Heidenhain’s
d) Weigert’s - Verhöeff’s hematoxylin is used to stain:
a) Collagen
b) Elastin fibers ✔
c) Reticulin
d) Mucins - Which artifact is caused by incomplete dehydration after eosin staining?
a) Faded nuclei
b) Water spots ✔
c) Precipitates
d) Over-stained cytoplasm
- Prolonged formalin fixation may lead to:
a) Enhanced nuclear staining
b) Acidic tissue pH, impairing H&E ✔
c) Bright red RBCs
d) Improved eosin differentiation - Which step neutralizes acidic fixatives before H&E staining?
a) Blueing
b) Decalcification
c) Post-chroming
d) Buffered formalin treatment ✔ - Nuclear fading in H&E is often due to:
a) Insufficient hematoxylin
b) Over-oxidized hematoxylin ✔
c) Excessive blueing
d) Prolonged eosin staining
- Which mordant is used in lead hematoxylin for endocrine granules?
a) Aluminum
b) Iron
c) Lead ✔
d) Tungsten - Ethylene glycol in Gill’s hematoxylin prevents:
a) Oxidation
b) Precipitation ✔
c) Fungal growth
d) Acidic pH - The blueing step requires a pH of:
a) ≤5
b) 5–6
c) ≥7 ✔
d) Neutral
- Which structure is highlighted by mucin staining with hematoxylin?
a) Nuclei
b) Goblet cells ✔
c) Collagen
d) RBCs - H&E is NOT suitable for visualizing:
a) Tumor necrosis
b) Bacterial colonies ✔
c) Mitotic figures
d) Keratin - Which tissue component stains pink in H&E due to eosin?
a) DNA
b) Collagen ✔
c) RNA
d) Lipids - In cancer diagnosis, increased mitotic activity in H&E indicates:
a) Benign tumor
b) Poor differentiation
c) High proliferation
d) Inflammatory response
41. Which of the following is true regarding hematoxylin and eosin staining?
A) Hematoxylin is a basic dye; it binds to nucleic acids in the nucleus, imparting a deep blue to black color. ✔
B) Eosin is a basic dye; it binds to nucleic acids in the nucleus, imparting a deep blue to black color.
C) Both are true
D) None of the above
42. Which of the following is added as a preservative in Eosin solutions to prevent fungal growth?
A) Ethanol
B) Crystal of thymol ✔
C) Methanol
D) Picric acid
43. Which statement is false regarding the preparation and extraction of hematoxylin stain?
A) It is extracted from the heartwood of the tree Hematoxylin campechianum.
B) It is extracted with hot water and precipitated out using urea.
C) Hematoxylin is a stain by itself. ✔
D) None of the above is wrong.
44. Which of the following hematoxylins are obtained by natural oxidation?
A) Ehrlich’s hematoxylin
B) Delafield’s hematoxylin
C) Both A and B ✔
D) None of the above
45. What is the mordant used in Meyer’s hematoxylin?
A) Mercuric Oxide
B) Iron
C) Aluminium ✔
D) Sodium Iodate
46. Which of the following is used as a mordant in Harris hematoxylin?
A) Mercuric Oxide
B) Iron
C) Aluminium ✔
D) Sodium Iodate
47. Which mordant is most frequently used in routine histopathology staining with H&E?
A) Mercuric Oxide
B) Iron
C) Aluminium ✔
D) Sodium Iodate
48. Which hematoxylin is routinely used for Papanicolaou staining (PAP)?
A) Ehrlich hematoxylin ✔
B) Delafield hematoxylin
C) Meyer hematoxylin
D) Harris hematoxylin
49. Which hematoxylin is routinely used for frozen sections?
A) Meyer hematoxylin
B) Harris hematoxylin ✔
C) Cole hematoxylin
D) Carazzi’s hematoxylin
50. Which hematoxylin takes the longest time to stain?
A) Ehrlich hematoxylin
B) Delafield hematoxylin ✔
C) Meyer hematoxylin
D) Harris hematoxylin
51. What does it mean to regressively stain a slide?
A) Staining is done to a desired intensity and no more.
B) A slide is deliberately overstained and de-stained until the endpoint is reached. ✔
C) Both are true.
D) None of the above.
52. How to correct unclear nuclear staining after using Ehrlich’s hematoxylin?
A) Increase the duration of staining with hematoxylin.
B) Use Weigert’s hematoxylin or celestine blue sequence instead. ✔
C) Use a different lot of hematoxylin.
D) Rest the solution for ripening.
53. All are examples of iron hematoxylin except:
A) Weigert’s hematoxylin
B) Heidenhain’s hematoxylin
C) Verhoeff’s hematoxylin for elastin fibers
D) Ehrlich hematoxylin ✔
54. Which hematoxylin is ideal for staining myelin?
A) Weigert’s ✔
B) Heidenhain’s
C) Loyez
D) Verhoeff’s for elastin fibers
55. Mallory’s PTAH contains which mordant?
A) Mercury
B) Tungsten ✔
C) Iron
D) Aluminium
56. Which is routinely used for differentiation of hematoxylin?
A) 1% acid alcohol ✔
B) 100% ethanol
C) Scott tap water
D) Methanol
57. Which is routinely used for blueing?
A) 1% acid alcohol
B) 100% ethanol
C) Scott tap water ✔
D) Methanol
58. Verhoeff’s hematoxylin is used to stain:
A) Nucleus
B) Elastic fibers ✔
C) Myelin
D) Mucins
59. Correct sequence of hematoxylin staining:
A) Dewax paraffin sections → Rehydrate with increasing grades of alcohol → Stain with hematoxylin → Differentiate, wash, and blue → Stain with eosin → Dehydrate and coverslip. ✔
B) Dewax paraffin sections → Rehydrate with decreasing grades of alcohol → Stain with hematoxylin → Differentiate, wash, and blue → Stain with eosin → Dehydrate and coverslip.
C) Stain with hematoxylin → Differentiate, wash, and blue → Stain with eosin → Dehydrate and coverslip → Dewax paraffin sections → Rehydrate.
D) Stain with eosin → Dehydrate and coverslip → Dewax paraffin sections → Rehydrate → Stain with hematoxylin → Differentiate, wash, and blue.
60. Which of the following factors affect hematoxylin staining?
A) Age of the staining solution
B) Duration of ripening
C) Type of mordant used
D) All of the above ✔
