Special Stains

Table of Contents

Special Staining Techniques

Special stains are essential for identifying specific normal and abnormal substances within cells and tissues, which routine hematoxylin and eosin staining cannot distinguish. For example, Van Gieson’s special stain is employed to differentiate between connective tissue and muscle fibers, while Pearl’s stain is necessary for demonstrating iron in tissues.

Interpretation and Quality Control:

Interpreting special stains requires expertise, as artifacts or faulty techniques may yield false results. It is crucial to adhere to strict protocols, including staining negative and positive controls with each procedure. The following measures should be strictly observed:

  1. Blocks and unstained control slides should be available for running controls.
  2. All reagents should be freshly prepared for optimal results.
  3. Reagents should be stored in brown-colored bottles with tight stoppers.
  4. Proper labeling of reagent bottles with clear expiry dates is essential.
  5. Meticulously follow all steps in special staining procedures, including specific instructions for fixatives and precautions mentioned.

1. Periodic Acid Schiff Reaction (PAS):Periodic Acid Schiff Reaction PAS

Purpose:

PAS is required to demonstrate carbohydrates, such as neutral mucopolysaccharides, mucoproteins, and glycoproteins, in tissues. It can also be utilized to showcase fungi.

Principle:

Periodic acid oxidizes glycol groups in carbohydrates, forming aldehydes that react with Schiff’s reagent, producing a colored compound.

Requirements:

  1. 0.1% periodic acid
  2. 0.5% sodium metabisulphite
  3. Schiff’s solution
  4. Haematoxylin (used in H&E stain)

Procedure:

  1. Rehydrate for H&E staining.
  2. Rinse in tap water for 5 min.
  3. Oxidize in 0.1% periodic acid for 15 min.
  4. Wash well in tap water for 5 min.
  5. Treat with Schiff’s reagent for 10 min.
  6. Treat with 0.5% sodium metabisulphite for 2 min.
  7. Stain in hematoxylin as desired.
  8. Dehydrate, clear, and mount as for H&E stain.

Result:

Neutral mucopolysaccharides, mucoproteins, and glycoproteins stain pink to magenta, while nuclei stain blue. Fungi also stain magenta.

2. Periodic Acid Schiff with Diastase:

Purpose:

To stain glycogen.

Principle:

Diastase treatment removes glycogen, preventing staining with PAS.

Requirements:

  1. Buffered saline
  2. 0.5% MALT diastase
  3. Reagents for PAS stain

Procedure:

  1. Rehydrate the sections.
  2. Rinse in warm water (37°C) for 10 dips.
  3. Treat with diastase and proceed from step 4 as for PAS stain.

Results:

Tissues positive for PAS stain become negative after diastase treatment.

3. Alcian Blue and Alcian Blue PAS Combined Stain:

Purpose:

Used to distinguish between mucin-secreting adenocarcinoma and undifferentiated squamous cell carcinoma, and to identify carbohydrates in tissue.

Principle:

 Alcian blue stains connective tissue ground substance, while PAS reacts with epithelial mucin or glycogen.

Control:

Small intestine

Requirements:

  1. Alcian blue
  2. 3% Glacial acetic acid
  3. Nuclear Fast red counterstain
  4. 1% Periodic acid
  5. Schiff’s reagent

Procedure for Alcian Blue Stain:

  1. Rehydrate the section.
  2. Rinse in tap water for 2 min.
  3. Stain in Alcian blue for 20 min.
  4. Rinse in tap water for 10 min.
  5. Counterstain in nuclear fast red for 3-5 min.
  6. Dehydrate, clear, and mount.

Results:

Acid mucopolysaccharides stain blue, while other tissue elements stain red.

Procedure for Alcian Blue-PAS Combined Stain:

  1. Rehydrate the sections and rinse in distilled water.
  2. Treat with Alcian blue solution for 30 min.
  3. Wash well and proceed from step 5 as for the PAS stain.

Results:

Acid mucin is stained blue, neutral mucin is stained red, and a mixture of the two is stained purple.

4. Gomori’s Reticulin Stain:Gomori's Reticulin Stain

Purpose:

To stain reticulin fibers.

Principle:

Silver oxide precipitates on reticulin fibers in the presence of ammoniacal solution.

Requirements:

  1. Silver nitrate reagent
  2. Potassium permanganate 1%
  3. Potassium metabisulphite 3%
  4. Iron alum 2%
  5. Formalin 10%
  6. Gold chloride
  7. Sodium thiosulphate 3%

Procedure:

  1. Rehydrate the sections.
  2. Oxidize with potassium permanganate.
  3. Decolorize with potassium metabisulphite.
  4. Sensitize in iron alum.
  5. Impregnate in silver solution.
  6. Reduce in formalin.
  7. Tone in gold chloride.
  8. Rinse and proceed with fixation and mounting.

Results:

  • Reticulin fibers stain black.
  • Collagen fibers stain purple.
  • Nuclei and cytoplasm stain shades of grey.

5. Masson’s Trichrome Stain:Massons Trichrome Stain

Purpose:

To differentiate connective tissue elements.

Fixative:

 Tissues fixed in Bouin’s or Zenker’s fluid.

Requirements:

  1. Weigert’s iron hematoxylin
  2. Biebrich scarlet acid fuchsin solution
  3. Phosphomolybdic-phosphotungstic acid solution
  4. Aniline blue solution

Procedure:

  1. Stain with Weigert’s iron hematoxylin.
  2. Stain in Biebrich scarlet-acid fuchsin solution.
  3. Place in phosphomolybdic-phosphotungstic acid solution.
  4. Impregnate in aniline blue solution.
  5. Differentiate, dehydrate, and mount.

Results:

  • Nuclei stain blue-black.
  • Cytoplasm, muscle, and keratin granules stain red.
  • Collagen, cartilage, mucin, and basophil granules stain blue.

6. Van Gieson’s Stain:Van Gieson's Stain

Purpose:

To differentiate between muscle and collagen fibers.

Principle:

Collagen fibers stain red with picrofuchsin solution.

Requirements:

  1. Van Gieson’s Solution
  2. Weigert’s Iron hematoxylin

Procedure:

  1. Stain nuclei with Weigert’s hematoxylin.
  2. Counterstain in Van Gieson’s solution.
  3. Dehydrate, clear, and mount.

Results:

  • Collagen stains are bright red.
  • Muscles and cornified epithelium stain yellow.
  • Nuclei stain blue-black.

7. Verhoeff’s Elastic Stain:Verhoeff's Elastic Stain

Purpose:

To stain elastic fibers.

Principle:

Elastic fibers stain with Verhoeff’s solution in the presence of ferric salts.

Requirements:

  1. Verhoeff’s Solution
  2. Ferric chloride 2%
  3. Aqueous sodium thiosulphate 5%
  4. Van Gieson’s counterstain

Procedure:

  1. Stain with Verhoeff’s solution.
  2. Differentiate, counterstain, and mount.

Results:

  • Elastic tissue stains black.
  • Nuclei stain grey-black.
  • Collagen stains red.
  • Other structures are stained yellow.

8. Bennhold’s Congo Red Stain:Bennhold’s Congo Red Stain

Purpose:

To demonstrate amyloid.

Principle:

Amyloid stains salmon red with Congo red solution in the presence of a differentiating agent.

Requirements:

  1. Congo red solution
  2. Differentiating agent
  3. Harris hematoxylin

Procedure:

  1. Stain with Congo red solution.
  2. Dip in saturated lithium sulphate solution.
  3. Counterstain with Harris hematoxylin.
  4. Differentiate, dehydrate, and mount.

Results (Bright Field Microscope):

  • Amyloid appears salmon red.
  • Nuclei appear blue.

Results (Polarized Light Microscope):

  • Amyloid appears apple green.
  • Collagen appears yellow.
  • Nuclei appear blue.

9. Von Kossa’s Calcium Stain, Modified:Von Kossa’s Calcium Stain, Modified

Purpose:

To demonstrate Ca3(PO4)2 and Ca(CO3)2 in tissue.

Requirements:

  1. 5% aqueous silver nitrate
  2. 5% aqueous sodium thiosulphate
  3. Nuclear fast red stain

Procedure:

  1. Stain with silver nitrate.
  2. Rinse, counterstain, and mount.

Results:

  • Calcium appears black.
  • Nuclei appear blue.
  • The cytoplasm is stained pink.

10. Papanicolaou Staining:Papanicolaou Staining

Purpose:

To stain cells in cervicovaginal and sputum smears for cytology.

Principle:

Nuclei are stained blue, and cytoplasm is stained green or orange, depending on cell maturity.

Requirements:

  1. Harris’s hematoxylin
  2. Orange G (OG-6)
  3. EA 50
  4. Alcohol solutions

Procedure:

  1. Stain with Harris hematoxylin.
  2. Differentiate, blue, and counterstain with Orange G or EA 50.
  3. Dehydrate, clear, and mount.

Results:

  • Nuclei stain blue.
  • Cytoplasm stains pink, blue, yellow, or green, indicating cell maturity.
  • Trichomonas, if present, stain pale greenish-blue.

11. Modified Ziehl-Neelsen Stain:Modified Ziehl-Neelsen Stain

Purpose:

To demonstrate Mycobacterium tuberculosis.

Principle:

  1. tuberculosis retains carbolfuchsin in the presence of hydrochloric acid.

Requirements:

  1. Carbol fuchsin stain
  2. Differentiating solution (1% HCl)
  3. Methylene blue counterstain

Procedure:

  1. Stain with carbol fuchsin.
  2. Rinse, differentiate, counterstain, and mount.

Result:

Acid-fast bacilli (AFB) stain red against a blue background.

12. May-Grünwald-Giemsa Stain:May-Grünwald-Giemsa Stain

Purpose:

To demonstrate Giardia lamblia, Toxoplasma gondii, and hematopoietic tissue.

Requirements:

  1. Jenner’s solution
  2. Giemsa stain
  3. Rosin differentiating solution
  4. Buffered distilled water

Procedure:

  1. Stain with Jenner’s solution.
  2. Rinse and stain with Giemsa.
  3. Differentiate, dehydrate, and mount.

Results:

  • Nuclei stain blue.
  • Cytoplasm stains pink to rose.
  • Bacteria stain pale blue.

13. Perl’s Staining Reaction:Perl’s Staining Reaction

Purpose:

To demonstrate ferric iron in haemosiderin and asbestos bodies.

Principle:

Iron is stained by potassium ferrocyanide in the presence of HCl.

Requirements:

  1. Perl’s solution
  2. Counterstain

Procedure:

  1. Stain with Perl’s solution.
  2. Counterstain, rinse, and mount.

Result:

  • Haemosiderin appears blue.
  • Nuclei and other tissue elements appear red.

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