2027 L3.5 Common Special Staining Techniques

Gram Stain

• Purpose: Identifies bacteria causing infections (lung abscesses, wound infections, etc.).

• Mechanism: Categorises bacteria as gram-positive or gram-negative based on cell wall composition.

  • Process:

    • Staining with crystal violet (purple dye).

    • Application of iodine.

    • Rinse with acetone:

      • Gram-positive: Retain purple color due to thick cell walls (appear blue under microscope).

      • Gram-negative: Lose purple color; stained with a red counterstain

Ziehl-Neelsen (Acid-Fast) Stain

• Purpose: Stains lipid-rich bacteria like Mycobacterium tuberculosis (TB).

• Mechanism:

  • Uses carbol fuchsin (lipid-soluble primary stain).

  • Staining enhanced by heating, melting cell wall lipids for deeper penetration.

  • Staining process:

    • Decolorization with acidic alcohol (non-acid-fast bacteria lose color).

    • TB retains red/pink color; counterstained with methylene blue.

• Definition: "Acid-fast" refers to ability to resist acidic alcohol staining due to lipid-laden walls.

Wade-Fite (Acid-Fast) Stain

• Purpose: Stains bacteria causing leprosy (Mycobacterium leprae), which is less acid and alcohol fast and than TB. When stained with Ziehl-Neelson M. leprae is more prone to decolorisation, appears less distinctive than TB

• Method:

  • Modification of Ziehl-Neelsen, omitting alcohol.

  • Uses xylene/clove oil mixture instead of alcohol.

  • Non acid-fast bacilli decolorized with sulphuric acid.

  • Post-staining slides dried in an oven instead of immersing in alcohol.

Giemsa Stain

• Purpose: Identify Helicobacter pylori (H. pylori), linked to gastric ulcers; also used in hematological studies.

• Mechanism: A combination of methylene blue and eosin (Romanowsky dye), both polychromatic.

  • Produces a variety of staining colors.

  • H. pylori appears dark blue against a pale background.

  • For tissues and blood smears.

• Alternative Methods: Warthin-Starry method available for H. pylori.

Periodic Acid Schiff (PAS) Stain

• Purpose: Detect structures rich in neutral hexose sugars (glycogen, mucin, fungi, basement membrane).

• Mechanism: Treat tissues with periodic acid to oxidize glycols to aldehydes;

  • Schiff reagent reacts to produce magenta color.

• Applications: Diagnose Glycogen Storage Disease, adenocarcinomas, Paget Disease, α1-Antitrypsin Deficiency; fungal detection often uses Grocott's Methenamine Silver Stain (GMS).

Orcein Staining Method

• Purpose: Visualize Hepatitis B virus surface antigens (HBsAg) as markers of active infection. Viruses usually only visual under electron microscope but can form viral inclusion bodies, which are visible under light microscope.

• Mechanism:

  • Samples treated with potassium permanganate to oxidize sulfur-containing proteins;

  • Proteins react with Orcein for visualization.

• Clinical Significance: Presence of HBsAg in hepatocytes indicates chronic Hepatitis B infection.

Grocott’s Methenamine Silver (GMS) Stain

• Purpose: Visualize fungi by staining their argentaffin cell wall carbohydrates.

• Mechanism: Similar to PAS; oxidizes carbohydrates to aldehydes, detected using silver complex.

• Visualization: Turns fungal structures black, with light green counterstain for contrast.

• Advantages: Clear and precise identification of individual fungal hyphae, aiding diagnostics.

Warthin-Starry Stain

• Purpose: Detect slender, Gram-negative spirochaetes (Treponema pallidum, (Syphilis) and Borrelia burgdorferi (Lyme’s disease), Leptospira, H. pylori and two causative agents of cat scratch disease).

• Mechanism: Uses silver ions at acidic pH (3.6), enabling selective nucleation in bacteria, but not in most mammalian tissue. Initially invisible, enlarged by a physical developer

• Color Outcome: Bacteria appear dark brown to black on light golden background.

Mucins (Glycoconjugates) and Glycogen

• Functions: Demonstrate carbohydrates in tissues.

• Locations: Glycogen abundant in liver, cardiac muscleskeletal muscle, hair follicles, endometrial glands, and vaginal and cervical epithelium. Glycoconjugates include proteoglycans, vital for the extracellular matrix and cartilage formation when combined with collagen, and glycoproteins, found on the plasma membrane's exterior, playing roles in immunity, mucus production, cell communication, and reproduction

• Mucins Classification:

  • Acid Mucins: Negative charge, carboxylate (COO-) or -sulfonate (SO3) groups. Found in GI and respiratory tracts.

  • Neutral Mucins: No charge, observed in stomach epithelia, Brunner’s glands and prostatic epithelium.

PAS Staining Technique and Mucins

• Purpose: Illustrate carbohydrates and mucins; sensitive to neutral mucins bc it’s sensitive to structure of monosaccharide units

• Clinical Usage: Identify glycogen deposits in liver, important for glycogen storage diseases. Also for tumours in organs like bladder, kidney and pancreas. Can detect Schiff reactive glycoproteins in basement membranes, good for renal diseases as it shows thickness of glomerular basement membrane.

Diastase PAS Stain

• Purpose: Variant of PAS with amylase to digest glycogen, makes it water-soluble maltose and dextrose which can be washed away.

• Outcome: Enhances specificity for glycogen deposits in liver and neutral mucins for adenocarcinomas.

• Stains for certain epithelial mucins, basement membranes, and fungal walls.

Alcian Blue

• Usage: Differentiates between subtypes of acid mucins.

• Mechanism: Doesn’t stain neutral mucins. Has a pos charge, attracts to anionic sites in acidic mucins molecules. All acidic mucins ionise at pH 2.5 to create anionic groups, but Alcian blue only stains those with more acidic sulfonate groups. In contrast, mucins with primarily carboxylate carbohydrates stain at pH 2.5 but not at pH 1, as the carboxyl groups don't ionize at that lower pH.

Alcian Blue PAS

• Usage: Combines Alcian blue (acidic mucins) with PAS (neutral mucins). For tissues from GI, esp in diagnosing Barrett’s oesphagus.

• Outcome: Tissues show dark blue/purple for both mucin types.

Connective Tissue Overview

• Role: Maintains shape and support of the body and organs.

• Components: Extracellular fibers (collagen, elastic), ground substance (clear, either viscous liquid or thin gel. made of large carbohydrates or protein carb complexes called proteoglycans), various cells ( stationary e.g. fibroblasts, adipose, motile e.g. mast cells, monocytes, lymphocytes, plasma cells, eosinophils, macrophages, etc.).

Jones Methanamine Silver (JMS) Stain

• Purpose: Visualize basement membranes in kidneys to assess function.

• Mechanism: Periodic acid reacts with carbohydrates to form aldehydes, selectively reduces MS to its metallic form aka argentaffin reaction.

Gordon Sweet Reticulin Stain

• Usage: Visualizes reticulin fibers; abnormalities can indicate conditions like cirrhosis and lymphoma.

• Mechanism: Argyrophillic reaction, tissue has affinity for silver. Impregnates fibres with silver ions which are reduced to visible metallic form under microscope.

Masson Trichrome Stain

• Purpose: For liver and kidney diseases esp. cirrhosis, determines fibrosis extent.

• Usage: Three dyes colour connective tissue based on molecular size and density; used for liver and kidney disease assessment.

• Mechanism: Tissues w fewer pores absorb smaller dye molecules, but larger dyes can replace them if they penetrate.

Van Gieson Stain

• Purpose: Identifies elastic fibers; helpful in diagnosing diseases affecting lung and vascular health e.g. emphysema and arteriosclerosis.

• Mechanism: Has two dyes, acid fuschin to stain collagen red and picric acid staning muscle yellow. Clear tissue differentiation.

Toluidine Blue Stain

• Mechanism: Stains acidic components through metachromasia.

• Usage: Important for detecting conditions related to mast cells, such as cancers and allergic inflammatory diseases, esp. oral cancer as it binds to DNA of dividing ce;;s making precancerous cells detectable by increased dye intake.

• Outcome: Nuclei stains dark blue, mast cells, granules and polysaccharides violet.

Lipids in Biology

• Types:

  • True Fats: Esters of fatty acids and glycerol e.g. Triacylglycerol which contains 3 fatty acids bonded to glycerol.

  • Lipids: Includes phospholipids, for cell membrane formation, cerebrosides and waxes.

  • Sterols: Like cholesterol, with C atoms in four rings, important for physiology

  • Hydrocarbons: Squalene and carotene.

  • Important as fat can appear in abnormal locations e.g. in bone fracture and injury to fatty regions, fat released into bloodstream can cause fat emboli. If found in liver and kidney, often linked to lipid metabolism disorders.

Oil Red O Stain

• Purpose: Detect fats in fresh/frozen tissue sections.

• Mechanism: Xylene and alcohol dissolve lipids, leaving unstained gaps in tissue when H&E is used. Dye is fat-soluble diaxo dye, is oil-soluble and colouring - doesnt bond with lipids and interacts with them.

Amyloid and Amyloidosis

• Definition: Abnormal protein deposition causing organ dysfunction. They aggregate and form insoluble fibrils, exhibiting beta sheet structure. Can replace native tissue.

• Types of Amyloidosis:

  • Primary: most common, affects heart and kidneys, etc.. Caused by bone marrow producing abnormal antibodies, forming amyloid deposits.

  • Secondary: mostly affects kidneys, linked to chronic diseases e.g. rheumatoid arthritis.

  • Hereditary: affects multiple organs, risk and progression determined by gene abnormalities.

  • Dialysis-related: in long-term dialysis patients, leads to joint pain and carpal tunnel.

  • Diseases such as Alzheimer’s, Type2 Diabetes, Mad cow disease associated also.

Congo Red Stain

• Purpose: Detect amyloid deposits; Pale-orange-red under bright field microscopy. isn’t conclusive because it misses undetected tiny deposits. For definitive diagnosis, examine under polarised light to reveal apple green birefringence=

Pigments

• Types:

  • Artefact - caused by tissue preservation, oftem from fixatives like fromalin or mercury

  • Exogenous - originating outside the body e.g. carbon, asbestos and tattoo ink

  • Endogenous - inside the body

    • haematogenous: related to blood e.g. haemaglobin and bile

    • non-haematogenous: melanin and aging pigment lipfuscin

Perl’s Prussian Blue Stain

• Purpose: Visualize haemosiderin (iron storage complexes). An increase could indicate sickle cell anaemia and thalassemia bc of chronic blood loss and frequent transfusions. Left ventricular failure can cause iron disposition in lungs, forming passive congestion. In lungs, asbestos bodies with iron stain positive.

  • Mechanism: Ferric iron reacts with dilute HCl and potassium ferrocyanide to form ferric ferrocyanide e.g. Prussian Blue

  • Reaction produces blue compound for tissue diagnosis, as in sickle cell anemia.

Vonkossa Stain

• Mechanism: Argyrophil stain. Identifies calcium deposits via silver ion replacement, turning black metallic silver.

• Clinical Significance: Important for conditions like atherosclerosis, sarcoidosis, tuberculosis, and certqain tumours.

• Using H&E, calcium appear as deep blue-purple, helps differentiation.

Rhodamine Stain

• Purpose: Detects indirect copper presence linked to conditions like Wilson’s disease, primary biliary cirrhosis.

• Mechanism: reveals the protein that copper binds to, indirectly showing copper presence

Hall’s Method for Bile Pigments

• Mechanism: Bilirubin conversion to biliverdin when oxidised in acid, showing a green colour, shows bile pigment presence. Can be intensified to emerald green with acid fuchsin in van Gieson.

• Usage: shows congenital enzyme defects, liver cell damage

Long ZN Stain for Lipofuscin

• Definition: Lipofuscin is a yellow-brown pigment associated with aging and certain metabolic disorders. Associated with lysosomal digestion. Found in tissues live liver, heart, muscle and nerve cells.

• Accumulation > adult neuronal ceroid lipofuscinosis (ANCL) a neurometabolic disorder, causing brain deterioration