List of Stains

From MedLabWiki

Types of Stains

Mechanisms of Staining

  • Ionic bonding
  • Hydrogen bonding
  • Histochemical
    • Use a non-coloured substance to produce a coloured, non-diffusable end-product in the tissue
  • Silver Staining
    • Argentaffin metallic impregnation
    • Argyophillic metallic impregnation

Summary of Conventional Staining Techniques

Stain Uses Tissue Components Targeted Mechanism of Staining (Primary Stain) Stains Used
  1. Primary
  2. Secondary
Other Reagents Controls
Hematoxylin & Eosin (H&E)
  • General tissue staining
  • Nuclei: blue
  • RBCs: red/dark pink
  • Muscle: pink
  • Collagen: light pink
  1. Harris Hematoxylin (nuclear)
  2. Eosin Y (cytoplasm)
  • Acid Alcohol
  • Scott's tap water substitute
Periodic Acid-Schiff (PAS)
  • Identifying neutral polysaccharides (Group I carbohydrates)
    • glycogen
    • starches
    • cellulose
    • fungi (chitin)
Alcian Blue (AB)
  • Identifying acid mucopolysaccharides (Group II carbohydrates)
  • Identifying glycoproteins (Group III carbohydrates)
  • pH 1.0: Sulfated mucopolysaccharides: pale blue
  • Acid mucins (mucopolysaccharides)
  • Sialomucins (glycoproteins)
  • Background: pink/red
Ionic bonding
Alcian Blue + Periodic Acid-Schiff (AB PAS) Identifying multiple different types of carbohydrate groups
Congo Red
  • Identifying amyloid deposits
Hydrogen bonding
Masson Trichrome
  • Identifying fibrotic tissue
Connective Tissue Stain
  • Nuclei: blue-black
  • RBCs: dark red
  • Muscle: red
  • Cytoplasm: red
  • Collagen: varies by stain (often green or blue)
Porosity and ionic bonding No primary stain.
  1. Iron hematoxylin (nuclear)
  2. Ponceau (dense connective)
  3. Light green or aniline blue (loose connective)
Gordon and Sweet's Reticulin
  • Identifies reticulin fibres (type III & IV collagen)
  • Differential diagnosis of certain tumour types
Connective Tissue Stain
  • Reticulin fibres: black
  • Nuclei and cytoplasm: varies by counterstain
Metallic impregnation
  • Argyophilic metallic impregnation & substitution

Counterstain

  • Ionic bonding
  1. a
  2. Varies (light green, nuclear fast red, neutral red, etc.)
Verhoeff's Van Giesen Connective Tissue Stain
  • Identifies elastin fibres
Connective Tissue Stain
  • Elastic fibres: blue-black/black
  • Nuclei: dark blue-black
  • Collagen: red
  • Muscle: orange
  • RBCs: yellow
  • Cytoplasm: yellow
Van der Waal's
  • Primary stain

Porosity and ionic bonding

  • Counterstain
  1. Verhoeff's Iron Hematoxylin (elastic fibres)
  2. Van Giesen
    1. Picric acid (RBCs and cytoplasm)
    2. Acid fuchsin (collagen and muscle)
Oil Red O Connective Tissue Stain
  • Identifying triglycerides, lipids, and lipoproteins
  • Fats: red
Selective solubility
Gram Differential Microorganism Stain
  • Gram +/- bacteria
Microorganism Stain
  • Gram positive: purple
  • Gram negative: pink
Ionic bonding
  1. Crystal violet
  2. Safranin
  • Lugol's iodine (mordant)
  • Acetone alcohol (decolourizer)
Positive: appendix, any tissues containing bacteria
Ziehl-Neelsen Differential Microorganism Stain
  • Acid fast bacteria
Microorganism Stain
  • Acid fast: pink
  • Non-acid fast & background: blue/pale blue
Ionic bonding
  1. Carbol fuchsin (basic fuchsin + alcohol + 5% phenol)
  2. Methylene blue
  • Acid alcohol (decolourizer)
Positive: any tissue with acid fast bacteria (e.g., M. tuberculosis)
Grocott Methenamine Silver Microorganism Stain
  • Fungi
  • Protozoa, encapsulated bacteria, fungal-like bacteria
Microorganism Stain
  • Fungi: black (with paler internal structures)
  • Background: light green
Argentaffin metallic impregnation
  • Induced - over-oxidizes everything except fungal walls
  1. Methenamine silver nitrate
  2. Light green or nuclear fast red
  • Chromic acid (oxidizer)
  • Sodium bisulphite (bleach)
  • Gold chloride (toner)
  • Sodium thiosulphate (fixative)
Positive: any tissue with fungi
Perl's Prussian Blue Iron Pigment Stain
  • Ferric iron (hemosiderin and ferritin)
  • Asbestos bodies
  • Iron: blue
  • Nucleus: red
  • Cytoplasm: pink
Histochemical
  1. Perl's solution (HCl + potassium ferrocyanide)
  2. Nuclear fast red or neutral red or safranin
Positive: spleen, bone marrow, liver
Masson Fontana Pigment Stain
  • Argentaffin substances
  • Melanin
  • Argentaffin granules
  • Neurosecretory granules
  • Melanin: black
  • Nuclei: pink/red
  • Other tissue: pale pink/brown /colourless
Argentaffin metallic impregnation
  1. Fontana's ammoniacal silver
  2. Nuclear fast red or light green
  • Gram's iodine (oxidizer)
  • Sodium thiosulphate (bleach)
  • Gold chloride (toner)
  • Sodium thiosulphate (fixation)
Positive: skin (melanin), small intestine (argentaffin granules)

Need 4 slides to confirm Melanin!

  • Pos QC
  • Neg QC
  • Unbleached patient
  • Bleached patient (melanin removed)
Von Kossa Pigment Stain for Calcium
  • Stains ions bound to calcium (phosphates, carbonates, etc.)
  • Mineralized bone and urates: dark brown /black
  • Non-mineralized tissue: varies by counterstain
    • Red (NFR)
Metallic substitution - silver impregnation
  1. von Kossa (silver nitrate)
  2. Nuclear fast red
  • Light or hydroquinone (reducer)
  • Sodium thiosulphate (fixation)
Positive: any calcified tissue (e.g., bone)

Can be used as negative control for:

  • Osteoid
  • Urates (use LiCO3)
  • Calcium (use HCl)

Need 4 slides for Ca neg control

  • VK
  • VK+HCl (neg)
  • Pos QC
  • Neg QC
Jones Methenamine Silver Connective Tissue Stain
  • Glomerular basement membrane
  • Glomerular basement membrane: black
  • Other tissue: varies
    • Green (LG)
Argentaffin metallic impregnation
  1. Methanamine silver nitrate
  2. Light green or nuclear fast red
  • Periodic acid (oxidizer)
  • Gold chloride (toner)
  • Sodium thiosulphate (fixation)
Positive: normal kidney
Toluidine Blue Connective Tissue Stain
  • Mast cells
  • Metachromatic granules: red/pink/purple
  • Nuclei and other components: varies
    • Blue (TB)
    • Colourless (1% acetic acid)
Metachromasia and hydrogen bonding
  1. Toluidine blue (0.1%)
  • Acetic acid dips
  • NO alcohols
Positive: tissue with mast cells (skin, small intestines)

Summary of Silver Staining Methods

Silver staining can be done via two methods: argentaffin or argyophillic staining.

  • Argentaffin staining has fewer steps as the tissue components can auto-reduce the silver and do not require sensitization.
  • Argyophillic methods require a sensitizing and reducing agent.
Argentaffin Methods* Argyophillic Methods Metallic Substitution
Stain Grocott's Methenamine Silver (GMS) Jones Methenamine Silver (JMS) Masson Fontana (MF) Gordon & Sweet's Reticulin (GSR) Von Kossa (VK)
Oxidation
  • Exposes reactive sites so they can bind with silver
  • Suppresses other tissue components
  • Affects specificity
Chromic acid
  • Aldehyde groups
Periodic acid
Gram's iodine
  • Phenolic ring
Bleaching
  • Removes discolouration from oxidizing agent
Sodium bisulphate Sodium thiosulphate
Sensitization
  • Helps silver attach to target
Impregnation
  • Impregnation reagent deposits silver
Methenamine silver Methenamine silver Fontana ammoniacal silver Silver nitrate
Reduction
  • Reduces reagent to metallic silver precipitate
Light or hydroquinone
Toning
  • Gold exchanged with silver to remove browning in non-target tissue
  • Improves contrast
  • Ion exchange/metallic substitution
Gold chloride Gold chloride Gold chloride
Fixation
  • Removes unreduced silver
Sodium thiosulphate (hypo) Sodium thiosulphate (hypo) Sodium thiosulphate (hypo) Sodium thiosulphate (hypo)
Counterstain
  • Provides contrast to background tissue
LG or NFR LG or NFR LG or NFR LG, NFR, or van Gieson
  • The bleaching step is only required when the reagents themselves are coloured or cause discolouration.
  • Sensitization step is not required in argentaffin methods as silver can attach directly to the target tissue.
  • Argentaffin methods involve auto-reducing tissue. The tissue reduces the silver itself, so no reduction agents are needed.

Summary of Immunohistochemistry Techniques

Technique

Gram Stain

  • Staining technique: Differential staining (ionic bonding to cell wall)
  • Primary Stain: Crystal violet
    • Targets: Gram positive bacteria (purple/blue)
  • Counterstain: Safranin
    • Targets: Gram negative bacteria (pink/red)

Ziehl-Neelsen

  • Staining technique: Differential staining/acid fastness (ionic bonding)
  • Primary Stain:
    • Targets: Acid-fast bacteria (e.g., Mycobacteria)
  • Counterstain:
    • Targets:

Grocott Methenamine Silver

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets:

Perl's Prussian Blue

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets:

Masson Fontana

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets:

Von Kossa

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets:

Jones Methenamine Silver

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets:

Toluidine Blue

  • Staining technique:
  • Primary Stain:
    • Targets:
  • Counterstain:
    • Targets: