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==== 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 ====
{| class="wikitable"
{| class="wikitable"
|+
|+
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!Uses
!Uses
!Tissue Components Targeted
!Tissue Components Targeted
!Mechanism of Staining
!Mechanism of Staining (Primary Stain)
!Stains Used
!Stains Used


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# Secondary
# Secondary
!Other Reagents
!Other Reagents
!Controls
|-
|-
|[[Hematoxylin & Eosin Staining|Hematoxylin & Eosin (H&E)]]
|[[Hematoxylin & Eosin Staining|Hematoxylin & Eosin (H&E)]]
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* General tissue staining
* General tissue staining
|
|
* Nuclei - blue
* Nuclei: '''blue'''
* RBCs - red/dark pink
* RBCs: '''red/dark pink'''
* Muscle - pink
* Muscle: '''pink'''
* Collagen - light pink
* Collagen: '''light pink'''
|
|
|
|
# Harris Hematoxylin (nuclear)
# Harris Hematoxylin (nuclear)
# Eosin Y (cytoplasm)
# Eosin Y (cytoplasm)
|Harris Hematoxylin
|
Acid Alcohol
* Acid Alcohol
Scott's tap water substitute
* Scott's tap water substitute
Eosin Y
|
|-
|-
|Periodic Acid-Schiff (PAS)
|Periodic Acid-Schiff (PAS)
|
|
* Identifying glycogen, starches, and fungi (chitin)
* Identifying neutral polysaccharides (Group I carbohydrates)
** glycogen
** starches
** cellulose
** fungi (chitin)
|
|
|
|
|
|-
| rowspan="4" |Alcian Blue (AB)
| rowspan="4" |
* Identifying acid mucopolysaccharides (Group II carbohydrates)
* Identifying glycoproteins (Group III carbohydrates)
| rowspan="4" |
* pH 1.0: Sulfated mucopolysaccharides: '''pale blue'''
* Acid mucins (mucopolysaccharides)
* Sialomucins (glycoproteins)
* Background: '''pink/red'''
|Ionic bonding
|
|
|
|-
|
|
|
|
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|
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|-
|-
|Alcian Blue (AB)
|
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|-
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|-
|-
|Alcian Blue + Periodic Acid-Schiff (AB PAS)
|Alcian Blue + Periodic Acid-Schiff (AB PAS)
|Identifying  multiple different types of carbohydrate groups
|
|
|
|
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|
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|Hydrogen bonding
|Hydrogen bonding
|
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|Connective Tissue Stain
|Connective Tissue Stain


* Nuclei - blue-black
* Nuclei: '''blue-black'''
* RBCs - dark red
* RBCs: '''dark red'''
* Muscle - red
* Muscle: '''red'''
* Cytoplasm - red
* Cytoplasm: '''red'''
* Collagen - varies by stain (often green or blue)
* Collagen: '''varies by stain (often green or blue)'''
|Porosity and ionic bonding
|Porosity and ionic bonding
|No primary stain.
|No primary stain.
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# Ponceau (dense connective)
# Ponceau (dense connective)
# Light green or aniline blue (loose connective)
# Light green or aniline blue (loose connective)
|
|
|
|-
|-
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* Differential diagnosis of certain tumour types
* Differential diagnosis of certain tumour types
|Connective Tissue Stain
|Connective Tissue Stain
* Reticulin fibres: '''black'''
* Nuclei and cytoplasm: '''varies by counterstain'''
|Metallic impregnation
|Metallic impregnation


* Argyophilic metallic impregnation & substitution
* Argyophilic metallic impregnation & substitution
Counterstain
* Ionic bonding
|
# a
# 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
|
# Verhoeff's Iron Hematoxylin (elastic fibres)
# Van Giesen
## Picric acid (RBCs and cytoplasm)
## Acid fuchsin (collagen and muscle)
|
|
|
|
|-
|-
|Verhoeff's Van Giesen
|[[Oil Red O (Stain)|Oil Red O]]
|Connective Tissue Stain
* Identifying triglycerides, lipids, and lipoproteins
|
* Fats: <span style="color:Red;">'''red'''</span>
|Selective solubility
|
|
|
|-
|[[Gram Staining (Histology)|Gram]]
|Differential Microorganism Stain
* Gram +/- bacteria
|Microorganism Stain
 
* Gram positive: <span style="color:Purple;">'''purple'''</span>
* Gram negative: <span style="color:Magenta;">'''pink'''</span>
|Ionic bonding
|
# Crystal violet
# Safranin
|
* Lugol's iodine (mordant)
* Acetone alcohol (decolourizer)
|'''Positive:''' appendix, any tissues containing bacteria
|-
|[[Ziehl-Neelsen Staining (Histology)|Ziehl-Neelsen]]
|Differential Microorganism Stain
* Acid fast bacteria
|Microorganism Stain
 
* Acid fast: <span style="color:Magenta;">'''pink'''</span>
* Non-acid fast & background: <span style="color:DodgerBlue;">'''blue/pale blue'''</span>
|Ionic bonding
|
# Carbol fuchsin (basic fuchsin + alcohol + 5% phenol)
# Methylene blue
|
* Acid alcohol (decolourizer)
|'''Positive:''' any tissue with acid fast bacteria (e.g., ''M. tuberculosis'')
|-
|[[Grocott Methenamine Silver Staining|Grocott Methenamine Silver]]
|Microorganism Stain
 
* Fungi
* Protozoa, encapsulated bacteria, fungal-like bacteria
|Microorganism Stain
 
* Fungi: '''black''' <span style="color:gray;">''' (with paler internal structures)'''</span>
* Background: <span style="color:LimeGreen;">'''light green'''</span>
|Argentaffin metallic impregnation
 
* Induced - over-oxidizes everything except fungal walls
|
# Methenamine silver nitrate
# 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 Staining|Perl's Prussian Blue]]
|Iron Pigment Stain
 
* Ferric iron (hemosiderin and ferritin)
* Asbestos bodies
|
* Iron: <span style="color:DodgerBlue;">'''blue'''</span>
* Nucleus: <span style="color:Red;">'''red'''</span>
* Cytoplasm: <span style="color:Magenta;">'''pink'''</span>
|Histochemical
|
# Perl's solution (HCl + potassium ferrocyanide)
# Nuclear fast red or neutral red or safranin
|
|'''Positive:''' spleen, bone marrow, liver
|-
|[[Masson Fontana Staining|Masson Fontana]]
|Pigment Stain
 
* Argentaffin substances
* Melanin
* Argentaffin granules
* Neurosecretory granules
|
* Melanin: '''black'''
* Nuclei: <span style="color:MediumVioletRed;">'''pink/red'''</span>
* Other tissue: <span style="color:Plum;">'''pale pink/brown'''</span> <span style="color:Silver;">'''/colourless'''</span>
|Argentaffin metallic impregnation
|
# Fontana's ammoniacal silver
# 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 Staining|Von Kossa]]
|Pigment Stain for Calcium
 
* Stains ions bound to calcium (phosphates, carbonates, etc.)
|
* Mineralized bone and urates: <span style="color:Maroon;">'''dark brown'''</span> '''/black'''
* Non-mineralized tissue: varies by counterstain
** <span style="color:Red;">'''Red (NFR)'''</span>
|Metallic substitution - silver impregnation
|
# von Kossa (silver nitrate)
# 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 Staining|Jones Methenamine Silver]]
|Connective Tissue Stain
 
* Glomerular basement membrane
|
* Glomerular basement membrane: '''black'''
* Other tissue: varies
** <span style="color:LimeGreen;">'''Green (LG)'''</span>
|Argentaffin metallic impregnation
|
# Methanamine silver nitrate
# Light green or nuclear fast red
|
* Periodic acid (oxidizer)
* Gold chloride (toner)
* Sodium thiosulphate (fixation)
|'''Positive:''' normal kidney
|-
|[[Toluidine Blue Staining|Toluidine Blue]]
|Connective Tissue Stain
 
* Mast cells
|
* Metachromatic granules: <span style="color:Purple;">'''red/pink/purple'''</span>
* Nuclei and other components: varies
** <span style="color:DodgerBlue;">'''Blue (TB)'''</span>
** <span style="color:Silver;">'''Colourless (1% acetic acid)'''</span>
|Metachromasia and hydrogen bonding
|
# 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.
{| class="wikitable"
|+
!
! colspan="3" |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 '''bi'''sulphate
|
|Sodium '''thio'''sulphate
|
|
|-
|'''Sensitization'''
* Helps silver attach to target
| colspan="3" |
|
|
|-
|'''Impregnation'''
* Impregnation reagent deposits silver
|Methenamine silver
|Methenamine silver
|Fontana ammoniacal silver
|
|Silver nitrate
|-
|'''Reduction'''
* Reduces reagent to metallic silver precipitate
| colspan="3" |
|
|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 ====
{| class="wikitable"
|+
!Technique
!
!
!
|-
|
|
|
|
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|
|
|-
|-
|Oil Red O
|
|
* Identifying triglycerides, lipids, and lipoproteins
|
|
|
|-
|
|
|
|
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|
|
|}
|}
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:

Latest revision as of 19:14, 23 April 2024

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:
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