Special stains

1. AruaInstituteofHealthScience
By
WilliamEdema
1

2.  H&E stain is routine stain.
- It is the preliminary or the first stain applied to the
tissue sections
- Gives diagnostic information in most cases.
A special stain is a staining technique to
highlight various individual tissue
component once we have preliminary
information from the H&E stain
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3. CLASSIFICATION
1. Stains for carbohydrates
2. Stains for amyloid
3. Nucleic acid stains
4. Lipid stains
5. Stains for microorganisms
6. Connective tissue stains
7. Stains for pigments and minerals
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4. CARBOHYDRATES
SIMPLE CARBOHYDRATES
(molecules composed purely of
carbohydrates)
•Monosaccharides
(glucose,mannose,galactose)
•Oligosaccharides
(sucrose,maltose)
• Polysaccharides
(glycogen,starch)
GLYCOCONJUGATES
(molecules composed of
carbohydrates and other
molecules such as protein
and lipid)
• Proteoglycans
• Mucins
•Others glycoproteins
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5. 90-95% of their molecular weight is due to the
carbohydrate component
 The carbohydrate component is known as
glycosaminoglycans(GAG)
A GAG is composed of repeating disaccharide units , each
made up of 2 different monosaccharides
Each disaccharide is composed of a carboxylated uronic
acid (glucuronic or iduronic acid) and a hexosamine such
as N-acetylglucosamine or N-acetylgalactosamine
TYPES OF GYCOSAMINOGLYCANS
o Chondroitinsulfate
o Dermatan sulfate
o Keratan sulfate
o Heparin sulfate
o Heparin Hyaluronic acid
PROTEOGLYCANS
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6. MUCINS
1. Neutral mucins : surface epithelia of gastric
mucosa
brunner’s glands
prostatic epithelia
2. Sialomucins : bronchial submucous
glands goblet cells
salivary glands
3. Sulfomucins : bronchial mucous glands
Sialomucins and sulfomucins are acidic mucins
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7. 1. Periodic acid schiff (PAS) technique
2. Alcian blue method
3. Combined alcian blue- PAS techhnique
4. Mucicarmine technique
5. Colloidal iron technique
6. Metachromatic staining
7. Iodine staining for glycogen
8. Enzymatic digestion technique
Diastase digestion, Sialidase
digestion, Hyaluronidase
digestion
CARBOHYDRATE STAINING
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8. CANDIDA IN
PAS STAIN
ALPHA
ANTITRYPSIN
IN PAS STAIN
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9. PAS REACTIVE CELLS AND
TISSUE COMPONENTS
1.GLYCOGEN
2. STARCH
3. MUCIN
4. BASEMENT MEMBRANE
5. RETICULIN
6. FUNGI(CAPSULES)
7. PANCREATIC ZYMOGEN GRANULES
8. THYROID COLLOID
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10. Diagnosis of several medical conditions:
Glycogen storage disorder
Staining macrophages in Whipple's
disease
Mucins in adenocarcinoma of large
intestine
Demonstration of fungi
Seminoma,rhabdomyosarcoma,ewing’s
sarcoma contain glycogen
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11. REAGENTS :
1. Alcian blue
2. Aluminium sulfate
3.Nuclear fast red
RESULTS
 sulfomucin,sialomucin
 Proteoglycans
 Hyaluronic acid
 Nucleus  red
Blue
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12. GOBLET CELLS BY ALCIAN BLUE
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13. COMBINED ALCIAN BLUE- PAS TECHNIQUE
PRINCIPLE
 Demonstrate presence of mucin
 Differentiate acid mucin from neutral mucin
1st stain all acid mucin with alcian blue (blue)
Those acid mucin which are PAS +ve will not be stained
on PAS reaction only neutral mucin will be
stained(magenta)
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14. ALCIAN BLUE WITH VARYING
ELECTROLYTE CONCENTRATIONS
This technique is based upon phenomenon known as
CRITICAL ELECTROLYTE CONCENTRATION (CEC)
 CEC is defined as point at which amount of electrolyte
such as MgCl2 is sufficient to prevent staining from AB
Competition between cations of salt and dye occurs for
polyanionic sites within tissue
 Different acidic carbohydrates have different CEC value
 So can differentiate acidic mucins and proteoglycans
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15. MUCICARMINE
 Active dye molecule is aluminium – carminic acid
complex known as CARMINE
carminic acid produced from dried bodies of
female Coccus Cacti insects
Carmine complex has a positive charge and so
attracts polyanions such as sialomucins and
sulfomucins
Useful for identification of adenocarcinoma
( especially of GIT)
 Capsule of fungus – ID of C. neoformans
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16. REAGENTS :
1.Southgate’s mucicarmine solution
2.Alcoholic hematoxylin
3.Acidified ferric chloride solution
4.Weigert’s iron hematoxylin solution
5.Metanil yellow solution
RESULTS :
Acidic mucins – deep rose to red
Nuclei – black
Other tissue elements – light yellow
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17. COCCUS CACTI
CRYPTOCOCCUS
STAINED BY
MUCICARMINE
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18. GOBLET CELLS BY MUCICARMINE
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19. NUCLEIC ACIDS
2 nucleic acids are :
1. DNA ( In the nucleus)
2. RNA (In the cytoplasm)
 They consist of: Sugar (Deoxyribose / Ribose),
Phosphate and Nitrogenous base
Demonstration of Nucleic acids depends upon either:
1. Reaction of the dyes with the phosphate groups , or,
2. Production of aldehydes from the sugar (deoxyribose)
 No histochemical methods are available to demonstrate
the nitrogenous bases (C,G,T,U, & A).
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20. 1. Feulgen technique ( demonstrate sugar)
2. Methyl green pyronin technique
(demonstrate phosphate)
3. Acridine orange (by fluorescent method)
4. Gallocyanin-chrome alum method
does not separate the 2 nucleic acids (stains both
DNA and RNA blue)
suitable extraction technique must be used
DNA IS DEMONSTRATED BY
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21. DNA BY FEULGEN STAIN
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22. METHYL GREEN PYRONIN METHOD
Reagents :
1. Methyl green
 impure dye contains methyl violet
 removed by washing with chloroform
 pure methyl green specific for DNA
 NH2 of dye reacts with phosphate of
DNA
2. Pyronin
• binds to any negatively
charged tissue
constituent
• apart from RNA, binds to acid
mucins and cartilage
RESULTS – DNA : green-blue
RNA : red
NUCLEIC ACIDS
BY METHYL
GREEN PYRONIN
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23. LIPIDS
SIMPLE LIPIDS
- FATS
- OILS
- WAXES
COMPOUND
LIPIDS
- c/o fatty acids,
alcohol and one
more group such as
phosphorus or
nitrogen
DERIVED LIPIDS
-Derived from
simple or
compound lipids
by hydrolysis
- cholesterol
- Bile acids
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24. Lipids with melting point below staining temperature
can be stained with fat stains
So only lipids which are liquid at staining temp. are
stained. Those in solid or crystalline state remains
unaffected
Melting point of a lipid is inversely related to its fatty
acid chain length
Simple lipid is best demonstrated with fresh frozen
sections
Best fixative – Formal calcium (2% calcium acetate +
10% formalin)
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25.  1st Sudan dye was Sudan 3
 Most sensitive of all fat dyes is – Sudan black B
Sudans must be dissolved in organic solvents to
penetrate fats
 Some organic solvents used are –
1. 70% ethanol
2. Isopropanol
3. Propylene glycol
4. Triethyl phosphate
SUDAN BLACK B
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26. AMYLOID
Extracellular , amorphous , eosinophilic
material
Composed of protein
In H&E stain , can be confused with hyaline
and fibrinoid substances
Earliest special stain used for amyloid was
Iodine by Virchow
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27. CONGO RED STAIN
 Acidic dye and composed of 2 identical halves
Each half has a phenyl ring bound to a
naphthalene moiety by a diazo group
2 phenyl groups bound by a diphenyl bond -
gives a linear dye molecule
It stains amyloid by hydrogen bonding and other
tissue components by electrochemical bonds
Electrochemical staining of other tissues
can be suppressed by –
using alkaline-alcoholic solvents
using competitive inhibition by salt solution 27

28. AMYLOID BY CONGO RED
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29. ALKALINE CONGO-RED TECHNIQUE
 High concentration of NaCl is used
Background electrochemical staining
is reduced
hydrogen bonding of congo-red to amyloid
is enhanced
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30. METHYL /CRYSTAL VIOLET METHOD
 Methyl violet contains a mixture of tetra- , penta- ,
and
hexa- methyl pararosaniline
Amyloid stained due to selective affinity for one of
the colored fractions
 Hence, polychromasia seen
Ammonium oxalate accentuates polychromatic
effect
RESULT :
AMYLOID, MUCIN , HYALINE – red-
purple BACKGROUND - blue
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31. STAINS FOR
MICROORGANISMS
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32. Gram staining of Bacteria
( MODIFIED BROWN-BRENN METHOD)
Reagents :
(1) Crystal violet stain
(2) Gram’s iodine solution
(3) Ethyl alcohol – acetone solution(decolorizer)
(4) Acetone-xylene solution
(5) Basic Fuchsin
(6) Picric acid, 0.1% in acetone
RESULTS :
GRAM POSITIVE BACTERIA – blue
GRAM NEGATIVE BACTERIA – red
NUCLEI – red
OTHER TISSUE ELEMENTS - yellow 32

33. Acid Fast Staining for Bacteria
 Mycobacteria cannot be demonstrated by gram’s
stain
– possess a capsule containing long chain fatty acid
(mycolic acid) that makes them hydrophobic
 Can be stained by a strong stain like carbol
fuchsin
Fatty capsule resist the removal of stain by
acid- alcohol solution (acid and alcohol
fastness)
Mycobacteria are PAS positive due to
carbohydrate content of their cell wall
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34. Ziehl Neelson (ZN) stain
Reagents
(1) Carbol fuchsin solution
(2) 1% acid alcohol
(3) 0.1%Methylene blue solution
RESULTS
Acid fast bacilli
Other tissue
Caseous material
bright red
Pale blue
very pale grayish blue
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35. Warthin Starry Method for Spirochetes
REAGENTS :
1. Acetate buffer pH-3.6
2. 1% silver nitrate
RESULTS :
SPIROCHETES – black
BACKGROUND – golden -yellow
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36. SPIROCHETES BY WARTHIN STARRY
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37. FUNGAL STAINS
Fungal cell walls are rich in
polysaccharides which can be converted
by oxidation to dialdehydes
Dialdehydes are then detected by
silver solution
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38. Gomori methenamine silver
nitrate(GMS) technique
Reagents
(1) 4% chromic acid
(2) 1% sodium bisulfite
(3) 5% sodium Thiosulfate
(4) 0.21% Silver nitrate(stock)
(5) Gold chloride 0.1% aqueous solution
(6) Light green solution
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39. Results
Fungi , Pneumocystis, melanin - Black
Mucin & Glycogen - dark grey
Background - Pale green
Hyphae & yeast form - sharply delineated in black
against green background
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40. CRYPTOCOCCUS BY GMS STAIN
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41. MISCELLANEOUS STAINS
Cresyl violet acetate method for
helicobacter pylori
Macchiavello’s stain for rickettsia and
viral inclusions
Lendrum’s phloxine – tartrazine stain for
viral inclusions
 Giemsa stain for parasites
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42. CONNECTIVE TISSUES
Provide a matrix that connects and binds
the cells and organs and ultimately gives
support to the body.
 Parent cell is embryonic mesenchyme
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43. COLLAGEN FIBRES
1. Masson ‘s trichrome technique
2. Van Gieson’s stain
3. Mallory’s Phosphotungstic Acid
Hematoxylin
4. MSB Technique
5. PAS
6. Heidenhain’s Azan stain
7. lillie’s allochrome method
8. Luxol fast blue G
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44.  Demonstrate collagen and muscle in normal
tissue
 Differentiate collagen and Muscle in tumors
 Identify an increase in collagenous tissue
 Indicate fibrotic change in cirrhosis of liver
 Indicate fibrotic change in pyelonephritis
Distinguish tumors that have arisen from muscle
cells and fibroblasts
Masson ‘s trichrome technique
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45. REAGENTS
1. Weigert’s iron hematoxylin
2. Acid fuchsin
3. Glacial acetic acid
4. Phosphomolybdic acid
5. Methyl blue
RESULT
 Nuclei – Blue/ Black
 Cytoplasm, muscle , RBC → Red
 Collagen → Blue
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46. Trichrome stain showing slight
mesangial prominance
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47. Van Gieson Technique
REAGENT :
 Weigert’s iron hematoxylin
 Saturated Picric acid
solution
 Acid fuchsin
RESULTS :
 Collagen – bright red
 Nuclei – Blue/Black
Cytoplasm, muscle, RBC , elastin , reticulin -
yellow
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48. PIGMENTS AND MINERALS
ENDOGENOUS
PIGMENTS
1. hematogenous
2. Non
hematogenous
EXOGENOUS
PIGMENTS
1. asbestos
2. silica
3. lead
4. carbon
ARTIFACT
PIGMENTS
1. formalin
2. malaria
3. mercury
4. schistosome
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49. Hemosiderin
 Breakdown product of haemoglobin composed of
ferric iron and protein
Seen as yellow-brown granules
3 methods for demonstration:
1. Perl’s prussian blue reaction – for ferric ion
2. Lillie’s method – for ferrous iron
3.Hukill and putt’s method – for both ferric and
ferrous iron
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50. PERL’S STAIN
Principle : unmasking of ferric iron in hydroxide
form by dilute HCl
PRUSSIAN BLUE REACTION –
Ferric Hydroxide + potassium ferrocyanide = Ferric
ferrocyanide (insoluble blue compound)
Reagents
2% aq. Potassium ferrocyanide
2% HCl
Counterstain with 1% neutral red or saffranin
Results
Ferric iron –
Blue Nuclei –
Red
50

51. Modified Fouchet’s technique: bile
pigment
BILE PIGMENTS BY MODIFIED
FOUCHET’S TECHNIQUE
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52. BILE PIGMENTS BY MODIFIED
FOUCHET’S TECHNIQUE
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53. MELANIN
Normally occur as light brown to black
granules in substantia nigra,hair , skin and
eye
 Found pathologically throughout the body
:benign nevus,malignant melanoma
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54. MELANIN DEMONSTRATED BY :
1. Reducing methods : a) Masson fontana silver
technique
b) Schmorl’s ferric-ferricyanide
reduction test
2. Enzyme methods – DOPA reaction
3. Solubility and bleaching characteristics
4. Fluorescent method
5. Immunohistochemistry
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55. MASSON FONTANA STAIN
ARGENTAFFIN REACTION – reduction of ammoniacal silver
solution to form metallic silver without the use of extraneous
reducer.
Masson’s method( using fontana’s silver solution)
rely on melanin’s argentaffin property
Melanins are blackened by acid silver nitrate solution
RESULT :
Melanin – black
Nuclei - red
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56. Schmorl’s ferric-ferricyanide reduction test
Schmorl reaction – Melanin reduce ferricyanide
to ferrocyanide with production of prussian blue in
the presence of ferric salts
RESULT :
Melanin – dark
blue Nuclei - red
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57. Special stains enhance detection &
localization of individual tissue component
But should not be substituted for routine H&E
CONCLUSION
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58. Thank you
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