Special

1. SPECIAL STAINS IN
HISTOPATHOLOGY
Presenter- Dr. Ruchi Khadayate
Moderator- Dr. Shilpi Sahu
Guidance- Dr. Piyush Sahu

2. A special stain or histochemical stain is a
staining technique by the help of various
chemical reactions to highlight various
individual tissue component once we have
preliminary information from the H&E stain.

3. Indications for special stains
1. Inflammatory lesions
 Pyogenic reaction
Gram’s stain for bacteria
Granulomatous reaction
Ziehl Neelsen for Mycobacteria
PAS :Fungus

4. 2. Lymph node biopsies
• Reticulin: general architecture
• PAS: histiocyte, vascular basement membrane
• Methyl green pyronine for plasma cells /
immunoblasts
• IF/IHC: lambda/kappa light chains: monoclonal/
polyclonal B-cell proliferation.

5. 3.Liver biopsies
• Reticulin: general architecture
• Masson’s trichrome: architectural integrity/
evidence of fibrosis
• Orcein method: Australia antigen
• PAS +/- diastase : retention globules in alpha-1
antitrypsin deficiency
• Perls stain: hemosiderin
• Rubeanic acid: for copper

6. 4.Kidney biopsies
• PAS and reticulin: basement membrane
• Martius yellow – scarlet blue(MSB) for fibrin
• Masson trichrome: interstitial fibrosis
• IF/IHC: complements, immune complexes

7. 5.Gastro-intestinal biopsies
• Alcian blue/ PAS : Identification of metaplasia /
mucin depletion.

8. 6.Muscle biopsies
• Enzyme histochemistry( succinic dehydrogenase,
ATPase) : for fibre types
• Methylene blue for motor end plates.

9. 7.Bone marrow
• Reticulin: for fibrosis
• Perls stain: hemosiderin
• Methyl green: plasma cells and immunoblasts

10. 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 andminerals

11. 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 suchas protein
and lipid)
• Proteoglycans
• Mucins
•Others glycoproteins

12.  The carbohydrate component is known as glycosaminoglycans(GAG)
 TYPES OF GYCOSAMINOGLYCANS
Chondroitin sulfate
Dermatan sulfate
Keratan sulfate
Heparin sulfate
Heparin
Hyaluronic acid
PROTEOGLYCANS

13. 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

14. FIXATIVES
Tissue placed in fixative promptly after removal – capable
of autolytic reaction
If immediate fixation not possible, tissue refrigerated until
adequate fixation possible
 Recommended fixatives : Rossman’s fluid
Alcoholic formalin withpicric
acid
Glutaraldehyde fixatives avoidedfree aldehyde groups
are capable of undergoing schiff reaction – so increased
background staining

15. 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

16. PERIODIC ACID SCHIFF METHOD
1st histochemical use was by McManus for
demonstration of mucin
Reagents – 1. Periodic acid(HIO4)
2. Schiff reagent
 0.5-1% solution of periodic acid (oxidant) used for 5-10
minutes oxidation of hydroxyl group within the
carbohydrate formation of two free aldehyde groups
free aldehydes react with schiff
reagent(Chromophores) Bright
red magenta endproduct

17. Result
• Glycogen: Magenta
• Nuclei: Blue
• Fungi: Red
• Control: Normal kidney tissue, appendix

18. • Dewax in xylene and rehydrate through graded
ethanols to distilled water.
• Oxidize with periodic acid for 5 minutes.
• Rinse in several changes of distilled water.
• Cover the sections with Schiff reagent for 15
minutes.
• Rinse in running tap water for 5–10 minutes.
• Stain the nuclei with hematoxylin. Differentiate and
blue the sections.
• Dehydrate in graded alcohol and clear with xylene.
• Coverslip.
STEPS

19. CANDIDA IN
PAS STAIN
ALPHA
ANTITRYPSIN
IN PAS STAIN

20.  Other oxidants like potassium permanganate/ chromic
acid not used – further oxidise aldehyde groups to
carboxylic groups – not reactive to schiff reagent
 MILD PAS TECHNIQUE –
0.01% periodic acid used for shorter period for N-acetyl
sialic acid containing mucins as the hydroxyl groups are
highly susceptible to periodic acid oxidation

21. PAS REACTIVE CELLS AND TISSUE COMPONENTS
1.Glycogen
2. Starch
3. Mucin
4. Basement membrane
5. Alpha anti trypsin
6. Reticulin
7. Fungi(capsules)
8. Pancreatic zymogen granules
9. Thyroid colloid
10. Corpora amylacea
11. Russell bodies

22. 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

23. • The distinction between mucins and glycogen can be
problematic when using the PAS technique and the
inclusion of a glycogen digestion step is necessary when
the diagnosis requires the correct identification between
these mucosubstances.
• α-Amylase may be used in this situation as it catalyzes
the hydrolysis of the glycosidic bonds of glycogen and the
breakdown of the large glycogen molecules to the water-
soluble disaccharide, maltose.
• The net result is the removal of glycogen from the tissue
section prior to the PAS technique.
PAS with Diastase Resistance

24. 1.Dewax two serial sections in xylene and rehydrate through
graded ethanols to water.
2.Place one slide in the diastase solution for 1 hour at 37°C.
The other slide is an untreated control and may remain in
water for 1 hour.
3.Wash both slides in running tap water for 5–10 minutes.
4.Proceed with the PAS technique.
• Human saliva and Malt Diastase can be used for
enzymatic reaction
STEPS

25. • Glycogen should demonstrate bright red/magenta
staining in the untreated slide. Glycogen staining
should be absent in the diastase treated
slide.
• A known positive control should be included to verify the
potency of the enzyme.

26.  Cationic dye.
 Alcian blue 8GX –recommended
Comprised of copper containing pthalocyanine ring
linked to 4 isothiouronium groups –strong bases -
account for cationic nature of the dye
Sulfate and carboxylate groups of proteoglycans
ionised at pH 2.5 and carry a negative charge
 Sialo- and sulfo mucins also reactive at pH 2.5
 So, they stain with alcian blue at pH 2.5
 Neutral mucins are not reactive with alcian blue
ALCIAN BLUE METHOD

27. REAGENTS :
1. Alcian blue
2. Aluminium sulfate
3.Nuclear fast red - counterstain
- RESULTS
 sulfomucin,sialomucin
 Proteoglycans
 Hyaluronic acid
 Nucleus  red
Blue

28. STEPS
• Dewax in xylene and rehydrate through graded
ethanols to distilled water.
• Stain in the alcian blue solution for 30 minutes.
• Rinse in running tap water for 5 minutes.
• Counterstain in nuclear fast red for 10 minutes.
• Wash in running tap water for 1 minute.
• Dehydrate in graded ethanols.
• Clear in xylene and mount in a miscible medium.

29. GOBLET CELLS BY ALCIAN BLUE

30. 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 bestained(magenta)
“ Pan Mucin Stain”

31. 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

32. MUCICARMINE
 Active dye molecule is aluminium – carminic acid
complex known asCARMINE
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 – cryptococcus neoformans is also
detected

33. 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

34. COCCUS
CACTI
CRYPTOCOCCUS
STAINED BY
MUCICARMINE

35. GOBLET CELLS BY MUCICARMINE

36. Control for Alcian blue and
Mucicarmine
Sections of Appendix and colon

37. AMYLOID
Extracellular , amorphous , eosinophilic
material
Composed of protein in an antiparallel  -
pleated sheet configuration
In H&E stain , can be confused with hyaline and
fibrinoid substances
Earliest special stain used for amyloid was
Iodine by Virchow

38. Stains for Amyloid
• Congo Red
• Van Gieson stain
• Alcian & PAS
• Thioflavin T

39. CONGORED 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 electrochemicalbonds
Electrochemical staining of other tissues can be
suppressed by –
using alkaline-alcoholic solvents
using competitive inhibition by salt solution

40. 2 factors are important to the congo red-
amyloid reaction
1.Linearity of the dye molecule
2.-pleated sheet configuration of theamyloid
If the spatial configuration of either is altered,
the reaction fails

41. Fixation – Not critical- Carnoy or Alcoholic.
Solution- 0.5 % Congo red in 50% alcohol
0.2% Potassium Hydroxide in 80% alcohol
TECHNIQUE
Counterstain- Meyers hematoxylin

42. Results-
Amyloid- red
Nuclei - Blue

43. AMYLOID BY CONGO RED

44. ALKALINE CONGO-RED TECHNIQUE
 High concentration of NaCl isused
Background electrochemical stainingis
reduced
hydrogen bonding of congo-red to amyloid is
enhanced

45. POLARIZING MICROSCOPY AND CONGO-RED
Under polarized light, congo red stained
amyloid exhibits apple-green birefringence
Most reliable diagnostic test for amyloid
currently

46. POINTS TO REMEMBER
Thickness of section is critical – 8-10 micro meter is
ideal
 Too thin section – show faint red color
 Too thick section – show yellow birefringent
 Other structures giving apple-green birefringence :
1. neurofibrillary tangles of alzheimer’s
2. intracellular inclusions seen in adrenal cortical cells
3. cellulose and chitin
4. dense collagen
 Thioflavin T – flourescent method

47. To differentiate AA and AL amyloid :
Section pretreatment with trypsin or
potassium permanganate done
AA amyloid lose their affinity for congo-red
but AL amyloid is resistant

48. 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

49. 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

50. 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)

51.  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

52.  Sudan black b has 2 fractions –
1st stains neutral fatsblue-black
2nd stains phospholipids gray

53. STAINS FOR
MICROORGANISMS

54. 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- counterstain
(6) Picric acid, 0.1% in acetone
RESULTS :
GRAM POSITIVE BACTERIA – blue
GRAM NEGATIVE BACTERIA – red
NUCLEI –red
OTHER TISSUE ELEMENTS -yellow

55. Dry picric acid is explosive – recommended to
purchase picric acid – acetone solution pre made
Do not allow sections to dry at any point in the
staining process – decolorization will be difficult
and uneven

56. Acid Fast Staining forBacteria
 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

57. 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 grayishblue

59. Blue counterstain may be patchy if extensive
caseation is present
Avoid over counterstaining – scant organism
can easily be obscured
Decalcification using strong acids may
destroy acid-fastness - so formic acid
recommended

60. MODIFIED FITE TECHNIQUE
REAGENTS :
1. Carbol fuchsin solution
2. 5% sulphuric acid in 25% alcohol
3. Methylene blue solution- counterstain
RESULTS:
M.leprae – bright red
nuclei and other tissue elements – pale blue

61. Fite stain Modified ZN
Uses mixture of xylol & liquid
paraffin prior to stain
Does not use
Incubation in ZN carbolfuchsin at
37ºc for 45 min
Incubation In preheated ZN carbol
fuchsin at 56ºc for 60min
Decolorize with 5% H2SO4 1% acid alcohol. Acid :20%
H2SO4
Demonstrates M leprae M tuberculosis
AFB :Fite vs modified ZN

62. Classification of silver staining techniques:
1. Argentaffin: Fontana Masson for melanin pigments.
2. Argyrophil: Warthin –Starry for spirochetes
AgNOR stain
3. Impregnation: Bodian’s method for nerve fibres
4. Oxidation-reduction
• Grocott’s methenamine silver for fungi(SM)
• Jone’s stain for basement membrane
• Reticulin stain
5. Autometallography Immunogold silver intensification

63. Warthin Starry Method for Spirochetes & Helicobacter
REAGENTS :
1. Acetate buffer pH-3.6
2. 1% silver nitrate
RESULTS :
SPIROCHETES – black
BACKGROUND – golden -yellow

64. SPIROCHETES BY WARTHIN STARRY

65. FUNGAL STAINS
Fungal cell walls are rich in polysaccharides
which can be converted by oxidation to
dialdehydes
Dialdehydes are then detected by silver
solution

66. 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

67. Results
Fungi , Pneumocystis, melanin - Black
Mucin & Glycogen - dark grey
Background - Pale green
Hyphae & yeast form - sharply delineated inblack
against green background

68. CRYPTOCOCCUS BY GMS STAIN

69. GMS aspergillus fungi

70. 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

71. 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

73. 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

74. FACTORS AFFECTING TRICHROME STAINING
1. TISSUE PERMEABILITY AND DYE
MOLECULAR SIZE
When protein component of a tissue exposed to a fixative –
insoluble protein networkformed
Structure of the protein network directly related to the
staining reactions
 Erythrocyte protein – dense network with small pores
 Muscle cells – larger pores
 Collagen – least dense network and quite porous

75. 2. Heat :
 Increases rate of staining and penetration
3. pH :
 LowpH ( 1.5 – 3)
4. Nuclear stain for trichrome
 Iron hematoxylin preferred
 More resistant to acidity of dye solutions
 Alum hematoxylins are decolorized
 Can use Celestin blue- alum hematoxylin sequence

76. EFFECT OF FIXATION
 10% NBF will not yield optimal results
Treatment of formaldehyde fixed tissue with picric
acid /mercuric chloride solution enhances intensity
and radiance of trichrome
 Recommended fixatives are: Bouin’s
Zenker’s,
Formal-mercury
Zinc formalin

77.  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

78. 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 →Green

79. Trichrome stain showing slight
mesangial prominance

80. 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

81. Gomori’s Reticulintechnique
• Principle: reticulin stains are silver stains based on
argyrophilic properties of reticulin fibres
• MC reticulin stains are Gomori’s and Gordon &
Sweet’s stain

82. Reagents:
0.5 % KMnO4
1% oxalic acid
2% ferric alum
10% formalin
0.2% gold chloride
2% sodium thiosulphate

83. • 1st step: Oxidation of hexose sugars in reticulin fibers to
yield aldehydes
• 2nd step: Sensitization: metallic compound like ammonium
sulphate is deposited around reticulin fibres
• Followed by silver impregnation in which an ammoniacal or
diamine silver solution is reduced by the exposed aldehyde
groups to metallic silver
• Further reduction of diamine silver is achieved by
transferring the sections to formaldehyde: this step is called
developing
• The last step is toning by gold chloride in which silver is
replaced by metallic gold and the colour of reticulin fibres
changes from brown to black.

84. • Dewax
• Bring sections to water
• Stain with 0.5% potassium permanganate for 5 min
• Wash with running tap water
• Bleach with 1% oxalic acid for 30 seconds
• Wash with tap water
• Put slide in 2% iron alum for 10 min

85. • Wash with distilled water
• Prepare ammoniacal silver nitrate solution.
• Take 4cc 10% silver nitrate ,add ammonia drop by
drop till precipitate just dissolves, impregnate in
ammoniacal silver nitrate solution for 30 min
• Wash with distilled water
• Reduce in 10 % formalin for 2 min

86. • Wash in water
• Tone in 0.2% gold chloride for 2 min
• Wash with tap water
• Fix in 2% sodium thiosulphate for 3 min
• Counterstain if desired with neutral red.
• Wash, blot, dry, dehydrate and mount in DPX.

87. Results
• Reticulin fibres: black
• Nuclei: black/unstained
• Collagen: if untoned
yellow brown and if
toned purple black
• Other elements:
according to
counterstain
• Control: liver tissue/
lymph node

88. Utility of reticulin stain
• Reticulin fibre network
particularly rich in liver
• Reticulin stain provides
important information
about the architecture
of liver .
• Areas of reticulin
crowding indicate focal
hepatocyte loss.
• When hepatocytes
regenerate, reticulin
fibres show thickening
of hepatic cell plates,
which appear as two or
three-cell thick plates

89. Silver nitrate solution
• Reticulin fibres: black
• Other structures: shades of pink

90. Phosphotungstic acid
hematoxylin:PTAH stain
• To stain connective
tissue
• Control: section of
striated muscle
• Results:
Nuclei and all fibrils:
blue
Cytoplasm: yellow pink
to brown red
Collagen: brown pink
Coarse elastic fibrils:
purple tinge
Mitotic figures: blue
Mitochondria: blue
Striated muscle fibers:
blue
Astrocytes
Amoeba

91. PTAH stain

92. 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

93. Hemosiderin
 Breakdown product of haemoglobin composed of
ferric iron andprotein
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

94. 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

95. Best positive control – postmortem lung tissue that
contains a reasonable amount of iron positive
macrophages (heart failurecells)
In Hb and myoglobin , iron bound tightly within protein
complex – cannot be demonstrated by traditional
technique
Treatment with hydrogen peroxide releases iron -
stained withperl’s stain

97. Perl’s Prussian Blue staining for
bone marrow iron
• Grade 4
• Increased marrow iron
stores

98. Calcium
1. Alizarin red S method(MCGee –Russel)
• To demo Ca
• Result: calcium salts: intense red orange
• Background: pale green
2. Von Kossa method
• Aim: to demo Ca
• Principle: indirectly demo Ca phophate, carbonate,
oxalate
• Result: calcium: dark green/black,backgound
depends on counterstain

100. Copper and Lead
• Copper: as in Wilson’s disease,PBC,
• Methods:
1. Mallory & Parker dye-lake method
2. DMABR method
3. Rhodanine technique
4. Rubeanic acid method of Uzman

101. Modified Fouchet’s technique: bile pigment
Demonstrates liver bilepigment
Most common routinemethod
• Reagents
Fouchet ‘s reagent : 25% aq trichloracetic acid
• 10% aq ferric chloride
• RESULT
Muscle : Yellow
Collagen: Red
Bile : Emerald green

102. BILE PIGMENTS BYMODIFIED
FOUCHET’S TECHNIQUE

103. 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

104. MELANIN DEMONSTRATED BY :
1. Reducing methods : a) Masson fontana silver
technique
b) Schmorl’s ferric-ferricyanide
reduction test
2. Enzyme methods – DOPAreaction
3. Solubility and bleaching characteristics
4. Fluorescent method
5. Immunohistochemistry

105. MASSON FONTANASTAIN
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 argentaffinproperty
Melanins are blackened by acid silver nitrate solution
RESULT :
Melanin –black
Nuclei - red

106. 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

107. 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 base

108. 1. Feulgen technique ( demonstrate sugar)
2. Methyl green pyronin technique
(demonstrate phosphate)
3. Acridine orange (by fluorescent method)
4. Gallocyanin-chrome alum method
Demonstra
tes both DNA
and RNA
The last staining method do not separate the 2 nucleic
acids (stains both DNA and RNA blue) and suitable extraction
technique must beused
DNA IS DEMONSTRATED BY

109. FEULGEN STAIN
SOLUTIONS USED ARE :
A) 1M HCL acid - used for acid hydrolysis to break the purine-
deoxyribose bond and yield an aldehyde.
- Done at 60̊ C (HCL should be preheated to 60 ̊C )
- Time (minutes) depends upon the fixative used
- For carnoy’s and formalin – 8 minutes used
B) Schiff reagent - The aldehydes are then demonstrated by
schiff’s reagent
C) Bisulfite solution
RESULT DNA : red-purple
CYTOPLASM : green

110. DNA BY FEULGEN STAIN

111. 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
1.Pyronin
- binds to any negatively charged tissue
constituent
- apart from RNA, binds to acid mucins and
cartilage
RESULTS – DNA : green-blue
RNA : red

112. Nucleic
acids by
methyl
green
pyronin

113. Special stains enhance detection &
localization of individual tissue component
But should not be substituted for routine H&E
CONCLUSION

114. References
• Bancroft’s theory and practice of histological
techniques 8th edition.
• Ramdas nayak histopathology techniques and their
management, 2018 edition.
• Carleton’s histological technique.

115. Thank You!