The document discusses various histological staining techniques. It begins by explaining hematoxylin and eosin staining, which provides basic diagnostic information. It then covers special stains that highlight specific tissue components, categorized by the structures they identify such as carbohydrates, amyloid, lipids, nucleic acids, and microorganisms. Carbohydrate stains discussed include periodic acid Schiff, alcian blue, mucicarmine, and others. Amyloid identification using Congo red and methyl violet is explained. Lipid stains using Sudan dyes are also summarized. The document provides details on techniques for staining nucleic acids and identifying bacteria by Gram staining.

1. DR. EKTA JAJODIA

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

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

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

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
Chondroitin sulfate
Dermatan sulfate
Keratan sulfate
Heparin sulfate
Heparin
Hyaluronic acid
PROTEOGLYCANS

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

7. 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 with picric
acid
 Glutaraldehyde fixatives avoidedfree aldehyde groups
are capable of undergoing schiff reaction – so increased
background staining

8. HEPATOCYTES SHOWING STREAMING
ARTIFACT
Fixation should be carried out at 4 ̊ C to minimize
streaming artifact

9. 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

10. PERIODIC ACID SCHIFF METHOD
 1st histochemical use was by McManus for
demonstration of mucin
Reagents – 1. periodic acid
2. schiff reagent
 0.5-1% solution of periodic acid (oxidant) used for 5-10
minutes oxidation of hydroxyl group within the
formation of two free aldehyde groups
free aldehydes react with schiff reagent
carbohydrate
bright
red magenta end product
RESULT – glycogen and mucins : magenta
nuclei : blue

11. CANDIDA IN
PAS STAIN
ALPHA
ANTITRYPSIN
IN PAS STAIN

12.  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

13. 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

14. 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

15. ALCIAN BLUE
 Alcian blue 8 GX – 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

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

17. GOBLET CELLS BY ALCIAN BLUE

18. 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)

19. 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

20. 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 – cryptococcus neoformans is also
detected

21. 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

22. COCCUS CACTI
CRYPTOCOCCUS
STAINED BY
MUCICARMINE

23. GOBLET CELLS BY MUCICARMINE

24. 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

25. 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 be used
DNA IS DEMONSTRATED BY

26. EXTRACTION TECHNIQUES
1. DIGESTION METHODS :
Pure deoxyribonuclease will digest DNA and pure
ribonuclease will digest RNA
2. CHEMICAL METHODS :
a) By perchloric acid : To remove RNA – 10% perchloric acid
at 4̊ C overnight
b) Trichloroacetic acid
c) Hydrochloric acid

27. 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

28. DNA BY FEULGEN STAIN

29. 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

30. NUCLEIC
ACIDS BY
METHYL
GREEN
PYRONIN

31. 1. Most suitable technique for identifying DNA is In-situ
hybridization
2. Bouin’s fixative is not suitable as it causes over hydrolysis of
the nucleic acid during fixation
3. RNA cannot be demonstrated by feulgen stain because ribose-
purine bond is unaffected by hydrolysis/ 1 M HCL
4. Control method for the standard feulgen technique is –
Naphthoic acid hydrazide (NAH) method – DNA is acid
hydrolysed by 1M HCL. Aldehydes are coupled with naphthoic
acid and then again coupled with diazonium salt, fast blue B.
results are identical to true feulgen reaction
5. Blue thionin-feulgen reaction – used for studying cancer cell
nuclear morphology and ploidy. Here DNA is stained blue and
cytoplasm remains unstained
POINTS TO REMEMBER

32. 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

33.  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)

34.  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

35.  Sudan black b has 2 fractions –
1st stains neutral fats blue-black
2nd stains phospholipids gray
 This gray reaction can be enhanced as a bronze
dichroism if section is viewed in polarised light
 It fails to stain crystalline cholesterol, lecithin and free
fatty acids
 Bromine pre treatment converts crystalline
cholesterol to oily derivatives and hence permeable to
Sudan dye

36. 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

37. 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

38.  2 factors are important to the congo red-
amyloid reaction
1.Linearity of the dye molecule
2.-pleated sheet configuration of the amyloid
If the spatial configuration of either is altered,
the reaction fails

39. Fixation – Not critical
Solution- 0.5 % Congo red in 50% alcohol
0.2% Potassium Hydroxide in 80% alcohol
Results- Amyloid - red
Nuclei - Blue
TECHNIQUE

40. AMYLOID BY CONGO RED

41. ALKALINE CONGO-RED TECHNIQUE
 High concentration of NaCl is used
 Background electrochemical staining is
reduced
 hydrogen bonding of congo-red to amyloid is
enhanced

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

43. 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

44. 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

45. 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

46. STAINS FOR
MICROORGANISMS

47. 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

48.  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

49. 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

50. Ziehl Neelson (ZN) stain
Reagents
(1) Carbol fuchsin solution
(2) 1% acid alcohol
(3) 0.1%Methylene blue solution
RESULTS
Acid fast bacilli bright red
Other tissue Pale blue
Caseous material very pale grayish blue

51.  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

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

53. 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

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

55. SPIROCHETES BY WARTHIN
STARRY

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

57. 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

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

59. CRYPTOCOCCUS BY GMS STAIN

60. 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

61. 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

63. 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

64. FACTORS AFFECTING TRICHROME
STAINING
1. TISSUE PERMEABILITY AND DYE
MOLECULAR SIZE
 When protein component of a tissue exposed to a fixative –
insoluble protein network formed
 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

65. 2. Heat :
 Increases rate of staining and penetration
3. pH :
 Low pH ( 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

66. 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

67.  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

68. 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

69. Trichrome stain showing slight
mesangial prominance

70. 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

71. 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

72. 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

73. 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)
Results
Ferric iron –Blue
Nuclei – Red
Reagents
2% aq. Potassium ferrocyanide
2% HCl
Counterstain with 1% neutral red or saffranin

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

75. Modified Fouchet’s technique: bile
pigment
 Demonstrates liver bile pigment
 Most common routine method
Reagents
a)Fouchet ‘s reagent : 25% aq trichloracetic acid
10% aq ferric chloride
b)Van Gieson stain :acid Fuchsin + saturated aq picric
acid
RESULT
Bile pigment : emerald to blue green
Muscle :yellow
Collagen :red

76. BILE PIGMENTS BY MODIFIED
FOUCHET’S TECHNIQUE

77. 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

78. 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

79. 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

80. 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

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