This document provides information and methods for special stains used to identify various tissues and microorganisms under the microscope. It discusses stains for amyloid, pigments, microbes and calcium. Specific stains covered include Congo red for amyloid, Perls' Prussian blue for iron, Fouchet's for bile pigments, Masson Fontana for melanin, von Kossa for calcium, rhodanine for copper, Gram stain for bacteria identification, Ziehl-Neelsen for acid-fast mycobacteria, and Warthin-Starry for spirochetes. Detailed protocols are provided for each staining method.

1. MODERATOR DR ASHIMA

2. SPECIAL STAINS FOR:
Amyloid
Pigments (Iron, bile, melanin, calcium, copper)
Microbes (gram positive bacteria, mycobacterium, spirochaete and
fungi )

3. AMYLOID-Congo red stain
Principle of congo red staining:
 Amyloids are homogenous and eosinophilic,
proteinaceous deposits, that are extracellular and may become
sufficiently large enough to cause damage to surrounding tissues.
When stained with the congo red stain the amyloid will show
bifringence an apple green color, under the polarizing microscope.

4. Bennhold's congo red staining:
Method
(1) Bring sections to water,
(2) Stain with Ehrliçh haematoxylin for 20 minutes,
(3) Differentiate with 1% acid alcohol.
(4) Wash in running water for 1 minute to remove the acid
(5) Stain with 1% aqueous Congo red for 20-30 minutes.

5. (6) Pour off stain and flood slide with a saturated aqueous solution of lithium carbonate;
leave for 15 seconds.
(7) Differentiate in 80% alcohol until excess Congo red is removed.
(8) Wash in running water for 10 minutes.
(9) Dehydrate, clear and mount
Results
 Amyloid -Pink to red
 Nuclei -Blue

6. With polarized light - amyloid exhibits bright green birefringence,
‘apple-green birefringence’
This property is shared by other β-pleated sheet proteins and specific
to that conformation.

7. Kidney section showing
amorphous eosinophilic amyloid
deposits on H&E

8. Congo red stained section of kidney
demonstrating amyloid deposition(red)

9. Same section viewed by polarized light showing
green birefringence against dark background

10. USES
It is used to demonstrate amyloid deposits in
Renal amyloidosis
Medullary carcinoma thyroid
Vessel wall in case of Alzheimer disease
Cardiac arrythmias

11. In upper dermis
there is deposit of eosinophilic
amorphous material (H&E)
Dermal amyloid deposits under
polarizing microscopy
(Thioflavin-T stain)
Other methods: 1)Flourescent
Microscopy Technique: Thioflavin T

12. 2)Metachromatic Staining
by Methyl or Crystal Violet
Metachromatically results in a red – purple colour
of amyloid

13. Perls’ Prussian blue
Hemosiderin is a brown pigment that is present in reticuloendothelial
cells of bone marrow, spleen and liver. It is formed by partial
degradation of aggregates of ferritin by lysosomes.
PRINCIPLE:
Treatment of tissue with an acid ferrocyanide solution will result in
the unmasking of ferric iron in hemosiderin, in the form of the
hydroxide, Fe(OH)3. The ferric iron then reacts with a dilute
potassium ferrocyanide solution to produce an insoluble blue
compound, ferric ferrocyanide (Prussian blue).

14. PURPOSE:
• To demonstrate ferric iron in tissue sections.
• Small amounts of iron are found normally in spleen
and bone marrow.
• Excessive amount are present in hemochromatosis,
with deposits found in the liver and pancreas,
hemosiderosis, with deposits in the liver, spleen, and
lymph nodes.

15. METHOD
(1)Bring sections to water.
(2) Transfer to a fresh solution of equal parts of 2% aqueous potassium
ferrocyanide and 2% hydrochloric acid, for 30 minutes.
(3) Wash thoroughly in water.
(4) Counterstain lightly with 1% neutral red for 10-15 seconds.
(5) Wash in water, dehydrate, clear and mount.
Results
 Ferric-iron-containing pigments (haemosiderin)- Blue
 Nuclei -Red

16. Hemosiderin, liver, iron stain.
RESULTS:
Iron -blue
Nuclei -red
Background -pink

17. H&E stain showing iron deposits in tubules

18. Liver section from a patient with
hemochromatosis stained for ferric iron with Perl’s
method. (Ferric iron is stained blue).

19. Fouchet technique for bile pigments
Bile pigments are endogenous breakdown products of haemoglobin-
bilirubin and biliverdin that are excreted in bile.
Principle of fouchets:
 The pigment is converted to the green color of biliverdin and
blue cholecyanin by the oxidative action of the ferric chloride in the
presence of trichloroacetic acid .

20. Solutions:
Fouchet’s solution :
25% aqueous trichloroacetic acid -36 ml
10% aqueous ferric chloride -4 ml
Van Gieson stain solution :
Dissolve 100 mg of acid fuchsin in 100 ml of saturated aqueous
picric acid

21. Method
1. Take sections to water.
2. Treat with the freshly prepared Fouchet’s solution for 10 minutes.
3. Wash well in running tap water for 1 minute.
4. Rinse in distilled water.

22. • 5. Counterstain with van Gieson solution for 2 minutes.
• 6. Dehydrate, clear and mount
Results
 Bile pigments- emerald to blue-green

23. Bile(yellow) in liver biopsy in cholestasis

24. Bile in liver section stained with fouchet’s
technique. Bilirubin is stained emerald green.

25. OTHER METHODS
• 1)GLENNER METHOD- treat sections with 3% potassium dichromate
bilirubin is stained emerald green
• 2)STEIN’S METHOD- treat sections with mixture of 3 parts lugol’s
iodine and 1 part tincture iodine, decolorise with sodium sulphite and
counterstain in neutral red. Bilirubin appears green

26. MELANIN
Melanin is non lipid, non hematogenous pigment.
It is a brown black pigment present normally in hair, skin, retina, iris
and certain parts of CNS.

27. Principle:
 The solutions of ammoniacal silver nitrate are reduced by
melanin to black metallic silver, basis for demonstrating melanin.

28. METHOD: Masson Fontana for melanin
1.Take sections to water.
2.Treat with the ammoniacal silver solution in a Coplin jar for 30–40
minutes at 56°C or overnight at room temperature.
3.Wash well in water.
4. Treat sections with 5% aqueous sodium thiosulfate for 1 minute.
5. Wash well in running tap water for 2–3 minutes.

29. 6.Lightly counterstain in 0.5% aqueous neutral red for 5 minutes
7. Rinse in distilled water.
8.Dehydrate, clear and mount.
Results
Melanin, argentaffin, chromaffin and some lipofuscins- black
Nuclei- red

30. Melanin pigment of skin showing black color
RESULTS:
Melanin,
argentaffin cells -black
Nuclei - red

31. USES
To identify melanin and argentaffin granules.
In diagnosis of malignant melanoma.
Argentaffin granules are found in carcinoid tumors.

32. Melanin(Brown) in stratum basale on H&E

33. Melanin pigment in cells of malignant
melanoma, fontana masson stain

34. OTHER METHODS- 1)ENZYME DOPA METHOD
Principle :
The enzyme tyrosinase is located within some cells producing melanin
will oxidize DOPA to form an insoluble brown – black pigment.
Purpose :
Cells that are capable of producing melanin can be demonstrated by
DOPA (dihydroxy- phenylalanine) method.
Result
DOPA oxidase – Brown
Nuclei - blue

35. 2)Fluoroscence methods-melanin precursors give yellow
fluorescence
3)Bleaching methods: melanin is bleached by strong
oxidising agents like hydrogen peroxide, potassium
chlorate or treatment with potassium permangate for
half hour followed by 1% oxalic acid.
4)Nile blue method: melanin stained dark blue

36. CALCIUM-VON KOSSA METHOD
Calcium is plentiful mineral found in human body in teeth, bones, blood and nerve cells. Abnormal
deposits of calcium may be found in any area of the body
Principle of von kossa method:
 Tissue sections are treated with silver nitrate solution, silver is deposited by
replacing the calcium and then it is reduced by the strong light and visualized as metallic silver.
o With H&E stain, calcium appear deep blue purple.
o On von kossa method it appears black.

37. Method
1) Bring sections to water.
2) Immerse section in pH 45 citrate buffer for 20 minutes
3) Wash well in distilled water.
4) Flood slides with 5% silver nitrate.
5) Expose to bright sunlight or to a 60-watt electric bulb at a range of 4-
5 inches for 30-60 minutes.

38. (6) Wash in several changes of distilled water
(7) Treat with 5% sodium thiosulphate for 2-3 minutes.
(8) Counterstain with neutral red
(9) Dehydrate, clear and mount
Result
Calcium deposits- Black

39. Calcification on H&E (Deep blue purple)

40. Coronary artery showing
brown calcified
atheromatous plaque-von
kossa

41. OTHER METHODS
1)FLUOROSCENT METHOD: Stain with morin in 85% alcohol containing
0.5% acetic acid and view under fluorescent microscope. Greenish
white fluorescence indicates presence of calcium.
2)ALIZARIN RED S METHOD:stain in alizarin solution(alizarin red S 1g
and 50ml distilled water. Calcium deposits are stained orange red.

42. Alizarin s method-orange red
calcium deposits in kidney
section.

43. COPPER
Copper is excreted in bile and accumulates in liver in chronic biliary
diseases
Rhodanine technique for copper
Principle:
 Rhodanine stain demonstrates protein to which copper binds rather
than copper itself. It is used to identify copper deposits in Wilson
disease

44. Rhodanine technique for copper
• Solutions
Rhodanine stock solution 5-p-Dimethylaminobenzylidene-
rhodanine 0.05 g
Absolute ethanol 25 ml
Working solution 5 ml of the stock rhodanine solution added to 45 ml of
2% sodium acetate trihydrate.
Borax solution
Disodium tetraborate 0.5 g
Distilled water 100 ml

45. Method
1. Take sections to water.
2. Incubate in the rhodanine working solution at 56°C for 3 hours or
overnight in a 37°C oven.
3. Rinse in several changes of distilled water for 3 minutes each.
4. Stain in acidified alum hematoxylin for 10 seconds.
5. Briefly rinse in distilled water and place immediately in borax
solution for 15 seconds.

46. • 6. Rinse well in distilled water.
• 7. Dehydrate clear and mount.
Results
 Copper and copper-associated protein red to orange-red
 Nuclei blue

47. Rhodanine stain for copper-
Wilson disease

48. OTHER METHODS
RUBEANIC ACID METHOD: Copper deposits greenish black
VICTORIA BLUE STAIN: Copper stained blue

49. GRAM STAIN
• Gram positive bacteria have thick cell wall without an outer membrane and
stain purple with gram stain.eg: cyanobacteria
• Gram negative bacteria have thin cell wall with an outer membrane and
stain red with gram stain .eg: salmonella
Principle of gram stain:
The structure of organism’s cell wall determines whether the organism is
gram positive or negative. Those bacteria which retain the primary stain by
resisting decolorization are gram positive and those which get decolorized
and then get counterstained are called gram negative.

50. Gram's method
Reagent
Lugol's iodine
Iodine 1 g
Potassium iodide 2 g
Distilled water to 100 ml
• Dissolve the potassium iodide in 4-5 ml of water; dissolve the iodine
in this. Dilute to 100 ml to make Lugol's iodine

51. Method
(1) Bring sections to water.
(2) Stain with 0.5% aqueous methyl violet 6 B for 1-3 minutes.
(3) Rinse with water.
(4) Pour on Lugol's iodine for 1-3 minutes

52. 5) Differentiate rapidly with acetone (1-2 seconds) and wash
immediately in running water.
6) Counterstain with 1% neutral red for 1 minute.
7) Wash in water.
8) Dehydrate, clear and mount.
Results
Gram-positive organisms- Blue-black
Other tissue structures -Shades of red

53. Gram Positive
Gram Negative

54. Ziehl-Neelsen (ZN) Stain
Mycobacteria are difficult to demonstrate by the Gram
technique because they possess a capsule containing a
long chain fatty acids, mycolic acid that make them
hydrophobic.
Phenolic acid or heat may be used to reduce the surface
tension and increase the porosity.

55. Ziehl-Neelsen method
Principle:
 Mycobacterias (tubercle bacilli) have a lipid rich cell wall
which is capable of taking up strong phenol dye solutions (eg.carbol
fuschin solution) such that the dye is retained upon subsequent
differentiation in acid or alcohol called as acid and alcohol fast(AAFB=
acid and alcohol fast bacilli)

56. Ziehl-Neelsen method
Reagent
Carbol-fuchsin
• Basic fuchsin 1 g
• Absolute alcohol 10 ml
• 5% Phenol 100 ml
• Dissolve the basic fuchsin in the alcohol, then add the 5%
phenol.

57. METHOD
1) Bring section to water.
2) Stain in hot carbol-fuchsin, either in a Coplin jar in a 56 °C
oven for 30 minutes, or flooding the slide with stain, heating until
the stain steams and leaving for 10 minutes.
3) Wash in water to remove excess stain
4) Differentiate in 1 % acid alcohol, 10 min.

58. 5) Wash in water.
6) Counterstain lightly in 0.1% méthylène blue for 10-15 seconds
7) Wash in water
8) Dehydrate, clear and mount.

59. Results
 Acid-fast bacilli Red
 Nuclei Blue
 Other tissue constituents Pale blue

60. OTHER METHODS
1) Modified Zeihl Neelsen method
Lepra bacillus is not as acid fast as M.
tuberculosis, so conc. of H2SO4 is less.
Decolorise with 5%-8% H2SO4 ( instead of 20%
in ZN method).
Results : lepra bacilli - red
other tissue - blue
2)Modified fite procedure:
lepra bacilli - red
other tissue - blue

61. 3)Bleach Concentration Technique (cytology)
Pus in tube
mixed with 2ml of 5% sod hypochlorite
incubated for 15min at 37c
2ml distilled water added
centrifuged at 3000rpm for 15 min
air dried smear using 1 drop sediment
stained by ZN stain

62. Mycobacteria seen as red rods

64. SPIROCHAETE
Spirochaetes are elongated motile, flexible bacteria twisted spirally along the long
axis with a periplasmic flagella.eg: Treponema pallidium(syphilis), borrelia
burgodorferi (lyme disease), leptospira (leptospirosis)
Principle of warthin-starry stain method:
 Spirochaetes have ability to bind silver ions.It involves
impregnation of spirochaetes in tissues with silver ions with subsequent
reduction of these ions to metallic silver using a developer containing
hydroquinone.

65. SPIROCHAETE
Warthin-Starry method for spirochetes
Solutions
Acetate buffer, pH 3.6
• Sodium acetate 4.1 g
• Acetic acid 6.25 ml
• Distilled water 500 ml
Silver solution 1% silver nitrate in pH 3.6 acetate buffer
Developer Dissolve 0.3 g of hydroquinone in 10 ml pH 3.6 acetate buffer, and
mix 1 ml of this solution and 15 ml of warmed 5% Scotch glue or gelatin;
keep at 40°C. Take 3 ml of 2% silver nitrate in pH 3.6 buffer solution and keep
at 55°C. Mix these two solutions immediately before use.

66. Method
• 1. Bring section to water.
• 2. Celloidinize in 0.5% celloidin, drain and harden in distilled water
for 1 minute.
• 3. Impregnate in preheated 55–60°C silver solution for 90–105
minutes.
• 4. Prepare and preheat developer in a water bath.
• 5. Treat with developer for 30 minutes at 55°C. Sections should be
golden-brown at this point.

67. • 6. Remove from developer and rinse in tap water for several minutes
at 55–60°C, then in buffer at room temperature.
• 7. Tone in 0.2% gold chloride.
• 8. Dehydrate, clear, and mount.
Results
 Spirochetes black
 Background golden/yellow

68. Warthin-Starry method showing
spirochetes

69. H&E of spirochetosis of colon

70. SILVER METHENAMINE STAIN FOR
FUNGI
o GMS staining is a silver staining technique for demonstrating
fungi in tissue sections.
o It is primarily based on staining the polysaccharides in fungal
cell walls.
• PRINCIPLE
This method depends upon the reduction of the silver by the
aldehyde groups produced after oxidation of fungal wall
components with chromic acid.

71. Solution
REAGENTS:
4% chromic acid commercially available
1% sodium bisulfite
5% sodium thiosulfate
0.2% light green

72. 0.21% silver nitrate, stock solution A
Silver nitrate 2.1 g
Distilled water 1000 ml
Methenamine-sodium borate, stock solution B
Methenamine 27 g
Sodium borate decahydrate (borax) 3.8 g
Distilled water 1000 ml
Methenamine-silver sodium borate working solution Equal parts of
solutions A and B.

73. Method
• 1. Bring section to water.
• 2. Oxidize in 4% aqueous chromic acid (chromium trioxide) for 30
minutes.
• 3. Wash briefly in distilled water.
• 4. Dip briefly in 1% sodium bisulfite.
• 5. Wash well in distilled water
• 6. Place in preheated (56–60°C water bath) working silver solution for
15–20 minutes. If section is ‘paper bag brown’ then rinse in distilled
water.

74. • 7. Tone in 0.1% gold chloride for 5 seconds. Rinse in distilled water.
• 8. Place in 5% sodium thiosulfate for 5 seconds.
• 9. Rinse well in running tap water.
• 10. Counterstain in light green solution until a medium green
(usually 5–15 seconds).
• 11. Dehydrate, clear and mount

75. • Fungi -black
• Hyphae and yeast-form cells sharply delineated in black of fungi
• Background-pale green

76. Fungi on H&E

77. GMS showing light green counterstain for
Histoplasma capsulatum, dimorphic fungus.

78. Periodic - acid – Schiff Stain (PAS)
Principle:
Certain tissue elements are oxidised by the periodic
acid. One of the reaction products is an aldehyde.
which combine with Schiff's reagent to form an
insoluble magenta compound
Periodic acid

Tissue element Dialdehydes
( containing 1:2 glycol groups
or 1:2 aminohydroxyl groups ) Schiff reagent
Coloured end product

79.  Procedure :
Bring sections to water

Treat with periodic acid for 5 min

Wash with tap water for 5 min

Stain with Schiff’s reagent for 8-10 min

Wash with tap water for 5 min

counterstain with Haematoxylin for 3 min

Wash with tap water for 5min

Dehydrate, clear & mount

80. Results:
 PAS positive substance -
Bright red / pink
 Nuclei - Blue

81. Thank you