This document discusses histopathology staining techniques. It defines histopathology as staining tissue samples to examine cellular and intracellular structures microscopically. The two main categories of stains are natural dyes derived from natural resources, and artificial dyes produced through chemical reactions. Hematoxylin is a commonly used natural dye that stains nuclei blue, while eosin is an artificial dye that counterstains cytoplasm pink. The document outlines hematoxylin and eosin staining as well as other histological stains and their characteristics.

2. 2
Histopathology

3. Definition
3
 Staining is used to highlight important features of the tissue as well as to
enhance the tissue contrast.
 Due to their transparent nature, the cellular and intracellular structure of
tissue samples can not be microscopically examined until they are colored
by dyes.
 Dyes used to stain tissue samples in the histology laboratory for
microscopic evaluation are called biological dyes or biological stains.
Biological dyes can be grouped into the following two categories:
 Natural: Dyes that are derived from natural resources. The most
important natural dye in the histopathology laboratory is hematoxylin.
 Artificial: Dyes that are derived through chemical reactions. Artificial
dyes greatly outnumber natural dyes.

4. STAINING
 The sections, as they are prepared, are colourless
and different components cannot be appreciated.
 Staining them by different coloured dyes, having
affinities of specific components of tissues, makes
identification and study of their morphology
possible.
 If unstained slide is observed under microscope
we can make out only nucleus and cell boundary.
 Staining is a technique used to highlight
important features of the tissue as well as to
enhance the tissue contrast.

5. Dyes used in staining
1.According to source
a. Natural –Haematoxylin, Carmine
b. Synthetic-Aniline base and derived from coal tar
2. Affinity to tissues
a. Acidophilic -These are anionic dyes and stain mainly
cytoplasm, eosinophilic granules
b. Basophilic - These are cationic dyes and stain nuclei,
basophili granules or bacteria
3. Chemical composition
a. Thiazines
b. Azo-dyes
c. Rosailins Histopathology

6. Dyes classification
Histopathology

7. Cont.’
Histopathology

8. Counter stains
 A counter stain is the application to the original stain,
usually nuclear, of one or more dyes that by contrast will
bring out difference between the various cells and
tissues.
 A heavy counterstain is to be avoided lest it mask the
nuclear stain. It can be done either by using dilute stain
or cutting down the staining time.
 Some counterstains which are acidic may lighten or
remove the nuclear stains.
Histopathology

9. Mordants
 Substance that causes certain staining reactions to take place by
forming a link between the tissue and the stain. The link is referred
as lake. Without it, dye is not capable of binding to and staining the
tissue.
e.g. Ammonium and Potassium alum for haematoxylin
 The mordant dye complex ‘lake’ combines with tissue to form
tissue-mordant-dye complex, which is insoluble.
Histopathology

10. Mordant-dyes application
Mordant is applied first, followed by the
dye.
e.g Heidenhain’s iron hematoxylin
Mordant and dye are mixed together and
then applied.
Commonly done in histo-technology
e.g Alum hematoxylin solutions
Dye applied first, followed by the mordant.
Hardly done in histo-technology.
Histopathology

11. Accentuators:
Accentuators:
Substances which increase the staining power of dye.
 They increase the intensity & selectivity of stain.
 These are substances that causes an increase in the selectively or in the staining
power of dye. Thus they lead to more intense staining.
e.g KOH in Lofflers methylene blue phenol in carbol fuschin &
carbol thionin.
Accelerators
Accentuators used in metallic impregnation technique for the
nervous system.
e.g chloral hydrate
Trapping agents
Agents which holds dye combination with tissue or bacteria .
e.g tannic acid/iodine
Histopathology

12. Types of stains reactions
Absorption or direct staining – tissue penetrated
by dye solution.
Indirect staining – using intermediate treatment
with mordant
Physical staining – simple solubility of dye in
element of tissue.
Chemical staining – formation of new substance
e.g. PAS
Adsorption phenomenon – accumulation on the
surface of the compound.
Histopathology

13. Types of stains methods
Vital
Routine
Special
Regressive
Progressive
Histopathology

14. Types of stains methods
Histopathology

15. Cont.’
Histopathology

16. Hematoxylin
Hematoxylin solutions can be arbitrarily
classified
according to which mordant is used:
Alum hematoxylins
Iron hematoxylins
Tungsten hematoxylins
Molybdenum hematoxylins
Lead hematoxylins
Hematoxylin without mordant.
Histopathology

17. History of hematoxylin
 Waldeyer firmly established the use of
hematoxylin in histology in 1862.
 Bohmer combined hematoxylin with
alum as a mordant and obtained more
specific staining in 1864.
 Heidenhan introduced his classical Iron-
Alum- Hematoxylin method used as a
standard technique in cytology.
 Ehrlich overcame the instability of
hematoxylin and alum by adding glacial
acteic acid and simultaneously
produced his formula for hematoxylin
used today. Histopathology

18. i. Alum Hematoxylin
Used routinely in the hematoxylin and eosin stain and produce
good nuclear staining.
The mordant is aluminum in the form of ‘potash alum -
aluminum potassium sulfate or ‘ammonium alum -aluminum
ammonium sulfate
Alum Hematoxylin can be used progressively or regressively.
Haematoxylin and eosin staining :is the most common staining
technique in the histopathology. This uses a combination of two
dyes, haematoxylin and eosin used for demonstration of nucleus
and cytoplasmic inclusions in clinical specimens.
Histopathology

19. Classification of Alum Hematoxylin
Ehrilch’s haematoxylin
Mayer’s haematoxylin
Harris’ haematoxylin
Gill’s haematoxylin
Cole’s haematoxylin
Delafield’s haematoxylin
Carazzi’s haematoxylin
Histopathology

20. Ehrilch’s haematoxylin ( 1886)
Naturally ripened strong alum hematoxylin.
Stains nuclei intensely and crisply - stained sections fade
much more slowly .
Stains mucin in salivary glands, cartilage and cement lines of
bones
Suitable for tissues subjected to acid decalcification.
Suitable for tissues that have been stored for a long period in
formalin fixatives which have gradually become acidic over the
storage period.
Suitable for Bouin’s fixed tissue.
not ideal for frozen sections.
Histopathology

21. Mayer’s haematoxylin (1903)
A widely used hematoxylin stain.
Chemically ripened with sodium iodate.
More vigorous in action than Ehrlich’s hematoxylin.
Used as both progressive and regressive stain.
Used as a nuclear counterstain in the demonstration of
glycogen (PAS,mucicarmine) in various enzyme histological
techniques.
Stain applied for a short period - 5-10 mins until nuclei
are stained and then blued without any differentiation which
might destroy/decolor the stained cytoplasmic components.
Histopathology

22. Harris’ haematoxylin (1900)
This alum hematoxylin was traditionally chemically ripened
with mercuric oxide (sodium or potassium iodate is frequently
used as a substitute for oxidation).
It gives particular clear nuclear staining.
It is used as a regressive stain in routine histology practice.
It is used as a progressive stain in diagnostic exfoliative
cytology.
When using it as a progressive stain, an acetic acid-alcohol
rinse provides a more controllable method in removing excess
stain from tissue components and the glass slide.
Histopathology

23. Gill’s haematoxylin (1974)
Available in 3 concentrations –
◦ Gill’s I (normal)
◦ Gill’s II (double)
◦ Gill’s III (triple) most concentrated.
More frequently used than Mayer’s hematoxylin for
routine H&E staining.
More stable than Harris’s hematoxylin, as auto-
oxidation is inhibited to the extent.
Histopathology

24. Cole’s haematoxylin (1943)
Alum hematoxylin, artificially ripened with an alcoholic
iodine solution.
Has good keeping qualities and is suitable for use
especially in sequence with celestine blue unlike Ehrlich’s
hematoxylin
Preparation
Hematoxylin 1.5 g
Saturated aqueous potassium alum 700 ml
1% iodine in 95% alcohol 50 ml
Distilled water 250 ml
Histopathology

25. Carazzi’s haematoxylin (1911)
Alum hematoxylin which is chemically ripened using potassium
iodate
Used as a progressive nuclear counterstain.
Largely confined to use with frozen sections because it gives
excellent and clear nuclear staining with a very short staining time.
Preparation –
Hematoxylin 5 g
Glycerol 100 ml
Potassium alum 25 g
Distilled water 400 ml
Potassium iodate 0.1 g
Histopathology

26. A naturally ripened alum hematoxylin with
similar longevity to
Ehrlich’s hematoxylin.
Preparation
Hematoxylin 4 g
95% alcohol 125 ml
Saturated aqueous ammonium alum 400
ml (15 g/100 ml)
Glycerin 100 ml
Delafield’s haematoxylin (1885)
Histopathology

27. ii. Iron Hematoxylin
Weigert’s Hematoxylin.
Heidenhain’s Hematoxylin.
Verhoeff’s Hematoxylin.
Loyez Hematoxylin.
Histopathology

28. Weigert’s Hematoxylin
An iron hematoxylin used as a nuclear stain in techniques
where acidic staining solutions are applied to the sections
subsequently
e.g Van Gieson stain – picric acid is a constituent which have
marked decolorizing action on nuclei stained with alum
hematoxylin.
It is a useful stain, with eosin, for CNS tissues.
Preparation
a) Hematoxylin solution
Hematoxylin 1 g
Absolute alcohol 100 ml
This is allowed to ripen naturally for 4 weeks before use.
b) Iron solution
30% aqueous ferric chloride (anhydrous) 4 ml
Hydrochloric acid (concentrated) 1 ml
Distilled water 95 ml

29. Heidenhain’s Hematoxylin.
This iron hematoxylin uses ferric ammonium sulfate as oxidant/mordant
It is used as the differentiating fluid.
It is a cytological stain.
It is used regressively.
After staining, all components are black or dark gray-black.
The hematoxylin staining is removed progressively from different tissue
structures at different rates using the iron alum solution.
It may be used to demonstrate
Chromatin
Chromosomes
Nuclei
Centrosomes
Mitochondria
Muscle striations
Myelin

30. Loyez Hematoxylin.
This iron hematoxylin uses ferric ammonium sulfate as
the mordant.
Differentiation is by Weigert’s differentiator (borax and
potassium ferricyanide).
Used to demonstrate myelin.
Can be applied to paraffin, frozen, or nitrocellulose
sections.
Histopathology

31. This iron hematoxylin is used to demonstrate elastic
fibers after all routine fixative.
Ferric chloride is included in the hematoxylin staining
solution, together with Lugol’s iodine, and 2% aqueous
ferric chloride is used as the differentiator.
Coarse elastic fibres stain black, but the staining of fine
fibers may be less than satisfactory.
The differentiation step is critical to the success of this
method.
Verhoeff’s Hematoxylin.
Histopathology

32. iii. Tungsten hematoxylins
 Widely used tungsten hematoxylin is PTAH (Phosphotungstic
acid hematoxylin Technique).
 Used to demonstrate fibrin, muscle striations, cilia and glial
fibres.
 Myelin can also be demonstrated Widely used as a CNS stain.
Preparation
PTAH solution using haematin
Haematin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
Stain is ready to use immediately, but short-lived.
PTAH Solution (KMnO4)
Haematoxylin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
0.25% Aqueous KMnO4 25 ml
Peak staining activity after 7 days

33. Tungsten hematoxylins
Results
• Muscle striations / neuroglia fibres / fibrin /
amoeba – ---------------------Dark Blue.
• Nuclei/cilia/RBC – ---------Blue
• Myelin – ---------------------Lighter blue
• Collagen /Osteoid / Cartilage / Elastic fibres – ---
---------------------------------Deep brownish red.
Cytoplasm – --------------------Pale pinkish brown.
Histopathology

34. iv. Molybdenum hematoxylins
Hematoxylin solution using molybdic acid as
mordant.
Rare stain
Used in demonstration of collagen, coarse reticulin.
Also stains Argentaffin cell granules.
Preparation
a. Hematoxylin solution
Hematoxylin 2.5 g
Dioxane 49 ml
Hydrogen peroxide 1 ml
b. Phosphomolybdic acid solution
Phosphomolybdic acid 16.5 g
Distilled water 44 ml
Diethylene glycol 11 ml
The resultant dark violet solution is allowed to stand for 24 hours before use.
Histopathology

35. Molybdenum hematoxylins
Results –
Collagen and coarse reticulin - violet to
black
Argentaffin cells - black
Nuclei pale - blue
Paneth cells - orange
Tissue fixed in dichromate do not give good
results.
Histopathology

36. v. Lead hematoxylin
Used in demonstration of granules in endocrine cells of ailmentary
tract and other regions.
Most practical diagnostic application is in identification of
endocrine cells in tumors of doubtful origin.
Also used in localization of gastrin secreting cells in stomach.
Histopathology

37. EOSIN
Xanthine dyes which stains connective tissue and
cytoplasm in varying intensity and shades (red to pink).
Available in the following types :
Eosin Y ( Eosin Yellowish, Eosin water soluble) –
most widely available.
Ethyl Eosin (Eosin S, eosin alcohol soluble).
Eosin B ( Eosin Bluish, Erythrosine B).
Ethyl eosin and eosin B are now rarely used, although
occasional old methods specify their use – e.g the Harris
stain for Negri bodies.Histopathology

38. EOSIN
Eosin Y
Most commonly used eosin.
Readily soluble in water.
Satisfactorily soluble in alcohol.
Preparation
Eosin Y, water soluble 5 gm
Distilled water 1000 ml
Crystals of Thymol added to inhibit fungal
growth.
Addition of little acetic acid (0.5 -1000 ml stain)
sharpens the staining.

39. The Hematoxylin and eosin
staining technique
Principle
Hematoxylin and Eosin are principle stains used for demonstration
of nucleus and cytoplasm.
Alum acts as a mordant and the hematoxylin containing alum stains
the nucleus light blue which turns red in the presence of acid.
The cell differentiation is achieved by treating the tissue with acid
solution.
The counterstaining is performed using eosin which imparts pink
color to cytoplasm.
Histopathology

40. H & E Stain of parraffin section
1. removal of wax.
2. hydration with graded alcohols.
3. staining.
4. differentiation
5. blueing
6. counterstain with eosin
7. dehydration through graded alcohol.
8. clearing in xylene
9. mounting under a cover slip.
Histopathology

41. H & E staining
Differentiation
 In regressive staining differentiation is the removal of washing out of the
excess stain until the colour is retained only in the tissue components to be
studies.
 Differentiation may use pH in acidic differentiation we use HCL,acetic
acid,etc.
Ripening or oxidation
 It is conversion inactive natural extract like hematoloxylin to active
principle(hematin) by oxidation
 It is essential that correct amount of oxidation occurs
Blueing
 After stain with hematoxylin the slide is kept in running tap water till it
become deep blue in color.blueing take 5 to 10 minutes and make the
nuclear morphology more clear

42. Haematoxylin and Eosin
staining
Procedure
♦ Deparaffinize in hot air oven.
♦ Hydrate the section.
i) 3 dips in xylene (2 Min. each)
ii) 3 dips in acetone / alcohol (2 Min. each)
iii) In running tap water for 5 Minutes.
♦ Mayer's haemotoxylin for 15 minutes.
♦ Wash in running tap water for 20 minutes
♦ Counter stain with eosin for 2 minutes
♦ Dehydrate the section in 95% and absolute alcohol/ acetone
2 changes (2minutes each).
♦ Clear in xylene 3 changes (2 minutes each)
♦ Mount in DPX
Histopathology

43. Haematoxylin and Eosin staining
Results
Nucleus - blue
Cytoplasm and background - pink
Histopathology

44. 4
4
SPECIAL STAININGS
Histopathology

45. 1.Van gieson method
This stain is used for staining of collagen fibres
Principle :
 It is based on the differential staining of collagen and other tissues
(e.g muscle) depending upon the porosity of tissue and the size of
the dye molecule.
 In the routine staining method collagen, elastic fibres and smooth
muscle appear pink or reddish in colour.
 In the Van Gieson stain, collagen and most reticulin stain selectively
with acid aniline dyes (acid fuchsin).
 Picric acid acts as counter stain for muscle and cytoplasm and form
complex with the dyes. This complex has special affinity for
collagen.
Histopathology

46. Cont.’
Reagents
1. Solution A
(a) Haematoxylin 1.0 gm
(b) Alcohol 95% 100 ml
2. Solution B
(a) 29% (w/v) ferric chloride 4 ml
(b) Conc. Hydrochloric acid 1.0 ml
(c) Distilled water 95.0 ml
3. Weight's iron heamatoxylin solution :mix equal quantities of solution
A and solution B. Colour of this reagent should appear violet black.
4. Van Gieson’s solution
(a) Saturated aqueous picric acid – 10 ml
(b) 1% (m/v) acid fuchsin – 1.5ml
It should be freshly prepared
Histopathology

47. Cont.’
Procedure
(1) Deparaffinize with xylene
(2) Hydration take sections to water
(3) Stain with Weigert’s haematoxylin for 20-40
minutes
(4) Wash in distilled water
(5) Van Gieson stain 1-3 min
(6) Rinse well in distilled water
(7) Dehydrate in absolute alcohol (2 changes)
(8) Clear in xylene (2 changes)
(9) Mount in DPX
Histopathology

48. Results
Results
1. Collagen --------------------------------Red
2. Muscle and
Cornified epithelium -----------------Yellow
3. Nuclei -----------------------------------Blue to Black

49. Clinical significance
 Several pathological changes are associated with
cellular changes in connective tissues.
 Histological diagnosis of collagen diseases is
based on the study of the section of various
connective tissues.
Histopathology

50. 2.Gomori’s method for reticulum
This is used for demonstration of reticulin
Principle :
 In the connective tissue, reticulin appears as a fibrillary extracellular framework.
 Reticular fibres have low natural affinity for silver salts and require pretreatment
with heavy metal solutions like fernic ammonium sulphate to enhance the
selectivity of impregnation Silver in alkaline solution is in a state readily able to
precipitate as metallic silver.
 Upon treatment with a reducing agent, silver taken up by the tissue in unreduced
form is, converted to metallic silver which is deposited at the sensitised site.
Histopathology

51. Cont.’
Reagents
1. Ammonical silver solution
To 10 ml of 10% silver nitrate solution add 2.5 ml of 10% aqueous
solution of potassium hydroxide, add 28% ammonium hydroxide drop
by drop while shaking the container continuously until the precipitate
in completely dissolved. Add again 4 drops of silver nitrate solution for
every 10 ml of silver nitrate used. Make the solution with distilled
water to twice its volume use acid clean glassware
2. 0.5% potassium permanganate –
Potassium permanganate –0.5 gm
Distilled water 100 ml
Histopathology

52. Cont.’
3. 2% potassium metabisulphite
Potassium metabisulphate 2.0 gm
Distilled water 100 ml
4. 2% ferric ammonium sulfate solution
Ferric ammonium sulphate 2 gm
Distilled water 100 ml
5. 20% formation solution
Formaldehyde 20 ml
Distilled water 80 ml
6. 0.2% gold chloride solution
Gold chloride solution 1% - 10 ml
Distilled water - 40 ml
7. 2% sodium thiosulphate solution
sodium thiosulphate 2 gm
Distilled water 100 ml
Histopathology

53. Cont.’
Procedure
1. Deparaffinze and hydrate to distilled water
2. Oxidize in potassium permagnate solution for 1 minute
3. Rinse well in tap water – 2 min
4. Differentiate with potassium metabisulphite solution for 1 minute.
5. Wash in tap water for 2 min
6. Sensitize in Ferric ammonium sulphate solution for 1 min.
7. Wash in tap water for 2 minutes follow with two changes of distilled water 30 seconds 8.
Impregnate in the silver solution for 1 minute
9. Rinse in distilled water for 20 seconds
10. Reduce in formalin solution for 3 minutes
11. Wash in tap water for 3 minutes
12. Tone in gold chloride solution for 10 minutes
13. Rinse in distilled water
14. Reduce in potassium metabisulfite solution for 1 minute
15. Fix in sodium thiosulfate solution for 1 minute.
16. Wash in tap water for 2 minutes.
17. Dehydrate in 95% alcohol, absolute alcohol and clear in xylene 2 changes
18. Mount in DPX.
Histopathology

54. Results
Results
Reticulin fibers –----------------------------- black
Background –--------------------------------- grey
Histopathology

55. Clinical significance
Clinical significance
 This type of staining is used for tumors of
uncertain origin.
 The pattern of deposition of reticulin in a tumor
is characteristic to identify the origin of tumor.
 Mild degree of fibrosis in organs can also be
recognized by reticulin stain especially in liver to
assess cirrhosis.
Histopathology

56. 3.McManus for glycogen (pas)
 This stain is used for demonstration of glycogen and muco
polysaccharides.
 PAS positive substances are glycogen, amyloid, colloid,
neutral mucin, basement membranes and hyaline cast
Principle :
 Tissue structures like liver & heart, striated muscles are
studied by Periodic acid Shiff stain.
 Periodic acid reacts with aldehyde group of the carbohydrates
and afterwards reaction with the schiff’s reagent produces a
red or purple red colour.
Histopathology

57. Reagents
Reagents
1. 0.5% w/v periodic acid solution
2. Schiff’s reagent
(a) Dissolve 1.0 gm of basic fuchsin in ≈100 ml of boiling
distilled
water cool to about 60 degrees and filter.
(b) Add 20 ml of 0.1 N hydrochloric acid, cool further and add
1.0 gm of sodium metabisulphite and mix well.
(c) Keep in the dark for 24-48 hours. When the solution
becomes
straw coloured, add 300 mg of activated charcoal, shake
vigorously, filter and store.
3. 1 N Hydrochloric acid
4. 0.1 gm of light green in 100 ml of 0.1% (v/v) acetic acid.
5. Harris haematoxylin stain Histopathology

58. Procedure
Procedure
1. Deparaffinize and hydrate to distilled water.
2. Oxidize in periodic acid solution for 5 minutes
3. Rinse in distilled water
4. Schiff’s regent solution for 15 minutes.
5. Wash in running water for 10 minutes for pink colour to develop.
6. Harris haematoxylin for 6 minutes or light green counter stain for a few
seconds.
7. Wash in running water.
8. Differentiate in 1% acid alcohol solution 3-10 quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue the sections
11. Wash in running water for 10 minutes
12. Dehydrate in 95% alcohol, absolute alcohol, clear in xylene two changes
each.
13. Mount in DPX. Histopathology

59. Results
Results
With hematoxylin counterstain
1. Nuclei --------------------------------------blue
2. Glycogen, mucin, hyaluronic acid,
reticulin, colloid droplets, amyloid
infiltration, thrombi. –------------------- purple red
3. Fungi – -------------------------------------Red
4. Background –------------------------------ pale green (with light green counter staining).
Histopathology

60. 4.Mayer’s mucicarmine method
To demonstrate mucin in a tumor or epithelium
Principle :
 The rationale for specificity of mucicarmine for mucin is not fully
understood by the probable mechanism is that the aluminium salts in
the solution form a chelate compound with carmine, thus producing
a net positive charge on the molecule and consequent binding to the
tissue polyanions.
 The compound has a large molecular size and allows that dye
complex to penetrate and bind to acidic substrates of low density
like mucins other acidic substances like nucleic acid are of high
density hence exclude the mucicarmine.
Histopathology

61. Reagents
Reagents
(1) Weigert’s Iron haematoxylin solution
(a) Solution A
Hematoxylin crystals 1.0 gm
Alcohol 95% 100 ml
(b) Solutions B
Ferric chloride 29% aqueous 4 ml
Hydrochloric acid conc. 1 ml
Distilled water 95 ML
Working solution – Add equal parts of solution A and solution B
(2) Mucicarmine solution
Carmine 1 gm
Aluminium chloride anhydrous 0.5 gm
Distilled water 2 ml
(3) 0.25% Metanil yellow solution
Melanil Yellow - 0.25 gm
Distilled water - 100 ml
Glacial acetic acid - 0.25 ml
Histopathology

62. Procedure
1. Deparaffinize and hydrate to distilled water.
2. Working solution of Weigert’s Haematoxylin for 7
minutes.
3. Wash in running water for 10 minutes
4. Diluted mucicarmine solution for 60 minutes
5. Rinse quickly in distilled water.
6. Metanil yellow solution for 1 minute.
7. Rinse quickly in distilled water
8. Dehydrate in 95% alcohol, absolute alcohol and clear in
xylene 2 changes each.
9. Mount with DPX.
Histopathology

63. Results
Results
Mucin --------------------------------------------------------------- Deep rose to red.
Capsule of cryptcoccus----------------------------------------- deep rose to red.
Nuclei --------------------------------------------------------------- Black
Other tissue elements ------------------------------------------ yellow

64. 5.FITE’s method for acid fast
organisms
to demonstrate weakly acid fast bacilli like mycobacterium Lepre and
Nocardia.
Principle:
 The mycobacteria are the bacteria which are relatively resistant to
staining because of the lipid capsule which surrounds them, when
stained by a strong stain (eg carbol fuchsin) they resist
decolourization by acid.
 In this method a red dye carbol fuchsin is forced into the bacteria
and other structures with heat and is then removed from other
structures with acid or alcohol ; tubercle bacillus because of lipid
capsule however resists decolourization.

65. Procedure
Procedure
1. Deparaffinize through 2 changes of xylene peanut oil solution for 12
minutes each.
2. Drain wipe off excess oil & blot to opacity.
3. Carbol fuchsin solution for 30 minute
4. Wash in tap water for 3 minutes & blot dry.
5. Differentiate slides with sulfuric acid solution till sections are faint
pink for 1-2 minutes.
6. Wash in running water for 3 minutes
7. Counterstain lightly with working methylene blue solution (few
seconds).
8. Rinse off excess methylene blue in tap water.
9. Blot and let it stand for few minutes to air dry thoroughly.
10. Dip slides in xylene before mounting.
11. Mount with DPX

66. Results
Results
M. Leprae ------------------------------------------------------- red
Nocardia filaments -------------------------------------------- blue grey
Histopathology

67. 6.Gomori's method for iron
Staining of section for haemosiderin (a tissue
pigment)
Principle:
 Haemosiderin is a brown granular pigment
occurring at the site of previous haemorrhage.
 It is a product of the breakdown of haemoglobin.
 It reacts with potassium ferrocyanide in acid
medium and yields a Prussian blue colour.

68. Reagents
1. Solution A - 20% hydrocholroc acid solution (stock)
Hydrochloric acid (conc.) 20ml
Distilled water 80ml
Distilled water 80ml
2. Solution B - 10% potassium ferrocyanide solution (stock)
Potassium ferrocyanide 10 gm
Distilled water 100ml
3. Acidified potassium ferrocyanide solution.
Prepare fresh by mixing equal part of solution A and solution B and
leave for 20 minutes
4. Nuclear fast red stain.
Dissolve 5.0 gm of aluminium sulphate is hot distilled water and add
0.1 gm of nuclear fast red mix well and filter.
Add a crystal of thymol as a preservative.
Histopathology

69. Procedure
Procedure
1. Deparaffinize and hydrate to distilled water
2. Put the slides in acidified potassium ferrocyanide
solution for 30 minutes
3. Rinse in distilled water
4. Counterstain in nuclear fast red solution for 5 minutes.
5. Rinse in distilled water
6. Dehydrate in 95% alcohol, absolute alcohol, and clear in
xylene 2 changes each
7. Mount with DPX
Histopathology

70. Results
Results
Iron pigments --------------------------------------------------------- bright blue
Nuclei ------------------------------------------------------------------- Red
Cytoplasm ------------------------------------------------------------- Light pink.
Histopathology

71. 7.Von kossa's method for
calcium
Staining of the sections for calcium
Principle –
 The calcium salts in the form of phosphates, carbonates
or oxalates occur as components of oxalates occur as
components of laminated concentration in various
organs such as the kidneys, urinary bladder, lymph
nodes.
 Von Kossa's technique in a metal substituted for calcium
by metallic salt formation with the anion of the calcium
salt.
Histopathology

72. Reagent
Reagent :
1. 5% W/v silver nitrate solution (stored in amber coloured
bottle)
2. 5% W/v sodium thosulphate solution
3. Nuclear fast red stain. Dissolve 0.1 gm nuclear fast red
no 100 ml of
5% solution of aluminium sulphate with the aid of heat.
Cool filter add grain of thymol as a preservative.
Histopathology

73. Procedure
Procedure: (Use control slides and chemically clean glassware)
1. Deparaffinize and hydrate to distilled water
2. Silver nitrate solution expose to bright sunlight or under the light
of 100 watt bulb for 60 minutes
3. Rinse in Distilled water
4. 5% sodium thiosulfate solution for 2 minutes
5. Rinse well in distilled water
6. Counter stain with nuclear fast led solution for 5 minutes
7. Rinse in distilled water
8. Dehydrate in 95% alcohol, absolute alcohol and clear in xylene 2
changes each
9. Mount with DPX
Histopathology

74. Results
Results
Calcium salt ------------------------------------------------------ Black
Nuclei -------------------------------------------------------------- Red
Cytoplasm -------------------------------------------------------- Light pink
Histopathology

75. 8.Hall's method for bilirubin
To stain the bilirubin or bile pigment in tissue section
Principle
- Bilirubin is oxidized to biliverdin and stains olive green to
emerald green depending on the concentration of bilirubin in
solution.
Histopathology

76. Reagents
Reagents
1. Fouchet's Reagent
Trichloroacetic acid --------------------------------------------------- 25.0gm
Distilled water ------------------------------------------------------------100ml
Mix and add 10 % ferric chloride
2. 10% Ferric chloride solution
Ferric chloride --------------------------------------------------------- 10gm
Distilled water --------------------------------------------------------- 100ml
3. Van Gieson's solution
(a) Saturated aqueous picric acid ---------------------------------10ml
(b) 1% w/v acid fuchsin -----------------------------------------------1.5ml
Histopathology

77. Procedure
Procedure
1. Deparaffinize and hydrate to distilled water
2. Fouchet's reagent for 5 minutes
3. Wash in running water, then in distilled water
4. Van Gieson's solution for 5 minutes
5. Dehydrate in 95% alcohol, absolute alcohol and
clear in xylene 2 changes each
6. Mount with DPX
Histopathology

78. Results
Results
Biliverdin ------------------------------------------------green
Collagen -------------------------------------------------red
Muscle ---------------------------------------------------YellowHistopathology

79. 9. Sudan black/ oil red O stain
Both these stains are used for demonstration of fat
Principle:
 Both SB and oil red O staining are based on physical
combination of the stain with fat. It involves
differential solubility of stain in fat because these
stains are more soluble in fat than the solvent in which
these are prepared.
 The stain leaves the solvent and goes into the fatHistopathology

80. Result
Result:
Oil red O
fat -------------------------------------------------------------- bright red
nuclei ---------------------------------------------------------- blue
Sudan black
fat ------------------------------------------------------------- black
nuclei --------------------------------------------------------- red
Histopathology

81. 10.Methyl violet stain
 This is a metachromatic stain i.e. the tissues are stained in a colour
which is different from the colour of the stain itself.
 It is used for demonstration of amyloid in tissue. Other
metachromatic positive substances are mucin and hyaline
Principle:
 This depends upon the type of dye(stain) used and character of the
tissue which unites with dye
 Tissues containing S04,PO4,or COOH groups react with basis dyes
and cause their polymerisation which in turn leads to production of
colour different from the original dye
Histopathology

82. Result
Metachromatic positive tissue: --------------- red to violet
Other tissues and background : --------------- blue
Other Metachromatic stains used are crystal violet and toluidine blue
Histopathology

83. Knowledge checking
1) Which are the special stains used for
demonstration of fat in tissue sections?
2) Van Gieson stain is used for which purpose and
what is a positive colour?
3) What is meant by silver impregnation?
4) What are the applications of PAS stain?
5) What are the main diagnostic stains used for
demonstration of amyloid?
6) What is the principle of prussian blue stain?
Histopathology

84. Question?
Histopathology

85. References
1. Bancroft, J.D. and Stevens, A.: theory and practice of histological
techniques ed.3, Churchill livingstone inc. 1990. Edinburgh.
London, Melbourne and New York.
2. Lillie, R.D.: Histopathologic technique and practice histo-
chemistry ed. 3, New York, 1965 McGraw Hill Book co.
3. Manual of histologic and special staining techniques ed. 2, New
York, 1960, The Blakiston Division McGraw Hill Book Co.
Histopathology

86. BOOKS