h n e stains

1. HEMATOXYLIN
AND EOSIN
STAINS:

2. CONTENT
 Hematoxylin
• Principles of hematoxylin
• Oxidation
• Mordant
 Classification of hematoxylin
 Alum hematoxylin
• Method of use of alum hematoxylin
• Bluing
• Differentiation
• Deterioration of the hematoxylin
• Types of alum hematoxylin
• Staining time with alum hematoxylin
• Disadvantages of alum hematoxylin

3.  Iron hematoxylin
 Tungsten hematoxylin
 Lead hematoxylin
 Tungsten hematoxylin
 Molybedenum hematoxylin
 Hematoxylin without mordant
 Weigert Pal technique – of mordanting blocks
 Eosin
• Introduction
• Types of eosin commerrcially available
• Substitutes for eosin
• Differentiation
• Difficulties encountered

4. Staining:
• Is the process of coloring cells, cellular
constituent & tissue fibers to facilitate optical
differentiation by microscopic examination.
• Is the union between a colored dye & a tissue
substrate which resists simple washing.
.

5. • It involves visual labeling of some entity by
attaching, or depositing in its vicinity a marker
of characteristic color or shape.
• Stain is the marker or reagent used to
generate the marker.

6. 6
HC CH
CH
CH
HC
HC
Chromophore
Chromogen
Principles of dye chemistry:
• All the dyes have an aromatic hydrocarbon benzene as a central
component.

7. CHROMOGEN AUXOCHROME SUBSTRATE
• A dye/stain is a coloured compound that binds to a substrate.
• It consists of a chromogen (colour) and auxochrome ( substrate
binding component).
Chromogen = benzene derivative + chromophore (colouring agent).

8. CHROMOGEN AUXOCHROME
SUBSTRATE
• Auxochrome : give +ve or –ve charge to the chromogen.
• The ionized stain is capable of binding to cell structures with
opposite charges.

9. Electrostatic bonding
Hydrogen bonding
Van der Waal’s forces
Covalent bonding
Hydrophobic bonding
Dye aggregation
Tissue permeability
STAINING MECHANISMS
9

10. ELECTROSTATIC BONDING
‘Salt linkage’ & ‘Ionic bonding’
1. Elecrostatic bonding:
The affinity between opposite ionic groups of dye & tissue.
Forces involved – Coulombic attraction
Dyes are classified as
Acidic dyes – Have a negative charge
Basic dyes – Have a positive charge
10

11. 11
Acid dyes
Anionic Chromogen Cationic Auxochrome
Basic Dyes
Cationic Chromogen Anionic Auxochrome
Dyes thus carry an organic, charged moeity & an inorganic salt with an
opposite charge

12. Binding of dye to tissue:
• When dye goes into tissue, they ionize or
dissociate.
• Acid dyes provide available anions
(chromogen) & cations that represent the
auxochrome or salt.
• Basic dyes provide cationic chromogen &
anionic auxochrome.
• The degree of ionization is pH dependent.

13. Reactive tissue groupings consist of
• Bound moiety of one charge
• Mobile moiety of opposite charge
Staining occurs when a chromogen of one charge attracts to
bound tissue moiety of opposite charge
13

14. STAINING OF BASIC CYTOPLASMIC PROTEIN BY ACID DYE
EOSIN
Tissue Eosin Stained tissue Free salt
NH2 Cl + Chromogen Na = NH2 Chromogen + Na Cl
14
An acid dye has a coloured acid radical which attaches to a basic
tissue component

15. WHY ONLY DNA STAINS WITH HEMATOXYLIN
AND NOT RNA
The close proximity of phosphoric acid groups in DNA is
responsible for stronger staining of nuclear chromatin
As against almost unstained less dense carboxyl side
groups of dicarboxylic acids in cytoplasmic RNA.

16. INTRODUCTION
Hematoxylin & eosin stain
• Is the most widely used histologic stain.
• Hematoxylin component stains  cell nuclei blue/black with good
intranuclear detail.
• Eosin stains  cell cytoplasm & most connective tissue fibers in
varying shades & intensities of pink, orange & red.

17. HEMATOXYLIN
It is extracted from the core
of the tree
HAEMATOXYLON CAMPECHIANUM.
 Hematoxlin - Greek word
Haimato(blood) and
Xylon(wood), reffering to its
dark red color in natural state
and to its origin(wood).
The hematoxylin is extracted from logwood with hot water and then
precipitated out from the aqueous solution using urea.

18. For years it was used in textile
industry until WALDEYER
established its use in histology in
1862.
Two years later Bohmer
combined haematoxylin with
alum as a mordant and obtained
more specific staining.

19. In 1891 Heidenhain introduced his
classical Iron alum-haematoxylin
method which today is still the
standard technique of the
cytologist.
Ehrlich (1886) who overcame the instability of
hematoxylin and alum by the additions of
glacial acetic acid and at the same time
produced his formula for haematoxylin as it is
used today.

20. Hematoxylin is a misnomer!!!!
• natural extract obtained from the logs, hematoxylin is not an active
dye.
• Hematoxylin is extracted and it is oxidised to haematein.
• Haematin is responsible for staining properties.
In this process of oxidative conversion to haematin, hematoxylin loses 2
hydrogen atoms & assumes a quinoid arrangement in one of its rings.

21. Chemical Structures:
Hematoxylin & Hematein
Hematoxylin Hematein
Oxidation (loss of electron) is demonstrated by the loss of
hydrogen and its electron from the Hematoxylin structure

22. NATURALLY RIPENED
HEMATOXYLINS
CHEMICALLY RIPENED HEMATOXYLINS
Ripening by exposure to light & air Ripening by exposure to chemical oxidizing
agents.
Slow process (3-4 months) Ripening instantaneous, ready to use immediately
after preparation
Long shelf life, retain stability for a
long time
Shorter shelf life (because of continuing oxidation
process in air & light eventually destroys much of the
hematein converting it into a colourless compound)
Example
 Ehrlich’s hematoxylin
 Delafield’s hematoxylin.
Example
 Sodium iodate in Mayer’s hematoxylin (SIM)
 Mercuric chloride in Harris’s hematoxylin (MCh)

23. MARSHALL AND HOROBIN 1972 –said about
overoxidation
Over ripening leads to production of a large number of
compounds which are colorless and useless. So correct
amount of oxidant should be used.
GLYCEROL- added to prevent over oxidation and reduce
evaporation
STABILISER
Improve staining properties

24. MORDANTS
 “To bite”
 Biological staining – substance intermediate
between dye and tissue
+
acid , base or neutral Basic
24
Mordant Dye LAKE

25. PRINCIPLE OF MORDANT
Hematin is anionic.
Tissue  is also anionic.
Therefore  hematin has poor affinity for tissue
Making hematin inadequate as a nuclear stain without the presence
of a 3rd element (mordant).
Mordant forms a link between the “tissue and the stain”
Dye mordant
tissue complex

26. MOLECULAR PROPERTY OF MORDANTS
• Mordants are always di-valent and tri-valent salts or
hydroxides of metals.
• They combine as hydroxides with the dye by displacing a
hydrogen atom from the dye.
• The remaining valences of the mordant serves to attach/bind the
dye-mordant complex to the tissue components (phosphate
groups of the nucleic acid)
• Although simple salts such as sulfates and chlorides will do,
generally double sulfates or alums are used.
• The double sulfates have
A. An active usually trivalent metal such as iron, aluminium or
chromium,
B. Together with potassium or ammonium as a second cation.

27. DYE – TISSUE INTERACTIONS
• Covalent bonds between the metal ions and mordant dyes – are
thought to facilitate dye-tissue binding (mordanting)
INCORPORATION OF MORDANT
• It can be incorporated into the hematoxylin staining solution
(most common way).
• The tissue section can be pretreated with mordant before
staining Heidenhain’s iron hematoxylin.
ADVANTAGES OF MORDANT
The dye-mordant complex is virtually insoluble in most fluids.

28. MORDANTS USED WITH HEMATIN
Most Commonly Used
1) Salts of aluminium  in the form of potash alum or
ammonium alum.
2) Salts of iron
3) Salts of Tungsten
Less commonly used
• Salts of Lead
• Salts of molybdenum.

29. A diagram showing how a mordant can be used to link the
dye molecule to selected tissue elements
• A Mordant-Dye “lake” using aluminum
• The mordant allows attachment where otherwise there would
only be a weak affinity
• The colored property of the dye (chromophore) allows
visualization of the site under the microscope.

30. CLASSIFICATION OF HEMATOXYLIN
I. Based on the Oxidation Procedure
1. Natural oxidation – Ehrlich’s and Delafield’s
2. Chemical Oxidation - Mayer’s and Harris
II. Based on the Mordant Used
1.Alum hematoxylin
2. Iron hematoxylin
3. Tungsten hematoxylin
4. Lead hematoxylin
5. Molybedenum hematoxylin
6. Hematoxylin without mordant

31. Types of Alum hematoxylin
Ehrilch’s haematoxylin.
Mayer’s haematoxylin.
Harris’s haematoxylin.
Gill’s haematoxylin.
Cole’s haematoxylin.
Delafield’s haematoxylin.
Carazzi’s haematoxylin.

32. 32
ALUM HEMATOXYLIN
• Routinely used
• Mordant - “potash alum” (aluminium potassium sulfate) or
“ammonium alum” (aluminium ammonium sulfate)
METHOD OF USE OF ALUM HEMATOXYLIN
Alum hematoxylin can be used in 2 ways
Regressively - the section is over stained & then differentiated in
acid alcohol, followed by “bluing”.
Progressively – stained for a pre determined time so as to
adequately stain the nuclei but leave the background tissue
relatively unstained.

33. DIFFERENTIATION
•  provides a more controllable method in removing excess stain
from tissue component and glass slide.
• Traditional HCl/alcohol acts quickly and indiscriminately, is more
difficult to control, and can result in light nuclear stain.
• 1ml of 5 – 10% solution of acetic acid in 99ml of 70 – 95%
alcohol detaches dye molecules from the cytoplasm/nucleoplasm
while keeping nucleic acid complexes intact.

34. BLUING
Introduction
After differentiating the hematoxylin with acid alcohol, the nuclei in
tissue are red colour
This red colour is converted to blue black when section is washed in
weak alkali solution  “BLUING”.
Principle of bluing
ALUM ( POTTASIUM ALUMINIUM SULPAHTE)
(ACIDIC SOLUTION)

35. Principle of bluing
ALUM ( POTTASIUM ALUMINIUM SULPAHTE)
(ACIDIC SOLUTION)
INSOLUBLE ALUMINIUM
HYDROXIDE
BLUE IN COLOUR
OH FROM WATER
SULPHURIC ACID
FREE H ATOMS FROM
WATER COMBINES WITH
SULPHATE
LACK OF OH GROUPS
LACK OF INSOLUBLE ALUMINIUM
HYDROXIDE
RED COLOUR
SO TO NEUTRALIZE A ALKALINE SOLUTION IS ADDED- BLUE INK

36. Alkaline solutions used for bluing
• Tap water is alkaline enough to produce this colour change.
• Substitute for alkaline solutions
 Scott’s tap water substitute
 Saturated Lithium carbonate (disadvantage – lithium has a
tendency to form crystalline deposits unless the slides are
agitated in it and well washed afterwards).
 Ammonia in distilled water (disadvantage – ammonia is “hard”
on delicate tissues and will loosen sections from the slide).

37. DETERIORATION OF ALUM HEMATOXYLIN
• Deterioration is marked by the formation of a precipitate in the
stored stain.
• At this stage the stain should be filtered before use and the
staining time need to be increased.
• It is advised to prepare fresh batch of stain every month.
• Since it would be uneconomical it is prepared in small batches.
TYPES OFALUM HEMATOXYLIN
1. Ehrlich’s hematoxylin (Ehrlich 1886)
2. Delafield’s hematoxylin (Delafield 1885)
3. Mayer’s hematoxylin (Mayer 1903)
4. Harris hematoxylin (Harris 1900)
5. Cole’s hematoxylin (Cole 1943)
6. Carazzi’s hematoxylin (Carazzi 1991)
7. Gill’s hematoxylin (Gill et al 1974)

42. STAINING TIMES WITH ALUM HEMATOXYLIN
Time varies according to the factors such as
1.Type of hematoxylin used
• Erhlcih’s  20 – 45 mins
• Mayer’s  10 – 20 mins
2. Age of stain
• As the stain ages  staining time has to be increased.
3. Intensity of use of stain
• Heavily used hematoxylin will lose its staining power more rapidly
and longer staining times will be necessary.
4. Method of use of stain
• When used progressively  Mayer’s hematoxylin  5 – 10mins
• When used regressively  Mayer’s hematoxylin  10 – 20mins.

43. 5. Pre treatment of tissues or sections - Length of time
• In fixative
• In acid decalcifying solution or
• Whether paraffin or frozen sections
6. Post treatment of sections – subsequent acid stains such as van
Geison.
7. Personal preference.
8. General rule – Time
• Shortened  for frozen sections
• Increased  for decalcified tissues
• Increased for those that have been stored for a long time in non
buffered formalin.

44. DISADVANTAGES OF ALUM HEMATOXYLIN
 The sensitivity of these stains to any subsequently applied acidic
staining solutions.
• Van Geison and other trichrome stains.
• Application of picric acid fucshin mixture in van Geison stain
removes most of the hematoxylin so that the nuclei are barely
discernable.
 Rectification
a. Using iron mordanted hematoxylin such as Weigert’s
hematoxylin, which is resistant to the effects of picric acid.
b. Using a combination of Celestian blue staining solution with an
alum hematoxylin.
Commonly used
Effect of celestian blue solution
• Celestian blue is resistant to the effects of acid
• Ferric salt in the prepared Celestine blue solution strengthens the
bond between the nucleus and the alum hematoxylin to provide a

45. STEPS IN STAINING PROCEDURE FOR ALUM
HEMATOXYLIN
1) Dewaxing the sections (hot plate and then into xylene)
2) Hydrating the sections (through graded alcohols 100%, 90%,
80%)
3) Bring the sections to water
4) Nuclear stain (Hematoxylin – harris – 5 – 10 mins)
5) Differentiation (1% acid alcohol = 1% HCl in 99ml 70%
alcohol) – 5-10s
6) Wash well in tap water until sections are ‘blue’(10-15 minutes)
7) Bluing - Blue by dipping in an alkaline solution (eg.ammonia
water), followed by 5 min tap water wash.
8) Stain in 1% Eosin Y for 10 min
9) Dehydration
10) Clearing
11) Mounting

46. EOSIN
INTRODUCTION
• Most suitable stain to combine with alum hematoxylin.
• It has the ability for proper differentiation  to distinguish
Between the cytoplasm of different types of cells &
Between the different types of connective tissue fibers and
matrices, by staining them different shades of red and pink.
• Eosins are xanthine dyes (tetrabromofluorescein)
TYPES OF EOSIN - commercially available
 Eosin Y
 Ethyl eosin
 Eosin B

47. Eosin Y
• Eosin yellowish
• Most widely used
• It is water & alcohol soluble.
• Used as a cytoplasmic stain - 0.5-1% solution in distilled water
with a Crystal of thymol - prevent fungal growth.
• Addition of Acetic acid (0.5 ml to 1000 ml) - sharpens the
staining
Ethyl eosin (eosin alcohol-soluble)
Eosin B (eosin bluish, erythrosine B)

48. SUBSTITUTE FOR EOSIN
• Phloxine
• Bierbrich scarlet – gives a more intense red color to the tissues.
• They are rarely as amenable to subtle differentiation as eosin and are
generally less valuable
DIFFERENTIATION OF EOSIN
• Occurs in the subsequent tap water wash
• Further differentiation occurs during the dehydration through the
alcohols
DIFFICULTIES ENCOUNTERED  Under circumstances
Eosin staining is intense and difficulty may be experienced in obtaining
adequate differentiation (this may occur after mercuric fixation)
Over differentiation of the eosin may be continues until only red blood
cells and granules of eosniphil polymorph are stained red. This is
occasionally used to facilitate the location and identification of
eosinophils.

50. IRON HEMATOXYLINS
INTRODUCTION
• Here iron salts are used both as oxidizing agent and as mordant
• Most commonly used iron salts are ferric chloride and ferric
ammonium sulfate
TYPES OF IRON HEMATOXYLIN
• Weigert hematoxylin
• Heidenhan hematoxylin  muscle striation, mitochondria &
• Verhoeff hematoxylin for elastin fibers
• Loyez hematoxylin for myelin

51. DISADVANTAGE OF IRON HEMATOXYLIN
1. It has strong oxidizing ability - resulting in over oxidation of the
hematoxylin.
To overcome this
 Separate mordant /oxidant and hematoxylin solutions are prepared
 2 solutions are
Mixed immediately before use (example – Weigert hematoxylin)
Used consecutively (example – Heidenhan’s and Loyez
hematoxylin).
2. Time consuming
3. Since the staining technique incorporates a differentiation stage it
needs microscopic control for accuracy.

52. DIFFERENTIATION OF HEMATOXYLIN
Because of the strong oxidizing ability of the solution containing
iron salts  the same iron solutions are used as a
 Mordanting fluid  before hematoxylin staining
 Differentiating fluid after hematoxylin staining.
Method
 Iron solution is used first  acts as a mordant.
 The section is then treated with the hematoxylin solution until it is
over-stained
 It is then differentiated with same iron solution under microscopic
control – acts as a differentiating agent.
ADVANTAGE OF IRON HEMATOXYLIN
Capable of demonstrating a much wider range of tissue structures
than alum hematoxylin

56. APPLICATION OF HEIDENHAIN’S HEMATOXYLIN
It can be used to demonstrate many structures according to the degree
of differentiation.
• After staining  all components are black or dark grey black.
• The hematoxylin staining is removed progressively from different
tissue structures at different rates using the iron alum solution.
• The black color disappears first from mitochondria  then from
muscle striations  then from nuclear chromatin  more
prolonged differentiation will remove the stain from almost all
structures, although red blood cells and keratin retain the stain the
longest.
• Therefore  Mitochondria, muscle striations, nuclear chromatin
and myelin can all be demonstrated.

57. TUNGSTEN HEMATOXYLIN
Only one widely used Tungsten hematoxylin  Mallory’s
PTAH.
MALLORY PTAH
COMPOSITION
• Hematoxylin
• 15% aqueous phosphotungstic acid  mordant

58. OXIDATION
Hematoxylin oxidation can be achieved by 3 ways
1. No ripening
Hematein can be used directly instead of hematoxylin
Oxidation process is unnecessary
Staining solution can be used immediately.
But its activity is short comparatively.
2. Chemical ripening
Hematoxylin can be oxidized chemically by using potassium
permanganate solution.
Solution can be used within 24 hours.
3. Natural ripening-
Of tungstein hematoxylin solution in light and air.
Most satisfactory method of preparation
But time consuming because it may take months to ripen.
But will remain usable for many years.
Applications – demonstrate CNS material and general tissue
structure.

59. Modifications of Mallory PTAH  based on the oxidation process
1. PTAH technique using hematein  no oxidation
2. PTAH solution  chemically oxidized with Potassium
permanganate
3. PTAH solution  naturally oxidized.

62. WEIGERT-PAL technique -
• For demonstration of myelin
• Is a hematoxylin method in which the tissue block is mordanted
in a chromate solution before embedding and sectioning
• Further mordanting of the section in a copper acetate solution is
often performed before the hematoxylin is applied.
• The major mordant is chromium compound.

63. The H & E stain is popular
• It can be applied to tissues differing widely in nature
and pretreatment (e.g., fixation)
• technically simple to use.

64. How does H & E staining relate to an ‘‘ideal routine histological
stain’’?

65. Theory and practice of histological techniques – Bancroft 5th edition
Cellular pathology technique – CFA Culling 4th edition
Histological staining methods – Disbray and Rack
Koss Diagnostic Cytology and its Histopathologic basis -
Leopold G Koss : volume one : 5th edition
Manual & Atlas of Fine Needle Aspiration Cytology : Svante R Orell
REFERENCES
67

66. Thank you……

68. Qtns:
• Which is the hematoxylin used in ihc ? - mayers.
• Which is the eosin used in ihc? – Eosin y
• What do u mean by1% acid alcohol?- 1% acid
alcohol = 1% HCl in 99ml 70% alcohol)
• What h stain used in our lab? – Harris
• Harris is regreesively or progressively stained ? –
both done, regressively for histology & progresively
for cytology.
• Which is better for bluing ? Tap water or scotts tap
water ?- tap water is not stable always…scott tap
water more alkaline….
• Composition of scott tap water?

69. Scott’s Tap Water/Bluing
Magnesium sulfate (MgSO4)30.0 gm
Sodium bicarbonate 2.0 gm
Tap water 3000.0 ml
Ammonia Water
Ammonium hydroxide 5.0 ml
Distilled water 1000.0 ml
0.05% Lithium Carbonate
Lithium carbonate 0.5 gm
Distilled water 1000.0 ml