Dental subject

1. HEMATOXYLIN AND EOSIN

2. CONTENTS

3. DYE is a single chemical reagent that chemically bonds to the
substrate to which it is being applied, to highlight a specific
component of a tissue
STAIN is a mixture of selected dyes, which when added to living cells
or structural components makes them clearly visible, by highlighting
different components
STAINING is treating tissues or cells with a reagent or series of
reagents so that it acquires a colour, usually no particles of dye are
seen and the stained element is transparent

4. INTRODUCTION
 The tissue section is colourless because the fixed protein has the same refractive
index as that of glass. We use dyes that have specific affinity with the different
tissue proteins and colour them differently.
 Colour is seen by the eye as a result of the effect of certain electromagnetic waves
on the rods and cones of the retina. These waves, which have a varying length, will
determine the colour that is seen.
 White light being composed of all the colours of the visible spectrum varies in
wavelength from 4,000 Â to 8,000 Â.
 If light of a specific wavelength is absorbed from white light the resultant light
will then be coloured, the colour being dependent upon the particular wavelength
that has been removed.

5. • White light being composed of all the colours of the visible spectrum varies in wavelength from 4,000
 to 8,000 Â.
• If light of a specific wavelength is absorbed from white light the resultant light will then be coloured,
the colour being dependent upon the particular wavelength that has been removed.

6. • To see the organism better in contrast with
background
• To differentiate one organism from another
• To determine particular structures.
THE PURPOSE OF STAINING:
For a successful staining there should be high specificity (ability to
distinguish between individual cell components) and high sensitivity (capacity
of a stain to demonstrate tissue substance at low concentration).

7. AN IDEAL STAIN SHOULD ALLOW FOR:
• Evaluation of the architectural pattern of the tissue fragments.
• Proper evaluation of nuclear morphology.
• Details of cytoplasmic characteristic.
• Visualization and identification of diagnostic features in the background.

8. DYE CLASSIFICATION

12. STRUCTURAL COMPONENTS (NATURE) OF STAINS:
Stains (dyes) usually have complex molecular structure and are chiefly benzene
derivatives.
A stain consists of three constituents. The organic compound containing a :
 Benzene ring
 Chromophore
 Auxochrome

13. • When 2 of the hydrogen atoms are replaced by oxygen, a readjustment of the
double bonds takes place and a new compound, quinone (C6H402), is formed,
compounds from which are called quinoid compounds.
• The change from benzene to quinone is important, because benzene derivatives
absorb light only in the UV band and are colourless, whereas quinoid
compounds have absorption bands in the visible spectrum and are coloured.

15. CHROMOPHORE
• Certain chemical group introduced into benzene
ring by substitution induce colour to the
compound -referred as chromphore – resultant
structure –chromogen.
• A covalently unsaturated group responsible for
absorption in the UV or visible region is known as
a chromophore.
E.g:
• Active chromophore group -C=C-
• nitro grouping -NO2-
• Azo coupling N=N

16. AUXOCHROME
•This part of the dye helps to intensify the light.
It is an ionizing group that also helps to stick the
stain with the tissue.
•The auxochrome group augments more free
electrons in the chromophore groups.
•The increased number of electrons in the
system helps to absorb light of longer
wavelength in the visible range.

17. • The chromophore group absorbs light and imparts colour to the
stain.
• The auxochrome group donates more electrons to the
chromophore group and helps to absorb light of longer
wavelength in the visible range.

18. CONVERSION

19. TYPES OF STAINING:
• Direct staining
• Indirect staining
• Progressive staining
• Regressive staining
• Intravital staining
• Supravitral staining

20. Direct Staining:
• When a staining procedure colours the cells present in a preparation, but
leaves the background colourless.
• Dyes such as eosin stain tissues perfectly when in alcoholic or aqueous
solutions.

21. Indirect Staining:
• When a procedure colours the background, but leaves the cells
colourless, with the presence of mordant.
• Stains such as hematoxylin require additional substance known as
mordant before satisfactorily binding to the tissue

22. Progressive Staining :
• Tissue section is immersed in a dye bath until such time as only the
desired structures are stained.
• Difficult process to control
E.g Mayer’s Haematoxylin: first stains the nuclei

23. Regressive Staining :
 Tissues are over stained and the excess dye is removed until the desired
tissue component is selectively stained
• The dye is selectively removed from unwanted tissue groups –
differentiation
E.g In H/E staining, HCl is used as a differentiator

24. The differentiation is done by using:
a) Acid in basic dye or base in acid dye such as in Papanicolaou’s staining.
Haematoxylin is removed from the cytoplasm by using 1% acid alcohol
b) Oxidizing agent: The oxidizing agents are used to oxidize the dye and make it
a colourless material such as picric acid, potassium permanganate.
c) Mordant: Here the dye-mordant complex at first binds with the tissue.
Subsequently excess mordant is used that attracts the attached dye in the
tissue. The mordant thereby removes the excess dye from the tissue.
d) One dye is replaced by other less affinity dye.

26. Intravital Staining: Stain may be injected into a living
animal and the stained tissue removed and examined.
Eg: Toluidine Blue.
Supravital Staining: The living tissue may be removed
directly and subsequently stained.
Eg: Janus Green for staining mitochondria

27. THEORIES OF STAINING
THEORIES
OF
STAINING
PHYSICAL
THEORIES
CHEMICAL
THEORIES

28. Physical theories depend upon:
1. Solubility: Examples of this method of staining are the fat stains which are
effective simply because the stain is more soluble in the fat than in the 70 %
alcohol or other solvent in which it may be dissolved.
2. Adsorption: This is the property by which a large body attracts to itself
minute particles from a surrounding medium, and is a phenomenon well
known to chemists.

29. CHEMICHAL THEORY

30. ELECTROSTATIC BONDING:
•The affinity between opposite ionic groups of dye and
tissue helps in staining.
•Dyes are classified as acid or basic
•For example, eosin is an acid dye with an affinity for
the basic protein of cytoplasm whereas methyl green is
a basic dye which has an affinity for the phosphate
groups of deoxyribonucleic acid of the nucleus.
•Salt linkage and ionic binding are alternative terms to
electrostatic binding; the correct name for the forces
involved is Coulombic attraction.

31. Acids have a negative charge(-ve)
They attach to positive charges, especially
hydrogen (H+
).
In an electrical field they migrate to the anode.
They are anions.
Tissues carrying a positive charge will attract
dyes with a negative charge (i.e. acid dyes); and
are termed acidophilic.
Examples : phosphates of nucleic acids
sulphate groups of acid mucopolysaccharides

32. Bases have a positive charge (+ve)
They attach to negative charges especially
hydroxy groups (OH-).
They migrate to the cathode. They are cations.
Tissues carrying a negative charge will attract
dyes of positive charge and are termed
basophilic.
Examples : amino acid such as lysine and
arginine

33. Vander Waal's Forces –
- Attractions b/w dipoles (i.e
molecules with areas of opposing
charges, for e.g H†CI) dipoles behave
similar to magnets
- These polar attractions are week,
effective over a short distance. These
occur b/w all reagents & tissue
substrates, but more important in
cases with molecules having large
dipoles
Eg :congo red/ orcein - elastic fibres.

34. Include - dipole dipole interactions-
- dipole induced dipole interactions-
- dispersion forces
a.Dipole Dipole Interactions:
• When both dye & tissue exists as dipoles (+ve & -ve charges separate) +-
b. Dipole Induced Dipole Interactions:
• When a dipole in one causes the polarization of another polarizable group.
c. Dispersion Forces:
• More imp in situations where dye with large, asymmetric molecular
structure in a non-aqueous solution are attracted to poorly hydrated,
strongly polarizable tissue group.

35. HYDOGEN BOND:
Hydrogen bonding is a weak bond. It occurs
readily in water. It is a type of covalent bond
that occurs between hydrogen and a strong
electronegative atom commonly O, N or F.
Water forms hydrogen bond and so competes
with stain-tissue bonds. Therefore, hydrogen
bonding in dye-tissue less likely occurs in
aqueous solution. Eg: Best’s carmine dye to
stain glycogen

36. COVALENT BONDING:
• Covalent bonding involves sharing
electrons.
Eg. In water each of two hydrogen atoms
shares an electron with oxygen, and oxygen
atom likewise shares the two hydrogen
electrons.
Here 2 atoms share 2, 3 or more electron
pairs, leading to multiple covalent bond.
Significance in mordant dying

37. HYDROPHOBIC BOND :
• A major contribution to stain-tissue affinity when using organic reagents
or dyes in aqueous solution is the hydrophobic effect.
• This is the tendency of hydrophobic groupings (such as leucine and valine
side chains of proteins; or biphenyl and naphthyl groupings of enzyme
substrates and dyes) to come together, even though initially dispersed in an
aqueous environment.

38. • The process occurs because water is a highly structured liquid. They
aggregate in solution and then penetrate tissue.
• The dye-dye aggregate increases when the dye concentration is high,
the molecular size of the dye is bigger and temperature is low.

39. DYE AGGREGATION
• Dye molecules may interact with each other forming
dye-dye interaction.
• They aggregate in solution and then penetrate into
tissue.
Factors increasing dye aggregation:
• Dye concentration is high
• The molecular size is bigger
• Temperature is low
Eg: Metachromatic staining and metallic
microcrystals after silver impregnation

40. METACHROMASIA:
• A dye which has the ability to change its colour without changing its
chemical structure is said to be metachromatic.
• Staining reaction is known as metachromasy, the tissue is said to exhibit
metachromasia, and the dye is known as a metachromatic dye.

41. Mechanism of dye aggregation
in metachromasia:
• The cationic (positively charged) dye
in aqueous solution is neutralized
with the polyanions of the tissues.
• Subsequently the nonpolar aromatic
ring of the dye binds with the other
dye by van der Waals force.
• The dye absorbs shorter wavelength of
light, and the visible colour changes

42. THREE TYPES OF METACHROMASIA

43. Toluidine blue
- low concentration- nucleus – blue
- high concentration - cartilage - purple
Section of loose CT stained with
toluidine blue and demonstrating
both orthochromasia (bright blue
color) and metachromasia (dark
purple color)

44. FACTORS INFLUENCING METACHROMASIA
The following factors may influence on metachromasia:
1. Dye concentration: High concentration of dye enhances metachromasia.
2. pH: Low pH increases metachromatic effect.
3. Temperature: Decreased temperature augments metachromatic effect.
4. Aqueous solution: Water enhances van der Waals force in between the dye
molecules and increases metachromatic effect.

45. MORDANT:
• Mordant is the salt and hydroxides of the metals that help in the attachment of
dye with the target tissue The metal used as mordant is either divalent or trivalent
such as aluminium, iron, copper, etc.
• The mordant binds with the dye by covalent or co-ordinate bonding.
• This dye and mordant combination is also known as "lake".

46. • The mordant-dye combination is basic in action irrespective of the
character of the dye.
• Mordant is insoluble in most of the biological fluid, and therefore
staining is not altered even after subsequent treatment of tissue.

47. Mordant may be used in three ways:
1. Pre-mordanting: The tissue is at first treated with mordant followed by
dye.
2. Meta-mordanting: Mordant in combination with dye is used.
3. Post-mordanting: The dye material is applied first followed by mordant
Example: Haematoxylin itself is a poor dye.* However the combination of
mordant such as aluminium and haematoxylin makes a stronger dye.

48. ACCENTUATORS
• Accentuators are the group of substances that help to increase the
staining intensity of the dye.
• Accentuators neither form any dye lake nor do they take part in
any chemical reaction.
Example: Potassium hydroxide in methylene blue solution.

50. FACTORS INFLUENCING STAINING
1. Dye affinity to the target tissue specimen
2. Specimen geometry
3. Target concentration
4. Rate of reaction
5. Rate of stain loss

51. Dye affinity to the target tissue specimen:
• The tendency to bind a dye with the target tissue is known as dye
affinity.
• The acidic dye such as eosin binds strongly with acidophilic target, that
is, cytoplasmic protein.
• The acidic dye has very little affinity with baso philic substances.
• Affinity of the dye to the specific tissue is also influenced by the pH and
the presence of inorganic salt concentration of the solvent.

52. Specimen geometry:
Specimen geometry or topography also influences the staining.
 Thick tissue: If the tissue is thick, then the penetration of dye is difficult,
and the central part of the tissue takes poorer stain.
 Surface topography: The surface of the tissue of paraffin section is more
even than cryostat section, and so it takes better stain.

53.  Disturbance of microtopography of tissue:
The alcohol is a coagulative fixative that disturbs the topography of the cell
and tissue. The shattering effect of alcohol increases the dye penetration rate.
 Inner geometry of tissue: The inner geometry of the tissue may also
influence staining such as bone marrow canaliculi which are rapidly
stained by Schmor's thionine stain than the adjacent connective tissue.

54. Target concentration :
The concentration of the target tissue affects the staining intensity as
the more the amount of the target tissue, the more intense will be the
staining.

55. Rate of reaction:
Rate of reaction in the target tissue also influences the staining
pattern such as in Feulgen reaction, the short reaction time
exposes only a few aldehyde groups producing weak staining
pattern.

56. Rate of stain loss:
Staining pattern is greatly influenced by rate of stain loss. At times, the
stain loss may be intentional such as differentiation or destaining. The
differentiation often removes the excess stain from the cell and thus helps
to differentiate the organisms.

57. HEMATOXYLIN AND EOSIN STAINING:
The hematoxylin and eosin stain (H&E) is the most widely used histological stain.
• Its popularity is based on its comparative simplicity and ability to demonstrate
clearly an enormous number of different tissue structures.
• While automated staining instruments and commercially prepared hematoxylin
and eosin solutions are more commonly used in today's laboratories for routine
staining,
• Haematoxylin is the most common stain used in pathology.It remains the
primary technique for the demonstration of microscopic nuclear details of
cellular and tissue components.

58. HISTORY
The introduction of hematoxylin is attributed to Waldeyer in 1862 that used it
as a watery extract but without very much success.
Two years later Bohmer combined haematoxylin with alum as a mordant and
obtained more specific staining.
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.

59. HEMATOXYLIN:
• The word Hematoxylin is derived from old Greek word Haimato meaning
blood & Xylon meaning wood.
• A natural dye extracted from the core or heartwood of tree
Haematoxylon campechianum.
• The hematoxylin is extracted from logwood with hot water, and then
precipitated out from the aqueous solution using urea.
• The major oxidization product of hematoxylin is hematein, a natural dye
that is responsible for the color properties.

60. Hematin can be produced from hematoxylin in two
ways:
NATURAL OXIDATION ('RIPENING’)
• By exposure to light and air.
• This is a slow process, takes as long as 3-4months.
• Solution retains its staining ability for a longtime.
• E.g. Ehrlich's and Delafield's solutions.

62. CHEMICAL OXIDATION:
• Oxidation occurs instantaneously.
• These solutions have a shorter shelf life.
• Oxidation using sodium iodate - most commonly used (0.2gm oxidizes 1.0
gmhematoxylin)
Eg. Mayer's hematoxylin.
• Oxidation by mercuric oxide e.g. Harris'shematoxylin
• Ferric chloride and potassiumpermanganate can also be used.

64. Types of Haematoxylin :
Haematoxylin can be classified according to its
combination with different mordants such as:
1. Iron haematoxylin
2. Alum haematoxylin
3. Tungsten haematoxylin
4. Lead haematoxylin
5. Molybdenum haematoxylin
6. Hematoxylin without mordant

65. BLUING:
• Bluing is necessary to convert nuclear coloration fromreddish purple to a crisp
blue/purple* Bluing agents typically are alkaline with a pH range of7.5-9.0
optimally*
• Tap water*
• Scott’s tap water*
• Ammonia water*
• Lithium Carbonate*
• Buffered solutions*
Bluing agents enhance the contrast of the H&E stain byincreasing the crispness of
the hematoxylin* Hematoxylin coloration is changed from purple toblue/purple.

66. • Bluing agents enhance the contrast of the H&E stain by increasing
the crispness of the hematoxylin*
• Hematoxylin coloration is changed from purple to blue/purple.
 How does bluing work?
• Elevated pH–
• Fewer H+ in solution effects the structure
ofhematoxylin
• Loss of H+ from ring structure

67. Scott's tap water:
Sodium bicarbonate: 2 g
Magnesium sulphate (anhydrous): 10 g
Water: 11
• Designed to maintain a specific pH (8.0 –8.2).– Produces crisp
blue/purple chromatin stain.
• Gentle to tissue sections.
• Excellent for use with frozen sections
• used with any hematoxylin

68. METHODS
• Running tap water for several minutes
• Treating the section by Scott's tap water (pH is 8): 2-3 min
• Ammonium hydroxide (5%): 2-3 min
• Ammonium vapour few sec.

69. Tap water- pH may be unpredictable
Ammonia water- pH may be too high–
Section loss may occur
Scott’s tap water or buffered solutions are more effective at
maintaining the optimal pH–
Gentle for tissues

70. THANK YOU