staining and h and e staining

1. GENERAL PRINCIPLES OF
STAINING AND
H AND E STAIN
Dr.SHUBHAM PATEL
PG II YEAR
DEPT. OF ORAL PATHOLOGY AND MICROBIOLOGY

2. CONTENTS
• INTRODUCTION
• STAINS AND DYES
• MORDANTS
• ACCENTUATORS
• METACHROMACY
• GENERAL THEORY OF STAINING
• STAINING OF PARAFFIN SECTION
• HEMATOXYLIN AND EOSION

3. INTRODUCTION
• Colors are beyond the expression
• But helps to know things, differentiate
& express , particular entity
• Same is applicable to histopathology ………

4. WHY TO STAIN
• 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.

5. COMMON TERMINOLOGIES
• Basophilic
• Acidophilic
• Sudanophilic
• Argyrophilic
• Direct staining
• Indirect staining
• Progressive staining
• Regressive staining
• Differentiation

6. Theories of staining :
• Physical theories :
1. Simple solubility e.g. Fat stains are effective because the stain is more
soluble in fat than in 70% alcohol.
2. Absorption: This is a property by which a large body attracts to itself
minute particles from a surrounding medium.
• Chemical theories It is generally true that acid dyes stain basic elements
(Cytoplasm) and basic dyes stain acidophilic material (nucleus) however
this far from being complete truth, Indeed hematoxylin, which is an acid
dye, does not stain the cytoplasm, but (in the presence of mordant) is one
of the most widely used nuclear stains.
Mann in 1902 said that'dyes and tissue molecules can adhere chemically to
one another by their side chains'

8. Stains and dyes
• Stains: Stains are chemical substances used to achieve visible color
contrast in the microscopic picture of a prepared tissue.
• DYES: These are essentially aromatic benzene ring compounds or
derivatives that possess the twin properties of color and ability to
bind to tissue.
• Staining: Staining may be loosely defined as treating tissue or cells
with a reagent or series of reagents so that it acquires a color; usually,
no particles of dyes are seen and the stained element is transparent.

10. According to the origin of a dye
1)Natural: e.g.
hematoxylin, Carmine, and
Saffron
2)Synthetic: e.g.
Benzene, toluene and
naphthalene or phenols

11. Mordant
A substance which acts as an intermediary between dye and tissue.
The term mordant is strictly applicable to salts and hydroxides of
divalent and trivalent metals
Should not be used to indicate any substance that improves in staining
in some other manner (accentulators and accelators).
Mordant can be defined as polyvalent metal ion which forms
coordination complexes with certain dyes.
The complex of the mordant and dye is called a ‘lake’.
The mordant dye combines with tissue to form tissue mordant-dye
complex.

12. Accentuators
• They increase the staining power of the dyes with which they are
used.
• They do not form lakes with dyes and they are not essential for the
chemical union of the dye with the tissue.
• Accentuators are often acids or alkalies which are added to anionic
(acidic) dyes and cationic (basic) dyes respectively.
• e.g. Potassium hydroxide in Loeffler’s methylene blue phenol in carbol
thionin and carbol fuchsin. This increases the intensity and selectivity
of staining.

14. METACHROMASY
• The entities will stain in a color or hue different from that of staining
solution itself.
• E.g.,Mucin will stain red with toluidine blue, while the rest of the
tissue stains in shades of blue.
• Among the principal tissue components which exhibit metachromasia
are mucin, cartilage and mast-cell granules.

15. Why staining takes place ………….?
• Why do any tissue components stain ?
• Why do the stained components remain stained?
• Why are all the components not stained?
ANSWERS….

16. Why do any tissue components stain ?
• Dye -- tissue or reagent -- tissue affinities
• Uptake of dyes or reagent is multistep
• Initial reaction – coulombic attraction
• Later reaction – covalent bonding

25. Van derwaal’s forces
• Occur between reagent and tissue substrates
• Involves various intermolecular attractions
Dipole-dipole
Dipole induced dipole
Dispersion forces
• These forces are polar attractions
• Effective over a short distance
• Non symmetrical molecules posses stronger dipoles than symmetrical
molecules

26. • Hydrogen bonding
Form of dye tissue attraction
Arises when hydrogen atom lies between two electronegative atoms
Spontaneous thermodynamic changes towards disorganization leads to
attraction between dye molecule and tissue groups
Eg: Staining of amyloid and cellulose by various bi azo dyes

27. • Covalent bonding
Can occur between the stain and the tissue
In contrast to ionic bonding, Covalent bonding involves sharing of
electrons
Eg: in water, each of the 2 hydrogen atoms shares an electron with
oxygen, and the oxygen atom likewise shares two hydrogen electrons
This bonding is of significance in mordant dying process

28. • Solvent – Solvent Interaction
Hydrophobic bonding
It is the tendency of hydrophobic grouping to come together
Water molecules held in clusters - hydrogen bonding
Transient clusters stabilised –hydrophobic groups
Removing hydrophobic bonds – hydrophobic groups of substrate and
reagent becomes marked Eg: staining of fat by sudan dyes

29. Why stains remain in tissue after removing
from stain bath?
• 2 possibilities…
No affinity for processing fluids or mounting media
Dissolves in these substances slowly
Sections stained with basic dyes – should be dehydrated
rapidly
Sections stained with acidic or basic dyes are mounted in non
aqueous media to prevent loss of dye

30. Why are stains not taken up by every part of
the tissue?
• Number and affinities of binding sites
Acid dyes – affinity for basic tissues
Basic dyes- affinity for acidic tissues
This produces 2 tone staining pattern in which cytoplasm contrast the nuclei
Affinities are influenced by: pH, Concentration of inorganic salt, Rate of
reagent uptake, Dyes diffusing at different rates exhibit staining rates of
varying intensities.
Eg: Red cells- stain slowly
Collagen fibres stain rapidly
Muscle fibres are intermediate in staining rates.

31. • Rate of reaction
Selective staining depends on differential rates of reaction
At low pH, hydrolysis of an organic phosphate is rapid in tissues
containing acid phosphatases
Structures containing alkaline phosphatases, whose pH optima are
higher, the hydrolysis rates are slower.
• Rate of reagent loss
Factors affecting are: 1. Variation in section thickness 2. Temperature 3.
Stirring of the reagent solution 4. Presence of cavities in the tissues

32. 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

34. 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 IronAlum- 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
History of hematoxylin

35. Ripening
• The process of oxidation of hematoxylin.
• The major oxidization product is hematein, a natural dye that is
responsible for the color properties.
• Carried out in 2 ways – Natural oxidation. Chemical oxidation.

38. 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

44. • 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
STEPS IN STAINING PROCEDURE FOR ALUM HEMATOXYLIN

46. • Method
1. Iron solution is used first acts as a mordant.
2. The section is then treated with the hematoxylin solution until it is
over-stained
3. It is then differentiated with same iron solution under microscopic
control acts as a differentiating agent.

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

52. MOLYBEDENUM HEMATOXYLIN
• 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.

53. • 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.

54. 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 localisation of gastrin secreting cells in stomach.

55. HEMATOXYLIN WITHOUT A MORDANT
• Freshly prepared hematoxylin is used to demonstrate various
minerals in tissue sections.
• These methods are now replaced with more specific techniques.

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

57. 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.

58. REFERENCES:
• Theory and practice of histological techniques – Bancroft 7th edition
• Cellular pathology technique – CFA Culling 4th edition
• Histological staining methods – Disbray and Rack

59. Thank you!
(LISTEN TO THE COLOUR OF YOUR SLIDE)