Post on 15-Jul-2015
Hematoxylin & Eosin
staining Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.
Content
Introduction
General principles of staining (theory )
Theory of staining
Method of H & E staining
Theory of H & E staining
Types of hematoxylin
Introduction
Colors are beyond the expression
But helps to know things, differentiate & express , particular
entity
Same is applicable to histopathology ……….
Why to stain
The purpose of staining is that of outlining the tissue and cellular
components
To identify tissue
To establish the presence or absence of disease processes.
Most commonly used stains
Histopathology – Routine Hematoxylin(H) & Eosin(E),
In microbiology – Gram’s Method and Ziehl-Neelson’s method,
In hematology- Romanowsky stain ,
In cytopathology -Papanicoloau stain.
Some important terminology
Basophilic- The entities stainable with basic dye &
are substances which are usually acidic in nature.
Acidophilic- The entities stainable with acidic dye &
are substances which are usually basic in nature.
Sudanophilic - The entities stainable with oil soluble
dyes e.g. sudan III, IV
Argyrophilic- The entities stainable with silver nitrate solution.
E.g. AgNOR
Argentaffin- The entities staininable with silver nitrate solutions without chemical reduction procedures
Neuroendocrine cells
Metachromatic -The entities will stain in a color or hue different from that of staining solution itself.
Definition
Stains:
Stains are chemical substances used to achieve visible color contrast
in the microscopic picture of a prepared 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.
DYES
These are essentially aromatic benzene ring compounds
or derivatives that possess the twin properties of color and
ability to bind to tissue.
Classification
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
According to their affinity for certain tissue components.
Acidic dyes
Acid dyes usually stain basic components such as cytoplasm, acidophil
granules etc.
e.g. Eosin, Acid fuchsin
Basic dyes
Usually stain acidic stain acidic components such as nucleus, basophil
granules etc.
e.g. Hematoxylin, Basic Fuchsin, Methylene blue.
Neutral dyes
These consist of mixtures of basic and acidic dyes.
Both cations and anion contain chromophoric groups and both have colored
radicles.
e.g. Romanowsky dyes formed by the interaction of polychrome methylene blue
and eosin.
Types of Staining reactions
Absorption or direct staining – tissue penetrated by dye solution
Indirect staining– using intermediary 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 compound.
Staining methods
Vital staining
Routine staining
Special staining
Other classification
Regressive staining
Progressive staining
Vital staining
Applied to living tissue
Accomplished by injecting the staining solution into
some part of animal body
By mixing the stain with living cells.
Primarily used for research purpose
Routine Staining
One that stains the different tissues with little
differentiation except between nucleus & cytoplasm.
General relationship among cells, tissues & organs
are demonstrated.
Eg. Hematoxylin & eosin stain
Special staining
Special or selective staining demonstrate special
feature of tissue such as
particular cell products,
Microscopic intracellular & intercellular structure.
e.g. PAS stain for mucopolysaccharide
Techniques used in bacteriology ….
Simple stains:
They provide color contrast, but impart the same color to all bacteria.
E.g. methylene blue or basic fuchsin
Negative staining:
That provide a uniformly colored background against which the unstained bacteria stand out in contrast.
E.g. Demonstration of bacterial capsules,
Very slender spirochetes
Differential stains:
These stains impart different colors to different bacteria or bacterial structures.
The two most widely used differential stains are the Gram stain and the acid fast stain.
Impregnation methods:
Cells and structures too thin to be seen under the ordinary microscope may be rendered visible if they are thickened by impregnation of silver on the surface.
Such methods are used for the demonstration of spirochetes and bacterial flagella.
Differentiation
Removal or washing out of excess stain until the color
is retained only by tissue component that are to be
studied.
Generally done with acid alcohol, ethyl alcohol.
Exposure to air may oxidized & improve the process.
Regressive staining
In a regressive stain, the tissue is first overstained & then
partially decolorized.
The process of partial declourization is (differentiation).
Differentiation is controlled visually by examination with
microscope.
Sharper degree of staining is obtained
Progressive staining
Once the dye taken up by the tissue it is not removed
Differentiation in progressive staining relies solely on
selective affinity of dyes for different tissue element
The tissue is left in dye solution only until it retains
the desired amount of coloration.
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.
This is insoluble
Allowing subsequent counter staining and dehydration.
Methods mordant- dye applied Mordant and dye mixed together.
e.g. Haematoxylin with potassium alum in Ehrlich’s Haematoxylin.
Mordant is used first, followed by the dye.
e.g. The preliminary iron alum bath before Heidenhain’sHaematoxylin.
The dye is applied first, followed by the mordant.
e. g. application of the methylene blue dye and after that grams iodine mordant.
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.
Accelerators:
Accelerators are used in the metallic impregnation
techniques for the nervous system.
e.g. Chloral hydrate and Veronal in Cajal’s methods.
Depth of coloration is depends on….
Chemical affinity
Density of component
Permeability of component
ph of reagent
Method of fixation
Oxidation & reduction
Factors influencing staining reaction
The component of the fixative used
pH of fixative
pH of solutions
Mordants
Chemical or reagent which produces oxidation or reduction
Historical aspect of hematoxylin
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.
Historical aspect of hematoxylin
The credit for introducing the differentiation of
stained sections goes to the Bottcher (1869) who
used alcohol when staining nuclei with solutions of
rosanilin nitrate.
In 1891 Heidenhain introduced his classical iron
alum-haematoxylin method which today is still the
standard technique of the cytologist.
Theory of staining
Staining involves the visual labeling of some entity by attaching
or depositing in its vicinity, a marker of characteristic color and
shape.
Therefore, stain is the marker or the reagent used to generate
the marker .
32
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….
33
Why are stains taken into the tissues?
Dye -- tissue or reagent -- tissue affinities
Uptake of dyes or reagent is multistep
Initial reaction – coulombic attraction
Later reaction – covalent bonding
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Why are stains taken into the tissues?
Involves 3 different interactions
1. Stain -tissue interactions
2. Solvent -solvent interactions
3. Stain -stain interactions
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Stain-tissue interactions
Coulombic attractions
Van der waal’s forces
Hydrogen bonding
Covalent bonding
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Coulombic attractions
Also termed as Salt links or electrostatic bonds
Arise from electrostatic attraction of unlike ions
Eg:
Basic dyes –phosphated DNA and RNA
Acid dyes – sulfated mucosubstances
37
Coulombic attractions
Dye ion binding depends on
Charge signs of dye and tissue
Magnitude
Amount of non dye electrolyte present in dye bath
Ability of tissue substrate to shrink or swell
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Coulombic attractions
Such phenomena is important in
Bests carmine stain
When dyes go into the solution
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They ionise and dissociate
Acid dyes provide anions - chromogen
Positively charged ions - auxochrome
Coulombic attractions
Reactive tissue groupings consist of
Bound moiety of one charge
Mobile moiety of opp. Charge
Tissue dissociates when immersed in dye bath
Staining occurs….
When chromogen of one charge attracts the bound tissue
moiety of other charge
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Van der waal 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
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Van der waal ‘s forces
Eg: staining of elastic fibres by orceins
Orcein is a large molecular weight dye
It has stronger dipoles
Used in alcoholic solutions
Elastin is a hydrophobic protein
Has many polarisable amino acids
Hence the criteria for this force is met
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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
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Hydrogen bonding
Eg: Staining of amyloid and cellulose by various bi azo dyes
In the above examples, selective staining involves hydrogen
bonding substitutes in the dye molecules
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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
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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
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Stain- stain interactions
Dye molecule attracts each other – dye aggregates
These are classic sites for metachromatic staining
Eg: Toulidine blue – this effect is because dye aggregates have
spectral properties different from monomeric dyes
Eg: Microcrystals of metallic silver in silver impregnation
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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
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Why are stains not taken up by every part of the tissue?
Factors
1. Number and affinities of binding sites
2. Rate of reagent uptake
3. Rate of reaction
4. Rate of reagent loss
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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
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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
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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.
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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
Dyes are easily lost by permeable structures
Impermeable structures retain stain the longest
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Effect of tissue modification on staining
Effects of fixation
May enhance or reduce the ionic strength of reactive groups
Formalin and osmium tetroxide- induce basophilia
Acidic dichromate solutions – induce acidophilia
54
Effects of specimen geometry on staining?
Varied 3D features of specimen
1. Differences in few micro meter affects staining
2. Dispersed cells stain differently compared to that cut from a block
3. Thin sections stain differently from thick
4. Sections with irregular surfaces stain differently compared to smooth surface
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Effects of specimen geometry on staining?
Simple geometrical influence on staining
1. Thin specimen stain faster than thick
2. Irregular surface stain faster than smooth
3. Dispersed specimen stain faster compactly arranged .
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Effects of specimen geometry on staining?
Complex effects of specimen geometry
Results from swelling of tissue components
Such swellings increase the rate of staining
This probably contributes to
High selectivity of aqueous alcian blue for mucins
High selectivity of strong acid picro trichrome stains for collagen fibres
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Effects of resin embedding on staining?
Resin as stain excluders
Stain penetration is slower
Biological material is occluded – crooslinking increased –
staining reduced
Resin as stain binders
Can give staining artefacts
Resin may reduce the amount of reagent reaching the target
Trap excess molecules – antiplasticising effect58
Factors contributing to dye tissue affinities
Interactions Practical example
Solvent –solvent interaction
Hydrophobic bonding Staining systems using aq.solution of dyes or other organic reagente.g. enzyme substrate
Stain- stain interaction Metachromatic staining with basic dyes
Reagents-tissue interaction
Coulombic attractions Acid & basic dyes
Van der waal’s forces Elastic fibre stain
Hydrogen bonding Staining of polysaccharides
Covalent bonding Method such as PAS.
Armamentarium in staining
Specially designated bench
Staining bench Should be facing window
Slide washing tray made of stainless steel
Bunsen burner – to heat up the stain
Thermostatically controlled hot plate to melt the wax
Microscope to control staining reaction
Armamentarium in staining
Slides are stained in following ways
1. Using staining dishes
Small grooved couplin jars with glass lids
Large staining troughs
2. Using staining racks
Two pieces of stout glass rods 2-4 cm apart
3. Using staining machine
Same as processing machine but carry slide racks
Armamentarium in staining
Stock stain
Should be labelled
Pouring should be opp. side of label
Choice of containers depends on
Speed of evaporation
Frequency of use
Length of exposure of sections
Need of speedy access
Need of an elevated temperature
Requirements for staining
All glassware should be thoroughly cleaned
Correct solvent should be used
Silver and osmic acid solutions should be kept in dark bottles
Solutions like dilute ammonia should be freshly prepared
Constituents of stain dissolved should follow the formula
Alcoholic solutions of the stain should be kept in glass stoppered
bottles
All dyes should be filtered before use
Practice tips
Keep stains & solutions covered when not in use.
Filter stain after use.
After slides are removed from the drying oven allow them to come to room temperature before placed in xylene.
Once the slide are have been placed in first xylene to remove the paraffin do not allow them to dry out.
Maintain the proper level of stains
Renew water baths after each staining
Drain all slide before moving on to next solution
Use microscope.