H & e staining part 1

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 .

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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….

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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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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

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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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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


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

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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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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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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 of staining

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


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


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.

Thank You……

H & e staining part 1

Health & Medicine

Transcript of H & e staining part 1