HEMATOXYLIN AND EOSIN

STAINS:

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

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

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.

.

• 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.

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HC CH

CH

CH

HC

HC

Chromophore

Chromogen

Principles of dye chemistry:• All the dyes have an aromatic hydrocarbon benzene as a central

component.

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).

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.

Electrostatic bonding

Hydrogen bonding

Van der Waal’s forces

Covalent bondingHydrophobic

bondingDye aggregation

Tissue permeability

STAINING MECHANISMS

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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

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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

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.

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 charge13

STAINING OF BASIC CYTOPLASMIC PROTEIN BY ACID DYE EOSIN

Tissue Eosin Stained tissue Free

salt

NH2 Cl + Chromogen Na = NH2 Chromogen + Na Cl

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An acid dye has a coloured acid radical which attaches to a basic tissue component

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.

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.

HEMATOXYLIN

It is extracted from the core of the treeHAEMATOXYLON 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.

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.

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.

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.

Chemical Structures: Hematoxylin & Hematein

Hematoxylin Hematein

Oxidation (loss of electron) is demonstrated by the loss of hydrogen and its electron from the Hematoxylin structure

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)

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

MORDANTS

“To bite”

Biological staining – substance intermediate

between dye and tissue

+ acid , base or neutral Basic

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Mordant Dye LAKE

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

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.

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.

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.

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.

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

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.

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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.

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.

BLUINGIntroduction 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)

Principle of bluing ALUM ( POTTASIUM ALUMINIUM SULPAHTE) (ACIDIC SOLUTION)

INSOLUBLE ALUMINIUM HYDROXIDEBLUE IN COLOUR

OH FROM WATER

SULPHURIC ACID

FREE H ATOMS FROM WATER COMBINES WITH SULPHATE

LACK OF OH GROUPS

LACK OF INSOLUBLE ALUMINIUM HYDROXIDERED COLOUR

SO TO NEUTRALIZE A ALKALINE SOLUTION IS ADDED- BLUE INK

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).

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 OF ALUM HEMATOXYLIN1. 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)

STAINING TIMES WITH ALUM HEMATOXYLINTime 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.

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.

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 strong nuclear stain which is reasonable resistant to acid.

STEPS IN STAINING PROCEDURE FOR ALUM HEMATOXYLIN1) Dewaxing the sections (hot plate and then into xylene)2) Hydrating the sections (through graded alcohols 100%, 90%,

80%)3) Bring the sections to water4) Nuclear stain (Hematoxylin – harris – 5 – 10 mins)5) Differentiation (1% acid alcohol = 1% HCl in 99ml 70%

alcohol) – 5-10s6) 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) Dehydration10) Clearing11) Mounting

EOSININTRODUCTION • 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

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

stainingEthyl eosin (eosin alcohol-soluble)

Eosin B (eosin bluish, erythrosine B)

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.

IRON HEMATOXYLINSINTRODUCTION • 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

DISADVANTAGE OF IRON HEMATOXYLIN1. 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.

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

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.

TUNGSTEN HEMATOXYLIN

Only one widely used Tungsten hematoxylin Mallory’s

PTAH.

MALLORY PTAH

COMPOSITION

• Hematoxylin

• 15% aqueous phosphotungstic acid mordant

OXIDATION Hematoxylin oxidation can be achieved by 3 ways1. No ripening Hematein can be used directly instead of hematoxylinOxidation 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 preparationBut time consuming because it may take months to ripen. But will remain usable for many years.Applications – demonstrate CNS material and general tissue structure.

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

permanganate3. PTAH solution naturally oxidized.

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.

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

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

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

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Thank you……THANK YOU

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?

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