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 ……….

Whytostain
 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 commonlyusedstains
 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

Accordingtotheiraffinityforcertaintissuecomponents.
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’s
Haematoxylin.
 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.

Theoryofstaining
 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
34

Whyarestainstakenintothetissues?
 Involves 3 different interactions
1. Stain -tissue interactions
2. Solvent -solvent interactions
3. Stain -stain interactions
35

Stain-tissueinteractions
 Coulombic attractions
 Van der waal’s forces
 Hydrogen bonding
 Covalent bonding
36

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

 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
38

Such phenomena is important in
Bests carmine stain
When dyes go into the solution
39
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
40

Vanderwaal sforces
 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
41

Vanderwaal ‘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
42

Hydrogenbonding
 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
43

Hydrogenbonding
 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
44

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
45

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
46

Stain-staininteractions
 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
47

Whystainsremainintissueafterremovingfromstainbath?
 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
48

Whyarestainsnot takenupbyeverypartof thetissue?
Factors
1. Number and affinities of binding sites
2. Rate of reagent uptake
3. Rate of reaction
4. Rate of reagent loss
49

Numberandaffinitiesofbinding 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
50

Rateofreagent 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
51

Rateofreaction
 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.
52

Rateofreagent 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
53

Effectof tissuemodificationonstaining
 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

Effectsof specimengeometryonstaining?
 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
55

 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 .
56

 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
57

Effectsof resinembedding onstaining?
 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 effect
58

Factors contributing to dye tissue affinities
Interactions Practical example
Solvent –solvent interaction
Hydrophobic bonding Staining systems using aq.solution of dyes
or other organic reagent
e.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.

Armamentariuminstaining
 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

Requirementsforstaining
 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

Practicetips
 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.

H & E staining part 1

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H & E staining part 1