4_5839287714297417902.ppt

Chapter 5: Principles of staining

At the end of the session, students will be able to:
 define staining
 describe principle of H-E stain
 discuss how hematoxylin is oxidized
 know what is mordant and its uses
 mention types of alum hematoxylin staining
 explain about bluing and differentiation
 List down the H-E procedures
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5.1. Staining
 Tissue sections are commonly stained to enhance contrast in the normally
colorless tissue sections
 For light microscopic examinations, colored agents (chromophores) are
used.
 Some cell structures do not stain well with aqueous dyes and so routinely
appear clear.
 This is especially true for those which are hydrophobic, containing fat.
 Included in this category are adipocytes, myelin around axons, and cell
membranes of the Golgi apparatus.
Histopathology, 28-Dec-21

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Why are stains taken up in to tissues?
 Staining occurs as a result of interaction of cells and tissue
components with dye.
 The stains are taken up by particular tissue component
 or tissue component become surrounded by other stained tissue
component (negative staining).
 Stains are taken up or eaten up by live tissue component
(Supravital stains)
 Stains are taken up by tissue components with high affinity:
Affinity: - more intense staining of component

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 Differing affinity between the dye and the tissue binding sites
results in selective staining of certain tissue constituents.
 Certain dyes are either basic or acidic.
 This is because synthetic dyes are prepared so that the
coloring part of the dye is either acidic (anionic) or basic
(cationic) in its chemical behavior.
 The cationic or basic dye has an affinity for nuclei and
ribosomes
 that exist in the tissue with a net negative charge and are
termed basophilic.

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 The anionic or acid dye has an affinity for positively charged
cytoplasm
 and other components like mitochondria and cilia, which are then
termed acidophilic structures.
 Basic stain: [dye]+ OH-
stains basophilic structures
e.g., nuclei, ribosomes
 Acid stain: [dye]- H+
stains acidophilic structures
e.g., mitochondria, collagen, cilia

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Factors contributing to dye-tissue affinities
Factors that can adversely affect staining of tissues:
 Solvent -solvent interaction (Hydrophobic bonding)
- depends on tendency of hydrophobic groups to come
together
 Reagent-reagent interaction
- metachromatic staining- color shift in a dye
 Reagent-tissue interactions (Vander -Waal forces)
 Columbic attraction, Hydrogen bonding, Covalent bonding.

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A. Coulombic attractions (electrostatic bond) interaction of acidic and
basic dyes
B. Van der waal’s forces- for non ionic, poorly hydrated substances. eg.
Elastic fibers
C. Hydrogen bonding-formed when a hydrogen atom lies between two
electro negative atoms
- in non-aqueous solvents hydrogen bonding may play a more
significant role.
D. Covalent bonding - Dye metal ion +tissue → Dye metal ion-tissue
 Tissue modifications altering pattern of stains

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Effect of fixation
 Different fixatives retain to different extent a given tissue
component.
e.g 1. Fat preferably be fixed by osmium tetraoxide
e.g 2. Proteins preferably be fixed by formalin
 Tissue functions are differently modified by different fixative
e. g. Alcohol retains some enzymes for enzyme histochemistry
 Fixatives affect the crispy of the stain
e. g. Alcohols make more acidic stains and formalin more
basophilic

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5.1.1 H & E Stain Method
 Most widely used and important general purpose stains
combination.
 Its popularity is due to its simplicity and ability to demonstrate
clearly different tissue structures.
 Hematoxylin, a natural dye product, acts as a basic dye that stains
blue or black.
 The basophilic structures are usually the ones containing nucleic
acids, such as the ribosomes and the chromatin-rich cell nucleus

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 Eosin is an acidic dye that stains cytoplasm, muscle, and
connective tissues various shades of pink and orange.
 This difference in staining intensity is useful in differentiating one
tissue from another.
 The eosinophilic structures are generally composed of intracellular
or extracellular protein.
 Most of the cytoplasm is eosinophilic.
 Red blood cells are stained intensely red.

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 The structures do not have to be acidic or basic to be called
basophilic and eosinophilic
 The terminology is based on the affinity for the dyes.
 Other colors, e.g. yellow and brown, can be present in the sample;
they are caused by intrinsic pigments, e.g. melanin.

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 Some structures do not stain well.
 Basal laminae need to be stained by PAS stain or some silver
stains, if they have to be well visible.
 Reticular fibers also require silver stain.
 Hydrophobic structures also tend to remain clear; These are
usually rich in fats, eg. adipocytes, myelin around neuron axons,
and Golgi apparatus membranes.

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Hematoxylin (H)
 Hematoxylin is a natural dye extracted by boiling the heartwood
of the tree called Haematoxylon campechianum with hot water.
 The word hematoxylin is derived from the old Greek words
Haimato (blood) and Xylon (wood), referring to its dark red color
in the natural state.
 Can demonstrate clearly enormous number of d/t tissue structures.
 is the most popular & widely used histologic stain.

Leaves and flowers of Hematoxylin campechianum

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 It is a brownish chocolate powder which is poorly soluble in
water
 and somewhat more soluble in ethyl alcohol (1 gm/100ml
water and 30-40 gm/100 ml alcohol).
 The active dye is not hematoxylin itself, but its oxidized
product, hematein.

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Hematoxylin Hematein

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 Hematoxylin should be oxidized to hematein- which can be done in
two ways
1. Natural oxidation (ripening)
 By exposing to light and air, takes 3-4 months
 E.g. Ehrlich’s and Delafield’s Hematoxylin
 It was common practice in the past to use natural oxidation in the
belief that it was the best,
• in the sense that it gave a more reliable and longer lasting
solution.

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 Natural ripening is accomplished by putting the solution in an
oversize flask, so it can be shaken, with the top plugged
loosely with cotton wool, allowing air to enter.
 This is left in a warm, light and airy place for oxidation to take
place.
 The flask is shaken periodically.

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 Oxidation may take several months, and is determined by
testing the solution from time to time.
 When the solution gives a satisfactory depth of staining, it is
transferred to a brown bottle and tightly stoppered for storage
in a dark cupboard.

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2. Chemical oxidation
 Hematoxylin oxidized in this way (chemical oxidation) is ready
to use immediately,
 but has shorter useful life due to continuing oxidation
converting the hematein into colorless cpd.
 Sodium iodate - Mayer’s hematoxylin
 Mercuric oxide - Harris’s hematoxylin
 Alcoholic iodine - Coles hematoxylin
 Potassium iodate - Carazzi’s hematoxylin

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 The most commonly used is sodium iodate, at about 200 mg per
gram hematoxylin.
 Others have also been suggested for particular formulas,
 but sodium iodate can be substituted for just about all of them if
used at the stated amount.
 Mercuric oxide was often recommended as an oxidant in the past.
 It is no longer recommended now a days.
 It is very toxic, and should be avoided whenever possible.

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 If it must be used, then full safety precautions should be taken,
 and the used solution must be disposed of in compliance with
government regulations to avoid contamination of the environment.
 If mercuric oxide is used as oxidant, the solution must be boiled
for the reaction to take place.
 Although this is also the case with sodium iodate, solutions to be
oxidized with it (mercuric oxide) may be brought to the boil for a
few minutes, then cooled.

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 Other oxidants do not need to be boiled, and oxidation will
take place at room temperature over a few days.
 This causes ripening to proceed faster and more efficiently,
and the solutions are usable immediately after they have
cooled.
 The addition of glycerin to several formulas is said to guard
against over-oxidation and perhaps to retard fungal growth.

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Mordants
 Hematein has poor affinity for tissue and is inadequate as a
nuclear stain without the presence of a mordant.
 Mordants are salts and hydroxides of divalent and trivalent metals
that form link between tissue and stain.
 A mordant is a metal with a valence of at least two.
 A mordant is a metallic ion bound to a dye that is involved in the
binding of the dye to tissue.
 It increases affinity of the dye to tissues.

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 The two commonest metals used in histotechnology are
aluminum and ferric iron, both with valencies of three.
 The attachment of mordants to dyes is by means of a covalent
and a coordinate bond.
 The complex formed from a mordant (metal) and is called a
lake.

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A lake can be defined as:–
 A coordination complex formed between a polyvalent metal ion
and certain dyes.
 Two types of bonds are involved in the fundamental reaction
between a mordant dye and a mordant.
 One is a covalent bond with a hydroxyl oxygen.
 The other is a coordinate bond with oxygen (the electron donor).

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Adjacent hydroxyl and carbonyl
groups of hematein
Co-ordination complex between
hematein and aluminum

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Classifications according to the Mordant used
 Alum Hematoxylin
 Iron Hematoxylin
 Tungsten Hematoxylin
 Molybdenum Hematoxylin
 Lead Hematoxylin
 Hematoxylin with out a mordant

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 The Hematoxylin in the H & E is invariably an aluminum
mordanted solution (a hemalum)
 Mordants and dyes may be applied in three ways.
 The terms used are defined below.
 The suffix -chrome in these terms refers to chromium, which
is a common mordant in textile dyeing.
 The terms are borrowed from that industry.
 In histotechnology chromium is only rarely used, but the terms
are still used.

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Mordant vs dye application
 1. On chrome: The mordant is applied first, followed by the dye.
- Heidenhain’s iron hematoxylin is a classic example.
 2. Meta chrome: Mordant and dye are mixed together, then applied.
- Alum hematoxylin solutions are applied like this. It is probably the
commonest way to use mordant dyes in histotechnology.
 3. After chrome: The dye is applied first, followed by the mordant.
This is hardly ever done in histotechnology.

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Progressive vs. regressive staining
 All alum hematoxylin, whatever their formula may be used as
either progressive or regressive stains.
 Progressive staining means that the tissue is left in the stain
just long enough to reach the proper endpoint.
 Therefore, it may be necessary to examine the slides at several
different intervals to determine when staining is dark enough
but not too dark.

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 Regressive staining means that the tissue is deliberately over
stained and then destained (differentiated) until the proper
endpoint is reached.
 The difference between methods is largely one of convenience.
 Progressive hematoxylins are generally less concentrated and
work slowly to avoid overshooting the endpoint.

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 Regressive hematoxylins are more concentrated and many can
achieve over staining in a matter of less than a minute,
 while differentiation (removal of excess unwanted stain)
requires only a few seconds.
 Also, timing is not so important in regressive procedures.
 As long as the slide is over stained, it doesn't matter whether it
was in the staining dish for 1 minute or 10.
 Regressive procedures are therefore faster and more
convenient than the progressive ones,

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 And they have the added advantage that differentiation also
removes hematoxylin from the gelatin or other slide adhesive,
producing a clear, transparent background.
 Differentiation is done in dilute acid (usually acid alcohol because
of hematoxylin's greater solubility in alcohol).
 Differentiation is stopped immediately by simply washing the
slides in water.

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 If too much hematoxylin has been removed, a few more
seconds in the stain will correct the problem, and the whole
process can be repeated.
 If too little has been removed, a few more dips in acid alcohol
will produce the correct endpoint.
 Note: With all hematoxylins, progressive or regressive, the
endpoint is the same!

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Bluing
 Because most alum hematoxylin formulae are fairly acid, the
nuclei will at first be stained the purplish color of the acid dye.
 Changing their color to blue gives a much better contrast with
the usual red counterstains.
 When the endpoint has been reached by either progressive or
regressive methods, nuclear color can be changed in one of
two ways.

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1. The slides may be dipped for a few seconds into a weakly
alkaline solution such as ammonia water or dilute sodium
carbonate.
Note that differentiation stopped the second you rinsed the slide
in water, so the alkaline solution is not necessary for that.

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2. They may be washed for 2-5 minutes in tap water.
3. Depending on the geographic area and the local method of water
treatment, tap water tends to be slightly acid, with a pH in the
range of 6.0 - 6.8.
However, this is considerably more alkaline than the pH of most
alum hematoxylins (2.6 - 2.9), so bluing results.

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Eosin (E)
 Eosin is one of the xanthene dyes which are derived from
xanthene.
 This class of dyes is divided into three subgroups:
 1. Fluorenes
 Pyronins
 Rhodamines
 2. Fluorones
 3. Rhodols

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Fluorone Dyes
 The fluorones are sometimes referred to as the eosins, and
include many dyes used as counterstains to alum hematoxylin.
 The most suitable stain to combine with alum hematoxylins
 Its particular value is its ability, with proper differentiation to
distinguish between the different types of cells,
 and between the different types of connective tissue fibers ,by
staining them differing shades of red and pink.

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Types of Eosins
 Eosin Y (yellowish)- the most widely used
 Eosin S (Ethyl eosin),
 Eosin B (Eosin bluish)
Eosin staining
 A good counterstain will not only contrast sharply with the blue
nuclei,
 but it will allow the non-nuclear tissue components to be clearly
differentiated from each other

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 Note that eosin Y is normally dissolved in 95% ethanol.
 Therefore an eosin-stained slide will be decolorized if it is left
very long in 95% ethanol before cover slipping.
 Eosin solubility in 100% ethanol is considerably less, but here,
too, stain can be lost slowly.
 To avoid these problems, slides should be run quickly from the
eosin dish to the clearant dish.

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 The shade of the red counter stain is important for optimum
contrast with the blue nuclei.
 Eosin Y normally has a somewhat yellow-orange color, but adding
a little acetic acid to the solution will cause it to become redder.
 That improves the contrast with the blue nuclei between the non-
nuclear tissue elements.

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 The density of the red color is important for differentiating the
non-nuclear components.
 Too little color will make the slides look pale and washed out
and will cause a certain amount of glare.
 On the other hand, too much dye will blur the distinction.

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Judging endpoints
 The endpoints of both hematoxylin and eosin staining involve
color density and contrast, neither of which is measurable by a
clock.
 The human eye, however, is very good at both measurements.

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 At first, endpoint judgment will have to be under the
microscope, which will take a little time.
 However, with a little experience and a standardized control
slide, judgment can be made accurately by the naked eye in
just a few seconds.

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Hematoxylin-Eosin staining procedure
1. Dewax/ deparaffinization with xylene
2. Hydrate through decreasing grades of alcohols
3. Stain with an alum hematoxylin of choice for a suitable time.
4. Wash well in running tape water until sections ”blue” for 5 min.
5. Differentiate in 1% acid alcohol for 5-10sec.

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Staining procedure........cont’d
6. Wash well in tap water
7. Stain in 1% eosin for 10 min.
8. Wash in running water
9. Dehydrate through increasing grade of alcohols
10. Clearing using xylene, and
11. Mounting using DPX

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RESULTS
 Nuclei – blue / black
 cytoplasm – pink
 RBC – orange red
 Fibrin – deep pink
 If an H&E slide shows any colors other than purple/blue and
red/pink – such as yellow or brown, the additional color is
probably due to an intrinsic pigment such as melanin.

Histopathology, 28-Dec-21 51
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GALATOOMAA !
Histopathology, 28-Dec-21 52
7/4/2023

4_5839287714297417902.ppt

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