BASIC PRINCIPLES

OF STAINING

Presented by
Lakshmi S Anand
II MDS
 STAINING

• Staining always involves the visual labeling of some

biological entity by attaching, or depositing in its
vicinity, a marker of characteristic color or form.
• 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.
• STAIN
Stain is defined as “any chemical substance which when
added to the living cells or to fixed structures or
structural components, makes them clearly visible or
detectable.”
A stain is the marker, or the reagent used to generate
the marker.
Outlines tissues and cellular components.

Identification of tissues.

Establishes the presence or absence of

disease processes.
 HISTORY

• A VAN LEEUWENHOEK (1714 – 1719) : alcoholic

saffron dyes
• H F LINK (1807) : iron sulfate – tannic acid in leaves
• GOPPERT & COHN : carmine staining
• JOSEPH VON GERLACH : cerebellum with
ammoniacal carmine
• R FEULGEN : poineering work in histochemistry
• K ZEIGER : physicochemical basics of histological
and histochemical staining methods
• T HARTIG,C WEIGERT,J GERLACH,P EHRLICH,
H GIERKE : natural and synthetic dyes
 Classification of dyes

CLASSIFICATION I
• Basic dyes
• Acidic dyes
• Neutral dyes
CLASSIFICATION 2
• Natural dyes
• Synthetic dyes
BASIC DYES
• Basic dyes are cationic and will stain anionic or acidic
materials such as carboxylates, sulphates and
phosphates.
• Most are used as nuclear stains.
• Acidic substances that stain with basic dyes are
termed basophilic.
BASIC:
• Haematoxylin
• Acridine red
• Aniline blue
• Azure
• Basic Fuschin
• Crystal violet
ACIDIC DYES
• Acidic dyes are anionic and will stain positive charged
groups in tissue
• Most are used to stain proteins in the cytoplasm and
connective tissues.
• Substances that stain with acid dyes are called
acidophilic.
ACIDIC:
• Eosin
• Erythrosine
• Picric acid
• Alizarin
• Acid fuschin
• Bismarck brown
NEUTRAL DYES
• Neutral dyes are simply compounds of basic and acid
dyes.
• In this case, both ions are coloured.
• Such dye complexes will stain both nucleus and
cytoplasm from a single dye bath.
• Romanowskystains are neutral dyes made from more
complex mixtures.
• These are the commonest dyes used in haematology.
• They are less common in histology but still very useful
and include Giemsa, Leishman and Wright’s stains.
NATURAL DYES
• Natural dyes are simply dye substances extracted from
natural sources.
• They have largely been replaced by synthetic dyes, which
are usually more reliable, cheaper and can be supplied
more readily.
• Natural dyes still in use include haematoxylin, carmine,
orcein and litmus, although synthetic varieties are also
available for some of these.
• Haematoxylin
• Carmine
Synthetic or Artificial:
• Nitroso (Naptholgreen)
• Nitro (Picric acid)
• Azo(Congo red, Orange G, Sudan III & IV)
• Xanthine (Eosin, Rose Bengal)
• Thiazole(Titan yellow)
• Quonolin
• Alcian blue
 Dye Based
on Chemical Structures
and Chromophore Groups
1. Azo dye: These dyes contain –N〓N– chromophore
group. Majority of the azo dyes are anionic (acid)
dye. Example: orange G and Congo red
2. Thiazine dye: Thiazine dye contains –C–N〓C– and
–C–S〓C chromophore group.Example: toluidine
blue and methylene blue
3. Triphenylmethanes: Triphenylmethanes contain〓N–
chromophore group. Example:methyl violet, light
green and malachite green
4. Azin dye: This group of dye contains C–O〓C and C–
N〓C chromophore. Example: Celestine blue and
Nile blue sulphate
5. Diphenylmethanes: They contain –NH chromophore
group. Example: auramine
6. Xanthene dyes: Example—eosin, Rose Bengal and
phloxines
7. Oxazine dyes: They contain C–O〓C chromophore
group. Example: cresyl violet and Celestine blue
8. Acridine dyes: The dyes of this group are derived
from acridine. Example: acridine orange
9. Anthraquinone dyes: These dyes are derived from
anthraquinone. Example: carminic acid
 Components of stain

• Benzene ring
• Chromgengroup: Property of colour comes from this
group
• Auxochrome group: Ability to bind the tissue comes
from this group
Principles of dye chemistry:
All the dyes have an aromatic hydrocarbon
benzene as a central component.
HC CH

HC CH

HC CH

Chromophore

Chromogen
6
 CHROMOGEN AUXOCHROME SUBSTRATE

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.
How dye produces color????

Chromophore
THE COLOUR INDEX
•To overcome all of the confusion there is a standard list of all dyes,
their synonyms and their structures.
This is called the Colour Index (CI).
•This is a monumental work of reference produced by The Society of
Dyers and Colourists and each dye is given an individual number and
listed along with its name(s) and properties
•Since each dye on the list has a unique number to identify it, this list is
the most reliable way of identifying a dye.
•When naming a dye in the description of a techniques, the CI number
should be given to avoid ambiguity, e.g. eosin Y (CI 45380).
•CI numbers are arranged according to their structure, with the most
important feature being their chromophoric group
 MORDANT
A substance which acts as an intermediary between
dye and tissue, thus increasing the affinity between
them .
Mordant – to bite
 MORDANT

• The dye can only bind strongly to the tissue when the
mordant acts as a link between the two.
• Salt and hydroxides of metals
Mordants and dyes may be applied in three ways:-
• Pre mordanting :
• The mordant is applied first, followed by the dye.
E.g. Heidenhain’s iron hematoxylin.
• Meta – mordanting
• Mordant and dye are mixed together then applied.
E.g. Alum hematoxylin solutions.
• Post mordanting
• The dye is applied first, followed by the mordant.
• The combination of dye & the mordant forms a
compound which called as “ lake.”
• These lake are capable of attaching themselves to
tissue.
ACCENTUATORS
• These are group of substances which while not acting
as mordants and forming lakes with dyes or taking
part in any obvious chemical union, will increase the
selectivity or staining power of the dyes.
• Effect of this group is due to the change in pH of
staining solutions.
Eg.
• potassium hydroxide in Loeffler’s Methylene Blue
 Metachromasia

“meta” means altered and “chromasia”means colour

• Metachromasia is defined as staining phenomenon
when the tissue is stained in different colours from the
original dye colour.
• The metachromatic dye is defined as the alteration of
the original colour of the dye without any change of
the chemical structure of the dye
• Cationic dye: Toluidine blue, methylene blue, azure A
and B, methyl violet, brilliant cresyl blue, etc.
• Anionic dyes: Biebrich scarlet and bromophenol blue.
Mechanism
This means that the colour absorption shifts to shorter wavelengths,
leaving only the longer wavelengths to be seen.
This is believed to represent polymerization of the dye.
The greater the degree of polymerization, the stronger the
metachromasia.
 Factors influencing metachromasia

1. Dye concentration: High concentration of dye

enhances metachromasia.
2. pH: Low pH increases metachromatic effect.
3. Temperature: Decreased temperature augments
metachromatic effect.
4. Aqueous solution: Water enhances van der Waals
force in between the dye molecules and increases
metachromatic effect.
 Types of staining

PROGRESSIVE STAINING
• In case of progressive staining, the dye is allowed to
react with the tissue until it stains the target structure.
• difficult task to supervise
• other influencing factors should be controlled such as
pH of the dye solution, thickness of tissue,
concentration of dye.
• It is always necessary to check the staining at
frequent intervals to prevent overstaining or to have
light staining.
• E.g Mayer’s Haematoxylin: first stains the nuclei
REGRESSIVE STAINING
• Here the tissue is intentionally overstained by dye.
• The affinity of the different structures of the tissue
with dye is variable, and this particular property is
exploited to remove the dye from the unwanted part
of the tissue.
• This procedure is also known as differentiation
STAINING MECHANISMS

Electrostatic bonding
Hydrogen bonding
Van der Waal’s forces
Covalent bonding

Hydrophobic bonding
Dye aggregation
Tissue permeability

9
 STAINING MECHANISM

1. ELECTROSTATIC BONDING
• The affinity between opposite ionic groups of dye and tissue
helps in staining.
• Dyes are classified as acid or basic
• For example, eosin is an acid dye with an affinity for the basic
protein of cytoplasm whereas hematoxylin is a basic dye
which has an affinity for the phosphate groups of
deoxyribonucleic acid of the nucleus.
• Salt linkage and ionic binding are alternative terms to electrostatic
binding; the correct name for the forces involved is
Coulombic attraction.
2.HYDROGEN BONDING

•Hydrogen bonding is a force of attraction between a hydrogen

atom in one molecule and a small atom of high
electronegativity in another molecule.

•Weak bond
•It require two components:-a donor group and an acceptor group
arise when a hydrogen atom lies between two electronegative atoms.

•Eg, Best’s carmine stain for glycogen

3. VAN DER WAALS FORCES

• These intermolecular forces are polar attractions.

• They are weak and are effective over a very short
distance.
• Attraction are between dipoles, that is molecules
possess separated positive and negative charges.
When the electrons of an atom concentrate in one pole of the atom,
then a dipole is formed. This dipole is just like a magnet having two
poles.
Dipole-dipole interaction: The positive charge of a permanent dipole
interacts with other permanent dipoles, and electrostatic interaction
occurs.
Dipole-induced dipole interaction: Similarly a permanent dipole may
induce adjacent atom and induces dipole. This permanent dipole may
interact with induced dipole.
Dispersion or London force: Permanent dipole may induce the
adjacent atom as induced dipole. The induced dipoles further induce
a chain of induced dipole. In this way a large network of tissue may
undergo induced dipole interaction. This is known as dispersion or
London force.
4.COVALENT BOND
In case of covalent bond, the two electrically neutral atoms
share electron with each other to satisfy the outer shell’s
required number.

The covalent bond is a stronger bond.

Example: periodic acid Schiff’s staining

for glycogen and Feulgen reaction.
5. HYDROPHOBIC INTERACTION

Maintains the dye in the tissue by means the exclusion of

water from hydrophobic regions,stabilizing the two groups
involved.
6. DYE AGGREGATION

• Dye molecules –have affinity for each other.

They aggregate in solution and then penetrate into tissue
• Factors influencing dye aggregation
• large molecular size
• Increased concentration
• Ionic strength
• Low temperature
• Useful in case of metachromasia
 7. TISSUE PERMEABILITY

• Degree of dye penetration will depend on the permeability or porosity of

tissue.

• Highly permeable tissues are more easily stained and decolorized than
poorly permeable tissues.
 Factors influencing staining

• Dye affinity to target tissue specimen

• Specimen geometry
• Target concentration
• Rate of reaction
• Rate of stain loss
1. Dye affinity to target tissue

• The tendency to bind a dye with the target tissue is

known as dye affinity.

• Acid dye binds with positively charged acidophilic

tissue, and basic dye binds with negatively charged
basophilic tissue.
2.SPECIMEN GEOMETRY
• Thick tissue: Less penetration of dye.
• Surface topography: More even surface gives better
stain.
• Microtopography of tissue: Alcohol fixation disturbs
microtopography.
• Inner geometry of tissue.
3.Target concentration
• The more the amount of the target tissue, the more
intense will be the staining.
4.RATE OF REACTION:
Short reaction time often decreases stain intensity.
5.RATE OF STAIN LOSS:
Too much differentiation often removes the stain.
 Staining of paraffin section

• The most common method of histological study is to

prepare thin sections (3-5 micron) from paraffin
embedded tissues. These are then suitably stained
and mounted in a medium of proper refractive index
for study.
 PARAFFIN SECTION
The basic steps in staining and mounting paraffin sections are as follows:

1. Deparaffinisation
2. Hydration
3. Staining
4. Dehydration
5. Clearing
6. Mounting
1. Deparaffinisation
•Removal of wax is done with xylene. It is essential to remove the
wax completely, otherwise subsequent stages will not be possible.
•At least 2 to 3 changes in xylol are given for suitable length of
time.
•Sections of this stage should appear clear and transparent.
Presence of any patches indicates the presence of wax and sections
should be kept longer in the xylol.
2. Hydration
Most of the stains used are aqueous or dilute alcoholic solutions.
Hence it is essential to bring the section to them before the stains are
applied.
The hydration is done with graded alcohol for higher concentration
to lower concentration.
Alcohol and acetone are miscible with xylene.
First change is made to absolute alcohol or acetone followed by
90,70% alcohol
Sections now should appear opaque.
Presence of any clear areas are indicative of the presence of xylene.
To remove this xylene, sections should be returned to absolute
alcohol and rehydrated.
3.STAINING
Various staining procedures are applied from this hydration stage.
The most common stain applied for histological study is
Haemotoxylin and Eosin.
Various types of haemotoxylin formulations are used
4. DEHYDRATION AND CLEARING
Dehydration is done with graded alcohols or acetones from 70% to
absolute alcohol or acetone.
Dehydrating alcohol and acetones can remove some of the stains.
Time has to be suitably modified to minimize fading of stains. Since
alcohol and acetone are miscible in xylol, it is used for clearing the
sections.
Any sections from which water has not been completely removed
would give a milky appearance after the first xylene.
Such sections should be returned to abs. alcohol and the process
repeated.
Mounting is done after 2nd or 3rd xylol
5. COVERSLIPPING AND MOUNTING
Make quite sure that the sections are quite clear.
1. Hold the slide between the thumb and the forefinger of one hand
and wipe with a clean cloth both ends of the slides. Look for the
engraved number to make sure the side the sections is present.

2. Clean carefully around the section and lay on a clean blotting

paper with section uppermost along with appropriate coverslip which
has already been polished.
3. Place a drop of mountant on the slide over coverslip. Amount of
mountant should be just enough. Invert the slide over the coverslip
and lower it so that it just adheres to the cover slip quickly turn the
slide over the lay it on a flat surface to allow the mountant to spread.
Do not press or push the slide at all.
4. After the mountant has spread to the edge of the coverslip wipe
around it for neatness. If proper care has been taken there should be
no air bubbles. If many are present, slide should be returned to the
Xylene to remove the coverslip. It will slip off and remounting is done.
No attempt should be made to pull the coverslip. Slight warming of the
slide from below will make the small air bubbles to escape from the
slide of the coverslip.
5. Coverslip should be in the center of the slide with neatly written
label on one slide.
Mountants
Histological sections which need to be examined for any length of time
or to be stored, must be mounted under a cover-slip.
There are two types of mounting media :
1. Aqueous media - Used for material which is unstained, stained for fat,
or mechanically stained.
2. Resinous media - For routine staining
Aqueous media
There are used for mounting sections from distilled water when the stains
would be decolorised or removed by alcohol and xylene, as would be the
case with most of fat stains (Sudan methods). Some stains, e.g. methyl
violet, tend to diffuse into medium after mounting. This can be avoided
by using Highman's medium. Aqueous mountants require addition of
bacteriostatic agents such as phenol, crystal of thymol or sodium
merthiolate to prevent the growth of fungi.
1. Apathy's medium (R.I- 1.52)
A very useful medium for mounting sections for
fluorescent microscopy.
2. Farrant's medium (R.I. 1.43)
Recommended for fat stains.
3. Glycerine jelly (R.I. 1.47)
An excellent routine mountant for fat stains.
4. Highman's medium (R.I. 1.52)
Recommended with the metachroamtic dyes especially
methyl violet.
Resinous mounting media
Natural or synthetic resins dissolved in benzene, toluene or xylene. These
are purchase readymade. In case they become too viscous they may have
to be diluted with xylene. Following are some of these media.
1. Canada balsam - Natural resin (R.I. - 1.52)
It is used as 60% resin by weight in xylene. H.&E stained slides are
fairly well preserved but basic aniline dyes tend to fade and prussian
blue is slowly bleached. Slides take few months to dry.
2. D.P.X. (R.I. 1.52)
Polystyrene resin dissolved in xylene as a 20% solution. It is most
commonly used.
3. There are many other synthetic resins sold under various trade
names e.g. Coverbond (R.I. 1.53), H.S.R. (Harlew synthetic Resin),
Histoclad (R.I. - 1.54), Permount (r.I. 1.54), Pro-Texx (R.I. 1.495).
DPX Dibutylphthalate Polystyrene Xylene
Criteria of acceptable mounting media
1. Refractive index should be as close as possible to that of glass i.e.
1.5.
2. It should not cause stain to diffuse or fade.
3. It should be crack or appear granular on setting.
4. It should be dry to a nonsticky consistency and harden relatively
quickly.
5. It should not shrink back from edge of cover-glass.
6. It should be free flowing and free bubbles.
Cover glasses used in histopathology
Care has to be exercised in selecting cover glasses for mounting, these are
available in variable sizes and thickness Following sizes are commonly available
22 x 22 mm
25 x 50mm
22 x 30 mm
Circular
22 x 40 mm
Cover glass should preferably be the No. 1 thickness (0.13 - 0.16 mm)
Some basic rules for staining
1. Keep stains and solutions covered when not in use.
2. After the slides are removed from oven these should be cooled before
being put in xylene.
3. Filter stains before use.
4. Once the slides have been put in the xylene to remove paraffin they
should not be allowed to dry out. Particular care must be taken not to
let the sections dry at the time of mounting as the xylene easily
evaporates and if the section dried before mounting preparation would
become useless.
5. Care should be taken that level of any solution used during staining is
such as to cover the slides.
6. Drain the slides well and blot the bottom on filter paper before
putting into the next solution. This is particularly necessary in
transferring from 95% of Abs. alcohol and Abs. alcohol to xylene
7. Xylene used to remove paraffin should not get mixed up with the
clearing xylene. It also should be frequently changed as it tends to get
saturated.
 Conclusion
Staining is used to highlight important features of the tissue
as well as to enhance the tissue contrast. The principle of
histological staining relies on the treatment of tissue
sections with dyes in solution which will react more or less
specifically with defined cell and tissue structures. A
uniform theory of tissue staining does not exist because the
mechanisms of dye binding with the various cell
components are quite heterogenous. Correct understanding
of the mechanism and following correct procedure will
ensure good staining results.
 Reference

•Theory and practice of histological techniques – Bancroft 7th

edition
•Cellular pathology technique – CFA Culling 4th edition
•Basic and advanced laboratory techniques in histopathology and
cytology- P.Dey