SEMINAR

ON

PRINCIPLES OF STAINING

SUBMITTED BY,

Dr.Lakshmi S Anand

II MDS
INTRODUCTION

If sections of human tissue are examined under the microscope immediately after sectioning,
they appear very dull and uninteresting.

The tissue lacks contrast because all of the fixed materials have a similar refractive index and a
similar colour so that a dull grey colour is all that can be seen.

To bring out the structure of the tissues, it is essential to stain the cells to see the different parts
in contrasting colours

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 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.

PURPOSE OF STAINING

Outlines tissues and cellular components.

Identification of tissues.

Establishes the presence or absence of disease processes.

HISTORY

The first stainings were tried by A VAN LEEUWENHOEK (1714-1719) studying their objects
with alcoholic saffron dyes.

H F LINK (1807) used iron sulfate for determining tannic acidin leaves.
M RASPAIL (1825) used iodine for the microchemical detection of starch.

Carmine staining was 1st introduced by botanists GOPPERT and COHN(1849), but they only
achieved a diffuse colouration of tissues.

JOSEPH VON GERLACH(1858), he successfully stained cerebellum with ammoniacal carmine.

GERLACH also discovered regressive staining by differentiation in weak acetic acid.

Pioneering work in histochemistry was done by R FEULGEN, & the Feulgen reaction
(FEULGEN R and ROSSENBECK H, 1924) is such an example.

In its initial formulation, this stain was developed for the detection of DNA in the nucleus.

K ZEIGER (1938) has given an excellent overview of the physicochemical basics of histological
and histochemical staining methods.

Genuine histological work began much later when T HARTIG, C WEIGERT, J GERLACH, P
EHRLICH and H GIERKE started in the second half of the 19th century with their systematic
studies of natural and synthetic dyes (aniline derivatives).

BASIC COMPONENTS OF STAIN

Dyes are aromatic organic compounds, & are based fundamentally on the structure of benzene.

Benzene is the parent compound of all aromatic compounds.

It is usually illustrated as a hexagon with its points representing each of six carbon atoms.

Each of the carbon atoms is illustrated as being joined to its two neighbours by either a single or
a double line.

In addition to benzene , it also contains

1. Chromgen group: Property of colour comes from this group

2. Auxochrome group: Ability to bind the tissue comes from this group

CHROMOPHORES
Most simple organic compounds such as alkanes, benzene and alcohols are colourless to the
human eye but will absorb light outside the visible spectrum.

Benzene, for example, absorbs strongly in the UV region of the spectrum but appears water-
white to the human eye.

Benzene must be altered so that it will absorb visible light and so become a visible coloured
compound that can be a useful as a dye

Any group that makes an organic compound coloured is called a chromophore.

For example;

Adding a single nitro group gives nitrobenzene, which is a pale yellow colour; adding a second
and third group intensifies the yellow colour and trinitrobenzene is a strong yellow colour.

AUXOCHROMES

Trinitrobenzene, although coloured, is still not a dye, as it will not bind to tissues.

Treating the section with trinitrobenzene will temporarily colour it yellow in the same way that a
plastic sponge appears coloured when it is soaked in a coloured liquid but the colour will wash
out as soon as the tissue is rinsed in a solvent.

To turn a coloured compound into a dye requires the addition of an ionizable group that will
allow binding to the tissues.

Such binding groups are called auxochromes.

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

The chemical, which is required to bring about the staining reaction, is called a Mordant.

Basic mordant react with acidic stains and acidic mordant react with basic stains.

Example;

Phenol in carbol-fuchsin (Ziehl–Neelsentechnique) and iodine which is added after the

application of crystal violet in the Gram staining method.

MORDANTING
The dye can only bind strongly to the tissue when the mordant acts as a link between the two.

Mordants and dyes may be applied in three ways:-

Pre mordanting (ONCHROME):

• The mordant is applied first, followed by the dye.

E.g. Heidenhain’s iron hematoxylin.

Meta – mordanting (METACHROME):

• Mordant and dye are mixed together then applied.

E.g. Alum hematoxylin solutions.

Post mordanting (AFTERCHROME):

• 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. Phenol in carbol-fuchsin & in carbolthionin

• potassium hydroxide in Loeffler’s Methylene Blue

METACHROMASIA

Certain tissue substances combine with stains to produce a color that is different from original
color of stain, such reaction is known as metachromasy, and exhibiting metachromacyis called
metachromasia.

Dye is known as metachromatic dye.

 • Eg-Toluidine blue.

Toluidine blue is a strong basic blue dye that stains nuclei a deep blue colour; however, it will
also stain mast cell granules a pink colour.

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.

• 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

• Safranine

ACIDIC DYES

• Acidic dyes are anionic and will stain cationic or basic groups in tissues such as amino
groups.

• 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.

• A) Natural:

• Haematoxylin

• Carmine

B ) 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

PROGRESSIVE AND REGRESSIVE STAINING

Progressive Staining In case of progressive staining, the dye is allowed to react with the tissue
until it stains the target structure. In fact, this is a difficult task to supervise, and all other
influencing factors should be controlled such as pH of the dye solution, thickness of tissue,
concentration of dye, etc. It is always necessary to check the staining at frequent intervals to
prevent overstaining or to have light staining.

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.

Mechanism of staining

The staining is the combination of a coloured substance (dye) with the tissue that retains the dye
after washing. The staining is primarily a chemical reaction between the dye and the tissue
The following chemical reactions are
involved between the dye and tissue components
1. Electrostatic bond
2. van der Waals attractions
3. Hydrogen bond
4. Covalent bond
5. Hydrophobic bond
6. Dye aggregations

1. Electrostatic bond: The electrostatic bond occurs between two oppositely charged particles,
and coulombic forces work between the particles. The oppositely charged dye binds with the
tissue For example, acid dye containing the anionic (negative charged) chromogen binds with
the acidophilic tissue containing positive charge. Similarly the basic dye having a positively
charged cationic chromogen binds with the basophilic tissue containing negative charge.
The dye is the combination of chromogen and auxochrome that are oppositely charged.
In the solution, the dye is dissociated into oppositely charged chromogen and auxochrome, such
as basic dye which dissociates into cationic chromogen (positive) and anionic auxochrome
(negative). Now the cationic pospositively charged chromogen of the basic dye combines with
negatively charged tissue
Example: Eosin, the acid dye, stains the cytoplasmic proteins.
2. van der Waals attractions : This is a type of non-coulombic force. This is the weakest force
due to the intermolecular interactions. The strength of this force is only 0.4–4 kJ/mol compared
to 20 kJ/mol in ionic bond. 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 asinduced 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.
Example: elastin stain by Miller’s stain and Congo red stain.
3. Hydrogen bond: Hydrogen bonding is a weak bond. It is a type of covalent bond that occurs
between hydrogen and a strong electronegative atom commonly O, N or F. Water forms
hydrogen bond and so competes with stain-tissue bonds. Therefore, hydrogen bonding in dye-
tissue less likely occurs in aqueous solution. Example: Best’s carmine dye to stain glycogen.
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 bond: This is a misnomer as in standard chemistry, there is no such bonding. It is
probably a type of van der Waals force. When two hydrophobic molecules interact, then London
force, the dispersion type of van der Waals force, interacts. Therefore instead of hydrophobic
bonding, the better terminology is probably “hydrophobic interaction” Example: staining in
aqueous solution and metachromatic staining.
6. Dye aggregations: Dye molecules may interact with each other forming dye-dye interaction.
They aggregate in solution and then penetrate into tissue. The dye-dye aggregate increases when
the dye concentration is high, the molecular size of the dye is bigger and temperature is low.

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 and strong. Commonest mountains used are resinous substances of
refractive index close to that of glass. These are soluble in xylol. Hence sections are dehydrated
and cleared inxylol and mounted. Mounting in aqueous mounting media is done directly after
staining for sections which cannot be subjected to dehydrating and clearing agents.
The basic steps in staining and mounting paraffin sections are as follows:
1. Deparaffinisation
2. hydration
3. Staining
4. Dehydration and clearing
5. Mounting
1. Deparaffinisation
Removal of was is done with xylol. It is essential to remove the wax completely, otherwise
subsequent stages will not be possible. At lest 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. HydrationMost of the stains used are aqueous or dilute alcoholic solutions. Hence it is
essential to bring the section to what before the stains are applied. The hydration is done with
graded alcohol for higher concentration to lower concentration. Alcohol and acetone are miscible
with xylol. First change is made to absolute alcohol or acetone followed by 90,70% alcohol and
finally distilled water. Sections now should appear opaque. Presence of any clear areas are
indicative of the presence of xylol. To remove this xyolo, sections should be returned to absolute
alcohol and rehydrated.
4. Staining
Various staining procedures are applied from this hydrates stage. The most common stain
applied for histological study is Haemotoxylin and Eosin. Various types of haemotoxylin
formulations are used.
5. Dehydration and clearing
Dehydration is done is 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 xylol. Such sections should be returned to abs.
alcohol and the process repeated. Mounting is done after 2nd or 3rd xylol.
6. Coverslipping and mounting
Make quite sure that the sections are quite clear. Do not let the section go dry before mounting
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 thexylol 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).
Gome stains, e.g. methyl violent, tend to diffuse into medium after mounting. This can be
avoided by using Highman's medium. Aqueous mountains require addition of bacteriostatic
agents such as phenol, crystal of thymol or sodium merthiolate to prevent the growth of fungi.
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).
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 and are supplied usually in 10 gm
packings.
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), but
never more than No. 1 ½ thickness (0.16 - 0.19 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 in xylol.
7. Xylol used to remove paraffin should not get mixed up with the clearing xylol. It also should
be frequently changed as it tends to get saturated.
CONCLUSION
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. Thus, conventional histological stainings
must be seen in connection with the chemical and morphological behaviour of cell structures.
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