Last Update: 2 November 2017 Part I

Staining M - 33
Q. Why staining of tissues is necessary during histological studies? Distinguish between stain and dye. Discuss the general
characteristics of histological dye. What are main Components of dye? Add a short note on: Bathychromy, Resonance and Spectral
shift. What is chromophore? How many types of chromophore are there? What do you mean by dye Diversity? Classify dyes by
following modes: a. Chemical mode of classification and b. Metachromatia Mordanting Mechanism of mordanting in case of
haematin. Give example of anyone of the following important dye & its extraction procedure: Carmine Haematoxylin and Orcein.

Why do we stain tissues during histological studies?

Live or only fixed cells when examined under microscope, our eye failed to understand of the different
components in these cells, due to the constituents of the cells and its intracellular materials are usually transparent &
colourless and have similar refractive index, and therefore not distinguishable from one another. It is only seen a black
and white image of these cells by the different contrast of light and shadows, nothing else but cell contain different
organelles, this organelles only possible to see under microscope, when stained them. In histological studies, the tissues
sections are treated with dyes, which not only impart colour to the tissue-constituents but also introduce differences of
refractive indices amongst different constituents of tissues or cells and makes them easily distinguishable under the
microscope. So staining is a necessary step in the study of histology.
What are stains?
Stains are defined as colourful substances, which time ability to impart their own colouration in the tissue. Most
of them are aromatic substances technically known as dye.
Principle of staining
1 To change the refractive indices of the different organelles of a cell that makes these visible
under microscope.
2 To identify the localised certain differential chemicals constituents of these cells.

These two basic requirements in the study of Histology are fulfilled by the use of dye.
Definition of dye
According to Barker 1969 for the purpose of micro technique, dyes may be defined as aromatic, salt-
like, crystalline solids, dissolve in water or any aqueous solution in the forms of ions [either the cataions (+ ve) or
anions ( ve) or both]. These ions do not lose colour and usually they do not change it.
Discuss the general characteristics of histological dye.
All colouring substances that may impart colour to a tissue or its constituents may not be a true histological dye,
for instance, tissue lipids are stained by Sudan black dissolved in 70% alcohol, but this is not a true case of dyeing.
Actually, Sudan black is more soluble in lipids than 70% alcohol and hence, it passes from the alcoholic solution to the
tissue-lipids and the lipids look stained.
Only certain chemical compounds having certain specific characteristics are considered as histological dyes.
These characteristics are :
1) Ionising capacity:
Histological dyes are aromatic, salt like compounds which get ionised in water or in solution so that
either the cationic or anionic part can bind to specific components of the tissue.
2) Bathychromy :
A dye in solution has a characteristic colour and the solution absorbs light-rays of particular web-lengths
of the visible spectrum and transmits tight-rays of other wave-lengths. This property, called, as Bathychromy
is a function of the chromophoric group or colour-imparting group present within the dye molecules. For
example, an aqueous solution of the magenta coloured dye, basic fuchsin transmits most of the red and
orange lights and some violet and blue light but absorbs green and yellow light.
3) Clinging /adherens (ability to bind with the tissue):
A dye molecule can bind chemically with specific components of the tissue. This ability to bind with
tissue constituents is function of the auxochrome group present within the dye molecule.
4) Auxochromophoric system:
A dye must possess two functionally important groups, called as the auxochrome and the chromophore
groups. The chromophore group is responsible for the particular colour of a dye while the auxochrome group
is responsible for binding the dye to the tissue.
5) Colour stability:

-1-
 The linkage between a dye molecule and the tissue constituents is quite stable so that the colour imparted
to tissues does not fade away during post-staining treatment of tissue.

Component of dye
In other words, dye is an organic compound which contains as integral part of
(1) The chromophore (colouring part) and
(2) a clinging part that is auxochrome.
So dye auxochromophoric system.

1. CHROMOPHORE
It is a part of dyes, which exhibit different colouration in response to absorption visible spectrum. The
colour is different in different dyes, even same molecular from but different in structural formula. So each dye have a
specific colour to the relation of its own chemical structure this physical properties is known as Bathychromy.
Example
If a visible spectrum of light (400 nm 700 nm) passes through a chromophoric system, it transmits only a
small portion of the spectrum and absorbs all other then such chromophoric system exhibit a particular colour. As,
Haematoxylin, absorb the entire visible spectrum except 450 nm thus in exhibit blue colour. The visible spectrum shows
different colouration (violate 400 nm Red 700 nm) in response to its variation of wavelength along the spectrum.
Resonance
Chromophore is the arrangements of the following linkages as side effective branch of the aromatic cycle. The
double bond in response to the effecting radicals changes its position single bond to double or vice versa in the only
conjugated system. The bonding electrons are able to move from one atoms to another along the conjugated system. This
phenomenon is known as resonance.

C = C, C = O, C = S, C = N, N N, N = O, -NO2 (effective radical as side branch of aromatic cycle)

Spectral shift:
The bathychromic change due to resonance leads to change of spectrum or spectral shift of the refractive
colour and in turns change in the colouration of the dye linked cellular components. Therefore, different resonance
variety of the molecule gives the different colouration as example,
Fig:

Effective radicals (side branch) Quinoid A structure

The 3 type of molecule are same in their general formula but differ only in their configuration so called resonance
variety also give 3 different types of colour response.

Types of chromophore
Depending upon the chemical structure chromophore is divided into the following types.

1. Quinoid chromophore:
such chromophore have a quinoid ring which is unsaturated because, ring with double bonds as
( ) . They can exist in two forms Quinoid-A and Quinoid-B

fig:- Quinoid-A and Quinoid-B

These two structures are same but relative orientation of double bond (=) along the ring is different in position. It
is important in histology. The chromophore containing quinoid ring are most common and show different colour variation
due to such orientation. There are about 200 quinoid dyes in histology. Example, haematoxylin, eosin, methylin blue,
basic fuchsine, light green crystal violate, toludin blue, carmine (Anthroquinon) azocarmine (azine).
2. Azo-chromophore
-2-
 Such type of chromophore have no quinoid ring, the nitrogen atoms link the two-ring together (-N=N-). These
chromophore commonly used in textile industry & there are about 20 types of ago chromophore.

Example,
(1) Orange G , (G = GELB = yellow). It takes past in orange coloration of cytoplasm and RBC.
(2) Genus green, it stains mitochondria in living condition succinic dehydrogenase of mitochondria is
responsible to bind with the genus green to give its colouration. In dead condition, this enzyme destroyed and
mitochondria cannot be stain. It is VITAL DYE because, when it enters into the living cell, it can stain
without fixation.
3. Nitro-chromophore
Such type of chromophore contain effective radical as NO2. It is particularly found only one due i.e. picric acid
which has 3-NO2 groups and one OH group. It is primarily a dye but secondarily a fixatives (non-additive). NO2 gives
rise to colour (yellow) and OH combined with the tissue.

auxochrome

chromophore

Effective radicals Picric acid

These 3 type of chromophore are used most frequently. The less frequently used chromophoric system in biological stain
are.
(I) Nitroso group. (-N=0)
(II) Indemine group (-N=0) this is always forms part of a larger chromophoric system as present in the
chromophore of toludin group.
Dye Diversity : There are three main types of chromophores in three different types of dye but they produce deferent
colours, which is much considerable in number then the basic types this is known as dye diversity. This may be due to the
following reasons.
1. Resonance : Change of intramolecular configuration with the changing effecting radicals in an unsaturated
conjugated molecule to form new one with different colour responses is known as resonance. Due to resonance a single
dye can exhibit several configuration as well as several bathychromic property due to spectral shift.
2. Number and Types of chromophore : Colour diversity depends upon chromophore present in dye. If we introduced
more than one chromophore into dye-molecule it causes dye-diversity. The colour of a dye changes in relation to the
number of chromophore. Such change the number of chromophore cause spectral shift.
3. Substitution of radical : Generally in aromatic compound, the halogens can be easily substituted by another with the
change of radicals there is changes of Bathychromy which lied to spectral change in terns change of colouration.
Example :
Eosin (Yellowish) Erythrosine (pink)

When the four Bromine groups are present in eosin then it shows a particular colour but when these Br. Group is
substituted by the Iodine, then the Bathychromy of eosin is changed and spectral shift occurs and subsequently a new dye
known as Erythrosine involved.
4. Addition of radical

-3-
 Addition of extra-molecule in the structure of dye molecule cause the change of Bathychromy i.e. subsequently
changes the colouration.
Example :

+2CH3 + CH3 + H+

+ H2

Basic fuchsine (red) Malachite Green Crystal violet

When extra 2 methyl groups (-CH3) is added to the NH2 group of the basic fuchsine then it change to Malachite
green when this malachite green subjected to further methylation in remaining NH2 position then the Bathychromy
change into another and form a new dye Crystal violet which is violet in colour.
Not only changed the structure and spectral shift but also changes the Staining properly of such dye because,
1) Basic fuchsine stain the DNA.
2) Malachite green stain the
3) Crystal violet stains the chromosome.

2) AUXOCHROME
The auxochrome or Colligator is responsible for the attachment of chromophore with the substrate. Chemically
these are the ions and such ionic portion form bond with the substrate and totally chromophore clinged with the
substrate by these auxochrome. On the basis of ion auxochrome are two types.
a) Cationic auxochrome :
The auxochrome with a net positive charge (+ve) is called cationic auxochrome. The +ve charge are
responsible for basis characteristic of the dye. The basic auxochrome are amine which ionises thus

Primary amine : -NH2 + H+ - NH3+

Secondary amine: NH + H+ NH2+
Tertiary amine NH + H+ NH+
Quaternary amine N+ exist only in ionised form

It is a basic dye due to the presence of basic radicals (-NH2). It stains the
acidic portion of the cell i.e. nucleic acid (DNA & RNA).
b) Anionic auxochrome :
The auxochrome with a net negative change (-ve) is called anionic
auxochrome. The ve charge are responsible for acidic characteristic of the dye.
The acidic (-SO3H), hydroxyl (-OH) phosphoric (-H3PO4) etc.

1) COOH -COO- + H+
2) SO3H SO3- + H+

3) + H+

H3PO4 H2PO4 + H+
-
4)
-
5) OH

In eosin, where, quinoid-B-chromophore is associated with an acid radicals -COOH as a result it acts as a acidic stain and
stains the basic portion of the cell i.e. cytoplasm.
-4-
Classification of dye
The system of classification of dye is very simple classification made upon two basic principles.
1) Physical mode of classification
According to the ionic nature of the auxochromophoric system i.e. what type of auxochrome is attached to
the chromophore on the basis of it the dyes may be of two types.
a) Acidic dye
When the chromophore are associated with the acid radicals form acidic dye or anionic dye. Hence staining
property of the dye depends on such type of radicals present in auxochromophoric system. Those part of the
tissue / cells stain with the acid dye is known as acidophilic. So acid dye stain the basic part of the tissue / cell.
Most of the acid dyes are the salts of sodium. The anionic part remain associated with the auxochromophoric
system and sodium (Na+) remains as true ions in the whole dye molecule and these COOH- / COO- and Na+
balance the total molecule as a neutral natural salt except picric acid.
Example : eosin, methylin blue etc.

Eosin (Eosin) Neutral natural salt

b) Basic dye
When the chromophores are associated with the basic radicals, form basic dye or cationic dye. Hence the
staining properly of the dye depends on such type of radicals present in the auxochromophoric system and that
part of the tissue / cell stain with the basic dye is known as basophilic. So basic dye stain the acid part of the
tissue / cell.
In most of the cases the basic dye are salt of chlorine (chloride). Beside chloride, it may be Nitrite or acetate.
c) Amphotic dye
This type of dye may be either cationic or anionic.
Example : haematin is cationic below the pH 6.6 but anionic above this pH.
Practically no dyes are acidic or basic in nature but they are natural neutral salts having both
acidic and basic radicals except the picric acid, which is a true acid.
2) Chemical mode of classification
Depending upon the chemical nature of the chromophore the dye may be of three types
A) Quinoid : Chromophore having quinoid ring depending upon the resonance the
quinoid type further classified in to following types,
1) Triaryl methane dye: Such type of dye contains 3-amyl ring in its
chromophore, which is held together. This dye may be anionic or cationic.

Example : Anionic Methylin blue.

Acid fuchsine.
Cationic Basic fuchsine,
Crystal violet
Light green, etc.

2) Haematin: In this type of dye the two benzene rings remain attached side by
side and quinoid remain attached to benzene ring. Example : Haematoxylin

3) Xanthene: The chromophoric system contain R. R. R may be

hydrogen, alkyl or oryl radicals. Example, eosin, pyronine etc.

-5-
 4) Azine: In this type of dye the chromophoric system is a pyrazine ring sandwich between two
aromatic system. Azine may be two types.

a) Oxazine : The oxazine chromophore has very much similar with that of azine and exist
in O-quinoid form

b) Thyazine : The chromophore of thyazine is like that of the

oxyzen. Sulpher and nitrogen links two rings. These are
metachromatic dyes. Example, Toludi blue, thiamine blue, etc.

5) Anthro quinon : These are also quinoid A type dye where quinoid ring
present between the two benzene ring and produce anthro quniod
structure, example cannine, alzarine, etc.

B) Azo dye :- It is very large grump of dyes which is important to the textile
industry and in biology. The chromophore (-N=N-) of this group containing
two aromatic rings. This aromatic ring usually Benzene or Naphthalene the
azo linkage can exit in two isomeric forms. Cis and trance.

N=N and N=N

Cis form Trans form

In dyes trans configuration is due

to intramolecular hydrogen bonding
between one of the azo nitrogen atom
and a subsequent in the ortho position or
at least one of the aromatic rings.

1) Amino azo dye : It is mono azo compounds with sulphuric and

phenolic substituents. Example Orange Green, Genus green,
etc.

2) Mordant azo dye : These group of dye are capable of

chelating with chromium (Cr) or similar metals the
commonest arrangement are.
a) Hydroxyl groups at orthro position I both aromatic
benzene ring :

b) Hydroxyl and amino group at artho position of the

aromatic ring respectively

c) The salicylic acid arrangement on one of the

aromatic rings :-

3) Cationic azo dye : These are not used in histology but mainly
used for colouring of leather. Example Bismarck brawn.

C) Nitro dye : Chromophore of this dye contain

NO2 group. It shows resonance variety. Example
picric acid.

-6-
 Important feature :
1) It never form any stable neutral salt therefore it is acidic.
2) It is additive fixative itself.
3) Stains the muscle fibre.
4) It is only representative of nitro dye.

D) Unclassified dye :
This type of dye have no fixed position is a particular group. Example Orcein.

Metachromatia : Certain basic dye which have a particular colour in response to particular chromophoric
system but when such type of dye subjected to stain a section which gives rise to different stained component
with different colour except of the original dye-colour. Such type of dye is known as metachromatic dye and the
property is known as Metachromatia. The tissue are stain with different colour are called chromatroph.
Reaction in Metachromatia : The chromophore of the dye exhibit a certain coloration or bathocromic
property but when auxochrome clinged with the different components of the section the structure of the
auxochromophoric system is quickly quite modified in different components due to their different
chemical form. This different auxochromophoric system exhibit different bathocromic property therefore
we get different specific colouration in each components.
Another investigator suggested that metachromatic dye have a tendency to form dimmers and polymers
in a aqueous solution their frequent aggregation and mode of attachment cause the shift of coloration in
the different case.
Example : Methyl violet, thiamine, Methylin blue.
Advantage : We can get several colour of the different tissue component with the application of only are
dye rather double stains.
Misnomer about the dye conception :- Acid and basic dyes are misnomers because they are actually not
acidic or basic but are neutral salt.
Mordanting : This is a process by which a feeble colour dye is converted into a strong colour dye by using
strong cationic multivalent metals present in the double salt. These double salt are called mordents.
Some chif mordents :
1) Ammon alum : Aluminium ammonium sulphate [Al2(SO4)2 (NH4)2SO4 ].
2) Iron alum : Aluminium iron sulphate.
3) chrome alum : Potassium aluminium sulphate (KAlSO4).
Only metallic parts of the mordents are used for forming polyvalent (Co-ordinating and ionisable bonds.)
Use of mordents : Mordents are used in two ways, as
1) Simple bath method : Here mordents are directly mixed with haematin.
2) Double bath method : 1st tissue section is treated with mordant and then the complex is mixed
with haematin.

Mechanism of mordanting in case of haematin

1) Simple bath method : Amman alum is used as a mordant in this mordant, (Al) is strongly
multivalent, cationic metal because highly affixed with the OH grams of the haematin by the
special type of chelating bond when mixed with haematin.
As the aluminium (Al) becomes the integral part of the dye molecule it acts as a modifier and as
a result of spectral shift the feeble red colour of the dye is covered into bright dye use to stain the
tissue section.
As aluminium is a strongly cationic multivalent metal the whole dye molecule is now converted
into a cationic entity and behave like a basic dye. The mordant haematin is used in the laboratory
popularly called haematoxylin.

2) Double bath method : Iron alum is used

as a mordent is first mixed with the tissue
section and then haematin is mixed with
it which forms a mordent-dye complex
with the help of cheated bond between the
OH group and Fe++ .
Step (1) : At first nucleic acid is mixed with iron alum which
forms a nuclei acid iron alum complex. The iron
is attached to the phosphoric part of the nucleic
acid.
Step (2) : Haematin then add with the nucleic acid - iron alum
-7-
 complex and it forms nucleic acid iron haematin complex.
H3PO4 complex

Nucleic acid-

Haematin Haematoxylin

Waterners theory : According to this theory the metal ion is incorporated to the Benzene ring by the
process of chelation.

In double bath method shifting of colour is more vigorous. The feeble red colour is converted into deep bluish
black colour. It is excellent for microphotography and generally used for chromosome staining. Mordents attached with
dye molecule on one hand and with the tissue on the other hand. As the mordents and dye both are used in solution such
complex are called Lakes.
Chelation : If there are two adjustment -OH group in an benzene ring, the strongly cationic polyvalent metal is changed
their orientation due to bond between -OH and polyvalent metals. This type of bond is known as chelate bond and this
process is known as chelation. After the attachment of the strong cationic metal the whole dye molecule act as a basic
dye which is attaches to the negative group or negatively changed molecule of the tissue.
Example : chelation bonding generally occurred in the mordanting as haematin to haematoxylin. In such case aluminium
and iron generally used as polyvalent cationic mordant element in single and double bath method respectively.

Advantage of mordanting :
1) The colour is not easily removable by neutral fluid (liquid).
2) Progressive and regressive staining become possible.
3) Slow dehydration is possible.
4) Counter staining also possible.

Nomenclature of dye :
The most sensible nomenclature of dye to call them by their chemical name like
1) Amino methyl parasos anilin for crystal violet the crystal violet is most simple then any other type of dye. The
chemical name of other dyes becomes so lengthy that can not be easily remember or used.
2) The most applicable nomenclature of dye is according to their nature of colouration produce which is easily
applicable.
Example : - crystal violet ------- violet in colour.
Methylin blue ----- blue in colour.
Eosin -------------- yellow is pink
Genus green ----- green in colour.
3) The another useful nomenclature is by their chemical nature for example,
Napthol yellow -------------- contains napthol group.
Some times name of the some dyes gives wrong chemical information for example
1) Methylin blue --- does not contain any methylin group.
2) Azo carmine ---- does not contain any azo group

-8-
 But still we accept this nomenclature yet.
4) Sometimes some funny informations also given by nomenclature for example Congo dye (red) used for
histology, cytology, and also in textile industry the name came from Belgium Congo.
Vat dye : This type of dye important in textile industry mainly used on cotton the oldest vat dye are Indigo.

Some important dye & its extraction procedure :

1. Orcein :
Orcein dye is a plant product and extracted from lichens popularly known as Orchil weeds grown on the rocks of
Sweden sea. Two common species are on the rocks of found these are Rosellan and Leukonara sp.

Technique of extraction :
Lichen released Lekanoric acid this acid boiled with water and produce orcinol. When orcinol exposed into
atmospheric oxygen then oxidized into Orcein. It is much important to stain connective tissue arterial elastic fibre gives
black colouration by the stain it.
Lichen collection processed Lekaronic acid Boiled with water Orcinol + Atmospheric Oxygen
Orcein
When Orcein combined with acetic acid produce a stain, aceto-orcein which is important to stain the chromosome. In the
similar manner it also produce lacto-Orcein.

2. Carmine :
Carmine has so much interest then any other dyes in this respects that it is only the dye, which is extracted from
animal source. All other dyes are plant product.
Name of animal :- Doctylophious cacti
Family coccidae
Sub order Homoptera
Order Hemiptera
Class Insecta
It is popularly known as scale insect or acid insect, which is parasitic on sum succulent cactus (Nopalea
coccinollifora in central America. The dye produce by the fat body or ovary of the female insect. Male are not
produce any coloration.

Technique of extraction :
Nearly 1000 taken and dried this dried specimen known as Cochineal. Cochineal are first powered
and then boil in water to extract the dye them add subsequent by lead acetate, ethanol, sulphuric acid into the
extract. During the addition of H2SO4 carmine dye is precipitate down at the bottom and then carmine isolated
from it.
When carmine mixed with borax produce borax-carmine for whole mount preparation of invertebrates
larvae, Cyclops and other micro organisms were used. Acetocarmine produced with mixing of carmine and acetic
acid and used for chromosome staining. It has great commercial value.

3. Haematoxylin ;
It is the commonest histological dye belong to sub division haematin under the Quinoid group and is for plant
origin. Though it is commonly known as Haematoxylin, it should be called as Haematin when ready for use.

Characteristic of Haematoxylin :- Haematoxylin possesses number of characteristic features:

1) Haematoxylin possesses instant colouration.
2) Universally used for histology, cytology and pathological purposes.
3) It is a permanent stain which not faded by ordinary chemical reagent like water, xylol, ethanol
etc.
4) It able to stain the tissue section in very short time.
5) Stain chromosome and nucleus excellently.
6) Colour differentiations can easily be control for this reason this are useful for photomicrograph.
7) Never form salt.
Histochemical importance
It is used for histochemical detection there are two aspect which detected by it.
1) Some phospholipid in cell can be localised by Bakers acid Haematoxylin technique.
2) The neurosecretory cells can be stained very nicely by chrome alum haematoxylin.
3) Iron haematoxylin is good stain for chromosome staining.

-9-
What is Hematoxylin?

Hematoxylin is a natural dye which is extracted from the heartwood of the tree Haematoxylum campechianum, although
histotechnologists are probably more familiar with the name as Hematoxylon campechianum. The genus name
Hematoxylum is derived from two Greek words: haimatos which means blood, and xylon which means wood.

Extraction procedure:

The hematoxylin which we buy is extracted from

the heartwood of this bloodwood tree. There may be
some differences in method, but one is to chip the
heartwood of freshly logged trees, then boil the
Hematoxylin Hematein
chips in water. An orange-red solution is obtained,
which turns yellow, then black on cooling. The water is evaporated leaving crude hematoxylin. Depending on the genetic
line of the tree, hematoxylin content ranges from 0% - 10%. Further purification is undoubtedly done.

Hematoxylin is not a dye? Justify

Although it is common practice to use hematoxylin, it is not itself the dye. During the preparation of staining solutions
hematoxylin is converted into hematein. This is usually accomplished with chemical oxidising agents, but is sometimes
accomplished by atmospheric oxygen over time. Sodium iodate is the most commonly used oxidising agent for this
purpose (0.2g will oxidise 1g hematoxylin). Others are mercuric oxide (now strongly deprecated because it is poisonous),
potassium permanganate and iodine. Hematein may also be referred to as hematoxein, although it is not usually seen in
histotechnology references. Hematein is not incorporated directly into most staining solutions because it continues to
oxidise in solution and forms non-staining products. The quality of hematoxylin is usually higher and more consistent
than the quality of hematein, and solutions made with it are more easily standardised.

The dye is usually used in conjunction with a mordant, the two commonest being aluminum (as ammonium or
potassium alum), or iron (ferric chloride or iron alum). Other mordants are used much less frequently but include
chrome alum and phosphotungstic acid. The tissue component most frequently demonstrated is nuclear chromatin
using an aluminum mordant in the Hematoxylin and Eosin general oversight staining method. Using ferric salts as the
mordant, it is also used for acid resistant nuclear staining, the demonstration of muscle striations and numerous other
elements. With phosphotungstic acid it can demonstrate fibrin, muscle striations and some neuroglia fibres. There are
many published formulas.

Mechanism of mordanting in case of haematin

3) Simple bath method : Amman alum is used as a mordant in this mordant, (Al) is strongly multivalent,
cationic metal because highly affixed with the OH grams of the haematin by the special type of chelating
bond when mixed with haematin.
As the aluminium (Al) becomes the integral part of the dye molecule it acts as a modifier and as a result of
spectral shift the feeble red colour of the dye is covered into bright dye use to stain the tissue section.
As aluminium is a strongly cationic multivalent metal the whole dye molecule is now converted into a
cationic entity and behave like a basic dye. The mordant haematin is used in the laboratory popularly called
haematoxylin.

4) Double bath method : Iron alum is used as a mordent

is first mixed with the tissue section and then
haematin is mixed with it which forms a mordent-dye
complex with the help of cheated bond between the
OH group and Fe++ .

Step (1) : At first nucleic acid is mixed with iron alum which
forms a nuclei acid iron alum complex. The iron
is attached to the phosphoric part of the nucleic
acid.

- 10 -
 Step (2) : Haematin then add with the nucleic acid - iron alum complex and it forms nucleic acid iron haematin
complex.
H3PO4 complex

Waterners theory : According to this theory the metal ion is incorporated to the Benzene ring by the process of
chelation.

In double bath method shifting of colour is more vigorous. The feeble red colour is converted into deep bluish
black colour. It is excellent for microphotography and generally used for chromosome staining. Mordents attached with
dye molecule on one hand and with the tissue on the other hand. As the mordents and dye both are used in solution such
complex are called Lakes.

Chelation : If there are two adjustment -OH group in an benzene ring, the strongly cationic polyvalent metal is changed
their orientation due to bond between -OH and polyvalent metals. This type of bond is known as chelate bond and this
process is known as chelation. After the attachment of the strong cationic metal the whole dye molecule act as a basic
dye which is attaches to the negative group or negatively changed molecule of the tissue. Example : chelation bonding
generally occurred in the mordanting as haematin to haematoxylin. In such case aluminium and iron generally used as
polyvalent cationic mordant element in single and double bath method respectively.

Advantage of mordanting :
5) The colour is not easily removable by neutral fluid (liquid).
6) Progressive and regressive staining become possible.
7) Slow dehydration is possible.
8) Counter staining also possible.

Due to the widespread use of this dye in medical histology, it is important that a steady supply be available. This has not
always been the case. The last shortage occured in the early part of the 1970s. During this period several dyes were tested
as substitutes with some success. Unfortunately, none of them have the wide variety of uses that hematoxylin has.
Celestine blue B and mordant blue 3 are probably the most successful.

Hematoxylin has not yet been fully synthesised, but the compound has been split into some precursors, which have been
successfully re-converted to the original compound.

- 11 -