The document discusses the principles and applications of staining in biological tissues, including definitions, history, types of staining, and the chemistry behind dyes. Staining is essential for visualizing tissue components, and the proper selection of dyes is influenced by factors such as tissue affinity and dye properties. Various staining techniques, including direct and indirect staining, as well as the use of mordants and accentuators, are detailed alongside their effects on tissue visualization.

1. BY
DR. ISHITA SINGHAL
MDS SECOND YEAR

2. DEFINITIONS
INTRODUCTION
HISTORY
STAINING
TYPES OF STAINING
PHYSICAL THEORIES
CHEMICAL THEORIES
CLASSIFICATION OF DYES
PRINCIPLES OF SYNTHETIC DYE
CHEMISTRY
MORDANTS
ACCENTUATORS AND ACCELERATORS
METACHROMASY
REFERENCES
INDEX

3. DEFINITIONS

4. Dye is a single chemical reagent that chemically bonds
to the substrate to which it is being applied, to highlight a
specific component of a tissue.
Stain is a mixture of selected dyes, which when added
to living cells or structural components makes them
clearly visible, by highlighting different components.
Biological staining is the union between a coloured dye
and a tissue substrate which resists simple washing.
Staining is treating tissues or cells with a reagent or
series of reagents so that it acquires a colour, usually no
particles of dye are seen and the stained element is
transparent. -Culling et al
Staining is a visual labelling of some entity by attaching
or depositing in its vicinity, a marker of definite colour or
shape. -Bancroft et al

5. INTRODUCTION

6. • The tissue section is colourless because the fixed protein has the
same refractive index as that of glass. We use dyes that have specific
affinity with the different tissue proteins and colour them differently.
• Colour is seen by the eye as a result of the effect of certain
electromagnetic waves on the rods and cones of the retina. These
waves, which have a varying length, will determine the colour that is
seen.
• White light being composed of all the colours of the visible spectrum
varies in wavelength from 4,000 Â to 8,000 Â.
• If light of a specific wavelength is absorbed from white light the
resultant light will then be coloured, the colour being dependent upon
the particular wavelength that has been removed.

7. Stained materials absorb certain components of the white light
illuminating the specimen and therefore appear coloured.

8. HISTORY

9. Leeuwenhoe
k used a dye
as a
biological
stain when
he applied
saffron to
muscles.
Carmine
was
introduced
by Goppert
and Cohn.
Aniline dyes
were first
introduced in
Textile
industry.
Haematoxyli
n was
introduced
by
Waldeyer.
Heidenhain
devised the
iron
Haematoxyli
n technique.
1714
1849
1856
1863
1891

10. STAINING

11. The purpose of staining is:
To see the organism
better in contrast with
background
To differentiate one
organism from another
To determine particular
structures
For a successful staining there should be high specificity (ability to
distinguish between individual cell components) and high sensitivity
(capacity of a stain to demonstrate tissue substance at low
concentration).

12. An ideal stain should allow for:
Evaluation of the architectural pattern of
the tissue fragments.
Proper evaluation of nuclear morphology.
Details of cytoplasmic characteristic.
Visualisation and identification of
diagnostic features in the background.

13. Factors affecting staining:
Dye affinity to
the target
tissue
specimen:
The tendency to
bind a dye with
the target tissue
is known as dye
affinity. Affinity of
the dye to the
specific tissue is
influenced by the
pH and the
presence of
inorganic salt
concentration of
the solvent.
Specimen
geometry
• Thickness of
the tissue
• Surface
topography
• Inner geometry
of the tissue
Target
concentration:
The
concentration of
the target tissue
affects the
staining intensity
as the more the
amount of the
target tissue, the
more intense will
be the staining.
Rate of
reaction:
Rate of reaction
in the target
tissue also
influences the
staining pattern
such as in
Feulgen
reaction, the
short reaction
time exposes
only a few
aldehyde groups
producing weak
staining pattern.
Rate of stain
loss:
Staining pattern
is greatly
influenced by
rate of stain loss.
At times the stain
loss may be
intentional such
as differentiation
or de-staining.
The
differentiation
often removes
the excess stain
from the cell and
thus helps to
differentiate the
organisms.

14. TYPES
OF
STAINING

15. Direct Staining: When a staining procedure colours the cells present in a
preparation, but leaves the background colourless. Eg: Methylene Blue.
Indirect Staining: When a procedure colours the background, but leaves the
cells colourless, with the presence of mordant. Eg: Iodine in Gram stain.
Progressive Staining: Solutions stain to a desired intensity and no more.
Therefore they do not require differentiation in a dilute acid alcohol. Eg:
Mayers Hematoxylin.
Regressive Staining: The tissue is deliberately over stained and then de-
stained (differentiated) until the proper endpoint is reached. Eg: Harris
Hematoxylin.
Intravital Staining: Stain may be injected into a living animal and the stained
tissue removed and examined. Eg: Toluidine Blue.
Supravital Staining: The living tissue may be removed directly and
subsequently stained. Eg: Janus Green for staining mitochondria.

16. PHYSICAL
THEORIES

17. • Physical theories depend upon:
1. Solubility: Examples of this method of staining are the fat stains
which are effective simply because the stain is more soluble in the fat
than in the 70 % alcohol or other solvent in which it may be
dissolved.
2. Adsorption: This is the property by which a large body attracts to
itself minute particles from a surrounding medium, and is a
phenomenon well known to chemists.

18. CHEMICAL
THEORIES

19. There are
3 types of
interaction
s
Reagent-
Tissue
Interaction
Electro-
static
Bond
Van Der
Waal’s
Bond
Hydrogen
Bond
Covalent
Bond
Solvent-
solvent
Interaction
The
Hydro-
phobic
Effect
Reagent-
reagent
Interaction
Dye
Aggregatio
n
Dyes and tissue molecules can adhere chemically to one another by their side
chains. Acid dyes stain basic elements (cytoplasm) and basic dyes stain
acidophil material (nucleus). Their action is modified by pH of the solution in
which they are employed.

20. Electrostatic
bond:
The electrostatic bond occurs between two oppositely charged
particles, and coulombic forces work between the particles.
The affinity between opposite ionic groups of dye and tissue helps
in staining.
Dyes are classified as acidic or basic.
Eg: Eosin is an acidic dye with an affinity for the basic protein of
cytoplasm.

21. Schematic diagram showing
interaction of anionic and
cationic dye with the oppositely
charged tissue components.
Acid or anionic dye binds with
the cytoplasmic protein and
collagen, whereas the basic or
cationic dye binds with nucleic
acid of the nucleus.

22. Van der
Waal’s
bond:
This is a type of non-coulombic force.
It is the weakest force due to the intermolecular interactions and
are effective over a very short distance.
The strength of this force is 0.4–4 kJ/mol.
Attraction are between dipoles, that is molecules possess
separated positive and negative charges.
Eg: Elastin stain by Miller’s stain and Congo red stain.

23. Covalent
bond:
The two electrically neutral atoms share electron with each other
to satisfy the outer shell’s required number.
It is a stronger bond.
Significance in mordant dying.
Eg: Periodic acid Schiff’s staining for glycogen and Feulgen
reaction.

24. Hydrogen
bond:
Hydrogen bonding is a weak bond. It occurs readily in water.
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.
Eg: Best’s carmine dye to stain glycogen.

25. Hydrophobi
c effect:
It is probably a type of Van der Waal’s force.
In an attempt to isolate themselves from surrounding water
molecules, reactive hydrophobic dye molecules will become
bound to reactive hydrophobic tissue groups.
This reaction is possible between dyes in aqueous solution.
Alcohol in the water will form hydrogen bonds with the water thus
inhibiting hydrophobic effect.
Eg: Staining in aqueous solution and Metachromatic staining.

26. Dye
aggregation:
Dye molecules may interact with each other forming dye-dye
interaction.
They aggregate in solution and then penetrate into tissue.
Factors increasing dye
aggregation:
Dye concentration is high
The molecular size is bigger
Temperature is low
Eg: Metachromatic staining and metallic microcrystals after
silver impregnation

27. CLASSIFICATION
OF
DYE

28. BASED ON ORIGIN
NATURAL
CARMINE
ORCEIN AND
LITMUS
HEMATOXYLIN
SYNTHETIC BENZENE

29. BASED ON
CHEMICAL
STRUCTURE
AZO DYE
ORANGE G & CONGO
RED
THIAZINE DYE
TOLUIDINE BLUE &
METHYLENE BLUE
TRIPHENYLMETHANES
METHYL VIOLET &
MALACHITE GREEN
AZIN DYE CELESTINE BLUE
DIPHENYLMETHANES AURAMINE
XANTHENE
EOSIN & ROSE BENGAL
DYE
OXAZINE DYE CRYSTAL VIOLET
ACRIDINE DYE ACRIDINE ORANGE

30. BASED ON USAGE IN
HISTOLOGICAL STAINING
ACIDIC
BASIC
NEUTRAL

31. ACIDIC DYES/ANIONIC
DYES/NEGATIVELY
CHARGED
The acid component is
coloured and the base
colourless.
Acid dyes usually stain
basic components, such as
cytoplasm and collagen.
Eg: Eosin.
BASIC DYES/CATIONIC
DYES/POSITIVELY
CHARGED
In these the base contains
the colouring substance
combined with a colourless
acid.
Basic dyes usually stain
acid components, such as
nuclei and epithelial mucin.
Eg: Methylene blue and
Alcian blue.
NEUTRAL DYES
In this both the acid and
basic radicles are coloured,
it is a neutral stain, and
since certain tissue
elements have an affinity
for the composite neutral
stain these structures are
termed 'neutrophilic'.
Eg: Romanowsky stains
like Giemsa stain.

32. 32

33. BENZENE
+
CHROMOPHOR
E
+
AUXOCHROME
Organic colourless solvent
Chemical group that imparts colour to
benzene
Chemical group that intensifies the colour by
conveying the property of ionization to the
chromogen and enabling it to form salts and
binding to the biological substance
Coloured
compound
not a stain
(chromogen)
Stain
STAIN
CHROMOGEN
AUXOCHROME
SUBSTRATE
CHROMOP
HORE
Coloured
compound
not a stain
(chromogen)
Coloured
compound
not a stain
(chromogen)
BENZENE

34. BENZENE
• When 2 of the hydrogen atoms are replaced by oxygen, a
readjustment of the double bonds takes place and a new compound,
quinone (C6H402), is formed, compounds from which are called
quinoid compounds.
• The change from benzene to quinone is important, because benzene
derivatives absorb light only in the UV band and are colourless,
whereas quinoid compounds have absorption bands in the visible
spectrum and are coloured.
Formed by 6
carbon atoms
and 6 hydrogen
atoms to form a
ring
Each carbon
atom has a
double bond on
one side and a
single bond on
the other

36. CHROMOPHORES
• Certain chemical group
introduced into benzene
ring by substitution
induce colour to the
compound. This is known
as chromophore, and the
chemical compound
formed by the grouping is
known as chromogen.
• These groups have many
free electrons that absorb
the ultraviolet rays of light
which are not in the
visible range.
• Eg: Active chromophore
group, nitro grouping and
azo coupling.
AUXOCHROMES
• It helps to intensify the
light. It is an ionizing
group that also helps to
stick the stain with the
tissue.
• The auxochrome group
augments more free
electrons in the
chromophore groups.
• The increased number of
electrons in the system
helps to absorb light of
longer wavelength in the
visible range.
• Eg: Acidic, such as the
hydroxyl group or basic,
such as the amino group
(NH2).
SULPHONIC GROUP
• The sulphonic group is
important in dye
chemistry because it
forms acid salts.
• It is only slightly
auxochromic and is used
principally either to make
an insoluble dye soluble
in water, or to convert an
otherwise basic dye into
an acid dye.
• Eg: Basic fuchsin is
converted to acid fuchsin
by the introduction of a
sulphonic group.

37. Schematic
diagram showing
colour generation
by dye. The
chromophore
group absorbs
light and imparts
colour to the
stain. The
auxochrome
group donates
more electrons to
the chromophore
group and helps
to absorb light of
longer

39. MORDANTS

40. • Mordant is a metallic substance that forms a link between the tissue
and the dye.
• It is a substance that forms an insoluble compound with a stain and
helps to fix the colour to the cell components.
• + MORDANT LAKE
• Some lakes are unstable, and with such compounds tissue may be
soaked in the mordant immediately before treatment with the stain, or
even afterwards. Eg: Heidenhain's iron haematoxylin stain and
Gram’s stain respectively.
• The term mordant is only strictly applicable to salts and hydroxides of
di-valent and tri-valent metals.
DYE

43. ACCENTUATORS
AND
ACCELERATORS

44. • Accentuators are chemical substances which increase the selectivity
or staining power of the dyes. Eg: Potassium hydroxide in methylene
blue, and phenol in carbol thionin and carbol fuchsin.
• The effect on staining of this group is due to the change in pH of the
staining solution.
• Accentuators when used in the impregnation of the nervous system
with metallic salts have been called accelerators. Eg: Veronal in
Cajal's method for axis cylinders, and chloral hydrate in Cajal's
method for motor end-plates.

45. METACHROMASY

46. • A dye which has the ability to change its colour without changing its
chemical structure is said to be metachromatic.
• Staining reaction is known as metachromasy, the tissue is said to
exhibit metachromasia, and the dye is known as a metachromatic
dye.
• Eg:
1. Cationic dye/ Positively charged/ Basic dye: Toluidine blue,
methylene blue, azure A and B, methyl violet, brilliant cresyl blue,
etc.
2. Anionic dye/ Negatively charged/ Acidic dye: Biebrich scarlet and
bromophenol blue.
• Among the principal tissue components which exhibit metachromasia

47. Mechanism of dye
aggregation in
metachromasia.
The cationic (positively
charged) dye in aqueous
solution is neutralized
with the polyanions of
the tissues.
Subsequently the
nonpolar aromatic ring
of the dye binds with the
other dye by van der
Waals force. The dye
absorbs shorter
wavelength of light, and
the visible colour
changes.

48. The different types of
metachromasia are
highlighted in this
schematic diagram.
Factors enhancing
metachromasia:
1. Higher dye concentration
2. Low pH
3. Decreased temperature
4. Aqueous solution

49. REFERENCES

50. 1. Culling CF. Handbook of histopathological and histochemical
techniques: including museum techniques. Butterworth-Heinemann;
2013 Oct 22.
2. Suvarna KS, Layton C, Bancroft JD, editors. Bancroft's theory and
practice of histological techniques E-Book. Elsevier Health Sciences;
2018 Feb 27.
3. Dey P. Basic and advanced laboratory techniques in histopathology
and cytology. Springer Singapore; 2018 Jun 8.
4. https://www.biologydiscussion.com/micro-biology/staining/stains-
meaning-purpose-and-components-microorganisms/55080
5. Veuthey, T., Herrera, G., & Dodero, V. Dyes and stains: from