STAINING

TECHNIQUES
STAINING METHODS

Counter Staining
● Is the application of a different color or stain
to provide contrast and background to the
staining of the structural components to be
demonstrated.
Metachromatic Staining

• Entails the used of specific dyes

which differentiate particular
substances by staining them
with a color that is different from
that of the stain itself.
 Regressive Staining

With this technique the tissue

is first over stained to
obliterate the cellular details
and the excess stain is removed
or decolorized from unwanted
parts of the tissue, until the
desired color is obtained.
 Progressive Staining

■ Is the process whereby tissue

elements are stained in a
definite sequence and the
staining solution is applied for
specific periods of time or until
the desired intensity of coloring
of the different tissue elements
is attained.
 Decolorization

● Is the selective removal of

excess stain from the tissue
during regressive staining in
order that a specific substance
may be stained distinctly from
the surrounding tissues.
 Direct Staining

• The process of giving color to

the sections by using aqueous
or alcoholic dye solutions.
Indirect Staining

■ The process where the action of

dye is intensified by adding
another agent or mordant which
serve as a link or bridge
between the tissue and the
dye, to make staining reaction
possible.
 Vital Staining

■ Selective staining of living cell

constituents, demonstrating
cytoplasmic structures by
phagocytosis of the dye
particle.
 Intravital Staining

Intravital staining of living cells

is done by injecting the dye into
any part of the animal body,
producing specific coloration of
certain cells, particularly those
of the reticulo-endothelial
system.
 Supravital Staining

■ Is used to stain living cells

immediately after removal from
the living body.
 Divisions of Biological
Stains Prepared from dyes
■ Natural Dyes- are those
obtained from plants and
animals, previously utilized for
dyeing of wool and cotton.
■ Examples: Hematoxylin,
Cochineal dyes and its
derivatives, Orcein, Saffron
 Hematoxylin

■ Derived by extraction from the

core of the heartwood of a
Mexican tree known
Hematoxylin Campechianum
■ The active coloring agent is
hematin, formed by the
oxidation of hematoxylin.
■ Strong oxidizing agents:
Hydrogen peroxide, Mercuric
 Cochineal Dyes

■ Extracted from the female

cochineal bug (Coccus Cacti),
W/c is treated with alum to
produce the dye carmine.
■ When combined with aluminum
chloride (Bests’ Carmine stain)
is used for demonstration of
glycogen.
 Orcein

■ Vegetable dye extracted from

certain lichens
■ Used for staining elastic fibers
■ Litmus is also obtained from
lichens, treated with lime and
soda and exposed to ammonia
and air.
 Synthetic Dyes

■ Known as “Coal Tar Dyes”

■ Derived from the hydrocarbon
benzene and are collectively
known as Aniline dyes
■ Chromophores are substances
with definite atomic groupings
and are capable of producing
visible colors
■ Benzene contains chromogens
 Classification of
Chromophore Dyes
■ Acid Dyes- where the coloring
substance is found in the acid
component.
■ Basic Dyes- where the active
coloring substance is found in a
basic component that combines
with the acid radical.
■ Neutral Dyes- formed by
combining aqueous solutions of
acid and basic dyes, capable of
staining cytoplasm and nucleus
 Common Staining
Solutions Used
■ Hematoxylin
a. Aluminum Hematoxylin
Solutions
are recommended for
progressive staining of tissues
and can also be used for
regressive staining
b. Erlich’s Hematoxylin-
generally used for regressive
staining
c. Harris hematoxylin- a good
regressive stain. Used also for
routine nuclear stain, exfoliative
cytology and for sex
chromosomes
d. Cole’s Hematoxylin-
recommended for routine
purposes, especially used in
sequence with celestine blue.
e. Mayer’s Hematoxylin- Alum
■ Iron hematoxylin- Used for
differential or regressive
staining
■ Examples: Weigert’s solution-
using ferric ammonium chloride
and
■ Heidenhein’s solution using
ferric ammoniun sulfate as
mordants. Demonstrates
nuclear and cytoplasmic
■ Phosphotungstic Acid
Hematoxylin (PTAH)
-It is a progressive stain
- Demonstrates structures in
paraffin, celloidin and frozen
sections.
 EOSIN

■ Used for differentially staining

connective tissues and
cytoplasm
■ Used as counterstain after
hematoxylin and before
methylene blue.
3 FORMS OF EOSIN
1. Yellowish (Eosin Y)- shows a
green yellow fluorescence
2.Eosin B, Erythrosin B (Bluish)-
deeper red color
3. Ethyl eosin- Eosin S, Eosin
alcohol-soluble
 OTHER STAINS USED
■ Van Gieson’s Stain (Acid
Fuchsin-Picric Acid)- is a
mixture of picric acid and acid
fuchsin for demonstration of
connective tissues.
■ Acridine Orange- is a basic
acridine fluorochrome which
permits discrimination between
dead and living cells, giving
green fluorescence for DNA and
■ Acridine Red 3B- demonstrates
deposits of Calcium salts and
possible sites of phosphatase
activities.
■ Alcian Blue- is for specific for
connective tissue and epithelial
mucin.
■ Aniline Blue- is a cytoplasmic
stain used for counterstaining of
Basic Fuchsin- is a plasma stain
used for deep staining of acid-
fast organisms, for
mitochondria, for differentiation
of smooth muscles with the use
of picric acid.
Examples: Carbol-Fuchsin,
Coleman’s Feulgen Reagent,
Schiff’s reagent, Mallory’s