STAINING
Staining
Applying dyes on the sections to study
architectural pattern of the tissues and physical
characteristics of the cell.
Embedding process must be reversed to get the
paraffin wax out of the tissue and allow water
solubles to penetrate.
1. Histological Staining-Tissueconstituents are
demonstrated in sections by direct interaction
with dye.
Also called as Micro anatomical staining
2. Histochemical Staining- various constituents
of the tissues are studied through chemical
reaction.
3. Immunohistochemical staining- combination
of immunologic and histochemical techniques.
METHODS OF STAINING
1. Direct Staining- giving color to the sections
using aqueous or alcoholic dye solution.
2. Indirect Staining:
• The action of the dye is intensified by adding another
agent.
• MORDANT- link or bridge between tissue and dye.
• ACCENTUATOR- accelerates/hastens the speed of the
staining reaction.
3. Progressive Staining- tissue elements are
stained in definite sequence, stain is applied until
the desired intensity of color is attained.
4. Regressive Staining- tissue is first overstained
and decolorized until the desired color is obtained.
5. Metachromatic Staining- entails the use of dyes
which differentiate particular substances by
staining them with the color that is different from
that of the stain itself.
6. Metallic Impregnation- demonstrated not by
stains, but by colorless solutions or metallic
salts w/c are thereby reduced by the tissue,
producing an opaque, usually black deposit on
the surface of the tissue or bacteria.
7. Counterstaining- application of a different
color or stain to provide contrast and
background.
8. Vital Staining- selective staining of living
cell constituents
• A. Intravital Stain- staining of living cells is done by
injecting the dye into any parts of the animal body.
• Common dyes: lithium, carmine, india ink
• B. Supravital Stain-stain living cells immediately after
removal from the living body.
HEMATOXYLIN AND EOSIN
TECHNIQUE
Most common method for tissue examination.
Fixative- except Osmic Acid, inhibit
hematoxylin.
HARRIS HEMATOXYLIN- primary stain
ACID ALCOHOL- differentiator
AMMONIA WATER- blueing agent
EOSIN- counterstain
STAINS AND STAINING SOLUTIONS
1. Natural Dyes
• A. Hematoxylin- derived from Mexican tree
Hematoxylin campechianum
• Hematin- active coloring agent formed by the oxidation
of hematoxylin (ripening)
Natural Ripening- exposing substances to air or sunlight.
Artificial ripening- uses substances that will accelerate the
process.
Hydrogen peroxide, mercuric oxide, potassium
permanganate, sodium perborate, etc.
B. Cochineal Dye- dye extracted from the
female cochineal bug (Coccus cacti)
• Treated with alum to produce dye- Carmine
• Carmine + picric acid = picrocarmine
• For neuropathological studies
• Orcein- a vegetable dye extracted from lichens
Used for staining elastic fibers.
2. Synthetic Dyes- known as Coal Tar Dyes
• Chromophores- substance capable of producing color
(chromogen)
• Chromogen + auxochrome=dye
• A. Acid dyes- active coloring substance is found in the
acid component and the inactive base.
• B. Basic Dyes- active coloring substance is found in
basic component
• C. Neutral Dyes- capable of staining cytoplasm and
nucleus
COMMON STAINING SOLUTIONS
HEMTOXYLIN- most common for routine
histology
• Mordant: alum, iron
• EOSIN- routinely used as couterstain
A. yellowish (eosin Y)- most commonly used
B. Bluish ( eosin B, Eryhrosin B)- deeper red color
C. Ethyl eosin ( Eosin S, eosin-alcohol soluble)
