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)