STAINING

METHODS AND PRINCIPLES

STAINING
 Process of applying dyes on the sections
 To see and study architectural pattern of the tissue
and the physical characteristics of the cells
 To make the tissues and cells become more visible
 To easily identify morphologic changes in the
tissues/cells
 To establish presence or absence of a disease
process

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Three Major Groups of Staining
1. Histological Staining
 Tissue constituents are demonstrated in
sections by direct interaction with a dye or
staining solution
 Micro-anatomic stains, bacterial stains,
specific tissue stains
 Used to demonstrate the general
relationship of tissues and cells with
differentiation of nucleus and cytoplasm

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2. Histochemical Staining/Histochemistry
 Tissues are studied thru chemical reactions that
will permit microscopic localization of specific
tissue substance
 Perl’s Prussian Blue – Hemoglobin
 Periodic Acid Schiff – Carbohydrates
 Enzyme histochemistry
 The active reagent serves as a substrate where
enzymes act. The final color produced is from
the substrate not on the tissue.

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3. Immunohistochemical Staining
 Combination of immunologic and histochemical
techniques that allow phenotypic markers to be
detected and demonstrated under the
microscope, using a wide range of polyclonal or
monoclonal, fluorescent labeled or enzyme
labeled antibodies.

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TWO CATEGORIES
NaturalDyes
Synthetic Dyes

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Natural Dyes
 Obtained from plants and animals
 Staining of basophilic cell structures
such as nuclei and some cytoplasmic
substances (cationic)
 Hematoxylin
 Hematoxylin campechianum
 not a stain
 HEMATIN – active coloring agent
 Most frequently used with mordant – alum, iron,
copper

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  Cochineal Dyes (Carmine)
 scarlet dye made from the ground bodies
of female cochineal bug (Coccus cacti)
 A powerful chromatin and nuclear stain
for fresh material and smear preparations
 Picrocarmine– used in neuropathological
studies
 Best Carmine stain – used for glycogen
demonstration

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  Orcein
 Vegetable dye extracted from certain
lichens which are normally colorless, but
when treated with ammonia and
exposed to air, produce blue/violet
colors.
 Used for staining elastic fibers

 Saffron

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Synthetic Dyes (Coal Tar Dyes)
 Derived from hydrocarbon benzene
 Collectively known as aniline dyes
 Consist of a chromophore and auzochrome
group attached to a hydrocarbon benzene ring
 Chromophore – substance with definite atomic
groupings and are capable of producing colors
 Auxochrome – substance which imparts to the
compound the property of electrolytic dissociation
to retain the color of the tissue

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 Chromophores
 Quinoid rings
 Azo groups
 Xanthene
 Quinone-amine group (OXAZIN and THIAZINS)
 Auxochromes
 CATIONIC (Amino group)
 ANIONIC (Hydroxyl and Carboxyl group)
 Dye modifiers (attached on benzene rings)
 Ethyl groups
 Methyl groups
 Sulphonic Acid

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Dye-to-Tissue Mechanisms
1. Electrostatic
 majority of tissue-dye reactions
 Neutral red and Light green
2. Hydrogen bonding
 Congo Red, Carmie, Weigert-type resorcinol dye

3. Van der Waals forces

 Alum hematoxylin solutions
4. Physical staining
 Sudan dyes
 SUDANOPHILIA – property of tissues to be stained with fat
soluble dyes regardless of the type of dye used due to their
essential lipid nature
5. Natural affinity
 Janus Green
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Classification of dyes depending on where
the chromophore is found
i. Acid Dyes
 the active coloring substance is found in the acid
component
 anionic dyes and stain mainly cytoplasm,
eosinophilic granules

ii. Basic Dyes

 the active coloring substance is found in the basic
component
 cationic dyes and stain nuclei, basophilic granules or
bacteria

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iii. Neutral Dyes
 Formed by combining aq. solutions of acid and
basic dyes
 Stains cytoplasm and nucleus simultaneously and
differentially

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 METHODS OF STAINING

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According to the presence of mordant:
A. DIRECT STAINING
 Process of giving color to the sections by
using aqueous or alcoholic dye solutions
 Methylene Blue
 Eosin

 No mordant is used
B. INDIRECT STAINING
 Process whereby the action of the dye is
intensified by adding another agent
 Uses mordant

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  MORDANT
Serves as a link or bridge between the tissue
and the dye
Potassium alum with hematoxylin (Ehrlich’s
hematoxylin); Iron (Weigert’s hematoxylin)
 ACCENTUATOR
Accelerates or hastens the speed of the
staining reaction by increasing the staining
power and selectivity of the dye
Potassium hydroxide (Loeffler’s methylene
blue); Phenol (Carbol thionine and Carbol
fuchsin)

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According to the presence of a
differentiator/decolorizer:
A. PROGRESSIVE STAINING
 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.
 No differentiator
 H/E for frozen sections

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B. REGRESSIVE STAINING
 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 intensity
of color is obtained.
 With differentiator
 H/E for routine histological staining

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 DIFFERENTIATION/DECOLORIZATION

 Selective removal of excess stain from

the tissue during regressive staining
 Done by washing the section in simple
solution (water/alcohol) or by the use
of acids and oxidizing agents.
 Alcohol acts as a differentiator for both
basic and acidic dyes

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According to resultant color:
A. ORTOCHROMATIC STAINING
 Substances are stained with a color that is the
same from that of the dye used.
B. METACHROMATIC STAINING
 Entails the use of specific dyes which
differentiate particular substances by
staining them with a color that is different
from that of the stain itself
 Employed for staining cartilage, CT, epithelial
mucins, mast cell granules, and amyloid.

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METACHROMATIC DYES
 Basicdyes belonging to thizine and
triphenylmethane groups
 Methyl violet or crystal violet
 Cresyl blue (reticulocytes)
 Safranin
 Bismarck brown
 Basic fuchsin
 Methylene blue
 Thionine
 Toluidine blue
 Azure A, B, C

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VITAL STAINING
Selective staining of living cell constituents,
demonstrating cytoplasmic structure.
A. INTRAVITAL STAINING
Injecting the dye into any part of the
animal body producing specific
coloration of certain cells particularly
those of the reticulo-endothelial system
 Lithium, Carmine, India Ink

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B. SUPRAVITAL STAINING
Stain living cell after removal from the
immediately after removal from the
living body.
 Neutral red (best vital dye)
 Janus green (mitochondria)
 Trypan blue
 Nile blue
 Thionine
 Toluidine blue

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COUNTERSTAINING
 Application of a different color or
stain to provide contrast and
background to the staining of the
structural components to be
demonstrated.

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METALLIC IMPREGNATION
 Process where specific tissue elements are
demonstrated, not by stains, but by colorless
solutions of metallic salts.
 Characteristics:
 Structures demonstrated are opaque and
black
 The colouring matter is particulate
 The deposit is on or around but not in the
element so demonstrated

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FACTORS INFLUENCING DYE BINDING
 pH of the solution
 Increase in temperature
 Increase concentration of dye molecules
 Presence of other salts

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 H and E Staining

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Hematoxylin
 A natural dye derived from the extraction
from the heartwood Mexican tree
 Ripening/Oxidation
 Done by:
 Exposing the substance to air and sunlight (slow)
 Adding oxidizing agents (H2O2, Mercuric oxide,
Potassium permanganate, Sodium perborate, Soidium
iodate)

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 ALUM HEMATOXYLIN
 The same mordant, different ripening agent
 Mordant: Potassium aluminum sulfate / “alum”
 Produce good nuclear stain
 Appears RED upon contact with acid
 Ehrlich’s
 Delafield’s
 Harris
 Gill’s
 Mayer’s

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 IRON HEMATOXYLIN
 Iron salts are used as oxidizing agents and
mordants
 Weigert’s - Ferric chloride
 In combination with van Gieson’s stain, can
demonstrate CT elements and Entamoeba hystolytica
sections
 Standard iron hematoxylin
 For muscles/CT fibers
*Van Gieson’s stain – good for demonstrating
collagen
 Heidenhain’s – ferric ammonium sulfate
 For mitochondria, muscle striations, chromatin and
myelin
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 TUNGSTEN HEMATOXYLIN
 Mallory’s PTAH
 To ripen, stand in the light for several weeks or use
potassium for immediate ripening
 For staining muscle striations
 COPPER HEMATOXYLIN
 Used for spermatogenesis studies

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Eosin
 Secondary stain, cytoplasmic stain, acidic stain
 Used as a counterstain after hematoxylin and
before methylene blue
 Three forms:
 Eosin Y (Yellowish) – most commonly used
 Bluish – gives deeper red color
 Ethyl eosin/Eosin S

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Routine H and E STaining
Steps:
1. 2 changes of xylol (for deparaffinization)
2. DESCENDING grade of OH (for rehydration)
3. WATER
4. Hematoxylin
5. Rinse with tap water
6. Acid OH (differentiator)
7. Ammonia water (bluing)
8. Rinse with tap water
9. Eosin
10. ASCENDING grade of OH
11. Xylol
12. Mount and label

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 Result:
 Nuclei – blue to blue black
 Karyosome – dark blue
 Cytoplasm, proteins in edema fluid – pale pink
 Calcium and calcified bone – purplish blue
 Muscle fibers – deep pink

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Other Stains Used:
 Benzidine
 for staining hemoglobin
 Acridine orange
 DNA (green fluorescence); RNA (red fluorescence)
 Crystal violet
 for staining amyloid in frozen sections and platelets in
blood
 Gentian violet
 formed by the mixture of CV, methyl violet and dexterin
 Congo red
 stain for axis cylinders in embryos; used as 4% aq soln in
Kraijan’s mtd (elastic tissues, amyloid and myelin)

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 Iodine
 Oldest stain; for amyloid, cellulose, starch, carotenes
and glycogen; widely used for removal of mercuric
fixative pimets
 Malachite green
 Contrast stain for staining Ascaris eggs and RBCs;
used also as a bacterial spore stain
 Janus green
 Used to demonstrate mitochondria during intravital
staining
 Night blue
 A substitute for carbol fuchsin in AFS

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 Van Gieson’s stain
 Mixture of picric acid and acid fuchsin used to
demonstrate CT
 Acridine Red 3B
 Used to demonstrate calcium salt deposits and
possible sites of phosphatase activities
 Alcian blue
 Stain acid MPS; more specific for CT and epithelial
mucin
 Bismarck brown
 For diphtheria organisms

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 Giemsa
 For staining blood to differentiate leukocytes
 Gold sublimate
 Used for metallic impregnation
 Orcein
 Excellent stain for elastic fibers; recommended in
dermatological studies
 Osmium tetroxide
 Stains fat
 Rhodamine B
 Stain blood and glandular tissues

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 Silver nitrate
 Used in identification of spirochetes, reticulum and
other fiber stains
 Toluidine blue
 Recommended for staining Nissl granules
 Victoria blue
 Used for the demonstration of neuroglia in frozen
tissues

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 Lysochromes (Oil soluble dyes)
 Not real dyes
 Do not have auxochrome groups
 Used for demonstration of intracellular fats
 Sudan Black B (black)
 Most sensitive
 Sudan III (orange)
 First sudan dye to be introduced in histochem
 For CNS tissues

 Sudan IV / Scharlach R (red)

 Addition of benzoic acid – intensifies fat and prevent
rapid deterioration of solutions
 Recommended for staining triglycerides

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Causes of poor quality of staining
1. Poor or inadequate fixation of tissue.
2. Over or under-ripened Haematoxylin.
3. Overused or worked out Haematoxylin.
4. Over or under differentiation of haematoxylin
5. Insufficient blueing following differentiation.
6. Failure to wash blueing agent out of section before
counter staining with eosin (especially when
ammonia is used).
7. Insufficient differentiation of eosin during washing or
dehydration.
8. Insufficient dehydration and clearing of sections.
9. Contamination of stains.

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