STAINING

LEARNING OBJECTIVES:
• TO DESCRIBE THE RELEVANCE OF STAINING IN
THE HISTOPATHOLOGY LABORATORY
• TO DIFFERENTIATE THE TYPES OF STAINS
• TO DESCRIBE THE RELEVANCE OF MOUNTING
AND RINGING AS PART OF THE
HISTOTECHNIQUE.
 STAINING
Natural Dyes - obtained from
plants and animals
• 1. Hematoxylin-
• 2. Cochineal
• 3. Orcein
• 4. Saffron
Synthetic dyes - “Coal tar
dyes”
• - derived from benzene and
collectively known as
“Aniline dye”
 NATURAL DYES
• 1. HEMATOXYLIN- a natural
dye derived from the core or
the heartwood of a mexican
tree known as “Hematoxylin
campechianum”
• Powerful nuclear and
chromatin staining capacity.
• Active coloring agent is
HEMATIN.
• Ripening – oxidation process
of hematin; accomplished
by exposing the substance
to air and sunlight.
 NATURAL DYES
• 2. COCHINAL DYES- extracted
from female cochineal bug
(Coccus cacti); treated with
alum to produce the dye
CARMINE.
• Powerful chromatin and
nuclear stain.
• Picrocarmine- carmine +
picric acid; neuropathological
studies
• Best’s carmine stain-
carmine+ aluminum chloride;
demonstration of glycogen.
 NATURAL DYES
• 3. ORCEIN – Is a vegetable
dye extracted from certain
lichens which are normally
colorless.
• Treated with ammonia and
exposed to air: produce blue
or violet colors.
• Staining elastic fibers.
• Demonstration of hepatitis B
surface antigen
• Litmus is also obtained from
lichens; treated with lime and
soda and exposed to
ammonia and air.
 SYNTHETIC DYES
• Sometimes known as “COAL TAR
DYES”
• Derived from hydrocarbon
benzene (C6H6) and are
collectively known as ANILINE
DYES.
• 1. Chromophores (Gk.”color
bearers”) – group of benzene
ring which confers color
• 2. Auxochromes (Gk.
“increasers”) - dyeing properties
• 3. Dye modifiers - substances
that affect either the color or
properties of a dye
• 4. Lake - the resultant complex
of stain-mordant-tissue
 CHROMOPHORE
• Chromophore may be classified in
3 groups:
• 1. ACID DYES- active coloring
substance is found in the acid
component and the inactive base.
E.g. acid fuchsin, is usually the
sodium salt of sulfonate of
rosaniline.
• Picric acid- fix, differentiate or
stain tissue all by itself.
• Basic cell structures (collagen,
eosinophilic granules of
leukocytes, etc. )have an affinity
for the acid dye ions and are
regarded as ACIDOPHILIC
 CHROMOPHORE
• 2. BASIC DYES- Coloring
substance is found in a
basic components that
combines with the acid
radical. E.g.
• METHELYNE BLUE- basic
nuclear stain which may be
used both as an indicator
and as a dye.
• Acidic cell structures
(chromatin, mucus, cartilage
matrix, etc.) have an affinity
for basic dye ions and
regarded as BASOPHILIC.
 CHROMOPHORE
• 3. NEUTRAL DYE- Formed by
combining aqueous
solutions of acid and basic
dyes, capable of staining
cytoplasm and nucleus
simultaneously and
differentially. E.g
• ROMANOWSKY DYE: used in
hematology, giemsa’s stain,
and irishman’s stain for
luekocyte differentiation.
 SYNTHETIC DYES
CHROMOPHORES
• Quinoid ring - basic fuchsin
• Azo groups - congo red
• Xanthene - Eosin
• Quinone - imine group
• Oxazin – cresyl fast violet
• Thiazins - toluidine blue
 SYNTHETIC DYES
AUXOCHROME
• Cationic auxochrome: Amino
group
• Anionic auxochrome: Hydroxyl
and Carboxyl groups
DYE MODIFIERS (attached on
benzene ring)
• Ethyl group
• Methyl group
• Sulfonic Acid
 DYE-TO-TISSUE MECHANISM
• Tissue will bind dyes by one of the following
mechanism
• 1. Electrostatic - majority of tissue-dye reactions
• e.g.: Neutral red and Light green
• 2. Hydrogen Bonding - Congo red, Carmine,
Weigert-type resorcinol dye
• 3. Van der waals Forces - Alum Hematoxylin
solutions
• 4. Physical Staining - Sudan dyes
SUDANOPHILIA - property of tissue to be stained with fat
or oil-soluble dyes, regardless of the type of the dye
used, due to the essential lipid nature
• 5. Natural Affinity - Janus green
 METHODS OF STAINING
ACCORDING TO PRESENCE OF MORDANT
• Direct - w/o mordant; methylene blue and
eosin; a staining process by which sections are
stained with simple aqueous alcoholic
solutions of dye
• Indirect - w/ mordant
 METHODS OF STAINING
ACCORDING TO THE PRESENCE OF DIFFERENTIATOR
• Progressive staining - w/o decolorizer
• Regressive - w/ decolorizer
• Counterstaining - application of different color or
stain to prove counterstain or background
ACCORDING TO RESULTANT COLOR
• Orthochromatic - color of dye – same color of the
tissue
• Metachromatic- color of dyes – different color of
the tissue
 METHODS OF STAINING
• Metallic Impregnation - specific tissue
elements are demonstrated by colorless
solutions of metallic salts
• Metallic saltsàreduced by tissueàblack
deposit on the surface
 METHODS OF STAINING
• 1. Intravital Staining (live)
• -Injections of dye to the
animal body
• e.g.: Lithium, Carmine, India
ink
• 2. Supravital Staining
• -The stain applied onto cells
after removal from the
animal body
• Neutral red - BEST vital dye
• Janus green - mitochondria
• Trypan blue
• Nile blue
• Thionine
• Toluidine blue
INTRAVITAL STAINING
 HEMATOXYLIN AND EOSIN
H and E Staining
• Hematoxylin
• -A natural dye derived from extraction from the heartwood of the
Mexican tree known as “Hematoxylin campechianum” or “Hematoxylon
campechianum”
• Jamaica Walteyer - First person to use Hematoxylin in Histology

• Ripening / Oxidation
• May be done by exposing the
substrate to air or sunlight (SLOW)
• -May be done by adding oxidizing agents
such as:
• H 2 O2
• HgCl2 - ripening agent of Harris Hematoxylin
• KMnO4
• Sodium perborate
• Sodium iodate - ripening agent of
Ehrlich’s hematoxylin
 ALUM HEMATOXYLIN
• A. Alum hematoxylins
• Mordant: Potash alum
(potassium aluminum sulfate or
simply alum)
• Produce good nuclear stain
(red)
• Examples:
• Erlich’s – slowly ripened /
sodium iodate
• Delafield’s – slowly ripened
• Harris – mercuric oxide
• Gill’s – sodium iodate
• Mayer’s – sodium iodate
 IRON HEMATOXYLIN
• B. Iron hematoxylins
• Iron salts are used as oxidizing
agents and mordants
Examples:
• Weigert’s – ferric chloride; in
combination with Van Gieson’s stain,
can demonstrate connective tissue
elements and Entamoeba histolytica
in sections
• Standard Iron Hematoxylin - for
nucleus/ connective tissue fibers;
Van Gieson’s stain - good for
demonstrating collagen
• Heidenhain’s - ferric ammonium
sulfate; for mitochondria, muscle
striations, chromatin, and myelin
TUNGSTEN HEMATOXYLIN
• Tungsten
Hematoxylin
• Mordant: Tungsten
• Mallory PTAH
(Phosphotungstic Acid
Hematoxylin)
• To ripen: stand in the
light for several weeks
or use potassium for
immediate ripening
• >For staining muscle
striations
 COPPER HEMATOXYLIN
Copper Hematoxylin
• Used for study of
spermatogenesis
• Spermatogonia à
Spermatid à Sperm cell
 EOSIN
• Eosin
• A red avid dye
• Routinely used dye in
histopathology as a counterstain
after hematoxylin and before
methylene blue
• 3 FORMS:
• Eosin Y (yellowish) – Most
commonly used
• Eosin B (bluish)
• Ethyl eosin
EOSIN
 H AND E STAINING STEPS
• 1. Xylol (2 changes) – deparafinized
• 2. DESCENDING GRADE OF ALCOHOL
• 3. Water
• *removal of pigments is done after rehydration and right before primary staining
• *removal of mercuric pigments:
• Place Weigert’s Iodine
• Wash in distilled water
• Remove iodine with 5% sodium thiosulfate
• Wash in running water
• Proceed with stain
• 4. Stain with Harris / Ehrlich’s / Delafield’s - red nucleus
• 5. Rinse slide in tap water
• 6. Acid alcohol (differentiator) - red nucleus
• 7. Ammonia water (ammonia hydrochloride, lithium carbonate, scott’s tap water) -
blueing agent
• 8. Wash well in running water
• 9. Stain with Eosin Y
• 10. Ascending grade of alcohol - dehydration
• 11. Xylol / xylene – dealcoholization - clearing
• 12. Mount then label
 RESULTS OF H AND E STAINING
• Nuclei - blue to black
• Karyosome - dark blue
• Cytoplasm, proteins in edema fluid - pale pink
• Calcium and calcified bone - purplish blue
• Muscle fibers - deep pink
• Hematoxylin – 1 stain (clear)
• Eosin - 2 counterstain – cytoplasmic
• Modified H and E staining - progressive staining,
no differentiation phase, frozen section
• Blueing step - bridging mordant and dye
 OTHER STAINS AND THEIR USES
1. Benzidine - used for staining hemoglobin
2. Acridine orange - DNA (green fluorescence), RNA (red
fluorescence)
3. Crystal violet - for amyloid in frozen sections and platelets in
blood
4. Gentian blue - formed by the mixture of crystal violet, methyl
violet, dextrin
5. Congo red - stain for axis cylinders in embryos, used as a 4%
aqueous solution in Krajen methods for staining, elastic tissue,
myelin
6. Iodine - probably the oldest of all stains, stains for amyloid,
cellulose, starch, carotenes and glycogen – widely used for
removal of mercuric fixative pigments
7. Malachite green - contrast stain for staining Ascaris eggs and
erythrocytes, bacterial spore stain
8. Janus green B - demonstrate mitochondria during intravital stain
9. Night blue - substitute for carbol fuchsin in acid fast staining
10. Victoria blue - demonstrate neuroglia in frozen sections
OTHER STAINS AND THEIR USES LYSOCHROME

11. Lysochromes (oil soluble dyes) - not real

dyes, no auxochromes
groups, give color to lipids simply because
they are more soluble
in lipid medium of the tissues than in their
medium of 70% alcohol
Examples of oil soluble dyes used for PERIODIC ACID SCHIFF
demonstration of
intracellular fats
-Sudan black B - black
-Sudan III - orange
12. Periodic Acid Schiff reaction - for fungal wall
>Red / magenta red
>Mucoproteins are most common PAS
positive substance GOMORI’S TRICHROME
13. Toluidine blue - used to demonstrate mast
cells in tissues
14. Gomori’s trichome - the best staining
procedure for
demonstration of reticulin
 ADHESIVES
• Adhesives are not necessary for routine
staining, provided that the slides are clean
and free from grease.
• Essential for methods that require exposure of
sections to acids and alkalis (ammoniacal
silver solution) during staining.
 ADHESIVES
1. Mayer’s egg albumin- most commonly used
adhesive.
2. Dried albumin
3. Gelatin (1%)-added to the water in a floating out
bath.
4. Gelatin-Formaldehyde mixture
5. Starch paste
6. Plasma-from outdated blood
7. Poly-L-Lysine- 0.1% solution, further diluted to
1:10with distilled water.
8. APES (3-aminopropylthrierhoxysilane)-
proteinaceous or bloody material.
MOUNTING
 GOOD MOUNTING MEDIUM
1. To avoid distortion of the image, the refractive
index if the mountant should be as near as
possible to that of the glass which is 1.518.
2. Should be freely miscible with xylene and toluene.
3. Should not dry quickly
4. Should not crack or produce artefactual
granularity.
5. Should not dissolve out or fade tissue sections.
6. Should not leach out any stain or affect staining.
7. Should not change in color or pH.
8. It should set hard. Thereby producing permanent
mounting of section.
 MOUNTING
Refractive index - ratio of speed of light in
air and speed of light in a specific medium
• Refactive index of glass - 1.518
A. Resinous media - greater than or equal
to 1.518
1. Eukitt
2. Entalian
3. DPX (1.532)
4. Histomount
5. XAM (1.52)
6. Paramount
7. Canada balsam - Abus balsamea
(1.524)
8. Clarite - (1.544)
 MOUNTING
B. Aqueous Media (usually for
lipids because resinous media
contain xylene which dissolve fats)
• 1. Glycerin (1.47)- Kaiser’s 1880
• 2. Gum arabic (Farrant’s
medium) (1.43)
• 3. Karo corn syrup
• 4. Apathy’s medium (1.52)
• 5. Brun’s fluid - recommended
for mounting frozen sections
from water
• 6. Water - evaporates easily
MOUNTING
 RINGING
• Is a process of sealing the
margins of the cover slip to
prevent the escape of fluid or
semi-fluid mounts and
evaporation of mountant, to
immobilize the coverslip and to
prevent sticking of the slides
upon storage.
1. Kronig cement- 2 parts
paraffin wax mixed with 4-9
parts powdered colophonium
resin.
2. Durofix- cellulose adhesives.