Metallic impregnation - specific tissue
elements are demonstrated not by stains
but by colorless solutions of metallic salts
which are reduced by the tissue into an
opaque, usually black deposit on the
surface of the tissue or bacteria.
● Ammoniacal silver by argentaffin
cells (eg., melanin and intestinal
glands) forming black deposits
● Unlike stains, it is not absorbed by
the tissue but held physically on the
surface as a precipitate or as a
reduction product in certain
components.
- Eg. gold chloride, silver
nitrate
● Precautions to avoid artifactual
metallic silver deposits in sections
- All reagents should be
chemically pure
- Glassware should be clean
- Formalin laden atmosphere
should be avoided
● Ammoniacal silver is potentially
explosives
- Use clean containers
- Avoid silvered glassware
- Use flexible plastic
containers
- Never expose to sunlight
- All unused reagents should
be inactivated by NaCl or
dilute HCL and discarded
- Avoid metallic instruments in
handling sections for metallic
impregnation
Vital staining
● Selective staining of living cell
constituents
● Cytoplasmic phagocytosis - ●
demonstrate cytoplasmic structures
by phagocytosis of dye particle
● Vital staining of RES with trypan
blue
● True vital staining - staining of
mitochondria by janus green
Nucleus of living cells is resistant to vital
stains
● Staining of nuclear structures
suggest permeability of membrane
to the dye signifying the death of the
cell
Intravital staining:
● Injecting dye into any part of the
animal body (IV, intraperitoneal or
subcutaneous) producing specific
coloration of certain cells particularly
the RES cells (chromoendoscopy,
lugols solutions, methylene blue)
● Lithium, carmine and india ink
Supravital staining
● Used to stain living cells
immediately after removal from the
living body
● Thin slices of tissues are places in
small staining dishes and covered
with enough staining solutions
● Eg. Neutral red - best vital dye
Janus green - for mitochondria
Typan blue - not allowed to stand for
more than hour, because it becomes
toxic to the cells
Nile blue, thionine, toluidine blue
Staining of paraffin sections:
● After the section is cut and mounted
on the slide - it must be drained and
dried thoroughly
- Ensure that all moisture
between the section and
slide has evaporated to firm
attachment
- Incomplete drying can cause
section to detach during
staining (most likely after
acid differentiation) especially
for bone and nervous tissue
Paraffin was poorly permeable to
most staining solutions
- Removed from sections prior
to staining by solvents
(xylene)
● Hydration - xylene is NOT miscible
with aqueous solutions and low
grade alcohol
 - Xylene is removed with
absolute alcohol followed by
descending grades of alcohol
to prevent detachment of
sections due to possible
production of diffusion
currents (oil droplets appear
fi clearant is inadequately
removed)
- Alcohol is then replaced with
water
● Staining
● Dehydration: increasing grades of
alcohol
● Clearing - with 2 changes of xylene
to prepare section for mounting
(mountants are miscible with xylene)
- Second xylene change will
raise refractive index
- Stained sections may be left
in xylene for an indefinite
period of time until it finally
mounted on a slide.
❖ Do let section stay in
alcohol - stains are
removed by alcohol
● Sections floating off the slide
- Dirty or greasy slides
- Sections have not been left
in the paraffin oven long
enough to dry and be fixed
on the slide
❖ Minimum of 30 mins.
MODULE 3 - The dye is hematein, and the
EXERCISE 6 mordant is aluminium salts (Al3+)
- Al3+ binds with the tissue, and
SPECIAL STAINS IN hematein binds with Al3+
HISTOPATHOLOGY Differentiation

- involves application of a chemical

OBJECTIVES
that removes excess stain from a
tissue section, leaving only the
Identify special staining techniques in tissue element of interest stained.
histopathology. - In general, if the primary stain used
Describe the staining reactions of is a basic dye, the differentiation is
each. carried out by an acid solution,
Correlate staining reactions with the while alkaline medium is used for
pathology. differentiation after applying an
acidic dye.
SPECIAL STAINS IN - Alcohol acts as a differentiator for
both basic and acidic dyes,
HISTOPATHOLOGY
probably by simply dissolving out
the excess dye.
Other than routine hematoxylin and
eosin stain, various special stains are 0.5 - 1% acid alcohol
now essential parts in routine
laboratory works. - Alcohol acts as a differentiator for
both basic and acidic dyes,
DEFINITION OF TERMS probably by simply dissolving out
Mordant the excess dye
- reagent that links a stain or dye to - Hematoxylin is not strictly a basic
a tissue element. dye, but in combination with the
- The mordant combines with a dye mordant can act as a basic dye.
to form a colored "lake", which in - Hematoxylin will remain bound to
turn combines with the tissue to acidic components (basic
form a "tissue mordant- dye-acidic component), while the
dye-complex" that is rendered coating other components will be
insoluble in ordinary aqueous and washed off by the differentiating
alcoholic solvents. agent
- allows subsequent counterstaining
and dehydration to be carried out
easily

Accentuator Blueing
- is not essential to the chemical
union of the tissue and the dye. - Blueing neutralizes the excess acid
- It does not participate in the where red soluble haemalum is
staining reaction, but merely converted to blue color insoluble
accelerates the reaction. form and this the sharpens nuclei
blue. The alkaline reagent
MORDANT neutralizes the free acid and makes
Hematoxylin stains hydroxyl group available to form
insoluble blue
- Contains both the dye and aluminium-hematin-tissue lake
mordant
 - Alum in watery solutions dissociate
such that aluminum combines
with –OH of the water forming
Aluminium hydroxide.
- Free hydrogen from water tends to
form sulfuric acid by uniting with
the sulfate from the alum
- If excess of acid is present then the
aluminium hydroxide cannot form
and in such situation, in an alum
hematoxylin dye, the insoluble dye
lake cannot form because of lack of
hydroxyl ions.
BLUEING
1% Lithium carbonate / 0.2%
Ammonia water
- During the routine staining when
the sections are dipped in acid
alcohol, pH of sections become
acidic and look pink.
- By placing the sections in the
blueing agent, or tap water which
is alkaline, pH will be neutralized
and sections appear blue.
- It is not possible to over-blue a
section as the bluing reagent can
only turn blue the amount of
hematoxylin in the tissue however,
excessive bluing can cause section
to lift due to the alkaline action on
the tissue.
Counterstaining
- the practice of staining tissue
components other than the tissue
element of interest to provide
greater visual contrast.
INDICATION FOR SPECIAL STAINING
1. Demonstration of various cellular
products for diagnosis
● Carbohydrates
● Proteins
● •Lipids
● •Pigments
● Tissue fibers
2. Demonstration of extracellular
material for the identification of
diseases such as amyloid
3. Identification of microbial
organisms
4. Estimation of DNA and RNA
content of the cell
ROUTINE STAIN
HEMATOXYLIN AND EOSIN
- It is a relatively simplistic staining
technique that takes advantage of
the acidic and basic properties of
the cell’s cytoplasm and nucleus to
stain a wide variety of tissues and
tissue structures.
● Nuclei - blue to purple
● Cytoplasm - pink
● Red blood cells, eosinophilic
granules, other tissue elements -
varying shades of pink
CARBOHYDRATES
- The presence of certain
carbohydrates in tissues is
indicative of certain diseases.
Glycogen
- Normal glycogen distribution
patterns may be disrupted in
diseases caused by carbohydrate
metabolism enzyme deficiencies
such as von Gierke’s disease and
Pompe’s disease.
Von Gierke’s disease
- Also known as Type 1 glycogen
storage disease
- an inherited disorder caused by
the buildup of a complex sugar
called glycogen in tissue cells
- caused by the deficiency of
glucose-6-phosphatase (G6Pase)
Staining Methods: Carbohydrates
1. Periodic Acid-Schiff (PAS)
 ● Glycogen, fungi, basement - Mucicarmine is used to
membranes and certain demonstrate acidic mucins
mucosubstances - Pink to red secreted by cells of epithelial
● Nuclei - Blue, if counterstained origin. It can also be used to stain
with hematoxylin the capsule of the Cryptococcus
● Other tissue elements - Green, if organism.
counterstained with light green
stains ● Mucins - Deep rose to red
● Nuclei - Blue or black, depending
2. Periodic Acid-Schiff (PAS) with on the hematoxylin used
Diastase ● Other tissue elements - Yellow
metanil yellow counterstain
- Because PAS stains other ● Cryptococcus capsule - Deep rose
carbohydrates apart from glycogen to red
such as mucins, the addition of
Diastase can help differentiate
distinguish glycogen granules 5. Colloidal Iron
from other granules
- Colloidal iron technique is more
CARBOHYDRATES sensitive when staining for acid
mucins that may present in small
MUCINS amounts, such as in some
- are secreted by a variety of mesotheliomas
epithelial and connective tissue ● Acid mucins - dark blue
cells ● Neutral mucins and other
- detection for mucins is divided into PAS-positive tissue elements - pink
two: neutral mucins, and acid to red if stained with PAS
mucins ● Mixtures of acid and neutral
- Abnormal systemic production of mucins - bluish to reddish purple
mucins is also found in diseases ● Nuclei - pink to red if stained with
caused by enzyme deficiencies nuclear fast red.
(Hurler disease, Schele disease,
Hunter disease). Alcian Blue/PAS
- The combination of the Alcian blue
3. Alcian Blue Stain (ph 2.5 and pH and the PAS techniques can be
1.0) used as a means of distinguishing
neutral mucins from acid mucins.
- stains acid mucin (in acidic pH 2.5),
such as sialomucin and Amyloid
sulphomucin. Alcian Blue does not - an intercellular material that is
stain neutral mucins deposited in various tissues such
● pH 2.5 Carboxylated and some as heart, muscle, skin, liver, spleen,
weakly sulfated acid mucins – Blue kidneys and brain.
● pH 1.0 Weakly and strongly - Amyloidosis may be associated
sulfated acid mucins – Blue with genetic predisposition,
● Nuclei – Red to pink chronic inflammatory diseases,
● Other tissue elements – Pale pink tumors or Alzheimer’s disease.

4. Mucicarmine Stain STAINING METHODS: AMYLOID

6. Congo Red
 - a dye with a selective affinity for
amyloid.
● Amyloid - pink to red (apple-green
with polarized light)
● Nuclei - blue
LIPIDS
- Lipids are defined as group of
naturally available organic fatty
substances that are soluble in
alcohol and insoluble in water.
- Lipids are the major components
of the cell membrane and the
membranous component of many
cellular organelles.
- The myelin component of the
nerve sheath is also made of lipid.
1. Oil Red O
2. Sudan Black B
STAINING METHODS: LIPIDS
1. Oil Red O
- Oil Red O stain is used for
demonstration of lipid material. It
is particularly useful to
demonstrate lipid in renal cell
carcinoma.
- Oil Red O stains lipid, lipoprotein
and triglycerides.
● Fat - Red
● Nuclei - Blue
2. Sudan Black B
- Sudan black B is a lipophilic dye
and is insoluble in water.
- This dye therefore is dissolved in
tissue fat and stains them.
- The slightly basic dye Sudan black
B combines with the acidic
component of the lipid.
● Lipid granules – Black
- Used in the diagnosis of AML –
indicated by the presence of black
granules in myeloblasts
NUCLEIC ACID AND PROTEINS
- There are two types of nucleic
acids: deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA). DNA
and RNA.
- DNA and RNA differ only in the
chemical structure of a sugar
group. DNA has a 5- carbon sugar
called deoxyribose, and RNA
contains a 5-carbon sugar called
ribose.
- The difference in these sugars is a
single hydroxyl group (OH-), and it
is this difference that allows for
selective demonstration of DNA
and RNA by special staining
techniques.
STAINING METHODS: NUCLEIC ACIDS
1. Feulgen Stain
- Takes advantage of the ability of
hydrochloric acid to hydrolyze or
chemically alter the deoxyribose
sugar of DNA into an aldehyde.
- The resulting aldehyde reacts with
fuchsin (Schiff’s reagent), which
specifically binds to aldehydes. This
combination of acid hydrolysis and
aldehyde staining is what
constitutes the Feulgen reaction
- Used to demonstrate DNA ploidy
● Nuclei - red-purple
● DNA - red-purple or magenta
● Background - Green
2. Methyl Green-Pyronin Y Stain
- The methyl green-Pyronin Y stain
is used to demonstrate RNA
- Methyl green preferentially binds
to DNA, and Pyronin Y to RNA
- is also utilized to detect the
presence of plasma cells and
lymphocytes. Useful in evaluating
plasmocytomas.
● DNA is stained green
● RNA is stained red
PIGMENTS

- Pigments play an important part in
the diagnosis of diseases and
conditions such as gout, kidney
and gallbladder stones, jaundice,
melanomas, Albinism, hemorrhage
and tuberculosis.
- In tissue sections, the term
pigment refers to a material that
has color and can be seen without
a microscope. It can be either
normal or pathological.
- Pigments are identified either by
their color, size and shape or by
chemical testing. For example, if
the chemical test normally gives a
blue color, and it is applied to a
yellow pigment, the results will be
a green color.
STAINING METHODS: HEMOSIDERIN
- Stains both bilirubin pigments and
collagen
● Bile pigment: green
● Muscle and cell cytoplasm: yellow
● Collagen: red
STAINING METHODS: CALCIUM
3. Von Kossa Calcium Stain
- This is an indirect staining method
for the demonstration of calcium
or calcium salt in tissue sections.
- This technique is not specific for
calcium. Other reducing
substances, such as formalin
pigment and melanin will also give
a positive reaction.
● Calcium salts – Black to
brown-black
● Nuclei – Red
● Cytoplasm – Light pink

1. Prussian Blue Reaction (Perls’ STAINING METHODS: MELANIN

Reaction) for Ferric Iron
4. Fontana-Masson stain
- Hemosiderin is the most common - Masson-Fontana stain
hemoglobin derivative and the demonstrates melanin and
iron-containing pigment of argentaffin granules.
hemoglobin. - Pathologically, melanin is found in
- Hydrochloric acid unmasks the conditions of malignant
ferric iron. melanomas, Addison’s disease,
- This ferric iron reacts with metabolism disturbances and
potassium ferrocyanide to from the benign nevus tumors.
insoluble blue-colored ferric
ferrocyanide ● Melanin, argentaffin granules,
lipofuscin - black
Ferric ferrocyanide - Blue ● Nucleus – red
Nuclei, cytoplasm - Red ● Background - pink
4. Fontana-Masson stain
STAINING METHODS: BILE PIGMENT
- Argentaffin granules are found in
2. Fouchet’s Stain carcinoid tumors. Argentaffin is a
term used for cells that take up
- The oxidizing action of Fouchet’s silver (including melanocytes). The
reagent converts the bile pigment argentaffin cell granules or
to green biliverdin (if it has been melanin will be stained black.
transported to the liver and ● Melanin, argentaffin granules,
reduced, it is referred to as lipofuscin - black
bilirubin). ● Nucleus – red
● Background - pink
FOUCHET-VAN GIESON STAIN (HALL’S
BILIRUBIN STAIN) STAINING METHODS: COPPER
 5. Rhodanine Stain
- The rhodanine dye binds to copper
or copper-associated proteins.
Tissue sections are then
counterstained in hematoxylin.
● Copper and copper-associated
protein - Red to orange-red
● Nuclei - Blue
STAINING METHODS: CARBON
- Carbon is the most commonly
seen exogenous mineral in tissues,
and is easily recognized in stained
tissue sections, such as lung and
adjacent lymph nodes of urban
dwellers and tobacco smokers.
- Black pigmentation of the lung
(anthracosis) is also seen as a result
of massive deposition of carbon in
coal workers.
- Carbon is extremely inert and
cannot be demonstrated with
conventional histochemical
methods. It may be confused with
melanin, which is dissolved by
bleaching agents while carbon is
not.
ARTIFACTS
1. Formaldehyde deposits
- Formalin deposits appear as fine,
dark-brown or black crystal-like
precipitates, often with no
relationship to the tissue (i.e., the
precipitate appears adjacent to
tissues or within interstices or
vessels) especially in postmortem
and blood-containing tissues fixed
with acid formaldehyde.
- The deposits are breakdown
products of laked hemoglobin,
found maximally around blood
vessels and congested tissues, e.g.
liver and spleen.
- They form when the formalin
buffer is exhausted and the tissue
becomes acidic, which promotes
the formation of a complex of
heme and formalin.
2. Mercuric chloride deposits
- Renal tissue fixated using mercuric
chloride. Note the brown granules.
CONNECTIVE TISSUES
- Reticular fibers are composed of
the protein collagen and are
coated with glycoprotein. They
form a delicate framework for
many soft organs and a network
around nerve fibers, fat cells,
lymph nodes and smooth and
skeletal muscle fibers.
- Collagenous fibers are composed
of the protein collagen and provide
the greatest strength of the three
fiber types. Collagenous fibers are
found in ligaments, tendons,
cartilage and bone.
- Elastic fibers are composed of the
protein elastin and offer the
greatest flexibility among the fiber
types. Elastic fibers allow tissue to
stretch and are located in the skin
and walls of blood vessels.
STAINING METHODS: RETICULAR FIBERS
1. Reticulin/Nuclear Fast Red stain
- Reticular fibers are commonly
demonstrated by the use of stains
involving silver solutions.
- These stains rely on the
impregnation of silver ions to the
fibers and subsequent reduction of
those silver ions to their visible
metallic form.
- The reticulin nuclear fast red stain
is used for visualization of reticulin
fibers in tissue section.
Reticulin fibers – black
Background – red
STAINING METHODS: COLLAGEN FIBERS
2. Masson Trichrome Stain
- Trichrome stains are used to
distinguish collagen from muscle
and aid in the diagnosis of fibrotic
changes, neuromuscular diseases
and tumors of muscle origin
● Muscle – red
● Collagen – blue
● Nuclei - black or blue
● Fibrin - red
3. Van Gieson Stain
- This stain demonstrates collagen
and is indicated for assessing the
extent of fibrosis, differentiation of
collagenous vs smooth muscle
tumors, and differentiating
amyloid and collagen
● Collagen fibers – red
● Nuclei - black
● Cytoplasm, muscle and fibrin -
yellow
4. Verhoeff-Van Gieson Stain
- The Verhoeff-Van Gieson (VVG)
stain is commonly used to
demonstrate elastic fibers. The
tissue section is initially
overstained with a solution of
hematoxylin-ferric chloride-iodine
and then differentiated for optimal
demonstration of elastic fibers.
- Ferric chloride and iodine act as a
mordant to link hematoxylin dye
molecules to tissue components.
They then act as oxidizers to
convert hematoxylin to hematein.
● Elastic fibers - blue-black to black
● Nuclei - blue to black
● Collagen – red
● Other tissue elements - yellow
STAINING METHODS: FIBRIN
5. Phosphotungstic Acid
Hematoxylin (PTAH)
- PTAH aims to stain fibrin, cross
striation of the muscle and glial
fibers
● Striated muscle fibers, fibrin, nuclei
and astrocytes - blue
● Cytoplasm - brown red
● Collagen and bone - brown pink
5. Phosphotungstic Acid Hematoxylin
(PTAH)
- preferred for demonstrating
cross-striations of skeletal muscle,
which may be lost in certain
muscle diseases.
- Used often without a counterstain
to demonstrate cross striations.
MICROBIAL ORGANISMS
- Routine hematoxylin and eosin
stain may not distinctly identify
microbial organisms in tissue or
smear, therefore special stain is
needed.
- Special stain helps to delineate the
morphology and characteristic
color of the organism:
1. Gram stain
2. Ziehl-Nielson Stain
3. Grocott’s Methenamine Silver
4. Warthin-Starry Stain
5. Alcian Yellow/Toluidine Blue stain
STAINING METHODS:
MICROORGANISMS
1. GRAM STAIN
- Gram-positive bacteria - Blue or
violet
- Gram-negative bacteria – Red
2. Ziehl-Nielson Stain (Acid fast
stain)
● Acid-fast bacteria - red
● Non-acid-fast bacteria - blue
3. Grocott’s Methenamine Silver
 - useful to identify a variety of
pathogenic fungi, including:
● Aspergillus fumigatus
● Blastomyces dermatitidis, Candida
albicans
● Coccidioides immitis
● Cryptococcus neoformans
● Histoplasma capsulatum
● Nocardia asteroids
● Pneumocystis carinii
● Sporothrix schenckii
● Background – Yellow to gold
2. Cajal stain for Astrocytes
- Astrocytes represent the major
supporting cells in the brain. They
respond to injury by producing a
dense network of processes,
somewhat analogous to the
fibrous scar that occurs elsewhere
in the body.

❖ Fungi and pneumocystis- black Note the star-shaped bodies.

❖ Background - light green

4. Warthin-Starry Stain

- useful in identifying Spirochetes

and some other bacteria, such as
H. pylori and two causative agents
of cat scratch disease, Bartonella
henselae and Afipia felis.
● Bacteria - black
● Background - yellow to light brown

5. Alcian Yellow/Toluidine Blue Stain

- The alcian yellow/toluidine blue
(AY/TB) stain is a common way of
screening patient specimens for
Helicobacter pylori.
- H. pylori is a spiral-shaped
bacterium implicated in gastric
inflammation, peptic ulcers and
gastric cancer.
● H. pylori - blue
● Mucin - yellow
● Background - pale blue

STAINING METHODS: NERVOUS TISSUE

1. Bielschowsky method

- particularly useful in the

identification of neurofibrillary
tangles and senile plaques, which
are the hallmarks of Alzheimer’s
disease. Senile plaques, also called
neuritic plaques, consist of an
amyloid core surrounded by either
axonal or dendritic processes.

● Neurofibrillary tangles and senile

plaques – Black