CHAPTER 4

4. Special Staining methods

Acknowledgements

 Addisa Ababa University

 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-
Ethiopia

2
Learning objectives

1. Describe method of demonstration of proteins.

2. Describe carbohydrate stain.

3. Describe special stain.

 Chapter outline
4.Special Staining methods

4.1 Connective tissue staining

4.2 Protein, Nucleic acid and Amyloid
4.3 Carbohydrates and Lipids
4.4 Pigments, Minerals and Bone
4.5 Microorganisms
4.1Connective tissue staining
 The three types of fibers present in connective tissue matrix are
reticular, collagenous and elastic fibers.

 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.
 
 Connective…
 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.

Reticular Fibers
Clinical Applications
 Reticular fibers perform a support function in the body and
are common in the liver, kidney and spleen.

 A cirrhotic liver shows disturbed patterns of reticular fibers,

explaining the routine use of reticular stains in liver biopsy
specimens.
 Connective…
 Reticular fibers also display characteristic patterns in tumors,
thereby aiding in the differential diagnosis of certain tumor
types.

Chemistry
 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.

 Reticular fibers are agyrophilic in that they possess the ability

to adsorb silver from solution but are unable to reduce it to
visible metallic form without the use of a reducing solution.
 Connective…
 An ammoniacal silver solution consists of a strong base
(ammonium hydroxide) added to an aqueous silver nitrate
solution to form a silver diamine complex.

 This method commonly calls for oxidation and sensitization

of tissue prior to application of this complex.

 Oxidation(potassium permanganate/periodic acid)

enhances subsequent staining, while the sensitizing agent
(uranyl nitrate/dilute silver nitrate) initially binds to the tissue
component of interest.

 Silver ions provided by the ammoniacal silver solution

impregnate the fibers and replace the sensitizer in the
metal-organic compound.
 Connective…
 Subsequently, by the action of a reducing agent (formalin),
the silver diamine complex is reduced to visible metallic form.
 Metallic silver is converted by use of a toning reagent (gold
chloride) to metallic gold, which is more stable and offers
better contrast and clarity.

 Unreduced silver and excess gold chloride are removed

(sodium thiosulfate), and the tissue section is then
counterstained, if desired.

 Nuclear fast red or light green counterstains are commonly

used.

 A section of normal liver or tonsil tissue may be used for

quality control.
 Connective…
Technical Considerations
 Non-selective silver precipitation on tissue sections may be
caused by use of improperly cleaned glassware.

 Silver solution should be made fresh with high quality water.

 Excess ammonium hydroxide added to ammoniacal silver

solution impairs impregnation of reticular fibers and results
in weak staining.

 Alkalinity of silver solution may contribute to loss of tissue

section from slide.
 The use of charged or adhesive-coated slides is
recommended.
Connective…
 Connective…
Basement Membranes
Application
 The basement membrane is a modified connective tissue
that provides support to epithelial cells, muscle fibers and
peripheral nerves.

 It also functions to permit diffusion of nutrients and wastes

between cells and capillaries in underlying connective
tissue.

 The Jones methenamine silver stain is excellent for the

demonstration of glomerular and tubular basement
membranes of kidney biopsies.

 It is routinely used as a component of kidney biopsy panels

to demonstrate evidence of renal disease.
 Connective…
Chemistry
 Basement membranes are commonly demonstrated with a
silver stain employing a methenamine silver solution.

 Methenamine silver methods rely on the oxidation of

carbohydrates within the tissue to form aldehyde groups.

 These groups will directly act to reduce silver ions from the
methenamine silver solution to metallic silver.

 The ability to bind silver ions from solution and independently

reduce silver to a visible metallic form is referred to as
argentaffin.

 The use of sensitizing and reducing solutions are not

necessary with this method.
 Connective…
 Oxidation by periodic acid or chromic acid enhances
subsequent staining.
 A working methenamine-silver nitrate solution provides silver
ions for impregnation.

 Gold chloride tones tissue sections and converts metallic

silver to metallic gold.

 The tissue section is then counterstained, if desired.

 Light green is commonly used.

 Normal kidney tissue may be used for quality control.

 Basement membranes may also be demonstrated using the

Periodic Acid-Schiff (PAS) technique. Technical
Connective…
 Connective…
 Considerations
 Glassware should be acid-cleaned and rinsed in distilled
water prior to use.

 Incomplete impregnation will result in weak, interrupted

staining patterns of the glomerular basement membrane.
Incubation time may need adjustment.

 Counterstaining should be delicate to avoid obscuring

positive reaction.
 Connective…
Elastic Fibers
Clinical Application
 Elastic fiber demonstration is useful to identify atrophy caused
by arteriosclerotic changes, evidence of other vascular
diseases, and vessel invasion by tumors.

Chemistry
 The Verhoeff-Van Gieson (VVG) stain, 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.
 Connective…
 They then act as oxidizers to convert hematoxylin to
hematein.
 A dilute solution of ferric chloride is then used to break the
tissue-mordant-dye complex, thereby differentiating structures
within the tissue.
 As elastic fibers have a strong affinity for the iron
-hematoxylin complex, they will retain dye molecules, while
other structures dissociate dye molecules.

 sodium thiosulfate remove excess iodine and ferric chloride

from tissue sections.

 Van Gieson solution acts as a counterstain.

 Common quality control tissues include lung parenchyma,

artery and skin.
Connective…
 Connective…
Technical Considerations

 Verhoeff solution should be made fresh for each

use.

 The differentiation step is critical to optimal results,

as over-differentiation results in loss of finer fibers.

 Prolonged staining in van Gieson counterstain will

continue to differentiate section due to its picric acid
component.
 Connective…
Trichrome Stains
Clinical Application
 Trichrome stains are used to distinguish collagen from muscle
and aid in the diagnosis of fibrotic changes, neuromuscular
diseases and tumors of muscle origin.

Masson’s Trichrome
Chemistry
 With the Masson’s trichrome stain , Bouin’s solution is used
initially as a mordant to link the dye molecules to the tissue
components of interest.

 Nuclei are stained with Weigert’s hematoxylin, which is

resistant to decolorization by subsequent acidic staining
solutions.
 Connective…
 Application of Biebrich-scarlet-acid-fuchsin stains all
acidophilic tissue elements such as cytoplasm, muscle and
collagen.

 Subsequent treatment by hosphomolybdic/phosphotungstic

acid serves as a decolorizer causing the Biebrich-scarlet-acid-
fuchsin to diffuse out of the collagen fibers while leaving the
muscle cells red.

 Subsequent application of aniline blue will stain the collagen

after which, 1% acetic acid is employed to properly
differentiate the tissue section.

 Most tissue will contain an internal quality control, but

appendix, fallopian tube, uterus or small intestine may be
used.
Connective…
 Connective…
Technical Considerations
 Viable Bouin’s solution used at the correct temperature will
ensure optimal staining.

 Proper differentiation by posphomolybdic/phosphotungstic

acid yields proper intensity of red-staining entities.

 Decreased red staining of muscle may indicate that Biebrich-

scarlet-acid-fuchsin solution has aged.

 Intensity of blue-staining entities is modified by

increasing/decreasing incubation time of aniline blue.

 Faded blue staining of connective tissue may indicate

overdifferentiation of sectionby 1% glacial acetic acid.
 4.2 Protein, nucleic acid and amyloid
 Protens, Nucl…
Staining of proteins
 Proteins are major constitutes of cells and tissues and
are made up of amino acids linked by peptide bonds.

Methods of demonstration of proteins in tissues

include:
 Histophysical method;
 Amino acid histochemical method;
 Enzyme histochemical method;
 Immuno-fluorescent method;
 Immuno histochemical method.

 Protein ,Nucl…
Histo physical methods

 Histo- physical methods are based on physical configuration

of their molecules and not on their chemical composition.

 These methods are based on selective staining with large or

small molecule dyes for example, trichrome method.

 Using these technique proteins present in collagen, fibrin,

elastic, reticulin, and amyloid can be demonstrated.
 Proteins Nucl…
Table : Histochemical reactions in protein demonstration.
 Protein ,Nucl…
Amino acid histochemical methods
 Amino acid histochemical methods are based upon
identification of exposed groupings and linkages with in
amino acid molecules.

 With this staining methods some of the amino acids in the

proteins and not the whole proteins are demonstrated.

Demonstration of Amino group by Ninhydrin Schiff

reaction for lysine
 At neutral pH and 370C, Ninhydrin reacts with alpha
aminogrops to produce aldehydes.

 The aldehyde can then be demonstrated using Schiff

reagent.
 Proteins Nucl…
 Fixation solution: Neutral formal saline or formaldehyde
vapor.
Method of staining
1. Take the section to 70 % alcohol.
2. Treat with Ninhydrin solution at 370C.
3. Wash with running tap water.
4. Place the slide in Schiff reagent for 45 minutes.
5.Wash with running tap water.
6. Counter stain with alum haematoxyline.
7. Wash with running tap water.
8. Dehydrate with graded alcohol.
9. Clean in xylene.
10. Mount in DPX.
 Protein ,Nucl…
Result: Amino group = Stain pinkish purple

Demonstration of phenyl group by Millon reaction for

tyrosine
 The section is treated with hot mercuric-sulphric-acid nitrate
mixture.
 A pinkish red color develops at the site of tyrosine containing
protein.
 Tyrosine is the only amino acid, which contains the
hydroxyphenyl group.
 This method can be regarded as specific for tyrosine.

 Tyrosine is invariably present in all tissue proteins.

 Therefore, Millon reaction is a suitable general protein
method.
 Proteins Nucl…
Fixative: Neutral formalin, formaldehyde vapor

Section: Paraffin, fixed cryostat, freez dried or collodin.

Staining solution
Solution A: Mercuric sulphate-sulphric acid mixture
1. Mercuric sulphate --------------------10 gm
2. Distilled water -------------------------90ml
3. Concentrated sulphric acid --------10ml

Preparation of mercuric sulphate-sulphric acid mixture

1. To 90ml of Distilled water, add concentrated sulphric acid.
2. To the mixture add 10 gm of Mercuric sulphate.
 Protein ,Nucl…
Solution B: Sodium nitrite solution
 Two hundred fifty mg (250 mg) of sodium nitrite is dissolved in
10 ml of distilled water.

Staining solution
 Five ml (5ml) of solution B is added to 50 ml of solution A.

Procedure
1. Take the section to water.
2. Immerse the section in staining solution in small beaker
3. Bring to boil gently; simmer for two minutes.
4. Allow cooling at room temperature.
5. Wash in three changes of distilled water two minutes each.
6. Dehydrate through graded alcohol.
7. Clear in xylol and mount in DPX.
 Proteins Nucl…
Result: Tyrosine containing proteins stain red or pinkish.

Nucleic Acids
Clinical Applications

 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.

 The two most common special staining techniques utilized for

visualizing nucleic acids are the Feulgen and methyl green-
pyronin Y stains.
 Protein ,Nucl…
Feulgen Stain
 The 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 Schiff’s reagent, which

specifically binds to aldehydes.

 This combination of acid hydrolysis and aldehyde staining is

what constitutes the Feulgen reaction.

 A light green counter stain is often applied to the tissue section

to achieve a better contrast.

 The ribose sugar of RNA is unaffected by this acid hydrolysis

and, therefore, will not stain.
Proteins Nucl…
 Protein ,Nucl…
Technical Considerations
 In the Feulgen stain, hydrolysis is the most critical
step.

 As the hydrolysis time is increased, an increasingly

stronger reaction can be achieved until an optimal
state is reached.
 Continued hydrolysis past this optimal state causes
the reaction to become weaker and ultimately fail.
 Proteins Nucl…
Methyl Green-Pyronin Y Stain
 The methyl green-pyronin Y stain is used to demonstrate RNA.
 Tissue sections are incubated in a solution of purified methyl
green and pyronin Y.
 Methyl green preferentially binds to DNA and pyronin Y to
RNA.

 The reason that two basic dyes bind preferentially is

suggested to be related to the different degrees of
polymerization observed in DNA and RNA.

 DNA, which is more highly polymerized, is bound only by

methyl green; whereas RNA, which is less polymerized than
DNA, is bound only by pyronin Y.
 Protein ,Nucl…
Depolymerization studies support this hypothesis.

Technical Considerations
 Typically, Carnoy’s fixative yields the best results, though
protocols have been modified to allow formalin fixed tissues
to be stained.

 After staining, it is imperative that the stained slides are not

exposed to water.

 The slides should be dehydrated in acetone or tertiary

butanol and mounted in a synthetic resin.
Proteins Nucl…
 Protein Nucl…
Amyloid
 Amyloid is an intercellular material that is deposited in various
tissues such as heart, muscle, skin, liver, spleen, kidneys and
brain.

 Clinical amyloidosis can be classified based either on the

underlying cause or by pattern of amyloid distribution.

 Amyloidosis may be associated with genetic predisposition,

chronic inflammatory diseases, tumors or Alzheimer’s
disease.

 Sometimes the progression of the amyloidosis can be halted

through treatment of the underlying condition, so early
diagnosis is extremely important.
 Protein Nucl…
Congo Red
Chemistry
 Congo red is a dye with a selective affinity for amyloid.

 It is believed that the linear shape of the molecule is

responsible for its ability to bind the β -pleated sheet
structure of the amyloid.

 The binding is thought to be through non-polar hydrogen

bonds.

 The staining methods used favor the formation of these

bonds over electrochemical bonds that may bind the Congo
red to other tissue elements.
 Protein Nucle…
 A combination of high pH and high sodium in the staining
solution favors selective binding of the dye and the amyloid.

 The binding arrangement between the dye and the amyloid

produces a unique effect (apple-green birefringence) when the
stained tissue is observed with polarized light.

 The presence of apple-green birefringence has become the

most reliable diagnostic feature of amyloid.

 Hematoxylin is usually the counterstain of choice for the

Congo red stain.
 Proteins Nucle…
Clinical Applications

 Amyloid deposits are formed in tissues spontaneously or in

association with a wide range of disease conditions.

 The deposits are intercellular and may become large

enough to cause damage to surrounding tissues.

 Depending on the type of underlying disease, amyloid

deposits may form in muscle, heart, skin, tongue, liver, spleen,
kidneys or adrenals.
 Proteins ,nucle…
Technical Considerations
 The high sodium concentration of the staining solution is
important in order to obtain specific staining.

 Some methods may use sodium hydroxide (1% w/v) either

before or after staining to reduce background.

 Tissue sections should be used within six months of cutting. Cut

sections stored for longer periods may exhibit weak or negative
staining.
 Sections should be cut at 7–10 microns in order to observe the
correct apple-green birefringence.

 Thinner sections will show a bluish or reddish birefringence while

thicker sections will appear yellowish under polarized light.
4.3 Carbohydrates and Lipids
 Carbohydrates and …

Carbohydrates
 Carbohydrates are widely distributed in both plants and
animals.

 They are often referred to as “starches” or “sugars” but can be

divided into numerous subtypes based on their chemical
structure.

 Over the years, diverse terminology has been used to

describe and classify tissue carbohydrates.

 For the purposes of this chapter related to the staining of

tissue carbohydrates, two main entities will be considered:
glycogen and mucins (also known as mucosubstances).
 Carbohydrates and …
Glycogen
 Glycogen is a simple polysaccharide that is widely distributed
throughout the body.

 It is found in greatest amounts in the liver, hair follicles,

endometrial glands, vaginal and ectocervical epithelium, and
cardiac and skeletal muscles.

 Glycogen may have diagnostic significance in several types of

tumors including carcinoma, mesothelioma and
rhabdomyosarcoma.

 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.
 Carbohydrates and …

Periodic Acid-Schiff (PAS)

Chemistry
 The PAS reaction demonstrates aldehyde groups formed by the
oxidation of certain tissue carbohydrates and glycogen.

 The oxidation of the tissue sections is performed using periodic

acid.

 After oxidation, tissue sections are treated with Schiff reagent, a

colorless mixture of basic fuchsin, HCl and sodium metabisulfite.

 During incubation, basic fuchsin binds to the newly formed

aldehyde groups in the tissue.

 Carbohydrates and …
 Several counterstains may then be used to visualize other
tissue elements.

 Hematoxylin counterstaining is very commonly used to

demonstrate cell nuclei, although other counterstains may
also be used.

 Some older methods include treating the sections in a

sulfurous rinse solution before the running water wash.

 This can serve to reduce background staining by removing

excess Schiff reagent from the tissue.
 Carbohydrates and …
Clinical Applications
 most commonly used to evaluate glycogen deposits in the liver.

 Tumors of the bladder, kidney, liver, ovary, pancreas and lung

may also contain glycogen granules of diagnostic significance.

 Abnormalities of the basement membranes revealed by PAS

staining can aid in the diagnosis of several disorders.

 Evaluation of the basement membranes can also be used to

measure the invasiveness of skin tumors.
 Carbohydrates and …

Technical Considerations
 Increasing or decreasing the incubation time of either the
periodic acid or the Schiff reagent can increase or decrease
the staining intensity.

 Incubation times should be optimized to obtain the desired

result.
 Extending the wash time in running water may also increase
the staining intensity.

 Schiff reagent is stored in the refrigerator so it is important to

allow the reagent to come to room temperature before use.

 Failure to do so may result in weak staining.

 Carbohydrates and …

Mucins
 Mucins are a large family of polypeptides that are secreted by
a variety of epithelial and connective tissue cells.

 The classification of mucins is complex but can, for purposes

here, be simply divided into neutral and acid mucins.

 Mucins are produced by many tumors including carcinoma,

liposarcoma and mesothelioma.

 Abnormal systemic production of mucins is also found in

diseases caused by enzyme deficiencies (Hurler disease,
Schele disease, Hunter disease).
 Carbohydrates and …

Mucicarmine
Chemistry
 The mucicarmine stain contains carmine (a red dye) bound in
solution to aluminum.

 It is believed that the aluminum acts as a mordant by binding

to the acid groups of the mucin.

 This binding causes the carmine dye, which is carried along

with the aluminum, to impart a deep pink or red color to the
mucin.

 Cell nuclei are usually stained with hematoxylin and the

remaining tissue elements are counterstained with metanil
yellow.
 Carbohydrates and …
Clinical Applications
 Mucicarmine is used to demonstrate acidic mucins secreted
by cells of epithelial origin.

 It can also be used to stain the capsule of the Cryptococcus

organism.
 Carbohydrates and …

Technical Considerations
 The working mucicarmine solution should be made just prior
to use and discarded after the procedure. Increase the
mucicarmine incubation time for darker staining.

 The incubation times for the hematoxylin and metanil yellow

can be optimized to obtain the desired color balance.
 Carbohydrates and …

Alcian Blue
Chemistry
 Alcian blue is a water soluble dye that derives its blue
color from the copper in the molecule.
 It is a basic dye and, therefore, has an affinity for tissue
elements containing anionic groups such as acid mucins
and other acidic carbohydrate moities.
 Alcian blue can be used as a comprehensive acid mucin
stain at pH 2.5.
 Alcian blue solutions of different pH will selectively stain
subgroups of acid mucins.

 Nuclear fast red is commonly used as a counterstain.

 Carbohydrates and …

Clinical Applications
 Alcian blue is used for the demonstration of acid mucins,
which can be produced by several types of epithelial and
connective tissue tumors.

 Acid mucins also play a role in collagen diseases and in the

early stages of atherosclerosis.

 The versatility of alcian blue staining methods is a valuable

tool for the evaluation of the various acid mucins.
Carbohydrates and …
 Carbohydrates and …

Technical Considerations
 The incubation times for alcian blue and nuclear fast red
can be optimized to achieve the desired results.

 Longer incubation times lead to darker staining.

 The nuclear fast red should be well-mixed before use to

assure good results.

 It is important to carefully follow the instructions for the

preparation of the alcian blue and pretreatment enzyme
solutions.
 This will ensure the specific demonstration of the various
acid mucins.
 Carbohydrates and …

Lipids
 Lipids are classified as simple, compound and derived.

 Lipids occur in cells either as microscopic droplets or

bound to other tissue elements.

 Staining of simple lipids with Sudan and oil red O dyes

will be discussed here.

Clinical Application

 Abnormal deposition of fat may develop because of

injury and from fat emboli, which may dislodge, relocate
and cause loss of function or death.
 Carbohydrates and …
 Liposarcomas may be differentiated from other tumor types
using lipid-staining techniques.

 Degenerative changes in myelin and cell membranes may

exude fat droplets demonstrated by these techniques.

Chemistry
 Simple lipids are demonstrated using oil red O and Sudan
black B stains.
 These “staining” techniques demonstrate physical
processes rather than chemical interactions displayed by
most stains.
 The dye (oil red O or Sudan black B) is dissolved in a lipid
solvent such as propylene glycol.
 Carbohydrates and …
 Other fat solvents, such as isopropanol, may be used,
but result in some lipid loss.

 Dye molecules are found to be more soluble in the

cellular lipid than in the dye solvent imparting a positive
stain color to the tissue lipid.

 Tissue sections are then stained with an appropriate

counterstain (hematoxylin/nuclear fast red).

 Tissue containing fat provide a source of quality control

material.
Carbohydrates and …
 Carbohydrates and …

Technical Considerations
 For demonstration of simple lipids, frozen sections must be
used.

 Frozen sections may be unfixed or post-fixed in 10% neutral

buffered formalin or formol-calcium.

 Tissue must not be exposed to fat solvents such as alcohols,

acetone, xylene or paraffin.

 Aqueous mounting media must be used when coverslipping

finished slides.
4.4 Pigments, Minerals and Bone
Pigments and Minerals
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.

 It can be either normal or pathological.

 Pigments are identified either by their color, size and shape or

by chemical testing.

 Pigments can be separated into three categories: artifact,

exogenous and endogenous.
 Pigments and …
 The endogenous pigments are described in this chapter.
Melanin
Clinical Applications
 The most recognized pigment is melanin.

 Melanin, a brown or black pigment derived from metabolism

and not directly associated with blood, is normally found in
skin cells (melanocytes), hair follicles, eyes (iris) and areas of
the embryonic brain.

 The distribution and amount of melanin is what accounts for

the wide variety of skin, hair and eye colors.

 Pathologically, melanin is found in conditions of malignant

melanomas,
 Pigments and ….
Chemistry
 Melanin possesses the strange ability to bind silver from silver
solutions and reduce it directly to metallic silver.

 It is also almost completely insoluble in organic solvents

because of its tight bond with proteins.

 Special staining techniques for melanin are varied in approach

and can be simple or complex.

 Schmorl’s reaction is a simple technique that uses melanin to

reduce ferric iron (Fe3+) to ferrous iron (Fe2+).
 Pigments and …
 The ferrous iron then combines with ferricyanide to form
ferrous ferricyanide, also known as Turnbull’s blue.

 More complex techniques use silver nitrate solutions.

 The Fontana-Masson method for melanin and the Warthin-
Starry method for melanin are two such methods.

 The Warthin-Starry method is further described.

 Silver nitrate solution first is applied to the tissue section.

 Melanin reduces the silver to metallic silver.

 The final step is application of nuclear fast red

counterstain.
 Carbohydrates and …
 As with all the special staining techniques for melanin, this
reaction is not melanin-specific and may stain other elements,
such as argentaffin, chromaffin and some lipofuscins.
 Pigments and …

Technical Considerations
 Melanin can be bleached over time by strong oxidizers.
 The melanin bleach method can also be used to remove
melanin.
 Lipofuscins are also removed from the tissue when using
this technique.
 For the melanin bleach method to be effective, it may be
necessary to oxidize in 0.25% potassium permanganate
solution an additional 5 to 20 minutes.
 Tissue sections are then briefly introduced to 5% oxalic
acid, which bleaches the melanin.
 Tissues can remain immersed until the sections become
clear.
 Sections should be washed for at least one minute to
remove traces of oxalic acid.
 Pigments and …

Hemosiderin
Clinical Applications
 Hemosiderin, a blood-derived pigment, is a crystalline
aggregate of proteins involved in iron storage.

 Some of the pathological conditions involving hemosiderin

are hemorrhage, hemolytic anemia, metabolic iron
disturbances, liver fibrosis, toxins, cirrhosis, heart failure
and diabetes mellitus.

Chemistry
 Appearing yellow to golden brown in unstained tissue,
hemosiderin is insoluble in alkalis and soluble in strong acid
solutions.
 Pigments and …
 Fixation in formalin alters hemosiderin so that it can be
solubilized slowly in weak acids.

 In Mallory’s and Perl’s methods for iron, the ferric ion in tissue
binds with ferrocyanide in solution forming ferric ferrocyanide,
otherwise known as Prussian blue.

 Because ferric iron in hemoglobin is tightly bound to

proteins, it is first necessary to use acid to release it from the
protein complex.
 A combination of hydrochloric acid (releasing solution) and
potassium ferrocyanide (binding solution) is able to release
and stain the ferric iron at the same time.

Nuclear fast red is the typical counterstain for these stains.

 Pigments and …

Technical Considerations

 Be careful not to overstain with the nuclear fast red, because

it may mask iron pigment that is delicately stained.
 Pigments and …

Lipofuscin
Clinical Applications
 Lipofuscin, a yellow-brown to reddish-brown pigment, is found
in many parts of the body including bone marrow, adrenal,
kidney, liver, heart, testis, ovary, cervix, brain and spinal cord.

 Lipofuscin is formed by the slow oxidation of lipids and

lipoproteins.
 It has been nicknamed the “wear-and-tear” pigment because
large accumulations of lipofuscin are found in people of
advanced age.

 When identified using special staining techniques, lipofuscin

can be foundin a variety of colors, shapes and sizes.
 Pigment and …
 The physical properties of lipofuscin are dependent upon
the length of time that the original lipid or lipoprotein has
undergone oxidation.
 It is recommended that more than one special staining
technique be performed to confirm the presence of
lipofuscin.
 Pathologically, lipofuscin is present in some lipid storage
diseases, such as Batten’s disease, and can be found at
the edges of cerebral hemorrhage or infarcts.

Chemistry
 Special staining techniques for lipofuscin include oil red O,
Sudan black B, Gomori’s aldehyde fuchsin , methyl green
basophilia, periodic acid-Schiff and Fontana-Masson.
 Pigment and ….
 Each stain has its own unique approach to staining
lipofuscin.
 Gomori’s aldehyde fuchsin will be further discussed.

 Tissue sections are oxidized in potassium permanganate.

 Oxalic acid is then applied to bleach the tissue section.

 The tissue is then stained with aldehyde fuchsin, which

binds to the lipofuscin.

 After rinsing in alcohol, the tissue section is counterstained

with tartrazine
 Pigment and …

Technical Considerations

It is important to note that results will vary based on the

state of oxidation.
 Pigments and …
Urates
Clinical Applications
 Nucleic acid metabolism creates uric acid, which is
normally excreted.
 Urate is the form of uric acid that circulates in the
bloodstream.
 Crystallized urate is in the form of monosodium urate.
 The color of crystalline urate is characteristically yellow
but can vary from colorless to brown, depending on the
thickness of the crystals.

 Pathologically, crystallization or increased urate in the

blood predisposes a patient to gout, poor kidney
function, kidney stones and arthritis.
 Pigments and …
 In other diseases, such as leukemia, urates can ultimately
be deposited in the kidneys.
 This happens because an extremely high number of cells
are destroyed, resulting in nucleic acid metabolism and
generation of uric acid.
Chemistry
 In tissue, uric acid crystals are present as sodium urate,
which is soluble in aqueous solutions and slightly soluble
in weak alcoholic solutions.
 The fixative of choice is 95% alcohol, which prevents the
dissolution of the urates.
 Sodium urate crystals can be visualized on a hematoxylin
& eosin (H&E) stained slide under a polarized light with a
red compensator.
 Pigments and …
 The urates will demonstrate a negative yellow or blue
birefringence, based on the alignment of the urate crystals.
 A special staining technique, Gomori’s methenamine-silver
method, can also be used to identify urate crystals in tissue
sections.
 Tissue sections are placed into a methenamine-silver
solution.
 Urates, like melanin, can bind silver from silver solutions and
reduce it directly to metallic silver.

 After reducing the silver, sodium thiosulfate is used to

remove the unreduced silver from the tissue sections.

 The section is then counterstained with light green.

 Pigments and …

Technical Considerations
 It is important to note that an alcoholic fixative must
be used in order to preserve the urates.
 Pigments and …

Bile
Clinical Applications
 One waste product of Red cell degradation is biliverdin.
 Biliverdin is transported to the liver, where is it further
reduced to bilirubin.
 Bilirubin is then removed from circulation in the blood and
is secreted as a component of bile.

 Biliverdin and bilirubin are considered bile pigments.

 Bile pigments can vary in color from yellowish-brown to
green.
Pathologically, excess bile pigment is seen in patients with
gallstones, sepsis, liver degeneration, congenital liver
enzyme disorders and liver tumors.
 Pigment and …
 When there is an obstruction in the flow of bile to the
gallbladder or colon, these pigments
 can be observed in hepatocytes, or liver cells, as yellow-
brown globules, and clinically, the patient will appeared
jaundiced.
 The obstruction can be within the liver or in organs other
than the liver.
 For example, a carcinoma at the head of the pancreas can
also cause
 an obstruction in the flow of bile and an accumulation of bile
pigments.
 Histologically, it is important to distinguish bile pigments
from lipofuscin, particularly in cases of possible sepsis in
liver transplant patients.
 Carbohydrates and …

Chemistry
 The most reliable and reproducible special staining
technique for demonstrating bile pigments is Hall’s
technique.
 In this technique bilirubin is easily oxidized to biliverdin in a
solution of ferric chloride and trichloracetic acid.
 The tissue section is then counterstained with Van
Gieson’s solution.
 Only bile and bile pigments in the liver are detected using
this special staining technique.

 To demonstrate bile pigments in other locations outside the

liver, such as hemorrhage, either the Gmelin technique or
Stein’s stain should be used.
Technical Considerations
 Small amounts of bile pigments are lost during routine
tissue processing and staining because of their slight
solubility in organic solvents.
 Large deposits of bile pigments, however, can resist
these processing procedures.
 It is recommended that two control sections should be
processed with the test section.
 Pigment and ….
Calcium
Clinical Applications
 Calcium is an insoluble mineral that is part of the normal
composition of bones, teeth and nails.
 It is one of the most important minerals used by the
human body, participating in such vital functions as blood
clotting, neuronal signaling, muscle response and heart
rhythm.
 Calcium deficiency cannot be identified using special
staining techniques.
 Pathologically, abnormal deposits of calcium can be
found associated with necrotic tissue areas in cases of
tuberculosis, atherosclerosis, kidney calcinosis,
sarcoidosis, hyperparathyroidism and kidney stones.
 Pigments and ….
Chemistry
 There are two well-known special staining techniques for
demonstrating calcium deposits:
 Von Kossa and alizarin red S.
 The Von Kossa calcium stain is an indirect method for
identifying calcium in tissue.
 The primary calcium salts in tissue are calcium phosphate
and calcium carbonate.
 In this technique, silver is substituted for calcium and is
then
reduced to metallic silver with sunlight.
 The stain of choice for identification of calcium is the
alizarin red S calcium stain.
Technical Considerations
 During the staining technique, the reaction must
be monitored microscopically to ensure that
diffusion artifact does not occur.
 Tissues preserved in non-acidic fixatives are the

best for demonstrating calcium deposits.

4.5 Microorganisms
 Microorg…
DEMONSTRATION OF MICROORGANISMS

Detection of infective microorganism can be done in

histologic examination though rarely sensitive.

Cause of difficulty in demonstrating microoraganisms:

Many infective microorganisms are too small to be seen in
light microscopy
Even many large organisms are not distinctive by HE.
  Some times the number of the microoraganisms are few
that even in special stain can be missed.
• In some necropsy specimen over growth of non important
organisms can obscure important pathogens.

• Despite these difficulties an experienced pathologist can

make a diagnosis of infection by correlating clinical and
morphololigc features.

• Detection methods
 Gram’s stain
 Ziehl-Neelson stain
 Silver stain (for spirochetes and fungi)
 Giemsa stain
 
Gram stain
 Bacteria appear on H and E as blue rods or cocci
regardless of gram reaction. Colonies appear as fuzzy
blue clusters.
  Structural differences affect the way these bacteria retain
dye complexes.
 With the Gram stain, all bacteria take up a crystal violet-
iodine complex.
 However, upon decolorization, Gram-positive bacteria
retain the dye complex, while Gram-negative bacteria do
not.
 Tissue gram stains are all basically the same as that used
in the microbiology lab except that neutral red is used
instead of safranin.
 Microorg…
AFB (acid fast bacilli) stain
 This stain uses carbol-fuchsin to stain the lipid walls of acid-
fast organisms such as M. tuberculosis.
 The most commonly used method is the Ziehl-Neelsen
method.
 A modification of this stain is known as the Fite stain and
has a weaker acid for supposedly more delicate M. leprae
bacilli.
 However, much of the lipid in mycobacteria is removed by
tissue processing, so this stain can, at times, be very
frustrating.
 The most sensitive stain for mycobacteria is the auramine
stain, which requires a fluorescence microscope for
viewing.
 Microorg…
 There are structures other than mycobacteria that
are acid fast.
 Included are
 Cryptosporidium
 Isospora
 hooklets of cysticerci.
• However, much of the lipid in mycobacteria is removed by tissue
processing, so at times, be very frustrating and causes to search
extensively for organsisms .
• The most sensitive stain for mycobacteria is the auramine stain
which requires a fluorescence microscope for viewing.
 
Silver Stain
• Fungi stain blue with H and E and red with PAS. The most
sensitive method for demonstrating them is Methenamine silver.
 
Methenamine Silver Stain
• The methenamine silver stain is useful to identify a variety of
pathogenic fungi, including Aspergillus fumigatus, Blastomyces
dermatitidis, Candida albicans, Coccidioides immitis,
Cryptococcus neoformans, Histoplasma capsulatum,
Pneumocystis carnii .
Chemistry
• Methenamine silver methods rely on the oxidation of
carbohydrates within the tissue/fungus to form aldehyde
groups.
• These groups will directly act to reduce silver ions from
the methenamine silver solution to visible metallic silver.

• Sodium thiosulfate is used to remove any unreduced silver.

Results
Fungi ---------------------------------Black
Background --------------------------Light green
 
Warthin-Starry Stain
 
• The Warthin-Starry stain is useful in identifying
Spirochetes and some other bacteria, such as H. pylori
 Chemistry
• The Warthin-Starry stain relies on the ability of certain
bacteria to bind silver ions from solution. Subsequent
addition of a reducing agent then converts this bound
silver to visible metallic silver.
• In the Warthin-Starry stain, the tissue is sensitized prior
to application of the silver complex.
• An aqueous silver nitrate solution combined with the
reducing agent, hydroquinone, is applied, and a silver
diamine complex is generated.
 Microorg…
Results
 Bacteria----------------------- Black
Background------------------- Yellow to light brown
 
Giemsa
A Giemsa stain may help demonstrate donovan bodies and
leishmania.
The giemsa stain can be helpful for identifying components in
a variety of tissues.
--belongs to a family of stains known as the Romanowsky
stains.
 
Another screening method for detecting H. pylori in tissue
sections is the Giemsa stain, which can also be used to
demonstrate the
 Gram-negative bacteria
 Rickettsia
 the protozoan
 Toxoplasma gondii.
Review questions
1.Discus Connective tissue staining
2 Elaborate the different methods of demonstration of
Protein, Nucleic acid and Amyloid
3. Discus Carbohydrates and Lipids stains
4. Describe demonstration of Pigments, Minerals and
Bone
5. Discus stining methods for tissue Microorganisms
References
1.Berhanu Seyum,Jemal YimamHaramaya
university ,Histopathology for Medical Laboratory
Technology students in Ethiopia ,Lecture note
series in collaboration with The Carter Center ,
2006 Addis Ababa.