LESSON 15: PRINCIPLES OF STAINING

STAINING
 The process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical
characteristics of the cells.
 To make the tissues and cells become more visible.
 To easily identify morphologic changes in tissues/cells.
 To establish presence or absence of a disease process.
PURPOSE OF STAINING: Differentiate and Identify proper structure of the cells/ tissues.

STAINING TISSUES CAN CLASSIFIED INTO THREE MAJOR GROUPS

1. HISTOLOGICAL STAINING
 the process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or
staining solution, producing coloration of the active tissue component
 Microanatomic stains, bacterial stains and specific tissue stains
 Used to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm

2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)

 process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic
localization of a specific tissue substance
 EXAMPLES: - Perl’s Prussian blue reaction for hemoglobin, and periodic acid schiff staining for
carbohydrates
 In enzyme histochemistry, the active reagent serves as a substance upon which the enzyme act, the final
opacity or coloration produced from the substrate rather than the tissue.

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, florescent labeled or
enzyme labeled antibodies.

TWO CATEGORIES OF STAINS

Biological stains or coloring substances are prepared from dyes which may generally be divided into two categories:

 NATURAL DYES- ex conchineal dyes, logwood dyes, and vegetables extracts

 SYNTHETIC DYES- ex aniline or coal tar dyes.
NATURAL DYES
 Obtained from plants and animals, previously utilized for dyeing of wool and cotton.
EXAMPLES:
A. Hematoxylin
B. Cochineal dyes and its derivatives
C. Orcein
D. Saffron

A. HEMATOXYLIN
 Derived from the core or heartwood of Mexican tree known as Hematoxylin Campechianum
 Most valuable staining reagent used by cytologist due its powerful nuclear and chromatin staining capacity.
 The active coloring agent is hematin, formed from oxidation of hematoxylin, a process known as “ripening”.
 This is accomplished by exposing the substance to air and sunlight (natural ripening) (THIS PROCESS IS
SLOW 3-4 MONTHS)
 Can be accelerated by adding strong oxidizing agent which converts hematoxylin to hematin (artificial
ripening)
 Hydrogen peroxide
 Mercuric oxide
  Potassium permanganate
 Sodium perborate/ sodium iodate

Ripened hematoxylin is seldom used alone due to its inherent low affinity of the tissue itself. Combined with:
 Alum
 Iron
 Chromium
 Copper salts
* Acts as mordants catalyzing or forming links between the hematin stain and the tissue
 Mordants are substances which combine with the tissue and staining solution, forming a “bridge” that allows
staining reaction to take place.

B. CONCHINEAL DYES (CARMINE)

 Old histologic dye extracted from the female cochineal bug (Coccus cacti), which is treated w/ alum to produce
the dye, carmine.
 It is widely used as a powerful chromatin and nuclear stain for fresh material and smear preparation.
 PICROCARMINE= PICRIC ACID + COCHINEAL DYES/CARMINE
 it is extensively used in neuropathological studies
 BEST CARMINE STAIN= ALUMINUM CHLORIDE + COCHINEAL DYES/CARMINE
 It is used for demonstration of glycogen

C. ORCEIN
 Vegetable dye extracted from certain lichens which are normally colorless, but which, when treated with
ammonia and exposed to alkali, produce blue or violet colors
 Used for staining elastic fibers

D. SAFFRON
SYNTHETHIC DYES
 AKA “Coal Tar Dyes”
 Derived from the hydrocarbon benzene and are collectively known as aniline dyes.
 Consist of : chromophore and auzochrome group attached to a hydrocarbon benzene ring.
 CHROMOPHORES are substances with definite atomic groupings and are capable of producing
visible colors
 AUXOCHROMES are substances which imparts to the compound the property of electrolytic
dissociation to retain the color of tissue.
IN SHORT:
CHROMOPHORES: “color bearer”/ coloring property
AUXOCHROME: “increasers”/ dyeing property

 Simple benzene compounds which contain such substances are known as chromogens.
 Before a chromogen can be properly called dye, it must have the property of retaining color in the tissue. This
property is acquired by adding auxochromes.
 A dye should composed of chromophore and auxochrome group attached to hydrocarbon benzene ring. The
coloring property is attributed to the chromophore, dyeing property to the auxochrome.

IN SHORT:
CHROMOGEN VS. DYE
CHROMOGEN- imparts color temporarily; made up of benzene and chromophore
DYE- imparts color to tissue almost permanently; made up of chromogen and auxochrome.

CLASSIFICATION OF DYE DEPENDING ON THE WHERE THE CHROMOPHORE IS FOUND

 ACID DYES
The active coloring substance is found in the acid component, and the inactive base (ex. Acid fuchsin is usually the
sodium salt of a sulfonate of rosaniline.)
Example: Picric acid

 BASIC DYES
The active coloring substance is found in a basic component that combines with acid radical.
Example: Methylene blue

 NEUTRAL DYES
 Formed by combining aqueous solutions of acid and basic dyes, capable of staining cytoplasm and nucleus
simultaneously and differentially

Examples: Romanowsky dyes used in hematology, giemsa stains and irishman’s stain for leuckocyte differentiation.

METHODS OF STAINING
I. ACCORDING TO THE PRESENCE OF MORDANT:

A. Direct staining
 The process of giving color to the sections by using aqueous or alcoholic dye solutions (methylene blue,
eosin)
 No mordant is used.

B. Indirect staining
 The process whereby the action of the dye is intensified by adding another agent or mordant which
serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.
 Uses mordant (by itself the dye may stain weakly)

MORDANT
 Serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.
 May be applied to the tissue before the stain, or it may be included as part of the staining technique, or it may
be added to the dye solution itself

Examples of mordants:
Potassium alum with hematoxylin in Erlich’s hematoxylin
Iron in Weigert’s hematoxylin.

ACCENTUATOR
 It is not essential to the chemical union of the tissue and the dye
 It does not participate in the staining reaction, but merely accelerates or hastens the speed of the staining
reaction by increasing the staining power and selectivity of the dye.
Examples of Accentuators:
Potassium Hydroxide in Loeffler’s methylene blue
Phenol in Carbon Thionine and Carbol Fuchsin

II. ACCORDING TO THE PRESENCE OF A DIFFERENTIATOR/ DECOLORIZER

A. Progressive staining
 The process whereby tissue elements are stained in a definite sequence, and the staining solution is
applied for specific periods of time until the desired intensity of coloring of the different tissue elements is
attained.
 No differentiator
(Once the dye is taken up by the tissue, it is not washed or decolorized . The differentiation or distinction of
tissue details relies solely on the selective affinity of dye for different cellular elements.)
  Less favored than regressive staining due to difficulty of producing sufficiently intense progressive
staining of cell structures without staining other parts, thereby resulting in diffused color and obscured
details

B. Regressive staining
 The 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

DIFFERENTIATION (DECOLORIZATION)
 Selective removal of excess stain from the tissue during regressive staining in order that a specific substance
may be stained distinctly from the surrounding tissues
 Done by washing the section on simple solution (water or alcohol), or by the use of acids and oxidizing agents
 In general, if primary stain used is a basic dye, differentiation is carried out by an acid solution, while alkaline
medium is used for differentiation after applying an acid dye
(Alcohol acts as a differentiator for both basic and acidic dyes, probably by simply dissolving out the excess
dye)

III. ACCORDING TO RESULTANT COLOR

A. Orthochromatic 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 dye which differentiate particular substances by staining them with a color that is
different from that of the stain itself (metachromasia)
 Tissue components combine with these dyes to form a different color from the surrounding tissue
 Employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules and amyloid.

Examples of Metachromatic Dyes

Are basic dyes belonging to the thizine and triphenylmethane groups
a. Methyl violet/ crystal violet
b. Cresyl blue
c. Safranin
d. Bismarck brown
e. Basic fuchsin
f. Methylene blue
g. Thionine
h. Toluidine blue
i. Azure A,B,C

IV. VITAL STAINING

 It is the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of
the dye particle.

A. Intravital Staining
 Done by injecting the dye into part of the animal body (either intravenous, intraperitoneal or subcutaneous),
producing specific coloration of certain cells, particularly those of the reticuloendothelial system
Common dye used:
a. Lithium
b. Carmine
c. India ink

B. Supravital Staining
 Used to stain living cells immediately after removal from the living body. Thin slices of tissues are placed in
small staining dishes enough staining solution is added to cover the tissue
Commonly used Dyes
a. Neutral red (probably the best vital dye)
b. Janus green (especially recommended for mitochondria)
c. Trypan blue (one gram of dye dissolve in 100 mL of sterile distilled water to be Used immediately,; it is
dangerous to allow the distilled water to stand for more than one hour, because it is likely to become toxic
to the cell.
d. Nile blue
e. Thionine
f. Toluidine blue
V. COUNTERSTAINING
The application of different color or stain to provide contrast and background to the staining of the structural
components to be demonstrated

A. Cytoplasmic Stains
Red Green
Yellow

Eosin Y Picric acid Light green SF

Eosin B Orange G Lissamine green

Phloxine B Rose bengal

B. Nuclear Stains
Red Blue

Neutral red Methylene blue

Safranin O Toluidine blue

Carmine Celestine blue

Hematoxylin

VI. METALLIC IMPREGNATION

A process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic
salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue
or bacteria
Characteristics:
 Structures demonstrated are opaquw and black
  The colouring matter is particulate

FACTORS INFLUENCING DYE BINDING

 pH of the solution
 Increase in temperature
 Increase concentration of dye molecules
 Presence of other salts

H AND E STAINING TECHNIQUE

 Routine H and E staining is the most common method utilized for microanatomical studies of tissues, using
the regressive staining which consists of over staining the nuclei, removal of superfluous and excessive color of
the tissue cinstituent by acid differentiation.

Hematoxylin
 A natural dye derived from the extraction of heartwood Mexican tree
 Most commonly used for routine histologic studies
 The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or
colored complexes, the color of which will depend on the salt used.

A. ALUMINUM HEMATOXYLIN SOLUTIONS

 Recommended for progressive staining of tissues
 Mordant: Potash alum
 Produce good nuclear stain

A.1 Erlich’s hematoxylin

 Suitable for tissues that have been subjected to acid decalcification, and is especially useful for tissues that
have been become acidic during prolonged storage in formalin.
 Ripening agent: sodium iodate
 Stabilizer: glycerine

A.2 Harris’ hematoxylin

 Widely used for routine nuclear staining, in exfoliative cytology, and for staining of sex chromosomes
 The usual staining time is 5-20 mins, depending on the batch and age of stain, the nature of tissue and the
degree of staining required
 Ripening agent: Mercuric oxide
 Stabilizer: 4%glacial acetic acid

A.3 Cole’s hematoxylin

 Ripening agent: sodium perbonate
 Used in sequence with Celestine Blue

A.4 Mayer’s hematoxylin

 Ripening agent: sodium iodate
 Regressive and progressive staining
 Cytoplasmic glycogen
A.5 Delafield’s hematoxylin
A.6 Gill’s hematoxylin

B. IRON HEMATOXYLIN
 Ferric salts ripen hematoxylin rapidly and are active oxidizing agents and mordant

B.1 Weigert’s Hematoxylin

 - ferric chloride; standard iron hematoxylin
 - used in demonstrating muscle fibers and connective tissues
 - combined w/ Van Gieson’s stain(GOOD FOR DEMONSTRATING COLLAGEN)
 MORDANT: Ferric Chloride
 Standard iron hematoxylinin the laboratory especially when demonstrating muscle fibers and connective tissue.

B.2 Heidenhains Hematoxylin

 MORDANT: ferric ammonium sulfate

 - for mitochondria, muscle striations, chromatin, and myelin

B.3 Tungsten hematoxylin

Mallory’s PTAH (phosphotungstic Acid Hematoxylin
- for staining muscle striations

B.4 Copper hematoxylin

- study for spermatogenesis

EOSIN
 Routinely used as counterstain after hematoxylin and before methylene blue
 Red acidic dye in three forms:
 Yellowish (Eosin Y) – most commonly used
 Bluish – deeper red color (eosin B, erythrosin B)
 Ethyl eosin – (eosin S, eosin alcohol soluble)

ROUTINE H & E STAINING

STEPS:
ROUTINE H and E STAINING FOR PARAFFIN EMEBEDED SECTION
FIXATION: most fixatives can be used except osmic acid solutions which inhibits hematoxylin.

PROCEDURE:
H and E staining Steps:
1. Xylol (2 changes)
2. Descending grade of alcohol
3. Water
4. Stain w/ Harris/Erlich’s/Delafield’s
5. Rinse slide in tap water
6. Acid alcohol
7. Ammonia water
8. Wash in tap water
9. Stain w. Eosin Y
10. Ascending grade of alcohol
11. Xylene
12. Mount and then label

RESULTS:
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
SMEAR STAINING
Steps:
1) Fix w/ 95% ETOH
2) Stain w/ Harri’s hematoxylin
3) Acid alcohol
4) Blueing step
5) Stain w/ OG-6
6) 70-95% ETOH (washing)
7) Stain EA 36 or 50
8) Dehydrate
9) Xylene
10) Mount and label

OTHER STAINS USED

1. Acid Fuchsin
Picric acid (Van Gieson stain) for demonstration of connective tissue.

2. Acridine orange
Permits discrimination between dead and living tissues

3. Acridine red 3B
Demonstrate deposits of calcium salts and possible sites of phosphates activities

4. Aniline blue
Used for counterstaining of epithelial sections

5. Basic fuschin
Plasma stain ultilized also for deep staining of acid fast organism, for mitochondria, for differentiation of smooth
muscles with the use of picric acid.

It is a main constituent of Fuelgen’s and schiff’s reagent for detection of aldehydes, of Van Gieson’s solution for
connective tissues, mucin and for elastic tissue staining

6. Bismarck Brown
Used as a contrast stain for gram’s technique, in acid fast and papanicolau method, and for staining diphtheria
organisms.

7. Carmine
Used as a chromatin stain for fresh materials in smear preparations.
It is combined with aluminun chloride to stain glycogen (best carmine solution)

8. Celestine blue
- Resistant to strong acid dyes, and is recommended for routine staining of fixed sections, giving a good nuclear
definition when used in conjugation with alum hematoxylin

9. Congo red
Best known as an indicator
Method of staining elastic tissues, amyloid and myelin

10. Crystal violet

Nuclear or chromatin stain used for staining amyloid in frozen sections and platelets in blood
11. Giemsa stain
Used for staining blood to differentiate leukocytes

12. Gold sublimate

-Used for metallic impregnation, made up of gold chloride and mercuric chloride

13. Iodine
 Oldest of all stain
 Used for microscopic study of starch granules
 It stain amyloid, cellulose, starch, carotenes and glycogen
 Widely used for the removal of mercuric fixative artefact pigments, and as a reagent to later crystal and methyl
violet so that they may be re-stained by certain bacteria and fungi
 Grams iodine – stains microorganisms
 Lugol’s iodine – used as a test for glycogen, amyloid and corpora amylacea

14. Janus green

Used for demonstrating mitochondria during intra-vital staining

15. Malachite green

Weakly basic dye used as a contrast stain for staining ascaris eggs and erythrocytes, and as a bacterial pore stain
Used as a decolorizer and as a counterstain

16. Methyl green

Stains chromatin green in the presence of an acid
Gives a false positive reaction with certain secretions such as mucin

17. Methylene blue

 Common basic nuclear stain employed with eosin to provide marked differentiation of various structures in the
tissue
 Usually contain some azures or methylene violet
 Valuable stain for plasma cells and may also be employed in cytological examination of fresh sputum for
malignant cells, as a bacterial organisms, for diagnosis of diphtheria and for vital staining of the nervous tissue

18. Methylene violet

 Metachromatic dye formed whenever methylene blue is heated in fixed alkali or alkalai carbonate, coloring
nuclei of leukocytes reddish-purple in the presence of methylene blue.

19. Neutral red

 - Basic dye recommended for observing cell granules and vacuoles of phagocytic cell.

20. Night blue

 Used as a substitute for carbol fuchsin in acid fast staining
21. Orcein
 Excellent stain for elastic fibers (Taenzer Unna Orcein Method)
 Recommended in dermatological studies due to its ability to demonstrate the finest and most delicate fibers in
the skin

22. Picric acid

Employed as a contrast stain to acid fuchsin, for the demonstration of connective tissue (Van Gieson), as a
cytoplasmic stain in contrast to basic dyes, as a counter stain to crystal violet, as a tissue fixative and as a
decalcifying agent.

23. Prussian blue

Used for microanantomical color contrast of specimens and for demonstration of the circulatory system by injection
(intravital stain)

24. Rhodamine blue

Used with osmic acid to fix and stain blood and grandular tissues

25. Silver nitrate

 Used in 10% aqueous solution to prepare various dilutions to be used in identification of spirochetes, reticulum
and other fiber stains

26. Toluidine blue

Nuclear stain for fixed tissues, used as substitute for thionine in fresh frozen tissue sections.
Recommended for staining of Nissl granules or chromophilic bodies.

27. Victoria blue

 Used for demonstration of neuroglia in frozen sections

28. OIL SOLUBLE DYES (LYSOCHROMES)

 Lysochromes (oil soluble dyes)
 Do not have auxochrome groups
 They give color to lipids simply because they are more soluble in lipid medium of the tissues, than in their
medium of 70% alcohol
Available in the form of:
 Sudan Black B
 Sudan III
 Sudan IV

A. Sudan Black
 Most sensitive of the oil soluble dyes
 Dye concentration, temperature and physical state of the fats are related to the ability of the fat to absorb dye
 Stains phospholipids and neutral fats
 Does not stain crystalline cholesterol, and free fatty acids tend to be soluble in ethanolic dye bath
B. Sudan IV (Scharlach R)
 Has no secondary amino group and it does not color phospholipids or the fine lipid droplets
 Recommended for staining triglycerides (neutral lipids), giving them a deep and intense red stain

C. Sudan III
 The first sudan dye to be introduced into histochemistry
 Fat soluble, good as fat stain for central nervous system, giving less deep and lighter orange stain compared to
the darker staining Sudan IV

CHIEF SOLVENTS USED FOR STAINS

1. Water
Should always be distilled unless otherwise stated
2. Alcohol
Ethyl alcohol may be used in various concentrations.
Methyl alcohol (usually absolute)
Blood stains (acetone free)
3. Aniline Water
10 mL, of aniline is added to every ½ to 1 liter of hot distilled water, shaken, cooled and filtered
4. Phenol
Is used in aqueous solution of 0.5 – 5%
PRECAUTIONS IN STAINING
 Stains on the skin should be avoided not only because thet are sign of poor technique but beacuase stains
are health hazards.
 Stains in the skin can be removed by topical application of 0.5% acid alcohol, followed by rinsing with
tap water.

RESTAINING OF OLD SECTIONS

Old, bleached or faded sections may be restained:
PROCEDURE:
1. The slide is usually immersed in xylene for 24 hours or gently heated until the mounting medium begins to
bubble.
2. Coverslip is removed by lifting it with dissecting needle.
3. The section is placed in xylene for 30 minutes to removed the remaining balsam then brought down to water
4. Placed it in the solution of Potassium Permanganate for 5-10 minute, rinsed with tap water and subsequently
immersed in 5% oxalic acid for 5 minutes or unil the section is decolorized.
5. After washing again in running tap water for another minutes, the section may then be restained with
appropriate staining technique.
BROKEN SLIDES
 Mounting a broken slide to another clean xylene moist slide wihth a drop of mounting media (clarite/permount)
may be sufficient for immediate examination.
 If an important slide is broken and replacement is not available, the section (if still intact) may be transferred
with another slide.
PROCEDURE:
1. The coverSlip may be removed by soaking in xylene, and placing the broken slide in incubator at 37C until all
mountant has been removed.
2. Cover the whole slide with mixture of 6 parts butylacetate and 1 part durofix and left in the incubator for 30
minutes until the mixture hardens into film.
3. Using a scalpel blade, the hardened film is cut around the section and the slide is placed is cold water until the
film and section float off.
4. The fil containing the section is mounted in a clean slide, placed in 37C incubator until dry, wahed gently with
butyl acetate , then washed with xylene and mounted in Clarite/ Permount.

SPECIAL STAINS
I. STAINS OF CARBOHYDRATES

 Carbohydrates are main sources of energy in the body, mobilized in the form of monosaccharides (glucose) and
stored in the form of polysaccharides, either in pure form (glycogen) or bound to other substances (mucin).

 GLYCOGEN- is made up of polysaccharides of glucose and is normally stored in the liver, heart and skeletal
muscle, but it may be abnormally present in certain diseases.

 MUCIN- is made up of hexosamines or mucus that is secreted by goblet cells, respiratory lining cells and
certain glands.

 BOTH mucin and glycogen can be Stained by PAS Technique.

1. Periodic Acid Schiff (PAS)

 It is a histochemical stain that will demonstrate carbohydrates and other substances.
 The staining procedure should not be done at temperature above 25C whihc markedly accelerates the reaction.
 Discard the solution if brown color appears.
 Most fixatives can be used except those that contain osmic acid, chromates and permanganates.
Result: PAS + red or magenta
 Nuclei- Blue
2. PAS with Diastase
 Method of choice for glycogen demonstration
 Result: Nuceli= blue black
 Glycogen: red

3. Best Carmine
 For glycogen demonstration
 Result: Nuclei: blue pr grayish blue
 Glycogen: bright red granules
 Mucin and Fibrin- weak red

4. Langhan’s Iodine
Oldest stain (obsolete)
Result: Glycogen- mahogany brown
Tissue constituents- yellow

5. Fresh Frozen Azure A for glycosaminoglycans

Result: glycosaminoglycans- red purple
Tissue background- blue

6. Metachromatic Toluidine Blue Staining

Results: Glycosaminoglycans- red-purple
Tissue background- blue

7. Alcian Blue Technique

Result: Acid mucin -blue
Nuclei- Red

8. Combined Alcian Blue-PAS Technique for acid and neutral mucin

Result:
Acid mucin- blue
Neutral mucin- magenta
Nuclei-pale blue

9. Mucicarmine Stain
Result:
Mucin-red
Nuclei- blue
Background- unstained

10. Hale’s Dialyzed Iron Technique

Result
Acid Mucin: dark blue
Nuceli- red

11. Fluorescent Acridine Orange

Result
Acid Mucopolysaccharides -black
Fungi- greenish red fluorescent
Background- reddish orange fluorescent

II. STAINS OF FATS

1. Sudan Black B
Most sensitive
Result:
Lipids: Blue-black
Nuclei- Red

2. Sudan III
First sudan introduced to histochemistry
For CNS tissues
Result:
Lipid- orange

3. Sudan Iv (Scharlach R)Lipids

Result:
(mainly TAG) - red
Nuclei- appear blue or black
4. Oil Red O in Dextrin
Results: Fats-brilliant red
Nuclei- blue

5. Osmic Acid
Result:
Fats-black
Nuclei- Yellow orange

III. STAINS OF PROTEINS, ENZYMES AND MUCLEIC ACID

1. Alkaline Fast Green
Results:
Histones and Protamines- Green

2. Gomori Calcium
Result:
Alkaline phosphatase activity - brwonish black
Nuclei- Green

3. Feulgen- for nuclear DNA

Result:
DNA- red purple
Cytoplasm- green

4. Methyl Green -Pyronin

For RNA and DNA
Result:
DNA : green or blue-green
RNA: rose red
Plasma cell cytoplasm: purple

IV. STAINS OF CONNECTIVE TISSUE

For Collagen:
1. Gomori’s Silver Impregnation Stain
Stain for reticulin
Result:
Reticulin fibers: black
2. Van Gieson’s Stain
For collagen
Result:
Collagen: pink, deep red
Muscle, RBC , cytoplasm and fibrin- yellow

3. Masson’s Trichrome Stain

For collagen
Result:
Collagen and mucus: blue
Muscle, RBC and keratin: red
Nuclei: blue-black

Other stains for Collagen: Mallory’s Aniline Blue, Azocarmine nad Kraijan’s Aniline Blue
For Amyloid:

1. Gram’s Iodine
2. Congo Red
3. Methyl Violet-Crystal Violet

For Elastic Fibers

1. Weigert’s Elastic Tissue Stain
Result:
Elastic fiber- bark blue or blue black

2. Verhoeff’s
Result: Elastic fibers: black
Nuclei: Gray to Black
Collagen: red
Cytoplasm: yellow

3. Taenzer-Unna Orcein
4. Gomori’s Aldehyde-Fuschin
5. Krajan’s

For Fibrin
1. Mallory’s PTAH

V. STAINS FOR MUSCLES AND BONES

1. Modified Gomori’s Trichrome Stain

Result:
Muscle fiber: red
Collagen: green
Nuclei: blue to black

2. Mallory’s PTAH
3. Heindenhain’s Iron Hematoxylin
4. Lissamine Fast Red-tartrazine method for muscles and bones.

For Bones: Schmori’s Picro-Thionin Method

VI. STAINS FOR BONE MARROW AND BLOOD ELEMENTS

1. Rapid Toluidine-Eosin Stain
2. Romanowsky
3. Wright’s Stain
4. Giemsa Stain
5. Peroxidase Reaction- for myeloid cells

VII. STAINS FOR CENTRAL NERVOUS SYSTEM

1. Bielschowsky’s Technique
For neurons, axons and nerve endings

2. Bosian’s Stain
For nerve fibers and nerve endings

3. Silver-Munger Technique
For neural tissues

4. Cresyl Fast Violet

For Nissl bodies

For Myelin Sheath:

1. Weigert-Pal Technique
2. Kluver and Barrera Luxol Fast Blue Stain
3. Weil’s Method

For Astrocytes:
1. Cajal’s Gold Sublimate
2. Modified PTAH
3. Modified Holzer’s Method

VIII. STAINS FOR TISSUE PIGMENTS AND DEPOSITS

For Hemosiderin:

1. Perl’s prussian blue

2. Gomori’s Prussian Blue
3. Turnbull’s Blue Reaction

For Hemoglobin:
1. Benzidine-Nitroprusside Stain

For Bile Pigments and Hematoidin

1. Modified Fouchet’s Technique
2. Gmelin’s Technique
3. Stain’s Iodine

For Lipofuchsin
1. Gomori’s Aldehyde Fuchsin Technique
2. Mallory’s Funschin Stain

For Melanin
1. Masson Fontana Technique- also for Argentaffin bodies
For Calcium
1. Von kossa’s Silver Nitrate Method

For Copper
1. Lindquist Modified Rhodamine Technique

IX. STAINS FOR MICROORGANISMS

For BACTERIA
1. Gram’s Method
2. Brown and Brenn
For Nocardia and Actinomyces

3. Ziehl Neelsen
For mycobacterium

4. Wade-Fite Technique
M. Leprae and Nocardia

5. Auramine-Rhodamine Technique
Mycobacteria

6. Toluidine Blue and Cresyl Violet Acetate

Helicobacter pylori

7. Dieterle Method
Legionella pneumophilia

8. Levaditi’s
Spirochetes

9. Modified Steiner and Steiner

Spirochetes

10. Warthin-Starry
Spirochetes

For FUNGI
1. Grocott Methamine Silver

For VIRUS
1. Lendrum’s Phloxine-Tartrazine Method- viral inclusion
2. Orcein Method- HBsAg

For PROTOZOA
1. Giemsa