Special Stains (PowerPoint)

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					Special stains

      Presented by:- Dr. Sourab Kumar
                    1st year PG student
Why are the special stains special ?
          Objectives of Special Staining

o To provide a definitive diagnosis.

o In a differential diagnosis of oral lesions in or
  around the oral cavity.

  e.g. To distinguish a tumor of salivary gland
  origin from an odontogenic tumor or a tumor
  arising from non-glandular epithelium
Broadly classifying the special stains under
following category…..
 Stains for collagen
 Stains for muscle
 Stains for elastic tissue
 Stains for reticulin fibres
 Stains for carbohydrates
 Stains for amyloid
 Stains for lipid
 Stains for pigments & minerals
 Stains for nerve tissue
 Stains for microorganisms
 Stains for decalcified bone
Demonstration of Collagen

o Massons trichrome stain
o Van Geisons stain

The term ‘trichrome stain’ is a general name for a number
  of techniques for selectively demonstration of muscle,
  collagen fibers, fibrin, and erythrocytes.

The general rule in trichrome staining is that the less
  porous tissues are colored by the smallest dye molecule;
  whenever a dye of large molecular size is able to
  penetrate, it will always do so at the expense of the
  smaller molecule.
• As a general rule, a three – dimensional, insoluble protein
  ‘network’ is formed (i.e.; protein component of a tissue +
  fixative in use) different proteins will form networks with
  different physical feature.

For example,
• Erythrocyte protein will produce a very dense network, with
  only small pores between the protein elements.
• Muscle cells will form a more open structure with large
• Collagen will show the least dense network and is quite
Another hypothesis is that the tissue is stained first
  with the acid dye, Biebrich Scarlet, which binds
  with the acidophilic tissue components. Then
  when treated with the phospho acids, the less
  permeable components retain the red color, while
  the red color is pulled out of the collagen. At the
  same time the collagen stained with the aniline
   The three dyes :

Aniline Blue (stains collagen and mucus to blue or blue green)

Beibrich Scarlet (stains cytoplasm, muscle and keratin to red)

Weigert's Iron Hematoxylin (stains nuclei blue to black).


- Used to differentiate between collagen and smooth muscle in tumors, and the
   increase of collagen in diseases such as cirrhosis.
- Routine stain for liver and kidney biopsies
Factors affecting trichrome staining
o Tissue permeability and dye-molecular size
o Heat (Increases the rate at which staining occurs)
   Acidic pH solution is used (pH- 1.5 to pH 3.0)


If the fixation in formaldehyde has been prolonged the
result will be even less satisfactory ,due to the saturation
of tissue group with formaldehyde

To improve the intensity of the staining reaction,
Zenker’s solution, formol-mercury,Bouin’s fixative or
picro-mercury alcohol are the most satisfactory fixative
for this stain.
Bouin’s solution              Biebrich Scarlet-Acid
Picric acid saturated aqueous   Fuchsin Solution
  solution             75cc   Biebrich Scarlet, aqueous 1%
Formaldehyde, 37- 40%                                 90 cc
                              Acid fuchsin, aqueous, 1%
  25cc                                               10 cc
Glacial acetic acid     5.0cc Glacial acetic acid    1.0 cc

Weigert’s Iron Hematoxylin
Solution A , Solution B and
  Working solution
Phosphomolybdic –            Aniline Blue Solution
  Phosphotungstic Acid       Aniline blue       2.5 gm
  Solution                   Acetic acid        2.0 cc
 Phosphomolybdic acid –      Distilled water 100 cc
Phosphotungstic acid -
                             Light Green Solution
                             Light green           5.0 cc
Distilled water -  200 cc
                             Distilled water      250 cc
                             Glacial acetic acid 2.0cc
1% Acetic Water Solution
                             Heat water, dissolve light
 Glacial acetic acid 1.0cc
                               green, cool, filter and
 Distilled water     100cc     add acid
Staining procedure
1.  Xylene
2.  Absolute alcohol
3.  95% alcohol
4.  Rinse in distilled water
5.  Mordant in Bouin’s fixative for 1 hour at 56°c
6.  Cool and wash in running water until yellow colour
7. Rinse in distilled water
8. Weigert’s iron haematoxylin solution for 10 min.
     wash in running water 10 min.
9. Rinse in distilled water
10. Biebrich Scarlet- acid fuchsin solution for 10 min.
11. Rinse in distilled water
12. Phosphomolybdic acid- Phosphotungstic acid solution
   for 10 – 15 min before aniline blue solution. Aqueous
   phosphotungstic acid 5 % for 15 min before light green
13. Aniline blue solution for 5 – 10 min or light green
   solution for 1 min.
14. Rinse in distilled water.
15. Acetic water 1 % for 3 to 5 min.
16. Alcohol, 95 %
17. Absolute alcohol - 3 changes
18. Xylene - 2 changes
19. Mount in DPX
Results                             Human skin

   Nuclei          – Blue- Black

  Cytoplasm, keratin,   – Red
muscle fibers,
intercellular fibers
 RBCs, Fibrin

  Collagen, mucous,     – Blue
 Cartilage, Amyloid,
adult or mature bone
        Van Gieson’s Stain (1889)

o Van Gieson’s mixture of picric acid and acid

o The simplest method for the differential staining
  of collagen.

 Picric acid is employed. It provides acidic pH and
 acts as a counterstain for muscle and cell
 cytoplasm. It forms with dyes a complex which
 has affinity for collagen.

o It is unable to stain young fibrils ,the deep red that is
  imparted to mature collagen
o The tendency for the red colour to fade ; whatever
  mounting medium is used

    To avoid this fading, Curtis (1905) suggested the
  use of ponceau S as a substitute for acid fuchsin but
  this dye, unfortunately, stains young collagen fibres
  less well than does acid fuchsin.
Any well-fixed tissue may be used.

Weigert’s Iron Hematoxylin
           Solution A
Hematoxylin           1.0 gm
Absolute alcohol      100.0 cc
           Solution B                Van Gieson’s Solution
29% ferric chloride    4.0 cc
Distilled water         95.0 cc      Acid Fuchsin, 1% aqueous
Conc. Hydrochloric acid 10.0 cc      solution      25 cc
          Working solution           Picric acid, saturated aqueous
                                        solution 97.5 cc
Equal parts of Solution A and
   Solution B
Staining Procedure
1.  Xylene
2.  Absolute alcohol
3.  95% alcohol
4.  Rinse in distilled water
5.  Stain in Weigert’s hematoxylin solution for 10
6. Wash in distilled water
7. Counterstain in van Gieson’s solution for 1-3
8. 95% alcohol
9. Absolute alcohol – 2 changes
10. Xylene – 2 changes
11. Mount in DPX

 Collagen     – deep red

 Muscle,           – yellow
cornified epithelium

 Nuclei        – blue to black

- Running water will remove van Gieson’s solution
- Nuclei stained with alum hematoxylin readily decolorized
  with picric acid in stain
     Demonstration of fibrin :
• Gram Weigert
• Phosphotungstic acid hematoxylin

• Early fibrin deposits may be stained by these dyes,
  although the phosphotungstic acid blocks the staining of
  muscle, collagen .and most of the connective tissue
Demonstration of muscle striations
- Haematoxylin and eosin and trichrome methods can
  demonstrate muscle striations.
- They may also be stained by using Heidenhain iron
  haematoxylin and Mallory’s phosphotungstic acid
  haematoxylin. Both these methods will give better
  definition of muscle striations than the trichromes.
                               Mallory's PTAH stain

skeletal muscle striations in tumor with rhabdomyoblasts.
Demonstration of elastic tissue fibres
- Numerous techniques have been evolved for the demonstration of
  elastic tissue fibres, although few are in current use.

-    Of these, the most popular are
 .   Verhoeff ,
 .   Orcein,
 .   Weigert’s resorcin-Fuchsin,
 .   Aldehyde fuchsin.
 Verhoff’s Elastic Stain

Verhoeff's Elastic stain is used to demonstrate
  pathologic conditions such as atrophy, breaks,
  thinning, loss etc. in elastic fibers.
• Principle

• The tissue is overstained with a soluble lake of hematoxylin-
  ferric chloride-iodine. Both ferric chloride and iodine serve
  as mordants, but they also have an oxidizing function that
  assists in converting hematoxylin to hematein.

•    The mechanism of dye binding is probably by formation of
    hydrogen bonds, but the exact chemical groups reacting
    with the hematoxylin have not been identified.

•    This method requires that the sections be overstained and
    then differentiated, so it is regressive.
• Differentiation is accomplished by using excess
  mordant, or ferric chloride, to break the tissue-
  mordant-dye complex. The dye will be attracted to
  the larger amount of mordant in the differentiating
  solution and will be removed from the tissue.

•    The elastic tissue has the strongest affinity for the
    iron-hematoxylin complex and will retain the dye
    longer than the other tissue elements. This allows
    other elements to be decolorized and the elastic
    fibers to remain stained.

• Sodium thiosulfate is used to remove excess iodine.

•    Van Gieson's solution is the most commonly used
    counterstain, but others may be used.
 Solution :
  Elastic tissue stain
       Hematoxylin                    - 1 gm
      Absolute alcohol               - 22 cc
      10 % Aqueous solution of FeCl 3 - 8 cc
      Iodine solution                - 8 cc
o Ferric Chloride solution
     Ferric Chloride - 2 gm
     Distilled Water- 100 cc
o Van Gieson’s Stain
     Acid Fuschin Aqueous solution 1%- 5cc
    Saturated Aqueous Picric acid solution – 100 cc
o Sodium Thiosulphate solution
     Sodium Thiosulphate – 5 gm
     Distilled water       - 100 cc
Staining Procedure
1.    Deparafinization and wash
2.    Verhoff’s Elastic tissue stain for 15 min
3.    Wash in Distilled water
4.    Differentiating 2% FeCl3 - only a few minutes
5.    Place in 5% sodium thiosulphate for 1 min
6.    Wash in tap water for 5 min
7.    Counterstain in Von Gieson’s stain for 1 min
8.    Absolute alcohol - two changes
9.    Xylene – two changes
10.   Mount in DPX
Results :

 Elastic Fibres – Bluish Black
                     to Black
 Nuclei          - Blue to Black
 Collagen        - Red
 Other tissue elements – Yellow
This section shows elastic cartilage stained with hematoxylin
and potassium iodine to reveal elastic fibers (arrow),
which are dark-stained linear structures embedded
in the cartilage matrix.
       Demonstration of reticulin fibres:
Reticulin fibres are demonstrated either by using dyes as means of
   coloring agent or by metal impregnation methods.

Dye techniques :1.Gordon and sweet’s method,
                2. Gomori’s method,

Reticulin fibre demonstration cannot be considered completely
 reliable by dye method

Silver impregnation is the best method because it provides good
   contrast enabling even the finest fibers to be resolved.
         Wilder’s Reticulin Stain

o Used to demonstrate reticular fibers in tissue
o Differential diagnosis of carcinomas, sarcomas, and
o Certain liver diseases where a change from normal
  reticular fiber pattern is seen.
Phosphomolybdic Acid solution
o Phosphomolybdic Acid – 10 mg
o Distilled water          - 100 cc
Uranium Nitrate solution
o Uranium nitrate – 1 gm
o Distilled water - 100 cc
Ammoniacal silver solution
o 10.2 % aqueous solution of silver nitrate- 5 cc
o 28 % ammonia water – drop by drop
o 3.1 % sodium hydroxide – 5 cc
Make the solution to 50cc with distilled water.
Reducing solutions
o Distilled water - 50 cc
o Neutral formaldehyde, 40% - 0.5 cc
o Uranium nitrate 1% aqueous solution – 1.5 cc
Gold Chloride solution
o 1% Gold Chloride solution -
o Distilled water – 40 cc
Sodium Thiosulphate solution
o Sodium Thiosulphate – 5 gm
o Distilled water - 100 cc
Nuclear fast red stain
o Nuclear fast red - 0.1 gm
o 5 % solution of aluminium sulphate – 100 cc
Staining Procedure

 1. Xylene, Absolute alcohol, 95% alcohol, Distilled
 2. Phosphomolybdic acid for 1 min (oxidizer)
 3. Rinse well in running water
 4. Dip in 1% aqeuous uranium nitrate for 5 seconds
  or less
 5. Wash in Distilled water for 10 to 20 min
 6. Place in ammoniacal silver solution for 1 min
 7. Dip quickly in 95% alcohol and then immediately
  into reducing solution for 1min
8. Rinse well in distilled water
9. Tone in Gold chloride solution for 1 min
10. Rinse in distilled water
11. Place in 5% Sodium Thiosulphate for 1 to 5 min
12. Wash well in tap water
13.Counter stain with Alum Hematoxylin and Eosin
   or Nuclei fast red. Rinse with distilled water
14.95% alcohol
15.Absolute alcohol - 2 changes
16.Xylene - 2 changes
17.Mount in DPX
o Reticulin fiber – Black
o Collagen – Rose color
o Other tissue elements –
  Depending on counter
  stain used
• Buccal mucosa. Reticulin stain delineates the
  cell membrane of nonkeratinized squamous
  epithelial cells and the delicate basement
  membrane. Papillae of the lamina propria
  contain blood vessels.
Stains for carbohydrates ?

Mechanism of PAS technique ?
Differentiation of the mucin by different stains ?

Clinical application of PAS stain?

o Periodic Acid Schiff Stain
o Alcian Blue
o Mucicarmine Stain – Southgate’s

• McManus, 1946)

• Glycogen is a simple intracytoplasmic polysaccharide found
  in greatest amount in liver, cardiac and skeletal muscle, with
  significant quantities also present in hair follicles,
  endometrial glands, vaginal and cervical epithelium, umbilical
  cord, neutrophil leucocytes and megakaryocytes.

• Tissue containing vicinal glycol groups or their amino or
  alkylamino derivatives are oxidized by periodic acid to form
  dialdehydes, which combine with Schiff's reagent to form an
  insoluble magenta compound.
• The chromphoric groups of basic fuchsin in Schiff's reagent
  are broken by sulphuration to form a colorless solution. In
  the presence of free aldehyde groups an insoluble colored
  compound similar to the original dye is formed.
PAS Technique:

- Periodic Acid Solution
            Periodic acid   - 1 gm
           Distilled water - 200 ml

- Schiff Reagent
                   Dissolve 1gm of basic fuchsin in 200 ml of
boiling distilled water. Allow the solution to cool 50 °c, and add
2-gm potassium metabisulphite with mixing. Allow to cool to
room temperature then add 2 ml conc. Hydrochloric acid, mix,
add 2 gm activated charcoal and leave overnight in the dark at
room temperature.
    Filter, the solution should be either clear or pale yellow
color. Store in a dark container at 4°c.
  Standard paraffin section fixed in 10% neutral buffered

  1. Deparaffinize and hydrate to distilled water.
  2. Oxidise with periodic acid for 5 minutes.
  3. Rinse in distilled water.
  4. Treat with Schiff's reagent for 5 minutes.
  5. Wash in running water for 10 minutes; this intensifies
  the colour reaction.
  6. Stain the nuclei with Mayer's haematoxylin for 1 minute
  (no need to blue).
  7. Wash in water.
  8. Dehydrate, clear and mount.

PAS positive substances
- magenta

Nuclei - blue

  Glycogen is digested with certain forms of amylase.
  Commercially available diastase, which á amylase or
  salivary amylase from saliva can be used to digest
  glycogen in tissue sections.

Purpose :
 - Glycogen is present in mucosa, skin, liver, parathyroid
   gland and skeletal & cardiac muscle.
  - The PAS stain is used for demonstration of basement
   membrane, mucosubstances secreted by the epithelia of
   various organs, fungi, etc.
    - Amylase Type VI-B from porcine pancreas (Sigma
    A 3176)- 0.25g (stored in fridge)
    - Distilled water 10mL
If larger quantity required, use 1g Amylase in 40mL
    distilled water. Prepare fresh just before use. Warm
    water for 5 sec in microwave oven then add amylase.

  (B) PAS TECHNIQUE for PAS reagents

  Place waste amylase into container and place into
  yellow biohazard bag for disposal by incineration..

• 1. Deparaffinise two positive controls and two test sections and
  hydrate to distilled water.
  2. Treat one control and one test section with Amylase for 25
  minutes to digest Glycogen.
  3. Wash in running water for 20 minutes.
  4. Proceed with P.A.S. technique as follows.
  5. Oxidise with periodic acid for 5 minutes.
  6. Rinse in distilled water.
  7. Treat with Schiff's reagent for 5 minutes.
  8. Wash in running water for 10 minutes, this intensifies the
  color reaction.
  9. Stain the nuclei with Mayer's haematoxylin 1 minute (no need
  to blue).
  10. Wash in water.
  11. Dehydrate, clear and mount.
  Presence of glycogen will be evidenced by loss of
  staining after enzyme treatment when compared to
  the untreated sections.

  PAS with Diastase                       PAS Stain
Glycogen in Ewing Sarcoma

o The most important PAS positive carbohydrates in
  tissues are polysaccharides (glycogen),Neutral
  mucopolysaccharides, mucoproteins, glyco proteins, and

o Acid mucopolyssacharides are only weakly positive or

o The PAS reaction can be used to demonstrate many
  other normal and pathological tissue constituents, the
  most important of which are amyloid, basement
  membrane, cartilage, cerebrosides, epithelial mucins,
  fungi, hyaline membrane of neonatal lung, lipochrome
  pigments, mucoid cells, mucoid granules, zymogen
  granules, starch, thyroid colloid etc.
The types of mucopolysaccharides are as follows:

Neutral - These can be found in glands of the GI tract and in prostate.
  They stain with PAS but not with Alcian blue, colloidal iron,
  mucicarmine, or metachromatic dyes.

Acid (simple, or non-sulfated) - Are the typical mucins of epithelial cells
   containing sialic acid. They stain with PAS, Alcin blue at pH 2.5,
   colloidal iron, and metachromatic dyes. They resist hyaluronidase

Acid (simple, mesenchymal) - These contain hyaluronic acid and are
   found in tissue stroma. They do not stain with PAS, but do stain with
   Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They
   digest with hyaluronic acid. They can be found in sarcomas.
Acid (complex, or sulfated, epithelial) - These are found
  in adenocarcinomas. PAS is usually positive. Alcian
  blue is positive at pH 1, and colloidal iron,
  mucicarmine, and metachromatic stains are also
  positive. They resist digestion with hyaluronidase.

Acid (complex, connective tissue) - Found in tissue
  stroma, cartilage, and bone and include substances
  such as chondroitin sulfate or keratan sulfate. They
  are PAS negative but do stain selectively with Alcian
  blue at pH 0.5.
PAS stain for fungi

o The fungal cell wall contains polysaccharides that are
  oxidized by the periodic acid to aldehydes.
o The aldehydes react with Schiff's reagent to stain the
  fungi rose pink.
                       Candida Hyphae in chronic hyperplastic Candidosis

materials can
simply be recognized
by their shape
(morphology) ,
eg.Fungal hyphae
Clinical Application of PAS Stain

o Salivary Gland Tumors

o Odontogenic Tumors
        Adenomatoid Odontogenic Tumor
        Calcifying Epithelial Odontogenic Cyst

o Soft tissue tumors
         Alveolar Soft-Part Sarcoma (Diastase resistant

o Fungal Infection

Alcian blue is a group of polyvalent basic dyes
 that are water soluble. The blue color is due
 to the presence of copper in the molecule.

Acid mucin - strongly sulphated connective
  tissue mucins react at low pH values with
  suitable cationic dyes and are usually PAS-

• Alcian blue is a copper phthalcyanin dye and
  contains positively charged groups capable of salt
  linkage with certain polyanions. These polyanions
  consist of the sulphate and carbocyl radicals of the
  acid mucins - the phosphate radicals of the nucleic
  acids do not react. Consequently, only the acid
  mucins are stained. By varying the pH of the
  solution of Alcian blue more information can be
  gained concerning the types of acid mucin present.

o    Alcian blue stains acid mucosubstances and acidic
o     Excessive amounts of non-sulfated acidic
  mucosubstances are seen in mesotheliomas, certain
  amounts occur normally in blood vessel walls but
  increase in early lesions of atherosclerosis.
 Standard paraffin section fixed in neutral buffered formalin

REAGENTS:                               • Nuclear Fast Red
• 3% Glacial Acetic Acid                   (Kernechtrot):
   Acetic acid          -3.0 ml         Aluminum sulfate 25.0 gm
  Distilled water      -100.0 ml        Distilled water 500.0 ml
• Alcian Blue Solution:
                                        Nuclear fast red 0.5 gm
  3% glacial acetic acid-
  100.0ml                               Dissolve the aluminum
  Alcian blue 8GX         -1.0 gm       sulfate in the water. Add the
Mix, adjust pH to 2.5, using            nuclear fast red, dissolve
  acetic acid. Filter, add a            with aid of heat. Filter,
  crystal of thymol, label with
  initial and date.                     add a crystal of thymol

1.   Hydrate slides to distilled water.
2.   3% acetic acid, 3 minutes.
3.   Alcian blue solution, 30 seconds.
4.   Wash in running water for 2 minutes, rinse in distilled.
5.   Nuclear-fast red, 5 minutes, wash in tap water.
6.   Dehydrate, clear, and coverslip.
Acid mucins
/mucosubstances     - blue
(using Nuclear fast red)
             -reddish pink
Background - Very pale red
or colourless

                             Submandibular salivary gland mucous cell showing
                             sulfomucin Brown black, sialomucin blue, -Iron
                             alcian blue technique
Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide stain purple
   Mixed minor salivary gland of the uvula. Acid
    mucopolysaccharides in mucous cells are stained turquoise
    (Alcian blue at pH 2.6).
The goblet cells of the gastrointestinal tract are filled with
abundant acid mucin and stain pale blue with this Alcian blue stain.


Mucicarmine has large molecular size which allows the dye to
  penetrate and bind to acidic substrates of low density, i.e.
  mucins. Neutral mucins and some strongly acidic sulphated
  mucins do not show appreciable staining.

o    To stain mucin which is a secretion produced by a variety
    of epithelial cells and connective tissue cells.

o Excess mucin is secreted by epithelial cells in certain
  inflammations and in certain intestinal carcinomas.

o    The mucicarmine technique is also useful in determining
    the site of a primary tumor. Finding mucin positive tumor
    cells in an area that does not contain mucin producing cells
    would indicate the tumor did not arise from that area.

o It is also useful in staining encapsulated fungi;
  Standard paraffin section fixed in 10% neutral buffered

o Southgate's MucicarmineSolution:
    Carmine, alum lake         -1.0 gm
    Aluminum hydroxide       -1.0 gm
    50% alcohol                -100.0 ml
    Aluminum chloride, anhydrous- 0.5 gm
o Mayer's Hematoxylin:
o Metanil Yellow Solution:
      Metanil yellow                 -0.25 gm
      Distilled water                -100.0 ml
      Glacial acetic acid            -0.25 ml

1. Deparaffinize and hydrate to distilled water.
2. Mayer's hematoxylin for 10 minutes.
3. Wash in running tap water, 5 minutes.
4. Mucicarmine solution, 45 seconds.
5. Rinse quickly in distilled water.
6. Metanil yellow, 30 seconds to 1 minute.
7. Dehydrate quickly in three changes of absolute
     alcohol, clear and coverslip
Mucin - deep rose
Nuclei - black
Other tissue elements- yellow

                                Colonic Mucosa showing sialomsucin stain   --
                                     - magenta
Mucoepidermoid carcinoma, mucin positive
Demonstration of amyloid
o   Congo red
o   Thioflavin T
o   Crystal Violet
Congo-Red - Putchler’s Modification –

Principle :
      Amyloid is homogeneous and eosinophilic, the deposits are
   extracellular and may become sufficiently large enough to
   cause damage to surrounding tissues. When stained with the
   congo red stain the amyloid, with aid of polarizing lenses, will
   birefringe an apple green color, under the microscope.

Purpose :
    To demonstrate amyloid deposits in tissue sections
                             1% Sodium Hydroxide
Mayer's Hematoxylin :         Sodium hydroxide 1.0 gm
  Hematoxylin - 1 gm          Distilled water 100.0 ml
 Distilled water- 1000 ml    Sodium Chloride,
 Sodium iodate -0.2 gm       Stock Solution, Saturated:
 Ammonium or Potassium          Sodium chloride 30.0 gm
  aluminum sulfate - 50 gm
                                Distilled water 200.0 ml
 Citric acid – 1 gm
                                Mix well, then add:
 Chloral hydrate – 50 gm
                                100% alcohol 800.0 ml
Working Solution:
o Stock sodium chloride 50.0 ml
o 1% sodium hydroxide 0.5 ml

Congo Red Stock Solution,Saturated:
o Congo red stain 1.0 gm
o Stock sodium chloride 500.0 ml

Working Solution:
o Stock Congo red solution 50.0 ml
o 1% sodium hydroxide 0.5 ml

1. Deparaffinize and hydrate to water.
 2.Mayer's Hematoxylin for 10 minutes.
 3.Wash in tap water until blue
 4.Working sodium chloride solution, allow slides to sit in
    solution for 2-5minutes.
5. Place directly into Working Congo red solution,
   Allow slides to sit in solution for 2-5 minutes.
6. Dehydrate rapidly in absolute alcohol, 10 dips, 3 changes.
7. Clear in xylene & mount.

Amyloid -red to pink

Nuclei -blue
   Puchtler and Sweat
   Congo Red Method

Polarised Light showing
“apple green”birefringence
of amyloid deposits
Positive red staining is present around the large central artery and
a smaller vessel to its upper right. The right panel shows the green
birefringence that is diagnostic of amyloid when the Congo red
stain is viewed with polarized light.
All amyloids have a fibrillar ultrastructure that gives this reaction.
Clinical Application

   Calcifying Epithelial Odontogenic Tumors:
      Extracellular amyloid of epithelial origin present in tumor.
  Keratin protein forms an important component of the amyloid
  in this tumor.
o Sections must be examined with a fluorescent
  microscope revealing a yellow to yellow green
In epidermis a cornoid lamella
without underlying granular
 layer and hydropic degeneration
of basal cell layer. In upper dermis
there are a mononuclear
inflammatory infiltrate, and a
 deposit of eosinophilic amorphous
 material (H&E, × 100).

Dermal amyloid deposits under
polarizing microscopy
(Thioflavin-T stain, × 200).
                 Crystal violet
    Metachromatically results in a red – purple colour
    of amyloid

APPLICATION: Amyloidosis (pink staining in a blue
Stains for
 of lipid and
     Stains for Lipid
- Oil Red O
- Sudan Black B

     Staining with oil-soluble dyes is based on the greater
     solubility of the dye in the lipid substances than in
     the usual hydroalcoholic dye solvents.

   To demonstrate fat or lipids in fresh tissue sections.

o Fat occurring in an abnormal place, such as fatty emboli
  that may develop after either a bone fracture or an injury
  that crushes a fatty body area.

o Tumors arising from fat cells (liposarcomas) can be
     differentiated from other types of tumors.
o 85% Propylene Glycol:
      Propylene glycol       -85.0 ml
      Distilled water         -15.0 ml
o Hematoxylin:
  Glycerin Jelly
o Oil Red O Solution:
       Oil red O               -0.7 gm
       Propylene glycol      -100.0 ml

o Dissolve oil red O in propylene glycol, slowly, while stirring.
  Heat to 100°C, but not over 110°C, for a few minutes,
  stirring constantly.
o Filter through Whatman #2 filter paper. Cool, and filter again
  through a frittered glass filter of medium porosity with
1. Fix slides in 10% formalin if fresh.
2. Wash well in tap, rinse in distilled, drain off excess water.
3. Propylene glycol, two changes, 5 minutes each.
4. Oil red O, 7 minutes, agitate.
5. 85% Propylene glycol, 3 minutes.
6. Rinse in distilled water.
7. Hematoxylin, 1 minute.
8. Wash in water.
9. Bluing solution, 20 dips, or running tap water.
10. Wash in tap water, rinse in distilled.
11. Mount with aqueous mounting media, Glycerin Jelly.
sebaceous gland stained
   with oil red O stain

    Cytoplasm – red

     Nuclei - blue
Oil red O stain of fat emboli in lung.
Lipid in skin
           FAT - SUDAN BLACK B -

 Sudan Black is slightly basic dye and will combine with acidic
  groups in compound lipids, thus staining phospholipids also.

     For the demonstration of fat.

o    85% Propylene Glycol:
         Propylene glycol    -85.0 ml
         Distilled water      -15.0 ml

o    Sudan Black B/Propylene:
        Sudan Black B     -0.7 gm
        Propylene glycol -100.0 ml

1. Fix slides in 10% formalin if fresh.
2. Wash well in tap water, rinse in distilled, drain off excess
3. Propylene glycol, two changes, 5 minutes each.
4. Sudan Black, 7 minutes, agitate.
5. 85% Propylene glycol, 3 minutes.
6 Rinse in distilled water.
7. Nuclear Fast Red, 3 minutes.
8. Wash in water.
9. Wash in tap water, rinse in distilled.
10. Mount with aqueous mounting media, Glycerin Jelly.

  Fat blue -black
  Nuclei -red
 Masson Fontana Method
 DOPA method
 Perl’s Prussian Blue Method
 Von Kossa stain for Calcium

• Melanin is insoluble in organic solvents but soluble in
  1M sodium hydrooxide.
• It is slowly bleached by strong oxidising agents.
• The solutions of ammoniacal silver nitrate are reduced
  by melanin to black metallic silver this is the basis of
  Massons Fontana method for demonstrating melanin.

  - To identify argentaffin granules and melanin.
Melanin is a nonlipid, non hematogenous pigment.It
  is a brown-black pigment present normally in the
  hair, skin, retina, iris and certain parts of the CNS.
Argentaffin granules are found in carcinoid tumors.
o Ammoniacal Silver              o Ammoniacal Silver
o Stock Solution:                o Working Solution:
  10% Silver nitrate 25.0 ml       Ammoniacal silver
  Add ammonium hydroxide            stock solution 12.5 ml
  drop by drop, until solution
  precipitates and clears           Distilled water 37.5 ml
  again.                         o 10% Silver Nitrate:
  10% silver nitrate 1.0 ml
                                   Silver nitrate 20.0 gm
o 0.1% Gold Chloride:
  1% Gold Chloride, Stock          Distilled water 200.0
  5.0 ml                           ml
o Nuclear-Fast Red:

1. Deparaffinize and hydrate to distilled water.
2. Working Silver Solution, 2 minutes.
3. Rinse in distilled water.
4. 0.1% Gold chloride, 10 minutes.
5. Rinse in distilled water.
6. Wash in tap water, rinse in distilled water.
7. Nuclear-fast red, 5 minutes.
8. Wash in tap water.
9. Dehydrate, clear and coverslip.

argentaffin cells -black

Nuclei         -red

                           Melanin pigment of skin showing black color
Melanin pigment in cells of malignant
 melanoma, Fontana-Masson stain.
                DOPA METHOD
  Principle :
The enzyme tyrosinase is located within some cells
  producing melanin will oxidize DOPA to form an
  insoluble brown – black pigment.

Purpose :
 Cells that are capable of producing melanin can be
 demonstrated by DOPA (dihydroxy- phenylalanine)
o pH buffer 7.4
    Sodium Cacodylate – 42.8 gm
    Hydrochloric acid – 9.6 mmol
    Distilled water – 1000 ml
o Primary fixative
    10% formalin in pH 7.4 buffer
    Sucrose   - 0.44 M
o Incubating medium
          0.1% DOPA in the pH 7.4 buffer
Procedure :
1.   Place section in distilled water for few seconds
2.   Place section in DOPA solution for 30 min at 37° C
3.   Replace with fresh solution and then examine
     microscopically every 30 min ( Color of solution
     changes from reddish to light brown)
4.   Wash section well in several changes of distilled water
5.   Stain in Mayer’s Hematoxylin for 2 min
6.   Wash well to “blue” the section, dehydrate, clear and
     mount in synthetic resin
DOPA oxidase – Brown
Nuclei       - blue
 Tissue sections are treated with silver nitrate solution,
 the calcium is reduced by the strong light and replaced
 with silver deposits, visualized as metallic silver.

 Abnormal deposits of calcium may be found in any area
 of the body. With the H&E stain, calcium appear deep
o 5% Silver Nitrate Solution:
    Silver nitrate 25.0 gm
    Distilled water 500.0 ml
o Nuclear Fast Red:

1. Deparaffinize and hydrate to distilled water.
2. 5% Silver solution, place in bright sunlight, or in front of a
   60-wattlamp, place foil (or mirror) behind the jar to reflect
   the light. Leave for 1 hour or until calcium turns black.
3. Rinse in distilled water, 3 changes.
4. Wash in tap water, rinse in distilled water.
5. Nuclear-fast Red, 5 minutes.
6. Wash in water,
7. Dehydrate, clear, and coverslip.
Calcium salts -black
Nuclei         -red
Cytoplasm      -pink

                       Coronary artery showing calcified atheromatous plaque


The reaction occurs with the treatment of sections in acid
solutions of ferrocyanides. Any ferric ion in the tissue
combines with the ferrocyanide and results in the formation
of a bright blue pigment called 'Prussian blue" or ferric

o To demonstrate ferric iron in tissue sections.
o Small amounts of iron are found normally in spleen
  and bone marrow.
o Excessive amount are present in hemochromatosis,
  with deposits found in the liver and pancreas,
  hemosiderosis, with deposits in the liver, spleen, and
  lymph nodes.
o 5% Potassium Ferrocyanide:
      Potassium ferrocyanide     - 25.0 gm
       Distilled water           - 500.0 ml
o Nuclear-fast Red:
o 5% Hydrochloric Acid:
      Conc. Hydrochloric acid,      - 25.0 ml
       Distilled water             - 475.0 ml
o Working Solution:
       5% potassium ferrocyanide -25.0 ml
       5% hydrochloric acid          -25.0 ml.

1. Deparaffinize and hydrate to distilled water.
2. Working solution. Allow slides to stand in solution for
   5 minutes.
3. Rinse in distilled water.
4. Nuclear-fast red, 5 minutes.
5. Wash in tap water.
6. Dehydrate, clear, and coverslip.

Iron (hemosiderin) -blue

Nuclei            -red

Background      - pink

                           Hemosiderin, liver, iron stain.
   Demonstration of Nerve tissue
o Bielschowsky’s Silver Stain
o Golgi cox potassium dichromate- mercuric chloride
o Holmes method for axons
o Weils haematoxylin method for myelin
o Luxol fast Blue method for myelin
Bielschowsky’s Silver Stain for
 Neurofibrils, Dendrites and Axons

Principle: The nerve fibers are sensitized with a silver solution.
   The sections are treated with ammoniacal silver, and then
   reduced to a visible metallic silver.
o Silver A
         20% silver nitrate
o Reducer A ( make fresh)
        Pyrogallol – 100 mg
        Formalin - 10 ml
        80% alcohol – 1200 ml
o Reducer B (make fresh)
          Formaldehyde – 5 ml
          Distilled water - 95 ml
o Silver B (make fresh)
          20% Silver nitrate - 5ml
          40% NaOH            - 200 µl
           Distilled water - 20 ml
          0.88 Ammonia         - 500 µl
o 5% Sodium thiosulphate
o 0.2% Gold chloride

o Section:
Paraffin sections at 10 um thick.
Method :
1.   Take section to distilled water
2.   Cover section with Silver A for 1 -2 hrs in 37° C in
     a moist chamber
3.   Rinse twice in distilled water and wash in a pot of
     reducer A for 3 - 5 min. Agitate regularly. The
     section should turn yellow
4.   Wash in 3 changes of distilled water
5.   Place in Silver B for 30 sec(Avoid prolonged
     treatment as it cause precipitation)
6.   Wash section in a pot of reducer for 2 – 5 min
7. Wash in distilled water – 3 changes
8. Wash section in a pot of reducer B until the
    neurons are dark brown
9. Wash in distilled water and tone in 0.2% Gold
    chloride for 2 – 3 min
10. Wash in distilled water
11. Fix in 5% Sodium thiosulphate for 5 min
12. Wash in tap water
13. Dehydrate, clear and mount in DPX
Result: Neurofibrils, dendrites and axon – black
     Staining for Microorganism
-   Gram staining
-   Ziehl- Neelsen (ZN) stain
-   Warthin-Starry for spirochetes
-   Gomori Methenamine (hexamine) silver for fungi
-   Giemsa stain for parasite
       Gram method for Bacteria

Gram staining differentiate bacteria into 2 classes depending
  on their cell wall structure and composition
 Gram positive
 Gram negative,
Crystal violet is first applied to the section as a primary stain,
  followed by iodine mordant forming a dye lake. The
  section are then decolorized using alcohol / acetone
Gram positive cell do not decolorize at this step and gram
  negative bacteria will not retain crystal violet
  Crystal Violet stains the nucleic acids of the bacteria
  (and background tissue) and after treatment with iodine,
  the sections are differentiated in acetone and
  counterstained with basic fuchsin. The tissue
  background and Gram-negative bacteria lose their blue
  staining and are subsequently stained with basic fuchsin.
  Gram-positive bacteria retain the crystal violet/iodine
  blue staining.ple:-
A .Gram method for bacteria in smear
Procedure :
1. Fix dry film by passing it 3 times through a flame
2. Stain for 15 sec in 1% crystal violet or methyl violet
3. Flood for 30 sec with Lugol’s iodine. Pour off excess
4. Flood with acetone for 2 – 5 sec, wash with water
5. Counter stain for 20 sec with dilute carbol fuchsin, or
    freshly filtered neutral red 1-2 min.
6. Wash with water and carefully blot section until it is dry

Gram positive organism
- blue-black

Gram negative organism - red

                               gram stain of the abscess shows thin, gram
                               positive rods in chains.
B .Gram method for paraffin sections

Crystal violet solution
     0.5% crystal violet in 25% alcohol
Gram’s & Lugol’s iodine
     Iodine         - 1 gm
     Potassium iodide - 2gm
     Distilled water - 10 ml
1% aqueous neutral red.
1.   Deparaffinize & rehydrate through graded alcohols to
     distilled water.
2.   Stain with crystal violet solution, 2 min.
3.   Rinse in tap water.
4.   Iodine solution, 2 min.
5.   Rinse in tap water, & flood with acetone, 1-2 sec.
6.   Wash in tap water.
7.   Counter stain in neutral red, 3 min.
8.   Blot, dehydrate rapidly, clear & mount.

Gram-positive organisms, fibrin, some fungi, keratohyalin,
   and keratin                - blue
Gram – negative organisms -red.
                Gram Positive

Gram Negative
Ziehl-Neelsen (ZN) Stain for Mycobacterium

 Mycobacteria are difficult to demonstrate by the Gram
 technique because they possess a capsule containing a long
 chain fatty acids that make them hydrophobic.
 Phenolic acid or heat may be used to reduce the surface
 tension and increase the porosity.
  Mycobacteria tuberculosis (tubercle bacilli) have a lipid-rich cell
  wall which is capable of taking up strong phenol dye solutions
  (eg. carbol fuchsin solution) such that the dye is retained upon
  subsequent differentiation in acid or alcohol ie. they are said to
  be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).
o Carbol – fuchsin
   1 g basic fuchsin dissolved in 10 ml of absolute
   alcohol; add 100 ml of 5% aqueous phenol. Mix
    well and filter before use.
o Acidified methylene blue
   0.25% methylene blue in 1 % acetic alcohol.
1.   Deparaffinize and rehydrate through graded alcohols to
     distilled water.
2.   Flood sections with freshly filtered carbol-fuchsin and
     heat to steaming with intermittent flaming, 15 minutes,
     or stain in Coplin jar at 56°-60°C, 30 min.
3.   Wash well in tap water.
4.    Differentiate in 1 % acid alcohol, 10 min.
5.    Wash well in tap water.
6.    Counterstain in methylene blue solution, 30 seconds.
7.    Blot and differentiate by alternate dehydration and
     rehydration until the background is a delicate pale blue.
8.   Dehydrate, clear and mount.
Mycobacteria, hair shafts, Russell bodies,
Splendore -Hoeppli immunoglobulins around
actinomycetes and some fungal organisms    -Red

Background                                -pale Blue
Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.
o The blue counterstain may be patchy if extensive
  caseation is present.
o Care should be taken to avoid over-counterstaining, as
  scant organisms can easily be obscured.
o Decalcification using strong acids can destroy acid-
  fastness; formic acid is recommended.
o Victoria blue can be substituted for carbol fuchsin and
  picric acid for the counterstain.
 Warthin-Starry method for spirochetes
                     (Warthin & Starry 1920)

  Organisms are demonstrated by silver impregnation technique.

Acetate buffer. pH 3.6
    Sodium acetate 4.1 g
    Acetic acid        6.25 ml
    Distilled water 500 ml
1 % silver nitrate in pH 3.6 acetate buffer.
    Warthin-Starry Method for
Developer :
    Dissolve 3 g of hydroquinone in 10 ml pH 3.6
 buffer, and mix 1 ml of this solution and 15 ml
 of warmed 5% Scotch glue or gelatin; keep at
 40°C. Take 3 ml of 2% silver nitrate in pH 3.6
 buffer solution and keep at 55°C. Mix these two
 solutions immediately before use.
1.   Deparaffinize & rehydrate through graded alcohol to
     distilled water.
2.   Impregnate in preheated 55-60°c silver solution, 90-
     105 min.
3.   Prepare and preheat developer in water bath.
4.   Treat with developer for 3 min at 55°c (section
     should be golden brown at this point)
5.   Remove from developer and rinse in tap water for
     few min at 55-60°c, then buffer at room
6.   Tone in 0.2% gold chloride.
7.   Dehydrate, clear and mount.

Spirochetes - black

Background - golden

                      Cat scratch associated bacilli
Long, spiraling spirochetes are seen with a Warthin-Starry silver
Spirochetes are black in a gold background.
Gomori methenamine silver for fungi
o GMS staining is a silver staining technique for
  demonstrating fungi in tissue sections.
o It is primarily based on staining the polysaccharides in
  fungal cell walls.

  This method depends upon the reduction of the
  silver by the aldehyde groups produced after
  oxidation of fungal wall components with chromic
 5% sodium tetraborate in distilled water (borax)
 Methenamine silver
Incubating solution
  borax                  - 5ml
  distilled water     - 25ml
  methenamine silver - 25ml
Arzac’s counterstain
  orange G             - 0.25 gm
  light green          - 1g
  50% alcohol          - 100ml
  glacial acetic acid - 1ml
1.  Deparaffinize & rehydrate through graded alcohol to
    distilled water.
2. Oxidize in 5% aqueous chromic acid, 1 hour.
3. Wash in tap water.
4. Rinse in 1% sodium metabisulfate.
5. Wash in tap water, 5 min.
6. Rinse in tap water, then place in preheated silver
    incubating solution in a dark place, upto 1 hour
7. Rinse well in distilled water.
8. Tone in 0.1% gold chloride for 4 min.
9. Place in 3% sodium thiosulfate, 5 min.
10. Counterstain in Arzac’s stain, 15-30 sec.
11. Blot, dehydrate, clear and mount.
Fungi, pneumocytstis,
melanin - black

Mucin and glycogen      - grey-black

RBCs - yellow

Background - pale green

                                       Pneumocystis carinii
GMS stain for Cryptococcus neoformans
                     Giemsa stain

A stain for hemopoietic tissue and hemoprotozoa consisting
  of a stock glycerol methanol solution of eosinates of
  Azure B and methylene blue with some excess of the basic
A mixture of glycerin, methanol, methylene azure, and eosin
  used to stain chromosomes, blood cell producing tissues,
  and certain species of spirochetes and protozoans
  This method is a modified version of the original Giemsa
  technique used for haematological smears and gives
  good results for sections. Giemsa is a Romanowsky
  stain which contains azure B and eosin Y and is capable
  of making subtle distinction in shades of staining. The
  acidic groupings of the nucleic acids and proteins of the
  cell nuclei determine their uptake of the basic dye azure
  B and the presence of basic groupings result in an
  affinity for acidic dyes and their staining by eosin.
o Giemsa stain is used for the histopathological diagnosis
  of Malaria and other parasites. It is named after Gustav
  Giemsa, an early malariologist.

o Giemsa stain is also a differential stain. It can be used to
  study the adherence of pathogenic bacteria to human

o It differentially stains human and bacterial cells pink and
  purple respectively. It can be used for histopathological
  diagnosis of malaria and some other spirochete and
  protozoan blood parasites.
o Giemsa stain is a classical blood film stain for
  peripheral blood smears and bone marrow specimens.

o Erythrocytes stain pink, platelets show a light pale pink,
  lymphocyte cytoplasm stains sky blue, monocyte
  cytoplasm stains pale blue, and leukocyte nuclear
  chromatin stains magenta.
Giemsa stock
Giemsa stain powder       - 4gm
Glycerol                  - 250 ml
Methanol                  - 250 ml
Working Giemsa for parasites
Giemsa stock                - 4 ml
Acetate buffered distilled water, pH 6.8   - 96 ml
Method :
1.   Deparaffinize and rehydrate through graded alcohols
     to water.
2.   Rinse in pH 6.8 buffered distilled water.
3.   Stain in working Giemsa, overnight.
4.   Rinse in distilled water.
5.   Rinse in 0.5% aqueous acetic acid until section is
6.   Wash in tap water.
7.   Dehydrate rapidly through alcohols, clear and
Designed to differentiate blood cells and to stain
intracellular parasites in red blood cells and plasma, e.g.
Plasmodium falciparum (malaria parasite).
Toxoplasma gondii
Demonstration of decalcified bone

  Masson’s trichrome stain
  PAS
  Silver impregnation
  Schmorl’s picro-thionin
  Weigert – van gaeson
• Masson’s trichrome stain:- adult or mature bone
  often shows areas of blue or green.

• PAS :- the +ve red PAS reaction demonstrate
  mucopolysaccharides in new bone.The PAS
  reaction is not affected by decalcification ,but
  prolonged treatment with strong acid should be

• Silver impregnation of reticulin fibers helps in
  diagnosis of bone tumors,tumor metastasis to bone
  and myelofibrosis.stain is not affected by
      Schmorl’s picro-thionin

• The major problem in demonstrating canaliculi is the
  attempt to show spaces too fine for identification by
  routine staining of surrounding bone so it is necessary to
  fill the spaces with a substance that appears dark
  against a lighter or unstained background.

• The picro-thionin method (Schmori 1934) depends on
  deposition of a thionin precipitate within the lacunae and
  canaliculi .
• Fixation
Any fixative, but avoid mercuric chloride
• Decalcification
Any decalcification solution

• Sections
Frozen or celloidin freshly cut.

• Solutions
• Stock solution
0.25% aqueous thionin.
• Working solution
(0.25% thionin):filter 50ml stock solution and dilute with 50ml
   distilled water .Add 1 or 2 drops concentration ammonia
   immediately before use .
• Saturated aqueous picric acid.
• Method

1. Wash sections in distilled water,10 min.
2. Stain in thionin solution ,5-20 min or longer.
3. Wash in distilled water
4. Immerse section in picric acid solution,30-60 seconds.
5. Wash in distilled water
6. Differentiate in 70% alcohol until the bluish-green
   clouds of stain cease to form, 5-10 min or longer.
7. Dehydrate rapidly, clear in xylene and mount in
   permanent mounting media.
• Note

• Agitate section gently during steps 3-6 .this is particularly
  important in stage 6 and change 70% alcohol frequently.

• If bone matrix is decolorized during differentiation (step
  6) restore yellow color by returning the section to the
  picric acid solution for a few seconds before proceeding
  with dehydration.
• Result

• Lacunae and canaliculi – dark brown-black
• Bone matrix – yellow or brownish – yellow
• Cells - red
Connective tissue and lipids

                                       Gomori's One-Step Trichrome Stain manual,
                                       Trichrome automated

Elastic fibers                         Verhoeff - Van Gieson stain
                                       Wilder's method, both manual and
Fat                                    Oil red O stain for frozen section

Glycogen/neutral mucin                 Lillie PAS stain manual, PAS automated

                                       Amylase digestion procedure, both manual
Glycogen digestion
                                       and automated.

Mucopolysaccharid-acid                 Modified Mowry's Colloidal iron stain

Mucopolysaccharid-acid                 Alcian Blue pH 2.5 or pH 1.0

Acid and neutral mucopolysaccharides   Alcian blue 2.5 and PAS

Sialomucin                             Mayer's mucicarmine stain

Amyloid                         Alkaline Congo Red stain

Amyloid                         Lieb's crystal violet method

Minerals, pigments and miscellaneous

Calcium                         Kossa's method

Iron                            Gomori's method

Melanin                         Fontana Masson Silver stain

Melanin removal                 Permanganate method

Charcott Leyden crystals        Luna stain

Muscle striations               Mallory's PTAH stain
                       NAME OF STAIN
Acid-Fast Organism      Fite stain
Bacteria, blood elementsMay - Grunwald Giemsa Stain
Bacteria                Gram stain
Bacteria/Protozoa       Lillie's Azure A Eosin B Stain
Helicobacter pylori     Toluidine Method
Helicobacter pylori     Alcian yellow, automated
                        Gomori's methanamine silver method, both
                        manual and automated
Fungi                   PAS green, both manual and automated
Fungi/Cyptococcus       Mucicarmine
Spirochetes             Steiner, automated.
Spirochetes             Warthin - Starry microwave method
Connective tissue and lipids

                                       Gomori's One-Step Trichrome Stain manual,
                                       Trichrome automated

Elastic fibers                         Verhoeff - Van Gieson stain
                                       Wilder's method, both manual and
Fat                                    Oil red O stain for frozen section

Glycogen/neutral mucin                 Lillie PAS stain manual, PAS automated

                                       Amylase digestion procedure, both manual
Glycogen digestion
                                       and automated.

Mucopolysaccharid-acid                 Modified Mowry's Colloidal iron stain

Mucopolysaccharid-acid                 Alcian Blue pH 2.5 or pH 1.0

Acid and neutral mucopolysaccharides   Alcian blue 2.5 and PAS

Sialomucin                             Mayer's mucicarmine stain

Amyloid                         Alkaline Congo Red stain

Amyloid                         Lieb's crystal violet method

Minerals, pigments and miscellaneous

Calcium                         Kossa's method

Iron                            Gomori's method

Melanin                         Fontana Masson Silver stain

Melanin removal                 Permanganate method

Charcott Leyden crystals        Luna stain

Muscle striations               Mallory's PTAH stain
                       NAME OF STAIN
Acid-Fast Organism      Fite stain
Bacteria, blood elementsMay - Grunwald Giemsa Stain
Bacteria                Gram stain
Bacteria/Protozoa       Lillie's Azure A Eosin B Stain
Helicobacter pylori     Toluidine Method
Helicobacter pylori     Alcian yellow, automated
                        Gomori's methanamine silver method, both
                        manual and automated
Fungi                   PAS green, both manual and automated
Fungi/Cyptococcus       Mucicarmine
Spirochetes             Steiner, automated.
Spirochetes             Warthin - Starry microwave method
Acid-Fast Organism       Fite stain
Bacteria, blood elements May - Grunwald Giemsa Stain
Bacteria                 Gram stain
Bacteria/Protozoa        Lillie's Azure A Eosin B Stain
Helicobacter pylori      Toluidine Method
Helicobacter pylori      Alcian yellow, automated
Fungi                    Gomori's methanamine silver method, both manual and automated
Fungi                    PAS green, both manual and automated
Fungi/Cyptococcus        Mucicarmine
Spirochetes              Steiner, automated.
Spirochetes              Warthin - Starry microwave method
Connective tissue and lipids
Collagen                 Gomori's One-Step Trichrome Stain manual, Trichrome automated
Elastic fibers           Verhoeff - Van Gieson stain
Reticulin                Wilder's method, both manual and automated
Fat                      Oil red O stain for frozen section
Glycogen/neutral mucin Lillie PAS stain manual, PAS automated
Glycogen digestion       Amylase digestion procedure, both manual and automated.
                         Wright stain
                                    RESULTS:
•   Erthrocytes yellowish-red
•   Polymorphonuclears:             Erthrocytes: pink-tan
•   Nucleus dark purple
                                    Eosinophiles:
•   Granules reddish-lilac
•   Cytoplasm pale-pink            Granules red
•   Eosinophiles:
•   Nuclei blue                     Lymphocytes:
•   Granules red to orange-red
                                   Granules red-purple
•   Cytoplasm blue
•   Basophiles:                    Cytoplasm blue
•   Nucleus purple to dark blue
•   Granules very dark purple       Neutrophils:
•   Lymphocytes:
                                   Granules purple-pink
•   Nuclei dark purple
•   Cytoplasm sky blue
•   Platelets violet to purple granules
         Stains for smears
•   Blood smears
•   Mucosal scrapes : buccal ,tongue
•   Mucinous sample : sputum
•   Fine needle aspiration
       Hematologic stains
• Romanowsky stains
Giemsa stain
Leishman stains
Wright’s stains
• Leukocyte alkaline phosphatase
• Tartrate-resistant acid phosphatase
• Myeloperoxidase
          Romanowsky stains

It will stain both nucleus and cytoplasm.

These histology stains are used for blood smear and bone

Examples of Romanowsky histology stains include Wright's
  stain, Giemsa stain and Jenner's stain.

These histology stains are based on a combination of eosin
  and methylene blue. .
             Giemsa Stain
Giemsa stain is a classical blood film stain
  for peripheral blood smears and bone
  marrow specimens..
• It is also used to visualize parasites and malaria.
• This is a Romanowski type stain. Methylene blue
  and eosin are used.

• Note that the acidic components of the cellular
  constituents such as the cytoplasm and chromatin,
  pick up the basic methylene blue azure
  compliments of the Giemsa stain, which reveals
  the characteristic blue coloration of this stain.
• Result :-

• Erythrocytes
- pink/red.

• Platelets and - blue
              Wright's Stain
  It is named for James Homer Wright, who devised the stain,
  a modification of the Romanowsky stain, in 1902.
• Because it distinguishes easily between blood cells, it
  became widely used for performing differential white blood
  cell counts, which are routinely ordered when infections are
• This histology stain is used for blood smears and bone
  marrow smears.
• This histology stain uses a blend of basic dyes, such as
  methylene blue derivatives and acid dyes, such as eosin.
• Streak thin (approx. one cell thick) smears across a sterile slide by
  means of a second slide or cover glass. Air-dry quickly.

1. Place 1.0 ml of the Wright-Giemsa Stain upon the smear, in
    sufficient quantity to cover the entire surface, for 3-4 minutes.
2. Add 2.0 ml distilled water or Phosphate Buffer pH 6.5 (B-149-2) and
    let stand twice as long as in step 1.
3. Rinse stained smear with water or the Phosphate Buffer pH 6.5 (B-
    149-2) until the edges show faintly pinkish red.
4. The film is allowed to dry in the air. The preparation may be blotted
    gently to hasten drying.
5. Permanent mounts can be made with mounting medium.
Erthrocytes - yellowish-red
                                             lymphocyte       basophils
Nucleus - dark purple
Granules - reddish-lilac
Cytoplasm - pale-pink

Nuclei - blue
Granules- red to orange-red
Cytoplasm- blue                         eosinophil        neutrophil

Nucleus - purple to dark blue
Granules - very dark purple

Nuclei - dark purple
Cytoplasm- sky blue
Platelets - violet to purple granules
Red blood cells - reddish/pink.
Eosinophilic granules -bright
white blood cells- purple.
          Leishmans stain
• Where to look for cells in a blood smear
  The density of cells varies across the smear.
• Cells will be "heaped and piled" close to the point
  were the drop of blood was placed on the slide.
• White blood cells appear shrunken, and some
  types are difficult to distinguish from each other.
•  There are fewer cells close to the tip of the smear.
  In this region, white blood cells are sometimes
  damaged and erythrocytes may be deformed.
• The best area to look at is between these two
  regions. Where it is located exactly and how wide it
  is will depend on the smear, but the middle of the
  smear is a good starting point
       Myeloperoxidase stain

• The myeloperoxidase (MPO) stain is helpful to identify
  cytoplasmic granules characteristic of myeloid cells.
• This is useful when there are large, immature white
  blood cells in the peripheral blood, and it is not clear
  whether they are of myeloid or of lymphoid origin.
• Staining with MPO in this setting suggests a myeloid
This peripheral blood smear is stained with the myeloperoxidase stain,
and there is red reaction product in these leukemic cells,
indicative that this is a myeloid leukemia.
   Leukocyte alkaline phosphatase stain
• The leukocyte alkaline phosphatase (LAP) stain is
  helpful in determining whether a high peripheral
  blood leukocytosis is a reactive process or a
  leukemia (chronic myelogenous leukemia, or
• The more differentiated cells in the reactive
  process will stain more readily with LAP, while
  leukemic cells will not.
• The cells on a smear can be assessed and an
  "LAP score" can be generated. A high score
  generally indicates a "leukemoid reaction" or
  reactive condition (with an infection or other
  inflammatory process) while a low score suggests
This peripheral blood smear is stained for leukocyte alkaline phosphatase.
The neutrophils have bright red reaction product in their cytoplasm
  Tartrate-resistant acid phosphatase stain

• The tartrate-resistant acid phosphatase (TRAP)
  stain has one major usefulness--to help diagnose a
  rare leukemia known as hairy cell leukemia.
• This neoplastic B lymphocyte proliferation affects
  mainly bone marrow and spleen.
• There is typically pancytopenia, so the peripheral
  WBC count is not high. The circulating hairy cells
  get their name from the cytoplasmic projections.
  However, positive staining with TRAP helps make
  the diagnosis.
This collage of cells shows the characteristic lymphocytes
of hairy cell leukemia with their indistinct cytoplasmic borders
and small projections. The cell at the lower right is stained for
tartrate-resistant acid phosphatase (TRAP) and is positive,
with red reaction product in the cytoplasm, characteristic for
hairy cell leukemia.
         Alkaline Phosphatase
This histology stain
  will stain endothelial cells.
• Sites of alkaline phophatase
activity will appear red.
 Nuclei will stain blue.
• Papnicolaou formula

• Harris’s hematoxylin             EA 50
Hematoxylin             - 5g       0.04 M light green SF -10ml
Ethanol                 - 50ml     0.3 M eosin Y            – 20ml
                                   Phosphotungstic acid - 2g
Potassium alum           - 100g
                                   Alcohol                 -750ml
Distilled water (50̊ C) – 1000ml   Methanol                -250ml
Mercuric oxide           – 2-5g    Glacial aceticmacid -20ml
Glacial acetic acid       - 40ml   Filter all stains before use.

• Orange G 6
Orange G (10% aqueous) – 50ml
Alcohol               – 950ml
Phosphotungstic acid   – 0-15g
• Specimen Collection:
• In the collection and preparation of smears for cytological
   examination, the major objectives are:
1. Specimens should have a sufficient number of cells from the
   area in question.
2. Smears should contain well preserved cells uniformly
   distributed so that each cell can be individually examined.
3. The staining procedure should clearly define the details of all

Cytological preparations are obtained from patient by approved
  methods and techniques.
Scraping, obtained from the vagina, uterus, cervix, mouth or
  ulcerated skin area is spread directly on a clean microscope
4.Sediment (obtained by centrifugation or filtration) from bodily
  secretions is spread on a clean microscope slide.
5.The smear is immediately fixed with a cytological spray
  fixative or in an alcohol-ether dip.
6.Fixation or preservation is one of the most important steps in
  the procedure. Drying of the cells prior to fixation will usually
  result in artifacts such as nuclear distortion and
7.After fixation there are no special handling requirements for
  cytological smears. However,smears which are to be mailed
  to a laboratory, should remain in the fixative for about one
  hour. A second clean glass slide may be placed on each
  fixed slide for protection.
• Procedure:

• Notes:
Filter the Harris Hematoxylin immediately before use.

1. Dip slide(s) gently 5-10 times in 95% ethanol.
2. Dip slide(s) gently 5-10 times in 70% ethanol.
3. Dip slide(s) gently 5-10 times in distilled water.
4. Stain 5 minutes in Harris Hematoxylin.
5. Place smears in distilled water. Rinse in successive changes of
   distilled water until the water remains colourless.
6. Dip slide(s) gently 5-10 times in 70% ethanol.
7. Dip slide(s) in a 1% solution of HCl in 70% ethanol until the
   smear shows a salmon colour.
8. Rinse slide(s) well in 2 changes of 70% ethanol.
9. Dip slide(s) gently in a 3% solution of ammonium hydroxide in
   70% ethanol until the smear takes on a blue colour.
10. Rinse the slide(s) in two changes of 70% ethanol.
11. Dip slide(s) 5-10 times in 95% ethanol.
12. Stain slide(s) in OG-6 for 2 minutes.
13. Rinse slide(s) in two changes of 95% ethanol.
14. Stain slide(s) in EA-50 or EA-65 for 3-6 minutes.
15. Rinse slide(s) well in two changes of 100% methanol.
16. Rinse slide(s) in one part absolute methanol one part xylene.
17. Clean smear in xylene.
• Mounting Procedure:
1. After the smear has been completely cleaned in xylene it is
   mounted with a microscope slide cover glass preferably
   22x40mm, #1 thinness.
2. A permanent clean mounting medium should be used.
3. The excess xylene should be drained, in order to avoid th
   appearance of air spaces when xylene evaporates.
4. Place the required amount of mounting medium along an edge
   of one of the longer borders of the coverslip.
5. Place the slide at right angles to the edge of the coverslip so
   that the side containing the cells is facing the mounting
6. Slowly lower the slide and permit the mounting medium to
   spread between the slide and coverslip
• Limitations:
1. Proper specimen collection and fixation of cells is essential for
• Results:
Nuclei are stained blue while cytoplasm displays varying
  shades of pink, orange, yellow and green
• Cytologic image of a scraping
 of the buccal mucosa. Intermediate
 squamous cell with sex chromatin
body (Barr body) (arrow) lying
against the inner nuclear membrane
(Papanicolaou stain).

 - Cytology smear showing clusters
 of keratinizing squamous carcinoma
indicating metastasis in the lymph
• : Cytology smear showing
metastasis of adenocarcinoma.
Note the neutrophils sticking
around the mucin of the tumor cells.

• Cytology smear showing the
streaking effect commonly seen with
 metastasis of nasopharyngeal
carcinoma, small cell carcinoma
 and lymphoma.
• : FNA smear showing metastasis of
  malignant melanoma. Note the presence
  of giant cells (inset) and the presence of
  melanin pigment. (MGG X 400)
                        Stains for biogenic amines

•   Types of stains for argentaffin include:
•   Diazo (diazonium salts)
•   Fontana-Masson
•   Schmorl's
•   Autofluorescence

•   Types of stains for chromaffin include:
•   Modified Giemsa
•   Schmorl's
•   Wiesel's

•   Types of stains for argyrophil include:
•   Grimelius (Bouin's fixative preferred)
•   Pascual's
                          Chromatin And Argentaffin

•   The distinction between chromaffin and argentaffin is artificial, since this
    depends upon the fixative used.
•   "Chromaffin" cells have cytoplasmic granules that appear brown when fixed
    with a dichromate solution.
•   "Argentaffin" cells reduce a silver solution to metallic silver after formalin
•   Either reaction can be produced depending upon which fixative was used.
•   Traditionally, chromaffin reaction is associated with adrenal medulla or
    extraadrenal paraganglion tissues (pheochromocytomas) whereas
    argentaffin reaction is associated with carcinoid tumors of the gut.
•   Using a pre-reduction step may get more cells to stain, but they are called
    "argyrophil" then.

This histology stain uses silver. Fontana-Masson stains
argentaffin granules and melanin black. The nucleus will
stain pink/red. The cytoplasm will stain light pink.
• Giemsa stain is also used to
  visualize chromosomes.
• Giemsa stain also stains the
  fungus histoplasma.
• It can also be used to identify Mast cells.[3]
• Paul Ehrlich had used mixtures of acidic and basic dyes for this purpose in
  1879: eg Fuchsine (acid) and methylene blue (base).[1] In 1891 Ernst
  Malachowski [2] and Dmitri Leonidovich Romanowsky [3] independently
  developed techniques using a mixture of Eosin Y and modified Methylene
  blue that produced a surprising hue unattributable to either staining
  component: a beautiful, distinctive shade of purple.[4][5] Requirement for
  the occurrence of the Malachowski-Romanowsky-Giemsa effect are: I A
  cationic dye: The best dye is azure B and, though azure A gives the nuclear
  purple colour, the cytoplasmic blue is inferior. No other cationic dye such
  as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is
  used.[6] Because the aqueous dye solutions were unstable, methanol was
  introduced as a solvent, and William Boog Leishman[7] and James Homer
  Wright[8] advocated use of methanol as a fixative prior to staining. Gustav
  Giemsa improved this technique by standardizing the dye solutions and
  adding glycerol to increase stability.[9]
• Giemsa-stained thin blood film showingPlasmodium
  falciparum infections[10]
• The demethylation of Methylene Blue in aqueous solution using heat
  and alkali produces a mixture of Azure A, Azure B, Methylene Violet
  and Methylene Blue. Eosin Y is then added to produce a "neutral"
  dye. The precipitate is then dissolved in a mixture of methanol and
  glycerol to form a stock solution; this is diluted with water or an
  aqueous buffer to form a 'working' solution that is used in the
  staining of pathology specimens. The 'working' solution is stable for
  3 hours.[11]
• Immunochromatographic capture procedures (Rapid Diagnostic
  Tests) are nonmicroscopic diagnostic options for the laboratory that
  may not have appropriate microscopy expertise available.[12]
      Mallory's Trichrome stain
• Solution A
Acid fuchsin-1g
Distilled water-100mL

• Solution B
Phosphomolybdic acid-1g
Distilled water-100mL

• Solution C
Orange G-2g
Methyl blue-0.5g
Oxalic acid-2g
Distilled water100mL
• Method
1. Bring sections to water via xylene and ethanol.
2. Place into solution A for 2 minute.
3. Rinse with distilled water.
4. Place into solution B for 2 minutes.
5. Rinse quickly with distilled water.
6. Place into solution C for 15 minutes.
7. Wash well with distilled water.
8. Dehydrate and differentiate with ethanol.
9. Clear with xylene and mount with a resinous
• Results
Nuclei – red
Erythrocytes – orange
Muscle – red
Collagen – blue

• Notes
  Phosphotungstic acid may be substituted for
  phosphomolybdic acid in solution B.
• This is the method that introduced the concept, if
  not the name, of trichrome staining.
    Gingiva (gum). The masticatory mucosa has tall rete ridges. A dense network of
    collagen fibers (blue) tightly anchors the epithelium to the underlying bone (not
    shown); the keratin layer (orange band) on the surface of the epithelium imparts
    further strength to it (Mallory's trichrome).
Buccal mucosa showing maturation of squamous epithelium: there is a row of small
  basal cells, larger cells of stratum spinosum, and parallel arranged flat surface cells.
  No keratinization is seen. Lamina propria shows delicate strands of connective
  tissue, blood vessels, and a few lymphocytes. (Mallory's trichrome.)
Endochondral ossification
 in Mallory stain. Cartilage
 is light blue and bone is
 dark blue. A thin layer of
 bone has already been
laid down on the surface
of the cartilage spicules
along the leading edge
 of cartilage. Blood cells
in the marrow cavity are red.
 The very dark blue at the
 lower left and right is
spongy bone of the
periosteal bone collar of the
shaft. This will later be
remodeled into Haversian
systems of compact bone
Periodic Acid Schiff Stain
(Mc Manus 1946)

     PAS reaction is a useful indicator of the
 presence of tissue carbohydrate, particularly for
 glycogen when the technique incorporate a diastase
 digestion stage.
o PAS with Diastase staining is used for the
  demonstration of glycogen in tissue sections.
o Diastase and alpha-Amylase are enzymes that digest
  glycogen polymer to maltose and glucose that are easily
  washed from the sections, thus giving a negative
  reaction with PAS.
- Diastase (amylase) specifically digests glycogen.

- Predigesting one section with amylase and comparing with
an undigested section.
(both of which have been stained by PAS) allows identification
of the glycogen.

         PAS with Diastase                                       PAS Stain

       The PAS stain is a histochemical reaction in
  that the periodic acid oxidizes the carbon to carbon
  bond forming aldehyde which react to the fuchsin-
  sulfurous acid which form the magenta color.

    Glucose + Periodic acid      - aldehyde
    Aldehyde + Schiff’s reagent - magenta
                                colored compound
PAS Technique:
   - Periodic Acid Solution
             Periodic acid   - 1 gm
            Distilled water - 200 ml
     - Schiff Reagent
                  Dissolve 1gm of basic fuchsin in 200 ml of
  boiling distilled water. Allow the solution to cool 50 °c,
  and add 2-gm potassium metabisulphite with mixing.
  Allow to cool to room temperature then add 2 ml conc.
  Hydrochloric acid, mix, add 2 gm activated charcoal and
  leave overnight in the dark at room temperature.
    Filter, the solution should be either clear or pale yellow
  color. Store in a dark container at 4°c.
o   Dewax sections and bring to distilled water.
o   Treat with periodic acid, 5 min.
o   Wash well with several changes of distilled water.
o   Cover with schiff’s reagent, 10-20 min.
o   Wash in running tap water, 5-10 min.
o   Stain nuclei with Harris’s haematoxylin or an iron
    haematoxylin. (Do not use Ehrlich’s haematoxylin,
    which will also stain some PAS positive compounds.)
o   Differentiate in acid-alcohol .
o   Wash in water, dehydrate in alcohol, clear in xylene
    and mount as desired.
MUCICARMINE STAIN –                                  SOUTHGATE’S – MUCIN

Aluminum is believed to form a chelation complex with the carmine, changing the
molecule to a positive charge allowing it to bind with the acid substrates of low density
such as mucins
           Stain for Mast Cells
Mast cell containing heparin and histamine in the form of
They are found in connective tissue around the blood
  vessels in many inflammatory conditions
Mast cell granules stains metachromatically with toludine
  blue, azure A, Bismarck brown and thionin.
Mast cells are usually visualized with alcian blue methods.
Alcian blue – safranin method for mast cells

Alcian blue       - 900 mg
Safranin         - 45 mg
Ferric ammonium sulfate - 1.2 gm
Acetate buffer (pH 1.4)    - 250 ml
1.   Take sections to water.
2.   Stain in alcian blue solution, 15 min.
3.   Rinse in tap water.
4.   Dehydrate in alcohol.
5.   Clear and mount.
Mast cell granules
containing biogenic
amines              – blue

Mast cell containing
heparin              - red

                             Mast cells are apparent in the upper dermis in
                             this section of skin because their cytoplasmic
                             Granules stain metachromatically, giving the
                             intense purple color seen here.
                TOLUIDINE BLUE
Toluidine Blue (Lariviere, 1999). Its specific role is as a metachromatic stain (meta=change

Toluidine blue is a polychromatic stain which stains many tissue components.

Toluidine Blue causes mast cells to stain purple in an otherwise blue background.
  Standard paraffin sections or 6æ frozen section

  Toluidine blue (CI 52040) 0.25 g
   Sodium acetate 2 g
    Sodium barbiturate 3 g
     Distilled water to 100 mL
  1. Paraffin or frozen sections may be used.
  2. Bring sections to water.
  3. Stain in toluidine blue solution, for 20 seconds .
  4. Blot with filter paper; allow to dry and clear in histolene.
  5. Mount in Safety Mount

  Metochromatic substances - Red, pink or purple
  Nuclei and other components - Blue
  Mast cell granules - Purple
            Staining reactions of mast cells

       Method                          Result
Toludine blue                  Purple
Azure A                        Red
Thionin                        Blue or red
Csaba’s alcian blue safranin   Purple to red
Aldehyde fuchsin               Yellow to brown
PAS                            Variable
• References:
• 1. Papanicolaou GN: Atlas of Exfoliative Cytology,
  Harvard University Press,
• Cambridge, 1954.
• 2. United States, Naval Medical School, National Naval
  Medical Center, Bethesda,
• Maryland, Manual of gynecological exfoliative cytology.
  US Government Printing
• Office, Washington, D.C., 1965.
• 3. A Manual for Cytotechnology, C. Keebler, J. Reagen,
  Editors, American Society
• of Clinical Pathologists Press, Chicago (IL), 1983.
• Lillie, R. D. :Factors influencing the staining of blood
  films and the role of methylene violet. J.
• Lab. Clin. Med. 29:1181, 1944.
• Lillie, R. D., H.J. Conn’s Biological Stains, 8th edition,
  William’s & Wilkins, Baltimore, p 438,1972.

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Description: Special Stains play a very important role in the detection of carbohydrates, lipids, connective tissues, DNA, RNA, and enzymes also. Thus, would find very useful staining techniques.