STAINING OF

CARBOHYDRATES
• Carbohydrates – main source of energy in the body, mobilize 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.

• Mucin - made up of hexosamines (neutral mucopolysaccharides) or

mucus that is secreted by the goblet cells of intestinal mucosa,
respiratory lining cells andcertain glands, or found in intercellular
substances and connective tissue fibers
PERIODIC ACID SCHIFF REACTION
• Periodic Acid Schiff ( PAS) is a histochemical stain that will demonstrate
carbohydrates and other substances in the tissue.
• It uses periodic acid to specifically oxidize the 1,2 glycol group of
polysaccharides and mucin, liberating aldehydes that are required for
the coloration of Schiff's reagent, thereby producing a red magenta or
purplish-pink color
• Since not all carbohydrates include this structure, the PAS is not a
method for carbohydrates in general but only for those which contain 1-
2 glycols or closely related structures.
• As a general rule, the intensity of PAS reaction is proportional to the
content of sugars present in the reacting substance.
• PAS-positive staining reaction, oxidation must occur to produce
aldehyde.
• Periodic acid is generally applied to the sections as a 0.5 to 1.0% aqueous
solution for 2 to 20 (average 5) minutes at room temperature.

General Principles of the PAS Stain

Reactivity of the PAS technique is based on the structure of the
monosaccharide units. Periodic acid ,an agent oxidize the carbon-to-carbon
bonds between two adjacent hydroxyl groups then produces aldehyde groups
,then reacts with Schiff reagent (made up of a mixture of basic fuchsin,
hydrochloric acid, and sodium metabisulphite). The basic fuchsin reacts with
newly formed aldehyde groups in the tissue then produces a bright magenta
color when the section is rinsed in water. The intensity of the color is
proportional to the concentration of hydroxyl groups originally present in the
monosaccharide
units.
SCHIFF REAGENT
• Basic fuchsin, which is an essential component
• Has a mixture of three dyes (rosanilin, pararosanilin and magenta
II).
• H. Schiff (1866) - Sulfur dioxide converts the magenta-colored
basic fuchsin into colorless leukofuchsin. Reoxidation by slow
exposure to light and air or by addition of periodic acid , restore
the colorless leukofuchsin to the magenta colored basic fuchsin.
• Schiff reagent can be prepared in different ways:
(1) by using thionyl chloride to release sulfur dioxide; (Barger and de Lamater method
1948)
(2) by adding sodium or potassium metabisulfite;
(De Tomasi – Coleman Method 1939)
(3) by using sulfur dioxide gas.
(Itikawa and Oguru method 1954)

• Both Schiff reagents are stable and may last up to 6 months, but it is preferable to
make fresh reagent every month.
• The reagent should be discarded when it begins to form a color.
• Reactivity of the Schiff reagent may be tested by adding a few drops of 10 cc of
37- 40% formaldehyde.
• Rapid development of a reddish purple ,the Schiff reagent is still good and usable.
• If the reaction is delayed and the resultant color is deep blue-purple, the solution is
breaking down and should be discarded.
STAINING OF GLYCOGEN
• Glycogen is very soluble in water and insoluble in alcohol.
• Alcoholic solutions are supposed to be the best fixatives.
• Bec. it penetrates the tissues slowly hence, preserves only the glycogen on the surface of
the block.
• For adequate demonstration, thin slices of tissues should be fresh as possible and
immediately placed in the fixative at 4 deg. Cel. to prevent rapid breakdown due to the action
of glycogenolytic enzymes (LIVER)
• For routine processing, recommended fixatives are acetic acid, alcohol formalin, Bouin's,
Brasil's, Kelly's and Gendre's solution. If minimal loss of glycogen is desired, it is advisable
to float the sections onto 75% ethanol when cutting, and also to mount them on slides from
75% ethanol.
• For staining, blocks should be fixed in absolute alcohol to prevent dissolving water soluble
glycogen.
• NOTE : RINSING WITH NORMAL SALINE BEFORE FIXING SHOULD BE AVOIDED
 PERIODIC ACID-SCHIFF REACTION
• Fixation: 10% neutral buffered formalin or Bouin’s solution.
• Sections: 4-5 μm
• Results:
• PAS-positive substances red or magenta red
• Nuclei - blue

NOTES:
• Mucoproteins are the most common PAS positive substances
• Carbohydrates, glycoproteins, glycolipids, unsaturated lipids and phospholipids are also PAS positive.
• Can also be found in certain bacteria, fungi, kerasin, connective tissue mucin, basement membrane,
thyroid and cartilage.
• useful indicator for glycogen when the technique incorporates a diastase digestion stage.
 PAS WITH DIASTASE METHOD FOR GLYCOGEN
DEMONSTRATION
• The section serving as control is treated with diastase (human saliva) or 0.1% malt diastase
in distilled water to remove glycogen.
• Glycogen is readily digested with amylase.
• Malt diastase is extracted from malt and contains both alpha- and beta- amylases. It is the
commonly employed enzyme for glycogen digestion.
• Saliva containing ptyalin (salivary amylase) is also a highly effective means of digesting
glycogen in tissue sections.
• most workers prefer the commercial diastase because it is easier to standardize
• Fixation: Helly's Fluid
• Solution:Diastase 1 gm and Distilled water 100 ml.
• Results: Nuclei- blue ,Glycogen – red

• NOTE:
Diastase digestion should be performed with solutions have been
preheated to 37 deg. Cel. For 1 hours prior to use.
• Strength of the diastase solution should be optimized to give
complete glycogen digestion.
• Concentrations (w/v) of 0.1% – 1.0%
• Should be dissolved in a phosphate buffer with a pH of around 6.0
for maximum effectiveness.
 • Other methods used for staining glycogen are
1. Best carmine method
2. Langham's iodine method

Best carmine method

 A good staining technique due to the affinity of alkaline carminic acid for
glycogen, producing a bright red color.

 Ehrlich's hematoxylin - counterstain, coloring the nuclei blue

 Carminic acid – essential ingredient,

 Potassium carbonate and potassium chloride salts
- added to the stock solution
- inhibit any non-specific background carmine carmine staining
due to electrostatic bonding between the negatively charged carmine
and the basic proteins found in tissue.

 This method is selective but not as highly specific for glycogen as the
PAS method with and without diastase.
Langham's iodine method
• Oldest stain
• Considered absolete
• Not specific for glycogen
• Colors amyloid and other protein substance
• Rapid stain but not permanent and fades after a few months

Fixation: Neutral 10% formol- alcohol, formol saline

Sections: Parrafin sect.
Results: Glycogen is stained mahogany
Tissues constituents – yellow
• Erlich hematoxylin – coloring nuclei (greenish blue)
• Origanum oil – decolorizer
STAINING OF MUCIN
• Mucins - polysaccharides bound to other substances forming the
ground substance of connective tissues primarily.
• Formalin and Carnoy's fluid are recommended for fixation
although prolonged storage in formalin tends to reduce the
strength of PAS reaction of mucin
• Hydration with 0.2.N NaOH for 10-15 minutes before staining may
be done to regain PA.S reaction
• The types of mucopolysaccharides
 Neutral mucopolysaccharides
 Acid (simple, or non-sulfated)
 Acid (simple, mesenchymal)
 Acid (complex, or sulfated, epithelial)
 Acid (complex, connective tissue)
ACID MUCOPOLYSACCHARIDES

• are polysaccharides with hexuronic acid as secondary

carbohydrate constituent, bound to sulfuric acid esters and
proteins
• only large group of carbohydrate compounds that are not
strongly PAS positive.
• They may, demonstrated by the following staining methods:
• 1. Metachromatic staining
1. Toluidine blue and Azure A
2. Uranyl nitrate- Azure method
• 2. Alcian blue technique
• 3. Colloidal iron technique
• 4. Aldehyde fuchsin stain
• 5. Mucicarmine stain
• 6. Fluorescent acridine orange technique
METACHROMATIC STAINING

• For Acid mucin

• Azure A - most useful metachromatic dye for acid mucin

• - intensity of staining appears to be due to the initial
potassium permanganate oxidation step which aids in uptake of
dye by the tissue.
• Uranyl nitrate Azure method
- gives excellent results with connective tissue mucins
- only method that is alcohol-fast, allowing permanent
preparations to be made easily.
• For metachromatic staining, mercurial fixatives are used.
• Toluidine blue
- a basic thiazine metachromatic dye with high affinity for acidic
tissue components and nucleic acids.
- used to highlight tissue components such as cartilage or
certain types of mucin.
Metachromatic Toluidine Blue Method
• Section: Frozen section
• Solution: 0.25% toluidine blue in pH 4.5 buffer
• Results: Mucopolysaccharide red purple
Tissue background blue

Alcian Blue Technique, pH 2.5 (Luna 1968; Carson 1983)

• histological dye that forms electrostatic bonds with certain tissue
components containing either carboxyl or sulfate groups
• most popular method for general demonstration of acid
Mucopolysaccharides, using 3% acetic acid at pH 2.5
• Fixation:10% neutral-buffered formalin or Bouin’s solution
• Sections: 4-5 μm paraffin sections
• Solution:
• Acetic Acid, 3% Solutionl
• Alcian Blue, 1% Solution
• Nuclear-Fast Red (Kernechtrot) Solution
• Results:Acid mucins blue , Nuclei red
Avoid celloidinization of slides as the alcian dyes are strongly retainedby
celloidin.

Uranyl Acetate – Azure A Metachromatic Tech.

To prepare stain : Azure A – 0.2g
Distilled water – 100ml
• Filter the staining solution before use, particullarly if the sol. Is more
than one month old. Discard sol.s that are more than 6 months old.
• Acid mucopolysaccharide are colored in crimson or red violet.
• Other tissue components are colored with various shades of blue.
Fresh Frozen Azure A Metachromatic
stainingforglycosaminoglycans
• Soln.s – Saturated azure A in absolute alcohol
Saturated azure A in 70% alcohol

Results : Glycosaminoglycans – red- purple

Tissue background - blue
Combined Alcian Blue-PAS-Hematoxylin Technique for
acid and neutral mucins

• useful for demonstrating the presence of any mucin,

especially for separating acid mucins and neutral mucins.
• only the neutral mucins will be stained by PAS, while acid
mucins will be stained by alcian blue.
• Fixative: 10% neutral-buffered formalin or Zenker’s solution
• Sections: 4-5 μm paraffin sections
• Results:
• Acid mucins -blue
• Neutral mucins - magenta
• Mixtures of above the color will range from blue-purplethrough purple to a violet or
mauve color, depending on dominant component
• Nuclei - pale blue
• It is important to stain only lightly with hematoxylin to distinguish it from alcian blue
staining. Ehrlich's hematoxylin should be avoided as counterstain
Gomori's Aldehyde Fuchsin Stain
• Gomori in 1950- introduce the Aldehyde Fuchsin as an elastic tissue
stain.
• demonstrate sulfur -containing compounds
• other tissue components are equally stained, including acid mucopoly-
saccharides, sulfated muco substances, pancreatic islets
ofLangerhans, thyrotrophic hormones, and secretory substances.
• It is also used to stain mast cells, particularly when no counterstaining
is done.
• has a greater affinity for sulfated mucins which are stained purple,
while the carboxylate forms stained blue after subsequent
counterstaining with alcian blue.
Combined Aldehyde Fuchsin – Alcian blue
• Alcian blue soln. – pH 2.5
• Aldehyde Fuchsin sol.
• Basic fuchsin
• Para aldehyde
• Conc. HCl
• Ethanol
• Distilled water
 Dissolve the basic fuchsin in alcohol- distilled water
 Add con. HCl and paraldehyde
 Allow to ripen 2- 7 days at room temp. (soln. change progressively from
magenta – purple color as the dye fraction becomes more soluble in the
solvent).
 Filter and store at 4 deg. Cel.
 Aldehyde fuchsin will keep for 2- 3 months
MUCICARMINE STAIN
• Adding aluminum hydroxide to carmine (Southgate 1927) improved
• the ability of carmine solution (Mayer 1896) to stain for mucin,
• aluminum salts in the solution form a chelate compound with
carmine, which binds to the mucin-containing tissue.
• Binds only to mucins
• Southgate's mucicarmine technique - is useful for staining
encapsulated fungi, e.g. Cryptococcus neoformans.
• Results: Mucins- red, Nuclei- blue, Background- unstained
• Ehrlich's hematoxylin should be avoided as a counterstain. Bec.
certain mucins pick up the hematoxylin and consequently will not
be stained.
COLLOIDAL (DIALYZED) IRON TECHNIQUE
• Principle: At low pH, colloidal iron will be adsorbed onto tissue containing acid
mucins, andsubsequently visualized by conversion to ferric ferrocyanide
(prussian blue) using the conventional Perl's technique.
• PAS reaction - give color differentiation between acid and neutral mucins.

• Greater sensitivity and intensity of reaction compared to alcian blue staining

• More complex and time-consuming method

• It requires formalin fixation.

• The actual blue color comes from a Prussian blue reaction.

• Can be pre-digested with hyaluronidase to provide more specificity.

 FLUORESCENT ACRIDINE ORANGE
TECHNIQUE
• Acridine orange – as a fluorochrome, use to demonstrate acid mucins.
• Stained with iron hematoxylin subdue the other fluorescence gives a color
black and acridine orange gives a selectibe brilliant orange fluorescence
• Disadvantage: temporary and will only last for about 2 hours once the
section is mounted.
• Fixation: Formalin and other fixatives, except heavy metals.
• Sections: Frozen or paraffin Sections
• Results:
Acid mucopolysaccharides Black
Fungi Greenish red flourescence
Background Reddish orange fluorescence
HALE’S ( DIALYZED) IRON TECH.
• Results :Acid mucin – dark blue , Nuclei – red

Neutral Mucopolysaccharides
• found in glands of the GI tract and in prostate.
• stain red with PAS and can be distinguished from acid
mucopolysaccharides
• do not stain with Alcian blue, colloidal iron, mucicarmine, or
metachromatic dyes.
• combination of the alcian blue and the PAS techniques can be
used as a means of distinguishing neutral mucins from acid mucins