This document discusses carbohydrates and their classification and identification. It describes: 1) Carbohydrates are abundant organic compounds in humans that originate from photosynthesis. Common types include monosaccharides like glucose and polysaccharides. 2) Polysaccharides can act as food storage or structural components. Mucins are large glycoconjugates found in connective tissue that contain carbohydrate chains. 3) Special stains like periodic acid Schiff stain carbohydrates pink and are commonly used to identify tissues like mucin or glycogen. Other stains target specific carbohydrates.

1. DONE BY: ASHWINI AJAY
GUIDED BY: Dr. DINESH

2. SYNOPSIS
INTRODUCTION
HISTORY
DEFINITON
CLASSIFICATION
STRUCTURE OF MONO AND POLYS
IDENTIFICATION OF CARBOHYDRATE
PERIODIC ACID SCHIFF

4. INTRODUCTION
Abundant
- organic
compoun
ds-
humans
Originate
- product
of
photosynt
hesis

5. HISTORY
Mid 19th century
1930’s -
Lyoglycogen and desmoglycogen - soluble and insoluble form.
Hyaluronic acid - Palmer and Meyer
Epithelial mucin silica or neuraminic acid

6. CLASSIFICATION

7. STRUCTURE
Structure of monosaccharides:
Basic and simple form
Formula - (CH2O)n
Basic monosacchride - 6 carbon atom
- glucose.
Neutral sugar - Glucose
Other neutral sugar - mannose ,
fructose and galactose.
Glucose is named as D (+)-glucose, D
represents the configuration
whereas (+) represents dextrorotatory
nature of the molecule.

8. Structure of polysaccharide:
Long monosaccharide unit - glycosidic linkage.
Act as food storage ( starch).

9. Is one of
polysaccharid
e
Only
polysaccharid
e in animalsOccupies a
Significant volume
of cytoplasm of
hepatocytes that
may form intra
nuclear inclusion.
Location:
Liver, skeletal
muscle,
cardiac
muscle, hair
follicle
Function:
storage form
of
carbohydrate
Disease
associated:
Ewings sarcoma
Rhabdomyosarco
ma
glycogen storage
disease

10. PROTEOGLYCANS
Referred to
as
connective
tissue
mucin
Larger glyco-
congugates that are
seen in higher
concentration with the
ECM
Carbohydrate
component -
glycosaminoglycans
GAGs- long
unbranched
polysaccharide
with repeating
disaccharide unit.

11. CHONDROITIN SULPHATE
Sulphate
d
glucosam
ine chain.
Location:
Cartilage,
tendon,
Ligament,
cell
membran
eHyaluronic acid
Anionic
nonsulfat
ed GAG.
Location:
Connecti
ve,
epithelial
and
neural
tissue.

12. CHARACTERISATION OF VARIOUS GAGs

13. • Carbohydrate content in mucin is 90% of its molecular weight.
• GAGs : Are acidic
• Polysacchride chains of mucins are: neutral or alkaline.
Polysacchrides covalently
linked with a protein core.
Funtionally different families (
Muc 1, 2, 3 etc)
Membrane bound mucins: Muc
1 and 4
Produced within the cell and
secreted :Muc 2 , 5a , 5b and 7
Neutral mucins: gastric mucosa, Brunner’s gland of duodenum and prostrate epithelia
Function:
Tissue
protection
Lubrication
and
protection
Pathology:
Presence of mucin in tumor: clue in
identifying malignancy.
Detectable mucin: carcinoma (epithelial
derived)
mucin neutral/acidic: identifying neoplastic
changes.

15. Tissue placed in fixative
promptly after removal is
capable of autolytic
reaction.
Immediate fixation is not
possible - refrigerated until
fixed.
Recommended fixatives:
1. Rossman’s fixative
2. Alcoholic formalin with
picric acid

16. CARBOHYDRATE
STAINING
1. Periodic acid schiff (PAS) technique
2. Alcian blue method
3. Combined alcian blue- PAS techhnique
4. Mucicarmine technique
5. Colloidal iron technique
6. Metachromatic staining
7. Iodine staining for glycogen
8. Enzymatic digestion technique
Diastase digestion, Sialidase digestion,
Sialidase digestion,
Hyaluronidase digestion

17. PERIODIC ACID SCHIFF
Was first
discovered by
McManus.
1947 Marches
demonstrated that
glycogen.
One of the most commonly
performed special staining
technique in histopathology
laboratory
Used to highlight molecules
with high percentage of
carbohydrate content such as
mucin, glycogen, fungi and
basement membrane in skin

18. PRINCIPLE OF PAS Oxidation( Periodic acid)
Chemical reaction(Schiff’s reagent with aldehyde)
Recolorization(water followed by sulphurous rinse)
Counterstain(Haemtoxylin and light green)

19. Preparation of
staining solutions
1) Periodic Acid Solution
:
Periodic Acid : 1 gram
Distilled water : 100 ml
2) Schiff’s Reagent :
Fuchsin Basic : 1 gm
Distilled water : 100 ml
Sodium metabisulphite : 2
gm
Conc. HCl : 2 ml
Charcoal activated : 0.3 g

20. PROCEDURE
Bring
sections to
distilled water
Periodic acid
for 5 minutes.
Rinse well in
distilled water
Schiff’s
reagent for 5-
15 minutes
Wash in
running tap
water for 5-10
minutes
Counter stain
with Herri’s
hematoxylin for
approximately
15 seconds.
Differentiate (if
necessary)
with acid
alcohol and
bluing as usual
Wash in tap
water
Rinse in
increasing
concentration
of alcohol
Clear in
xylene and
mount as
usual.
Formation of insoluble magenta colored complex denotes positive result

21. ALCIAN BLUE
Comprised of copper containing pthalocyanine ring
linked to 4 isothiouronium groups – strong bases -
account for cationic nature of the dye
Sulfate and carboxylate groups of proteoglycans
ionised at pH 2.5 and carry a negative charge
Sialo- and sulfo mucins also reactive at pH 2.5
So, they stain with alcian blue at pH 2.5

22. Alcian blueAluminium sulphate Nuclear fast red
REAGENTS

23. RESULT
• Sulfomucin,sialomucin
• Proteoglycans
• Hyaluronic acid
Blue
• Nucleus red

24. COMBINED ALCIAN BLUE- PAS TECHNIQUE
PRINCIPLE
Demonstrate presence of mucin
Differentiate acid mucin from neutral mucin
1st stain all acid mucin with alcian blue (blue)
Those acid mucin which are PAS +ve will not be stained on PAS
reaction
only neutral mucin will be stained(magenta)

25. MUCICARMINE
• Active dye molecule is aluminium – carminic acid complex known as CARMINE
Carminic acid produced from dried bodies of female Coccus Cacti insects
• Carmine complex has a positive charge and so attracts polyanions
• Useful for identification of adenocarcinoma ( especially of GIT)
REAGENTS :
1.Southgate’s mucicarmine solution
3.Acidified ferric chloride solution
RESULT:
Acidic mucins – deep rose to red
Nuclei – black
Other tissue elements – light yellow
CRYPTOCOCCUS STAINED BY MUCICARMINE