LAB TECHNIQUE

2. PAS TEST
SUDAN BLACK
FUELGEN REACTION
HISTOCHEMICAL
TESTS

3.  Histochemistry- chemistry of the tissues
 Cytochemistry - chemistry of cells
 Histochemistry - cover the properties of both
cellular and extracellular material
 Hischemistry is the investigation of tissue component by
applying to microscopical preparations method of
physical and chemical analysis which do not damage
the tissues

4. STAINING TECHNIQUE FOR
INSOLUBLE POLYSACCHARIDES
PAS TEST
 periodic acid schiff (PAS) is a staining method used to
detect polysaccharide such as glycogen and
mucosubstances such as glycoproteins,glycolipids and
musins in tissues
 The reaction of periodic acid oxidizes the vicinal diols
in these sugars,usually breaking up the bond between
two adjacent carbons

5.  PAS Staining is mainly used for staining structures containing
a high proportion of carbohydrate
macromolecules(glycogen,glycoprotein,proteoglycans)
 PAS staining can be used to assist in the diagnosis of several
medical condition
 glycogen reaction
 adenocarcinomas
alveolar soft sarcoma
 staining macrophages in whipples disease
 PAS is used to confirm the presence of glycogen
 PAS staining is also used for staining cellulose

6. Schiffs Reaction
 German chemist Hugo schiff (1834-1915) was first
recognize
 Schiff reagent is widely used in organic chemisrty and
histochemistry to detect aldehyde by forming the stable
red -magenta reaction product
 Periodic acid Schiff test is widely used for the
demonstration of vicinal glycol moieties in
carbohydrates

7.  PROCEDURE
 The tissue is first treated with periodic acid
 The 1-2 glycol linkage are then converted to aldehyde
groups
 Then treated with the clear Schiff reagent to form the
coloured (magenta) reaction product

8. Pas test

9. STAINING TECHNIQUE FOR
LIPIDS
SUDAN BLACK
 Sudan staining is the use of Sudan dyes to stain sudanophilic
substances, usually lipids
 Sudan lysochromes (Sudan II, sudanIII, sudanIV, Sudan
black B, oil red O are used
 Sudan dyes have high affinity to fats therefore they are used
to demonstrate triglycerides, lipids and lipoproteins
 For neutral fats sudan III, sudan IV are most commonly
employed
 The use sudan black B first introduced by Lison (1936)
 Sudan black is usually employed as a saturated solution in
70% alcohol

10. Sudan black

11. STAINING TECHNIQUE FOR NUCLEIC
ACID
FUELGEN REACTION
• Fuelgen reaction introduced by Fuelgen and Rossenback in
1924
• Fuelgen reaction is employed for the cytochemical
localization of deoxy ribonucleic acid
• Fuelgen stain is both a qualitative and quantitative stain for
DNA

12. Qualitative staining of fuelgen stain
for DNA
• The dye component of this stain is basic fuchsin (a chloride of
pararosaniline )
• This is prepared by boiling together small amount of aniline, p
toludine and mercuric chloride.then adding this mixture to 70%
alcohol.after this preparation, this forms a colourless solution
called lucobasic fuchsin or schiffs reagent
• This tissue should be hydrolyzed to enable it for staining.this is
most critical step in fuelgen staining
• During hydrolysis ,the purin bases are liberated from the
deoxyribose components liberating the aldehyde groups on the
sugar moiety.this insoluble product called apurinic acid reacts
with schiff’s reagentto stain the chromosome bright magenta

13. Quantitative staining of fuelgen stain
• By spectrophotometric analysis after proper standardization of
staining,the amount of DNA present in the chromosome can be
determined
PROCEDURE
 Take fresh, healthy, young root tips
 Wash them thoroughly in running water
 Cut them into suitable sizes
 Fix in acetic acid alcohol mixture for 45 min. to 1 hour
 Hydrolyze in normal HCl for 10-12 min. at 60 degree C in a water bath
 Rinse with distilled water
 Stain in leuco basic fuchsin solution for 30 min., till the tips become
bright magenta in colour
 Transfer to a clean glass slide
 Cut out coloured tip and then squash

14. Fuelgen reaction