feulgen reaction, chemistry

1. Feulgen stain

2. Feulgen stain
Is a staining technique
Discovered by Robert feulgen 1924
Used in histology to identify
chromosomal material or DNA

3. Depend on acid hydrolysis
therefore fixating agents using strong
acids should be avoided
The Feulgen reaction is a semi. quantitative technique
If the only aldehydes remaining in the
cell are those produced from the
hydrolysis of DNA then the technique
.is quantitative for DNA

4. The Feulgen reaction consists of two steps.
Initially, acid hydrolysis is performed, usually
for 8–12 min, resulting in the cleavage of the
nitrogen bases and formation of aldehyde
groups. The preparations are then placed in
light yellow Schiff reagent (fuchsinesulfurous acid), which forms bonds with these
groups. The red-violet product formed in this
step is evidence of the presence of DNA
RNA is not hydrolyzed by the HCl
treatment and, thus, the reaction is
. DNA-specific

5. Feulgen reaction mechanism&
definition
An aldehyde specific
reaction based on the formation of a
purple-colored compound when
aldehydes react with fuchsinsulfuric acid; deoxyribonucleic acid
gives this reaction after removal of
its purine bases by acid hydrolysis;
.used as a nuclear stain

6. The staining intensity is proportional to
the DNA concentration
.
hydrochloric acid does not hydrolyze
nucleic acids instantly therefore
prolonged gives more intense reaction

7. From the morphological point of view,
specific demonstration of DNA in cell
structures at the light microscopic
level is very little used nowadays. On
the other hand, application of the
Feulgen principles to electron
microscopy have recently allowed
specific DNA-staining procedures to
be

8. FEULGEN STAINING
PROTOCOL
.Bring sections to water.
.Rinse sections in M - HCL at room temperature 1 min.
Place sections in preheated M - HCL at 60°c (this hydrolysis time.
will vary and depends on the fixative. For formalin fixed tissues 8
.mins hydrolysis is required
.Rinse sections in M - HCL at room temperature 1 min.
.Transfer sections to Schiff's reagent 45 min - 1 hr.
.Rinse sections in bisulphite solution x 3 washes - 2 mins each.
.Rinse well in water.
.Counter stain with 1% light green in 1% acetic acid 40 sec 8
Mount sections in DPX . Dehydrate, clear.
RESULTS
DNA Magenta --Cytoplasm Green--

9. It is possible to use an instrument known as a microdensitometer or
microscpectrophotometer to actually measure the intensity of the pink
. feulgen reaction for a given organelle

10. Schiff's reagent
is prepared by pouring 200 mL of boiling distilled water
over 1-g basic fuchsin. Shake thoroughly, cool to
50°C, filter, and add 30 mL 1N HCl to the filtrate.
Cool to room temperature and add 1 g
potassiummetabisulfite
Allow the solution to stand overnight in the dark or
until a light straw or faint pink color develops. If not
completely decolorized, add 0.5 g charcoal powder,
shake, filter through a coarse filter, and refrigerate
in a tightly-stoppered bottle in the dark.
Commercially prepared Schiff's reagent can be
obtained from several companies

11. Schiff's reagent
is a very sensitive means of detecting aldehydes,
and can be used in a method to demonstrate
deoxyribosenucleic acid (DNA) specifically, in
contrast to unstained ribosenucleic acid (RNA)
because the acid hydrolysis causes it to
dissolve
or because of the hydroxyl group
present in ribose which has lost its oxygen in
deoxyribose
This method is the nucleal reaction of Feulgen.
, and Rossenbeck

12. it would seem that two things happen during
acid hydrolysis of DNA. The first is that the
purine bases are removed and aldehyde
groups on deoxyribose are formed. The
second is that histones (protein associated
with DNA) and apurinic acids (deoxyribose
without the base) are progressively
removed. Hydrolysis should be stopped and
Schiff's reagent applied when the first is
well on its way, but before the second has
. removed too much of the aldehyde