Materials and methods

Preparation of reagents
Phytohaemagglutinin (PHA-M: 5mg stock):-
Dissolve 5 mg stock of PHA-M in 5 ml of distilled water.
Colchicine (Stock-1gm):-
Dissolve 1mg of stock Colchicines (98%), (MW-399.45) in distilled
water to obtain desired quantity of 0.04micro gram/ml concentrations.
Hypotonic KCL Solution (0.075M):-
Dissolve 5.6 gm of KCL (99.9%), (MW-74.55) in 1000ml of distilled
water.
Fixative [Methanol and Glacial Acetic acid in the ratio 3:1 (v/v) ]:-
Dissolve three parts of absolute methanol (99.8%), (32.04g/mol) and
one part of glacial acetic acid (99.7%), (MW-60.05). Methanol acts as
a fixative and acetic acid as the scavenger. Better morphological
fixation occurs at low temperatures.
Sorenson’s Buffer
Solution A: 0.95 of potassium dihydrogen phosphate (KH2PHO4) was
dissolved in1000ml distilled water.
Solution B: 0.9% of Disodium dihydrogen phosphate (Na2HPO4) was
dissolved in 1000ml of dissolved water.
Then mix equal volumes of solution A and solution B was mixed to
get a pH 6.8.
Giemsa stain:
Stock solution
1 gm of giemsa powder was dissolved (E.Merk) in 66ml of Glycerin,
incubated in water bath at 60 ͦ C for 2 hours and 66mlof methanol was
added.
Working solution
10% Giemsa staining solution was prepared by dissolving 10ml stock
Giemsa stain in 90mlSorenson’s buffer.
CRITERIA FOR IDENTIFYING BINUCLEATED CYTOKINESIS
BLOCKED CELLS
The cytokinesis blocked cells scored for micronucleus frequency have
to satisfy the following criteria (Fenech, 2003);
 Cells should have two nuclei of approximately equal size.
 The two nuclei may be attached by a fine nucleoplasmic bridge.
 The two nuclei may overlap slightly or touch each other at the
edges.
 Cells should not contain more than six micronuclei

CRITERIS FOR IDENTIFYING MICRONUCLEI.

Micronuclei are morphologically identical to, but smaller than

normal nuclei.
They also have the following characteristics.
 Diameter between 6/12 and 1/3 that of main nuclei.
 They are non –retractile.
 They are not linked to the main nuclei via the nucleoplasmic
bridge.
 Micronuclei may sometimes overlap the boundaries of the main
nuclei.
CYTOKINESIS BLOCK MICRONUCLEI (CBMN) ASSAY
1. Fresh blood was collected by venepuncture under sterile
conditions and transferred to heparinised vacutainer.
2. Lymphocytes were isolated on lymphoprep (pharmacia)
gradients as follows: 2 ml of lymphoprep was added to a 10
ml centrifuge tube and carefully overlayed 4 ml of diluted
blood sample. Then it was centrifuged at 1000 rpm for 10
minutes.
3. Lymphocyte layer was drawn off using a sterile Pasteur
pipette and transferred to a 10 ml tube. The cell pellet was
suspended in RPMI 1640 medium and centrifuged for 10
minutes at 1000 rpm.
4. Supernatant was removed and the above step was repeated.
5. Isolated lymphocytes were cultured in sterile bottle
containing 10 ml of RPMI 1640 medium supplemented with
15% foetal calf serum. Lymphocytes were stimulated to
divide with phytohemagglutinin ( PHA , 10 µg/ml) and
incubated at 37 ͦ C for 72 hours.
6. 44 hour after PHA stimulation, cytochalasin – B was added to
the culture to give a
7. final concentration of 4.5 µg/ml.
8. At the 72th hour, the culture was harvested. The whole
content was transferred to a sterile centrifuge tube and was
centrifuged at 1000 rpm for 10 minutes.
9. The supernatant was discarded, and pellets were re-suspended
in prewarmed hypotonic KCL (0.075) and incubated for 10
minutes at 37 ͦ C
10. 2-3 drops of freshly prepared methanol: acetic acid (3:1)
was added and centrifuged at 1000 rpm for 10 minutes.
Supernatant was removed and pellets were broken.
11. The contents were fixed in freshly p9repared methanol :
acetic acid (3:1) mixture and kept in refrigerator for at least
10 minutes for proper fixation.
12. Fresh fixative was added, and centrifuged, and repeated
the process 3-4 times until the supernatant appear clear.
13. A cell suspension is prepared with the same fixative.
Dropped the cell suspension on to a pre cleaned, labelled and
chilled slides from a particular height.
14. The slides were air dried and then stained with 10%
Giemsa staining solution.
15. Observed the stained slides under a research microscope.
The slides were coded before scoring and examining at 100 X
magnification. The number of micronuclei (MN) in no less
than 1000 binucleiated cells was recorded.