MICRONUCLUES TEST

Preparation of Aqueous Extract

1. Dissolve lyophilized extract in sterilized distilled water to make a concentration of 10, 40, 70 and 100 mg/mL.

Indicator Plant
1. 18 onion bulbs (Allium cepa L.) of similar size and weight (about 30g) were selected (Mohammed, Aarey,
Tamkeen & Jahan, 2015).
2. Immerse bottom of the onion bulb in distilled water (50mL) for root growth in 2 days. Allow onion roots to grow
to 1-3 cm length at room temperature.
3. After the roots have grown, three bulbs showing acceptable growth were exposed into different concentration
of aqueous extract of lagundi (10, 40, 70 and 100 mg/mL) for 30 hours (Kataeva, Kotseruba, Terelhina, Kutlunina
& Beljaeva, 2012). Use distilled water for negative control and potassium dichromate for positive control
(Mohammed, Aarey, Tamkeen & Jahan, 2015).

Negative Positive
Lagundi aqueous extract
Control Control
Distilled H20 10mg/ml 40mg/ml 70mg/ml 100mg/ml Potassium
(20ppm) (80ppm) (140ppm) (200ppm) dichromate
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Slide Preparation
1. After exposure, root tips were harvested and hydrolyzed in 1N HCl for 10 minutes in water bath at 60 degree
celcius (Meneguetti at al., 2012)
2. Treat with carnoy’s solution (methanol:acetic acid, 3:1) for 4 minutes (Mohammed, Aarey, Tamkeen & Jahan,
2015).
3. Stain with acetocarmine/iodine stain for 30 minutes, then wash with sonicated distilled water and dry at room
temperature.
4. Observe under a microscope the formation of micronucleus in cell.

Indication of Micronucleus
The presence of micronuclei in a cell indicates anomalies generated by ruptures in DNA on failures at the level of
cell cycle (de Diana et al., 2018).
MICRONUCLUES TEST

Preparation of Aqueous Extract

1. Dissolve lyophilized extract of each plant sample in distilled water to make a concentration of 10, 40, 70 and 100
ppm to make 100 ml.

Indicator Plant
2. Mung bean (Vigna radiate) weighing approximately 1.50 mg were selected, with at least 4 mung bean per
concentration.
3. Soak mung bean for six (6) hours in distilled water for the negative control group (100 ml), in different
concentration of the plant aqueous extract for the test group and in Potassium dichromate for the positive
control group (Kumar & Singhal, 2009).
4. Remove the seedlings from the concentration substrate after six hours (negative control group, test group and
positive control group). Transfer the seedling into a petri dish with cover and keep moist by saturating cotton
with the respective substrates until the radicles had grown into 1.0 -1.5 cm.
5. The seedlings were maintained at room temperature under moist conditions for 48 hours (T48).Allow mung
bean roots to grow 1-3 cm.

Negative Positive
Lagundi aqueous extract
Control Control
Distilled H20 10mg/ml 40mg/ml 70mg/ml 100mg/ml Potassium
(20ppm) (80ppm) (140ppm) (200ppm) dichromate
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Slide Preparation
6. After exposure, root tips were harvested and hydrolyzed in 1N HCl for 10 minutes in water bath at 60 degree
celcius (Meneguetti at al., 2012)
7. Treat with carnoy’s solution (methanol:acetic acid, 3:1) for 4 minutes (Mohammed, Aarey, Tamkeen & Jahan,
2015).
8. Stain with acetocarmine/iodine stain for 30 minutes, then wash with sonicated distilled water and dry at room
temperature.
9. Observe under a microscope the formation of micronucleus in cell.

Indication of Micronucleus
The presence of micronuclei in a cell indicates anomalies generated by ruptures in DNA on failures at the level of
cell cycle (de Diana et al., 2018).