D3.1.

1; Biodiversity and Humans

D3.1.2; Life Science Solutions to address
Challenges in Food, Water and Energy

Section 6
Pollutants Cytotoxicity Evaluation
Using Allium cepa Test.
 Cell Division
Meristem and Differentiated Cells

Cells of Root Tip Cells of Onion Epidermis

Cell Division of Mitosis

Root Tip
 Mitotic Division

* Occurrence
* Purpose
* Stages

Interphase Prophase Metaphase Anaphase Telophase

Fig: Mitotic Division Phases under light microscope

Mitotic Division
 (1)
---------------------------------

(3) (2)
Late Telophase
Interphase
Prophase
Interphase

Telophase
Anaphase

Metaphase

Anaphase
Metaphase

Fig: Mitotic Division Phases under light microscope

Cytokinesis
‫؟؟‬
‫؟؟؟‬
‫؟؟؟‬
‫؟؟؟‬
 (G0 Exit from cell
cycle- nondividing cell)

Cell Cycle
 Some Abnormalities of Mitosis
Mutagens have the ability to induce a number of mitotic abnormalities. The
abnormalities could be formed spontaneously i.e. without man interferes but can also
artificially induce as a result of an action by many chemicals or the effect of various types
of radiation.

Three major classes of aberrations were distinguished;

(a) Spindle disturbance and its consequences (e.g., C-metaphase, lagging chromosome,
star metaphase/anaphase, multipolar anaphase/telophase, unequal distribution, phase
disturbance and non-oriented chromosome).
(b) Clastogenic aberrations on chromosomes (e.g., bridges, fragments or breaks,
micronuclei and ring chromosomes).
(c) Chromosome stickiness.
1- Lagging Chromosome ‫الكروموسوم المعلق‬
 ‫‪3- Chromosomal bridge‬‬
‫النواة الصغيرة ‪2- Micronucleus‬‬ ‫القنطرة الكروموسومية‬
 ‫اإلستوائى الكولشيسينى ‪C-Metaphase‬‬

‫‪4- Inhibition of spindle formation‬‬

‫تثبيط تكوين خيوط المغزل‬
‫اإلستوائى النجمى ‪5- Star Metaphase‬‬ ‫اإلنحراف أو الميل ‪6- Diagonal Phase‬‬
‫؟؟؟‬

‫؟؟؟‬
???
 cytotoxicity
 Many substances or pollutants have a toxic effect. Some are more toxic than
others, and it is vital that scientists know the effects and how severely they can affect
an organism. Toxicity is defined as the quality of a substance (for example, a
fungicide) has of being toxic or poisonous. Toxicity is dependent on dosage.
 Whilst toxicity is a more general term for how harmful a substance is to an
organism; cytotoxicity is the term for how toxic a substance is to cells. A cytotoxic
compound can cause cell damage or death, either through necrosis or apoptosis.
Some substances are more cytotoxic than others and researchers aim to measure a
chemical’s cytotoxicity levels to ensure that it is not harmful and/or fatal to patients.
Examples of cytotoxic agents include chemotherapy drugs and certain venoms.
 The mitotic index (MI) indicates the frequency of cell division,
and has been regarded as an important parameter to examine the
cytotoxicity of agents. Decreased MI which may reflect a disturbance
in growth could be indicative of the presence of cytotoxic agents in
the environment. Chromosome aberration is referred to the atypical
number of chromosomes or changes in the chromosomal structure in
the cells exposed to physical or chemical agents. Different types of
Chromosome aberration such as chromosome bridges and breaks,
losses, C-mitosis, etc.. can be evaluated using A. cepa test.
 Calculations 
Preparation of a permanent slide using Feulgen squash method for mitotic division study
(1) Cultivation: cultivate the onion bulbs (in distilled water as a control) until the roots become 2-3 Cm.
*** Treatment (when the roots were of 2 cm, bulbs were exposed to the treatment)
(2) Cut the root tips (1 Cm.) and rinse in changes of water.
(3) Fixation: Fix the root tips in fixative solution (3 parts absolute alcohol: 1 part glacial acetic acid) for 24 hours.
(4) Rinse in water, 2-3 changes.
(5) Hydrolysis: hydrolyse the root tips in in 1N HCl at 55-60˚C for 10 minutes.
(6) Staining: stain in basic fuchsin (Feulgen), for 3-4 hours.
(7) Squashing: (a) Cut out the root tips of the meristematic zone (about 2 mm), on a slide with a drop of 45%
acetic acid, and apply pressure under several thickness of blotting paper, allowing no sideways movement of
cover slip.
(8) Samples were then dehydrated in absolute ethanol for 2-3 minutes, cover slips were separated and made
as permanent preparation by mounting in D.P.X.
(9) Slides were allowed to dry at room temperature for two days then, examine using the high power lens of
light microscope.
Thank you for careful listening