EXPERIMENT NUMBER: 7

Object: to study different stages of mitosis by squash preparation of onion root tip

Requirements: Compound microscope, Acetocarmine stain, Water, Burner, N/10

Hydrochloric acid, Filter paper, Coverslip Aceto alcohol (Glacial acetic acid and Ethanol in the
ratio 1:3), Glass Slide, Onion root tip, Forceps,Blade, Watch glass, Dropper, Needle

Theory:
General Principle:
The genetic information of all organisms resides in the individual DNA molecules or
chromosomes. An onion cell possesses eight chromosomes whereas human cells possess forty six
chromosomes.

In 1842, C. Nägeli first saw chromosomes and in 1888 W. Waldeyer named them. Walther
Flemming studied and named the process of cell division as mitosis. Cell division occurs rapidly
in growing root tips of sprouting seeds or bulbs. An onion root tip is a rapidly growing part of the
onion and thus many cells will be in different stages of mitosis. The onion root tips can be prepared
and squashed in a way that allows them to be flattened on a microscopic slide, so that the
chromosomes of individual cells can be observed easily. The super coiled chromosomes during
different stages of mitosis present in the onion root tip cells can be visualized by treating with
DNA specific stains, like Feulgen stain and Acetocarmine stain.
All living organisms grow, differentiate and reproduce by cell division. There are two types of cell
division namely, mitosis and meiosis.

Mitosis
The type of cell division in which the number of chromosome in the daughter cells remain same
as in the parent cells is called Mitosis, it is known as duplication division, usually occur in somatic
cells of eukaryotes. It results in 2 daughter cells with same number of chromosomes. It is necessary
for growth and to replace were not cells. Each mitotic cell division has two events.

1. Karyokinesis (division of the nucleus)

2. Cytokinesis (division of the cytoplasm).

The events that occur during karyokinesis are:

1. Prophase: During this stage, the chromosomes super coil, condense and become visible for
first time during the cell cycle simply The chromatin network condense by uncoiling and
chromosomes become prominent. Each chromosome consist of two chromatids held together
by centromere. The spindle fibers start forming. The nuclear membrane starts disintegrating.

2. Metaphase: Spindle fibers are prominent, holding the chromatids at the equatorial plane so
during this phase the chromosomes arrange themselves at equatorial plane in the centre of cell.
3. Anaphase: (Movement of chromatids towards poles by splitting at the centromere.).
It begins when the centromere split and two chromatids of each chromosomes get separate.
The two sister chromatids face opposite poles. Each chromosomes in attach to the
discontinuous spindle fiber by centromere. Now spindle fibres contract and the sister
chromatids migrate towards opposite poles.

4. Telophase: it is the stage in which formation of daughter nuclei occurred. This phase begins
when all chromosomes have reached to the pole. Now chromosomes uncoil, become thin, long
and visible. Spindle fibres disintegrate, nucleolus reappear and nuclear membrane is formed
and each group of chromosome developed two daughter cell.

The meristematic cells of plants provide suitable material for studying mitosis. The root apices of
onion is used in the experiment.

Procedure:

1. Place an onion on a tile With the help of a sharp blade, carefully snip the dry roots of the onion
2. Place the bulbs in a beaker containing water to grow the root tips It may take around 4 to 6
days for the new roots to grow and appear Trim around 3 cm of the newly grown roots and
place them in a watch glass
3. With the help of forceps, shift it to a vial holding freshly prepared aceto-alcohol i.e., a mixture
of glacial acetic acid and ethanol in the ratio 1:3
4. Allow the root tips to remain in the vial for one complete day
5. With the help of forceps, pick one root and set in on a new glass slide
6. With the help of a dropper, allow one drop of N/10 HCl to come in contact with the tip of the
root. Additionally, add around 2 to 3 drops of the acetocarmine stain
7. Heat it lightly on the burner in such a way that the stain does not dry up
8. Excessive stain can be carefully treated using filter paper
9. The more stained part of the root tip can be trimmed with the help of a blade.
10. Discard the lesser stained part while retaining the more stained section
11. Add a droplet of water to it
12. With the help of a needle, a coverslip can be mounted on it
13. Gently tap the coverslip with an unsharpened end of a needle in order for the meristematic
tissue of the root tip present under the coverslip to be squashed properly and to be straightened
out as a fine cell layer
14. The onion root tip cells’ slide is now prepared and ready to be examined for different stages of
mitosis
15. Observe and study mitosis by placing the slide under the compound microscope. Focus as
desired to obtain a distinct and clear image
16. Switch to 40x for a better view.

Observation and conclusion: