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Sem1_P2

The document outlines a procedure for observing the stages of mitosis using onion root tips, detailing the necessary materials, theory behind mitosis, and step-by-step instructions for preparing root tip squashes. It explains the phases of mitosis (prophase, metaphase, anaphase, telophase) and the process of cytokinesis, as well as the advantages of using onion roots for this experiment. The procedure includes fixation, staining, and preparing slides for microscopic observation of actively dividing cells.

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yaragollanaagaanandini
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6 views5 pages

Sem1_P2

The document outlines a procedure for observing the stages of mitosis using onion root tips, detailing the necessary materials, theory behind mitosis, and step-by-step instructions for preparing root tip squashes. It explains the phases of mitosis (prophase, metaphase, anaphase, telophase) and the process of cytokinesis, as well as the advantages of using onion roots for this experiment. The procedure includes fixation, staining, and preparing slides for microscopic observation of actively dividing cells.

Uploaded by

yaragollanaagaanandini
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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P2.

Preparation of different stages of Mitosis (onion root tips)

Aim: To view different stages of mitosis using onion Root tips

Materials Required: Onions with growing roots, petri plate, blade, foreceps, microscope,
ethanol, acetic acid.

Theory:

All the eukaryotic cells are diploid, that is they have two sets of linear chromosomes. When a
new cell is born, it has two sets of chromosomes. Soon after its birth, a somatic cell takes a
decision whether to grow and divide or to grow and remain quiescent. If the cell takes the
decision to grow and divide, it enters the G1 phase of the cell cycle and progresses through S
phase , G2 phase and M phase. During S phase, all the chromosomes are duplicated due to the
action of replication machinery of the cells, resulting in 4 sets of chromosomes in its nucleus.
The cell then enter G2 phase which is the preparatory step to M phase. In M phase (mitotic
phase ), the cell undergoes karyokinesis followed by cytokinesis.

The mitotic phase can be discussed under four stages, 1. Prophase, 2. Metaphase, 3. Anaphase,
and 4. Telophase.

Prophase: The condensation of duplicated chromosomes from the diffuse network of chromatin
are hallmarks of the first stage of mitosis, called prophase

Metaphase: Attachment of spindle microtubules to the kinetochores indicates that the cell is
entering the metaphase of mitosis. During metaphase the duplicated chromosomes move to
positions midway between the spindle poles.

Anaphase: The sister chromatids of duplicated chromosomes are separated from each other
during the anaphase of mitosis. This separation is accomplished by shortening the microtubules
attached to the kinetochores

Telophase: Each set of chromosomes becomes enclosed by a nuclear membrane. The


decondensation of the chromosomes and the restoration of the internal organelles are
characteristic of the telophase of mitosis

Cytokinesis: When mitosis is complete, the two daughter cells are separated by the
formation of membranes between them. In plants, a wall is also laid down between the
daughter cells. This physical separation of the daughter cells is called cytokinesis.
I. Viewing mitosis in onion root tips.

Why use onion roots for viewing mitosis?


• The roots are easy to grow in large numbers.
• The cells at the tip of the roots are actively dividing, and thus many cells will be in
stages of mitosis.
• The tips can be prepared in a way that allows them to be flattened on
microscopes slide (“squashed”) so that the chromosomes of individual cells can be
observed.
• The chromosomes can be stained to make them more easily observable.

Regions of Onion Root tips

There are three cellular regions near the tip of an onion root.
1. The root cap contains cells that cover and protect the
underlying growth region as the root is pushed through the
soil.
2. The region of cell division (or meristem) is where cells
are actively dividing but not increasing significantly in size.
3. In the region of cell elongation, cell are increasing in
size, but not dividing.

Procedure for preparing root tip squashes

Fixation (use any one of the following four fixatives)

1) 3 volumes absolute ethanol : 1 volume acetic acid (v/v),

2) glacial acetic acid

3) 3 volumes dimethyl sulfoxide : 1 voume chloroform (v/v), and

4) 2 volumes acetone : 1 volume acetic acid (v/v) + 2% (w/v) polyvinylpyrrolidone (PVP) of


molecular weight 40,000.

The first step will be to ‘soften’ the roots so that they later can be spread on a
microscope slide.
1. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into a plastic
micro-tube. (One of the rots will be an extra one.)
2. Fill the tube about 2/3 full with 1N HCl from a dropper bottle.

3. Place the tube in a 60OC water bath, and allow the roots to incubate for 12 minutes.
4. After the 12 minute incubation period, remove the tube from the water bath.

Rinse the roots in H2O.


1. Using forceps, carefully transfer the root tips to a small petri plate.
2. Using a plastic ‘squeeze’ pipet, carefully remove the HCl from the micro-tube and transfer it
to
the “discard flask”.
3. Rinse the root tips 3 times with water from the dropper bottle, disposing of the rinses in the
discard flask.

Staining the chromosomes.


1. After removing the water from the third rinse, cover the root with the stain (Carbol. Fuschin)
2. Incubate the roots in the stain for 12 minutes. During this time the very tip of the root will
begin to turn red as the DNA stains the numerous small actively dividing cells at the tip.

Remove the stain and again rinse the roots.


1. Using a plastic ‘squeeze’ pipet, carefully remove the Feulgen stain and discard it in the discard
flask.
2. Again, rinse the root tips 3 times with water.

Preparing the root tip squash.


1. Transfer a root to the center of a clean microscope slide and add a drop of water.
2. Using a razor blade cut off most of the unstained part of the root, and discard it.
3. Cover the root tip with a cover slip, and then carefully push down on the cover slide with the
wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide sideways.
The root tip should spread out to a diameter about 0.5 – 1 cm.

Observations of onion root tip squash.


Scan the microscope under the 10x objective. Look for the region that has large nuclei relative to
the size of the cell; among these cells will be found cells displaying stages of mitosis. Examples
are shown in the figure to the right. Switch to the 40X objective to make closer observations.
Pictures to be pasted in the record

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