Cell Biology

Lecturer: Assist. Prof. Dr. Altijana Hromić-Jahjefendić

Assistant: Furkan Enes Oflaz

LAB EXPERIMENT 4: Mitosis in Onion Root Tip Cells

Objective
After completing this exercise, you should be able to:

1. Better understand the process and stages of mitosis.

2. Prepare your own specimens of onion root in which you can visualize all of the stages of
mitosis.

3. Apply an analytical technique by which the relative length of each stage of mitosis can
be estimated

1. Introduction

The genetic information of plants, animals and other eukaryotic organisms resides in
several (or many) individual DNA molecules, or chromosomes . For example, each human cell
possesses 46 chromosomes, while each cell of an onion possesses 8 chromosomes. All cells
must replicate their DNA when dividing. During DNA replication, the two strands of the DNA
double helix separate, and for each original strand a new complementary strand is produced,
yielding two identical DNA molecules. DNA replication yields an identical pair of DNA molecules
(called sister chromatids) attached at a region called the centromere.

DNA replication in eukaryotes is followed by the process called mitosis which assures
that each daughter cell receives one copy of each of the replicated chromosomes. During the
process of mitosis, the chromosomes pass through several stages known as prophase,
metaphase, anaphase and telophase. The actual division of the cytoplasm is called cytokinesis
and occurs during telophase. During each of the preceding stages, particular events occur that
contribute to the orderly distribution of the replicated chromosomes prior to cytokinesis.
The stages of mitosis
Prophase. During prophase, the chromosomes supercoil and the fibers of the spindle
apparatus begin to form between centrosomes located at the pole of the cells. The nuclear
membrane also disintegrates at this time, freeing the chromosomes into the surrounding
cytoplasm.

Prometaphase. During prometaphase, some of the fibers attach to the centromere of each
pair of sister chromatids and they begin to move toward the center of the cell.

Metaphase. At metaphase the chromosomes have come to rest along the center plane of the
cell.

Anaphase. During anaphase, the centromeres split and the sister chromatids begin to
migrate toward the opposite poles of the cell.

Telophase. During telophase, the chromosomes at either end of the cell cluster begin to
cluster together, which facilitates the formation of a new nuclear membrane. This also is when
cytokinesis occurs, leading to two separate cells. One way to identify that telophase has begun
is by looking for the formation of the cell plate, the new cell wall forming between the two
cells.

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 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.

Region of cell elongation

Region of cell division

Protective root cap

Viewing Chromosomes

Chromosomes generally are not visible as distinct entities in nondividing

cells, since the DNA is uncoiled, but the process of mitosis is facilitated by supercoiling of the
chromosomes into a highly compacted form. Supercoiled chromosomes can be visualized in
cells, particularly if they are treated with a DNA-specific stain, such as the Feulgen stain
2. Procedure for preparing root tip squashes

While actively growing onions are present in the lab for you to observe, you will be provided
with roots that have been previously harvested and treated with a fixative to stabilize the cells.
You will work in groups of two for this lab exercise.

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.

*** Caution: Work with the HCl carefully, it is a strong acid. ***
3. Place the tube in a 60 °C water bath, and allow the roots to incubate for 12 minutes.

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 Feulgen stain.

Caution: Although the Feulgen stain does not

appear colored, it will strongly stain skin and
clothing!!!!
 1. 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.

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. Since
prophase and prometaphase are difficult to
distinguish, classify all these cells as prophase

.
3. Results
Onion root tip squash

Find and draw a cell showing each stage of mitosis.

Prophase Metaphase Anaphase Telophase