Experiment 4 Identification of Different Stages of

Mitosis in Onion Root Tips

EXPERIMENT 4
IDENTIFICATION OF
DIFFERENT
STAGES OF MITOSIS IN
ONION TIPS

Structure

4.1 Introduction 4.4 Identifying features of

mitotic stages
Expected Learning Outcomes
4.4 Observations and Results
4.2 Materials Required
4.5 Precautions
4.3 Procedure

4.1 INTRODUCTION
Cell division is a fundamental process by which an organism is formed. In
multicellular organisms certain cells divide throughout life to account for wear
and tear and for immune defense. The division of somatic cells is by
mitosis. The cell cycle consists of two major phases: interphase (the period
between divisions) and the M phase (the period of active cell division). Mitosis
is the nuclear division that is conventionally divided into four stages, namely,
prophase, metaphase, anaphase and telophase. It is generally followed by
cytokinesis.

In this exercise you are going to visualize the stages of mitosis in squashes of
onion (Allium cepa) root tips. For lab exercises it is an ideal material because it
is (i) Easily available and (ii) Chromosomes are relatively large, few in number
(2n=16) and stain dark. The mitotically active cells in the root are localized in
the meristematic region, near the tip. Before performing this lab exercise, it is
advisable to revise the unit on cell division.

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Expected Learning Outcomes

At the end of the lab exercise, you will be able to:

 prepare stained squashes of onion root tips;

 describe the identifying feature (s) of each stage;

 calculate the mitotic index and;

 perform the experiment independently preferably with another material

starting from pre lab preparations.

4.2 MATERIALS REQUIRED

The following materials are required for the experiment:

 Onion root tips

 Carnoy’s fixative / Acetic alcohol(acetic acid 1 part and ethyl alcohol 3

parts v/v)

 2N hydrochloric acid

 70% ethanol

 Dropping bottle of acetocarmine or acetoorcein (2%)

 Dropping bottle of acetic acid (45%)

 Microscope slides and cover slips

 Pasteur pipettes,

 Teasing needles, fine forceps and razor blade

 Watch glass,

 Filter paper / blotting paper

 Nail polish or DPX mountant

 Compound microscope.

 Spirit lamp

4.3 PROCEDURE
The protocol has been broadly divided into two stages (A and B) each
involving multiple steps.

A) Pre lab preparations

i. Place onion bulbs with their root side down in contact with water in a flask
or Coplin jar. You can make few cuts in this region to induce better
rooting.
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 Experiment 4 Identification of Different Stages of
Mitosis in Onion Root Tips

ii. After 3-4 days / sometimes more depending on the season, roots (2-3 cm
long) can be observed.

iii. Excise the root tips with a razor blade and transfer them to Carnoy’s
fixative (acetic -alcohol; 1:3v/v) for 24 hrs.

iv. Once fixed transfer the root tips to a storage solution (70% ethanol) and
keep these vials at 4-50C until required for the experiment. These tips can
be used for more than one year.

Fig. 4.1: Rooting in onion.

B) Preparation of stained squashes

i. Transfer few root tips from the fixative (acetic alcohol) / storage solution
onto a watch glass and wash extensively with water.

ii. Drain off the water with a Pasteur pipette and add a few drops of 2N HCl.
The tips are softened due to hydrolysis in 2N HCl for 10 minutes at room
temperature or 2-3 min over a spirit lamp flame. HCl also depurinates
DNA which improves staining.

iii. After hydrolysis drain off the hydrochloric acid (HCl) and wash the root
tips repeatedly with water. Carefully pick few root tips and place them on a
clean slide.

iv. Remove water and add 2% acetocarmine and cover the slide with a watch
glass and leave it for 10mn. Do not allow the tissue to run dry. If
required add more stain.

v. Transfer carefully the stained tips to another slide. Very gently tease the
extreme tip of the root to dislodge some of the root cap cells.

vi. Add a few drops of 45% acetic acid and excise all excess debris with the
help of a needle, leaving behind only the very deeply stained root tips, in
which the dividing cells are found.

vii. Place carefully a cover slip on the stained root tips and transfer the slide
between the folds of filter paper to remove excess acetic acid. Apply
moderate pressure in a vertical direction with your thumb or with the flat
bottom of a pencil. Tapping flattens the cells and spreads the
chromosomes. A properly squashed tissue should be about three times its
original size.

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Do not allow the cover slip to move laterally as this would ruin the
squash. At no time sho
should
uld the squash run dry.

viii. Seal the edges of the cover slip with DPX or nail polish to prevent drying.

ix. underr the compound microscope at 10X and 40X.

Examine the slides unde

4.4 IDENTIFYING FEATURES OF MITOTIC STAGES

You are already familiar with the stages of mitosis. In this section the features
of each stage and how the chromosomes appear are summarised. The period
between divisions is known as interphase in which a typical cell spends most
the time. In a squash of onion root tips, you will find that majority of cells are in
interphase.. The nuclear membrane is intact and the chromatin is decondensed
interphase
in the form of a network. Individual chromosomes cannot be seen. It is at this
phase that DNA is replicated and the cell makes preparations for cell division.

Prophase marks the beginning of mitosis.

At this stage:

 Chromosomes become visible (sister chromatids),

 Centrioles migrate to the poles (only in animals),

 Nuclear membrane disappears,

 Nucleolus disappears.

Fig.4.2: Prophase.

Metaphase:

 The chromosomes are maximally condensed at metaphase.

 All the chromosomes line up at the equator of the cell (metaphase plate)

 Spindles from both poles attach to the centromeres of sister chromatids.

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Experiment 4 Identification of Different Stages of
Mitosis in Onion Root Tips

Fig.4.3: Metaphase.

Anaphase:

 Sister chromatids separate.

 Sister chromatids move towards the poles.

Fig.4.4: Anaphase.

Telophase

 Unwinding of chromosomes and appearance of chromatin,

 Reconstruction of nuclear membrane,

 Disappearance of spindle.

 Reappearance of the nucleolus.

Fig. 4.5: Telophase.

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4.5 OBSERVATIONS AND RESULTS

Carefully observe the cells under the microscope. Y ou should either sketch the
You
stages or take pictures from your mobile camera.

i. Identify cells in different stages of mitosis.

i. Record the characteristics of the various stages that you could clearly
observe.

ii. Count as many cells as you can and complete the table below. Analyze
the data.
Interphase Prophase Metaphase Anaphase Telophase Total

number of
cells

percent of 100
cells

a) In which stage of the cell cycle did you find maximum number of cells?

mitotic index) and how much

b) What percentage of the cells are dividing ((mitotic
time they spend in each stage of mitosis?

The mitotic index is calculated using the following relationship:

4.6 PRECAUTIONS
You should take the following precautions to get clean and clear results:

 Slides and cover slips should be clean and free from dust and fingerprints.

 Do not let the cover slip move laterally as this would ruin the squash.

 At no time should the squash run dry.

 The excess mounting fluids should be removed carefully from the sides of
the cover slip using the edge of a blotting paper.

 Place the cover slip gently to prevent trapping of air bubbles.

 The material and fluid should be placed centrally on the slide.

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