Looking at Chromosomes and the Stages of Mitosis
Introduction:
Mitosis is a process that is vital to the understanding of cell biology and the growth and
reproduction of multicellular organisms.
One of the best places to see cells undergoing mitosis is in root meristems, the zone of dividing
cells in a root tip. There are several reasons for this:
 the material is easy to grow
 the root tip meristem tissue contains a large congregation of small cells whose function is to
divide and provide new cells for new root growth
 cells need to be squashed (spread out) so that individual cells can be seen. In the root tip
the cells are relatively easily spread after treatment with acids.
How to grow roots to observe;
Three methods are recommended:
1. Broad bean lateral roots
2. Garlic bulb roots
3. Seedling roots
Broad bean lateral roots
It is easiest to grow roots from broad bean seeds in pots of pumice or vermiculite as a growing
medium. In a deep pot, plant as many seeds as you will need, water and allow to germinate.
Placing the pot in an incubator at 250C will speed up the process.
Inspect the seeds regularly; it will take 4-6 days. When the roots are about 3 - 5 cm long (left photo
below) uncover the seeds, wash and cut off about 1 cm from the root tip (centre photo). Replant
the seeds in the same pot and growing medium and water again.
In a matter of days each plant will have produced a large number of fine, lateral roots (Right photo
below). These lateral roots are much easier to use because they are finer and squash more easily.
Stages in the production of broad bean lateral roots.
Garlic bulb roots
This method is very simple. Garlic bulbs are separated from the
clove and dress-making pins are used to suspend the bulb in the
mouth of a boiling tube. The tube is filled with water so that the
bulb is half in and half out of the water. Roots normally grow
within a few days and are ready for examining under the
microscope in about 1 week.
One issue that might occur is the ‘no-sprout’ treatment
sometimes used to prevent the growth of bulbs in storage. The
same treatment is used for onions and potatoes. It involves using
plant hormones (auxins) that inhibit the growth of buds and roots.
If this is an issue choose a different clove of garlic or grow seeds
as below.
This method can also be used with onions suspended similarly in
a 250 ml beaker of water with toothpicks.
Seedling roots
Producing seedling roots is simple:
 Cut at least 12 layers of newspaper so that they just fit inside the base. Flood this base with
water and allow it to stand for 10 minutes. Water will saturate the paper and this will be
enough to supply the seeds as they germinate.
 Pour off any excess water, if seeds are flooded they will drown (due to lack of oxygen) and
not germinate.
 Seeds are simply placed on the damp paper. Small, fast growing seeds are best. Suitable
seeds include: cress, mustard, onion etc. Slightly larger seeds like radish work well too but
because the roots are thicker they can be more difficult to mount on a microscope slide.
 Place in an incubator at 250C.
Petrie dish lid
Air gap between lid and base
Petrie dish base
Seeds (12 is enough)
At least 12 layers of newspaper
cut to fit into base
Petrie dishes with germinated seeds: Radish (left) and Cress (right)
Preparing specimens for microscope examination.
This process has 2 aims:
 Staining the chromosomes so that they are visible under the microscope. The staining
process ‘fixes’ or kills the cells and you are looking for cells ‘caught in the act’ of mitosis.
 Breaking down the middle lamellae of the cell walls of the root tip cells so that the cells can
be separated into a single layer (squashing).
Staining
Instructions for two methods are given below:
Method 1:
1. Cut off the tip 2 mm of your roots (do a number of root tips at once).
2. Place tips a watch glass with 10 drops of ‘aceto-ocein’ stain and 1 drop of 1M HCl.
3. Heat in a flame. Many students make the over-heating their roots tips.
Hold the watch glass by hand and do not heat it so much that steam is visible. You will
need to keep the root tips warm (but not hot) for 10-20 minutes. As they cool reheat them in
the flame.
4. Transfer one root tip to a slide; add a drop of the ‘aceto-ocein’ stain. Add a cover slip and
squash (see below). If it does not squash, keep heating the rest of the tips and try another
after 5 minutes..
Method 2:
1. Cut of the tip 2mm of your roots
2. Place in a test tube of 0.5% ocein stain dissolved in white vinegar. Use a bung to seal the
tube.
3. Keep warm for 24-48 hours. An incubator is ideal but a hot-water cupboard is also
adequate. Time is flexible here. If you cut and warm a number of root tips try one and if it
does not squash, keep warming the rest and try again later.
4. Transfer root tips to a slide, add a drop of your ‘aceto-vinegar’ stain. Add a cover slip and
squash (see below).
Squashing:
The objective of this process is to spread the root tip cells into a single layer that will allow
observation. The root tips will be highly stained and because they are quite thick no light will be
transmitted through them and no individual cells, let alone any chromosomes will be visible.
There are several methods of ‘squashing’. All have the risk of cover slip ‘breakage’ and most
students will break one or two in this way.
Methods to try:
1. Tap the blunt end of a pencil on the cover slip immediately above you root tip. You will soon
see if you are being effective as the cells will spread out. Many light taps are better than
fewer, heavier ones. Stain will exude from under the cover slip and will need to be soaked
up with absorbent paper.
2. Make sure the slide is on a perfectly flat surface, with several layers of newspaper under it.
Fold another piece of newspaper about 4 or 5 times to make a thick wad. Place this above
the specimen and with your thumb apply pressure directly down onto the specimen. Lift to
see if you have been effective and repeat several times. In this method any stain that gets
pressed out from under your cover slip will be mopped up, so the slide will dry out quickly
once you start to observe it. You can prevent this by adding a small drop of stain along the
edge of the cover slip and allowing it to flow back under the cover slip.
How many cells will show stages of mitosis?
Many students are disappointed with this exercise because they prepare their slides well but see
few cells undergoing mitosis. There are several possible reasons for this:
1. Cells spend more time growing than they do dividing
Mitosis produces two smaller cells and before these cells are ready to divide again they
need time to grow (this happens during interphase).
As an example, if a cell spends 98% of its time in interphase (and it is usually more than
98%) then it will only spend 2% (or less) of its time in mitosis. This means that all things
being equal only 2% of the cells you see will be undergoing mitosis.
The implication of this is that you will need to search around you slide to find cells caught in
division.
Some stages of mitosis also occur very quickly. Anaphase, (when chromosomes are
separated on the spindle) is very rapid and ‘catching’ cells at early anaphase requires
patience.
If you find a slide that is particularly good and you want to keep it seal around the cover slip
with clear nail varnish to prevent desiccation. The slide will then be able to be kept for a few
weeks.
2. Biorhythms
Mitosis does not always occur equally throughout a 24 hour period. There are peaks and
troughs of activity. This means that at some times of the day no mitosis will occur and at
others lots of cells will be caught at various stages of mitosis.
This of course suggests an investigation. Root tips could be collected every 2 hours during
a 24 hour period and stained and squashed for later observation to determine when peaks
of mitosis occur.
Prepared slides showing cells undergoing Mitosis
The emphasis in the resource is to encourage students to observe the slides they have prepared
themselves. However due to the difficulties in preparing slides with cells undergoing mitosis the
use of prepared slides can be useful backup.
These slide are usually Longitudinal Sections (L.S.) of Onion or Lily root tips. They show the root
cap, meristem and some of the lower part of the root with slightly elongated cells above the
meristem. Purchase and use these if necessary.
Images: Garlic root tips
When you squash a slide,
air bubbles are often
sucked back in when the
pressure is released.
You will also see the more
elongated root cap cells
on the right of the
photograph.
The place to start
searching for cells
undergoing division is on
the left, look at the smaller
cells that are cubic in
shape
Medium Power (x100)
In these High
Power (x400)
photographs
can you
recognise any
of the stages
of mitosis?
Images: using prepared slides
These are pictures taken of an onion seedling
root tip. The Low Power (x40) picture on the
right shows the root cap and the rows of cells
produced when meristematic cells divide.
Columns of cells are produced and as the cells
move away from the meristem, they start to
elongate and differentiate.
You will be able to recognise some of the
stages of mitosis in the High Power (x400)
photographs below.