Core Practical 6: Prepare and stain a root tip squash to observe

the stages of mitosis

Objectives
 To know how to prepare a temporary slide of a root tip to observe mitosis
 To recognise the stages of mitosis in dividing cells
 To identify hazards, associated risks and control measures for the procedure
Safety
 Eye protection must be worn.
 Take care with glassware and scissors.
 Acetic orcein stain is corrosive, causes burns, has an irritating vapour and will stain.
Wear eye protection and avoid contact with skin. If contact does occur, wash the area
thoroughly with water for 10 minutes and tell your teacher. Avoid inhaling the vapour. If
you spill the stain, do not attempt to mop it up; tell your teacher instead.
 Avoid skin contact with the hydrochloric acid.
 If your microscope uses daylight illumination, be careful not to use it where sunlight
could strike the mirror.
 The water bath at 55 °C will scald your skin; cool under cold running water if you get
splashed.
 Do not handle electric plugs, sockets or switches with wet hands.
Maths skills
 Use ratios, fractions and percentages.
 Solve algebraic equations.
Equipment Diagram
 eye protection
 garlic clove with growing root tip
 glass slide and coverslip
 scissors
 water bath at 55 °C
 small bottle with lid or laboratory
stretch film
 hydrochloric acid 1 mol dm −3
 acetic orcein stain in a small bottle or
vial
 two dissecting needles
 paper towels
 microscope
 white tile
 fine forceps
 stop clock
 safety information sheet figure A Section of a root tip showing
meristem with dividing cells

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 Procedure
To see mitosis in action, you need to look at living cells. Garlic bulbs grow roots that have
actively dividing cells in their tips, in a region called the meristem (see figure A). Each cell
has only eight chromosomes, so it is relatively easy to see the chromosomes once they
have condensed. In order to see the chromosomes inside the cells, you need to separate
the cells and spread them out into a layer that is ideally just one cell thick. Plant cells are
glued together by a middle lamella of pectins. Hydrochloric acid will break down the pectins
that hold the cell together. Acetic orcein will stain the chromosomes dark red and fix the
cells, stopping mitosis.
You should examine your preparation carefully, looking for cells undergoing different
stages of mitosis. Identify the different stages by comparing your preparation with labelled
pictures or photographs of cells during mitosis. Bear in mind that mitosis is a dynamic
process, so cells may have been fixed in transition from one stage to the next. You will
have to interpret what you see.
Make sure you follow all necessary safety precautions. You should also complete your own
risk assessment before starting this practical. There is a risk assessment template for you
to fill in on page 33 of the Lab Book.
1. This first step may have been done for you. Fill a small bottle with 1 mol dm −3
hydrochloric acid and place it in a thermostatically controlled water bath set at 55 °C.
Leave the bottle for 15 minutes to allow the acid to warm to the temperature of the
water bath.
2. Place a garlic clove in the top of the bottle so that the roots are submerged in the
hydrochloric acid at 55 °C. Leave the roots in the acid for 5 minutes.
3. After 5 minutes, take out the garlic clove and rinse the roots thoroughly in tap water.
Use a pair of sharp scissors to cut off several root tips of 5–10 mm in length. Let them
fall into a small vial of acetic orcein standing on a white tile. Use the scissors to make
sure the root tips are immersed in the stain. Place a lid or laboratory stretch film over
the vial. Lids should have a pin-prick hole, or be slightly loose if they are screw caps,
to prevent the ejection of liquid during heating.
4. Place the vial containing the root tips in acetic orcein in the 55 °C water bath for
5 minutes to intensify the staining.
5. After 5 minutes, use forceps to remove the root tips from the vial and place them on a
microscope slide. Add a drop of water to the root tips on the slide. Tease each root tip
apart with needles (maceration), to spread out the cells a little. Cover with a coverslip.
Replace the lid on the vial of stain and return it to the teacher as instructed.
6. Wrap the slide in several layers of paper towel and press gently on the paper to
squash the tissues. Take care not to twist the slide as you press down, or the coverslip
will break.
7. Examine under the microscope on low power to identify the area of dividing cells or
meristem (see figure A). Position the cells in the centre of the field of view. Meristem
cells are small and square, have no obvious vacuoles and are usually found in rows.
8. Move to high power (×400). Identify as many stages of the cell cycle as you can in
your field of view.
9. Count the number of cells in each of the stages of mitosis, plus interphase, in the field
of view. Record your results in a table.
10. Draw and annotate one cell from each of the stages you have identified. Your
drawings will be simple outlines of the cells and the groups of chromosomes in them;
few other structures will be visible. Aim to show the relative sizes and positions of the
chromosomes and the cell accurately. Annotate your drawings to describe what is
happening.

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 Analysis of results
1. Calculate the percentage of the cells in each stage by dividing the number of cells in
the phase by the total number of cells and multiplying by 100. Add the percentages to
your table. You could use a spreadsheet to calculate and record these values.
2. Given that your preparation freezes the process of mitosis at one point in time, what do
the values calculated in analysis question 1 suggest to you about the length of time a
cell spends in each stage of mitosis?
3. If a group of cells is dividing rapidly, a high proportion of the cells will be undergoing
mitosis. A group of cells that is not dividing will have all cells in interphase of the cell
cycle (chromosomes will not be clearly visible). The amount of cell division occurring in
a tissue can be quantified using the mitotic index. The mitotic index is used to study
tumour growth in cancer patients. Use the formula below to calculate the mitotic index
for the root tip.
number of cells containing visible chromosomes
mitotic index 
total cells in field of view

Learning tips
 Before starting this practical, you may need to remind yourself about parts of the
microscope and how to use the instrument as detailed in the learning tips for Core
Practical 5.
 Cell counts for each stage of mitosis in the field of view should indicate the duration of
each of the stages. This will be a relative value – the more cells you can see in one
stage, the longer the duration of that stage in the cell cycle.
Questions
1. Explain why the root tip is heated with acid.
2. State the effect of maceration and pressing the slide preparation on the dividing cells.
3. Describe what information the cell counts give about each stage of mitosis.
4. Describe the role of mitosis in the life of an organism.

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 Exam-style questions
1. Observations of meristem cells of an onion root tip were made and the stage of the cell
cycle was recorded for each cell. The following results were obtained.

Phase Number of cells Cells in each phase (%)

prophase 25 10
metaphase 10 4
anaphase 5 2
telophase 10 4
interphase 200

(a) Calculate the percentage of cells that are in interphase.

(1)
(b) Assume that the mean total duration of the cell cycle is 18 hours for these
cells.
Calculate the actual time, in minutes, spent in anaphase. Show your
working.
(2)
2. Describe how cells from the root of a plant could be prepared in order to observe
mitosis.
(5)
3. Some students are planning to take measurements of root tip cells and compare the
size of cells in different stages of mitosis. They would like to make sure that their
results are valid, accurate and reliable.
(a) Explain what is meant by the term ‘valid results’.
(3)
(b) Explain how the students could ensure that their results are as accurate
(near to the true value) as possible.
(3)

© Pearson Education Ltd 2018. Copying permitted for purchasing institution only. This material is not copyright free.
Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment
information to local circumstances. This document may have been altered from the original. 4