ROOT-TIP SQUASH METHOD
PREPARE AND STAIN A ROOT TIP SQUASH TO OBSERVE THE STAGES OF
MITOSIS
Event
Cut final 5mm of root tip (tissue) from onion
or garlic (organism).
Place root-tip tissue in 1 moldm3 HCL for 5
minutes
Rinse in distilled water
Add a few drops of suitable stain; acetic
orcein, toluidine blue, Schiffs, Feulgens. This
takes 5 minutes
Warm the slide using a Bunsen Burner.
Gently break open the tissue using mounted
needle on a microscope slide (physical
maceration).
Place a cover slip and gently squash
between the slide and coverslip, ensuring
that bubbles are not trapped between them.
Use blunt end of pencil to squash. Blot away
any free stain using filter paper.
Warm using Bunsen.
Observe using a microscope; starting with
low power and then high power.
Reason
Stages of mitosis can only be seen in
rapidly dividing cells (meristematic cells)
found in meristem (root tip).
To soften it by breaking down the pectin in
middle lamella that holds the cells
together. This is chemical maceration.
To remove acid
To make chromosomes visible
Fix the stain in the tissue.
To spread cells
To get a single layer of cells in order to
allow microscope light to penetrate
through the cells to be able to observe
mitotic stages in these cells.
To intensify the staining to improve the
viewing.
Cells are microscopic (very small) hence
the need for microscope.
The smaller the magnification, the greater
the number of cells that will be observed
in the field of view.
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Safety Comment
Cutting the tissue away from oneself using
scalpel & forceps
Eye care; wear goggles.
Skin care; wear gloves because acid can burn
the skin.
Eye care from stain; wear goggles.
Skin care from stains; wear gloves.
Protect clothes from stain by wearing lab
coats
Take care from burning by wearing heat proof
gloves.
Careful not to break the glass slides that may
cause injury.
Careful not to break slides and cover slips that
may cause injuries; wear gloves.
Take care from burning; wear heat proof
gloves.
Avoiding placing the microscope near the
edges of benches.
NB.

Cell counts for each stage of mitosis in the field of view indicate the duration of each of the
stages. The more cells you can see in one stage, the longer the duration of that stage in the cell
cycle.

Safety
1. Eye protection must be worn
2. Take care with glassware and scissors
3. Acetin 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 mins and tell your teacher. Avoid inhaling the
vapour. If you spill the stain, do not attempt to mop it up; tell your teacher instead.
4. Avoid skin contact with HCL.
5. If your microscope uses daylight illumination, be careful not to use it where sunlight could strike the mirror.
6. The water bath at 60°C will scald your skin; cool under cold running water if you get splashed.
7. Do not handle electric plugs, sockets or switches with wet hands.

Questions

1. Why do scientists prefer using garlic in this experiment?

 Garlic bulbs have roots whose tips have actively dividing cells.
 The root tip is a meristem because it has rapidly cells (meristematic cells)
 Stages of mitosis can only be seen in rapidly dividing cells
 Each cell has only 8 chromosomes hence easy to see and count when they condense.

2. 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 pectins that hold the cells together. Acetic orcein will stain the chromosomes dark red and fix
the cells, stopping mitosis.

3. Toluidine blue stain is the most preferred over others and therefore the technique using toluidine stain is
most preferred. Why is this technique preferred?
a. It is simple
b. It is cheap
c. It is safe
d. It is reliable
e. It is quick.
4. When cutting the root tip, write two characteristics that should be observed.
They should be white.
They should have a round firm end.

NB: Any brown part will give poor results due to presence of xylem that is already dead.

5. What do we call the area visible under the microscope?

Field of view.

6. What does the field of view show?

It shows cells showing different stages of cell cycle (Interphase, Mitosis and cytokinesis)

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7. How can one know the length of any stage of mitosis from the field of view?
The length of different phases can be deduced from the number of cells in the field of view showing the
different phases. If many cells in the field of view show a particular phase, then that phase is the longest.

8. What is mitotic index?

This is the number of cells containing visible chromosomes divided by the total number of cells in the field
of view.

So mitotic index shows the amount of cell division occurring in a tissue.

9. A root tip squash was prepared and then observed using a microscope. The number of cells at each stage of
the cycle was counted. Suggest how the number of cells in each stage can be used to calculate the duration
of each stage.
 The number of cells at each stage is proportional to the duration of that stage i.e. cells
will spend different lengths of time at different stages.

10. The following table shows the percentage of cells in each stage of mitosis. The cell cycle time for these
cells was 1200 minutes:

Stage of mitosis Percentage of cells in this stage

Prophase 2.43

Metaphase 1.40

Anaphase 0.70

Telophase 2.78

a) The percentage of cells in a stage of mitosis is proportional to the duration of that stage. Use this
information to compare the duration of each stage of mitosis in these root tip cells.
 The longest process is telophase – 33.36 mins
 Followed by prophase- 29.16 mins
 Then metaphase – 16.80 mins
 And lastly anaphase– 8.40 mins

b) The duration of each stage of mitosis can be calculated using the equation below.

Percentage of cells ∈that stage× cell cycle time

Duration of stage=
100

Describe how you would use this data to determine the total duration of mitosis.
 Use the equation to work out the actual time of each phase.
 Get the total of all four times.

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 11. The table below shows the number of cells at each stage of the cell cycle in one sample of tissue taken from
the growing region of a plant root.

Stage of the cell cycle Number of cells in each stage Percentage of cells in each stage
(%)

Interphase 47 78.3

Prophase 3 5.0

Metaphase 2 3.3

Anaphase 1 1.7

Telophase 3 5.0

Cytokinesis 4 6.7

Total 60 100

a) Complete the table giving your answer to the nearest whole number.
b) Using the table, suggest which stage of the cycle takes the longest. Give a reason for your answer.
 Interphase
 The time duration of each stage is proportional to the number of cells or percentage of
cells in that stage. Interphase has the highest number.
c) Suggest one reason why the above answer may be unreliable:
 The data was only taken from one point in time.

The root tip squash procedure can be used to observe cells undergoing mitosis.
Explain the role of mitosis in the development of roots. (2)

 increasing cell number

 replacing damaged/dead cells

 producing genetically identical cells/cloning

 needed for asexual reproduction

More Questions

1. Explain why the root tip is heated with acid.

2. State the effects 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