Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
TEACHER/LECTURER GUIDE
Type and purpose of activity
This experiment can be used to:
• provide evidence for assessment of Outcome 3
(For advice on marking Outcome 3 report, please contact the SAPS Scotland office.)
• develop knowledge and understanding of the process of mitosis
• develop problem solving skills and in particular Outcome 2 PCs: (b) information is accurately processed, using
calculations where appropriate (d) experimental procedures are planned, designed and evaluated appropriately
Background information
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In this activity students will prepare and stain root tips. To achieve an Outcome 3 students must either have TWO
different sources of root tips OR stain one type of root tip with two different stains. A comparison between EITHER
the root types OR the stains will then be possible.
This practical is best carried out in the morning to get good results.
Two recommended sources of roots are garlic and hyacinth. The garlic cloves, bought normally for cooking purposes,
will produce roots at any time of year. Hyacinth bulbs can be bought at Garden Centres during autumn and winter.
Both garlic cloves and hyacinth bulbs will produce ample roots for the experiment.
Suitable stains for studying the stages of mitosis in root tips are lactopropionic orcein and toluidene blue.
The mitotic index is the fraction of cells in a microscope field which contain condensed chromosomes. This index will
be calculated for each slide prepared.
Preparation of the plant materials and the stains is covered in the Technical Guide.
To make this activity non-seasonal, it is possible to ‘fix’ the root tips when available and then store them until required.
Fixing of root tips is only covered in the Technical Guide.
Classroom management
Students are asked to mark the root tip one or two days prior to staining the root tips. This will enable them to link rate
of growth with mitotic index.
Microscopic examination of the slides:
Students should examine several slides and calculate the mitotic index for each one. Prepared slides could also be
available.
Supply of materials
In order to satisfy the core skill in problem solving, students will be required to identify and obtain resources required
for themselves. Further advice on supply of material is given in the Technical Guide.
Extension work
Try to vary the mitotic index of the plant tissue e.g. cutting the root tips and keeping them at 0°C for 24 hours may
increase the mitotic index. The experimental method can be varied e.g. varying the temperature or concentration of
acid; varying the time the root tip is in the acid; squashing the root tip with a coverslip instead of macerating; varying
the age of the root used; preparing the stains differently (e.g. different dilutions, different pHs); heating the
lactopropionic orcein slide gently; investigating a possible link between rate of growth of root and mitotic index.
Acknowledgements
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Information and advice from Dr Kwiton Jong, Royal Botanic Garden, Edinburgh, is gratefully acknowledged.
Information was also received from Ashby Merson-Davies, Sevenoaks School, Kent.
This experiment was produced by the SAPS Biotechnology Scotland Project. Funding for the project was provided by
SAPS, Unilever and The Scottish Office. Support was also provided by Edinburgh University, Quest International, the
Scottish CCC, the Higher Still Development Unit and SSERC.
Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
TECHNICAL GUIDE
The class will be varying EITHER plant material OR stain for this activity. The list of materials required will vary depending on
this decision.
Materials required
Materials required by each student/group:
gloves and eye protection
compound microscope (x100 - x400 magnification)
small beaker of 1 M hydrochloric acid (2 will be required if plant material is being investigated)
small beaker of water and dropper
microscope slides
coverslips
fine forceps
dissecting needle
scissors
soft tissue paper
ruler
fine thread
dropping bottle of lactopropionic orcein AND/OR (see below) dropping bottle of toluidine blue
garlic clove with suitable roots AND/OR (see below) hyacinth bulb with suitable roots
Materials to be shared:
water bath at 60°C
marker pen
timer
dropping bottle of 50% glycerol
dropping bottle of 70% ethanol
lens tissue
Preparation of materials
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If PLANT MATERIAL is to be varied prepare BOTH plant types below. If STAIN is to be varied prepare just any one of
the plant types.
To prepare hyacinth bulb roots: Place the bulb in a suitably sized container with water so that the root end is just in
contact with the water. It is best to change the water daily if possible. Roots of a suitable length (2-6 cm) will be
available within a week and perhaps sooner.
CARE! Hyacinth bulbs can cause allergies. Wear gloves if handling the bulbs regularly.
To prepare garlic clove roots: Carefully peel the cloves and place in holes in a foam or polystyrene float. Sit the float
in a beaker of water so that the base of the clove is in contact with the water. Roots of a suitable length (2-6 cm) will
be available after 2-4 days.
If STAIN is to be varied prepare BOTH stains, as detailed next. If PLANT MATERIAL is to be varied prepare just any
one of the stains.
CARE! - Wear gloves and eye protection when handling the stains.
Lactopropionic orcein should be prepared in a fume cupboard or well ventilated room. Dilute it to a 45% solution by
volume with distilled water.
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Toluidine blue is harmful if swallowed. Prepare a 0.5% solution in a citrate/phosphate buffer at pH4 (20 cm 0.1 M
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citric acid + 10 cm 0.2 M disodium hydrogen phosphate + 8 cm distilled water).
Fixing the roots
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This stage is required only if suitable roots are available but they are to be stained at a later date.
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Mix 6 cm absolute alcohol with 2 cm glacial acetic acid in a fume cupboard. This mixture is called Farmer’s fluid and
must be freshly prepared. Once added to the Farmer’s fluid, the root tips can be stored for many months in a
refrigerator.
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray system for each student/group. Equipment
and materials should be supplied in a way that students have to identify and obtain resources. Normal laboratory apparatus
Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
should not be made available in kits but should generally be available in the laboratory. Trays could be provided containing
one type of specialist equipment or materials.
1. Toluidine blue stain can be purchased in powder form from suppliers such as Philip Harris Education [tel: 01543 480077,
fax: 01543 480068]. Make up a 0.5% solution in McIlvaine buffer [0.1M citric acid, 0.2M sodium hydrogen phosphate at pH4].
This should keep for many months at room temperature.
2. Ideal dropping bottles can be purchased from chemists. These are the ones used for eye drops. They are very strong and
have a built in dropper.
3. Lacto-propionic orcein stain can be purchased from Philip Harris Education [tel: 01543 480077, fax: 01543 480068] as
Orcein Propionic C7A69849 100ml £10.10 (2006)
Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity
1. What is the aim of the activity?
2. Do you know if you are using two types of roots OR two types of stain?
3. What measurements are you going to make?
4. What safety measures are you required to take?
5. As a class, decide what a ‘nucleus’ should look like for it to be composed of condensed chromosomes.
6. In your group, decide how the activity will be managed by allocating tasks to each member. For Outcome 3 it is
important that you play an active part in setting up the experiment and in collecting results.
Recording of data
Prepare a table to record your results. You should use a ruler and appropriate headings.
Evaluation
1. If varying plant material, was rate of growth of the two roots similar. If not, is there a link between mitotic index and
rate of growth?
2. If varying stain, was there a difference in the ability of the root cells to absorb the stains? Were they absorbed too
much/insufficiently?
3. Does the mitotic index vary much between different results? Account for these differences, if possible.
4. Was the treatment in acid (step 4) sufficient to allow for both easy handling of the root tip and easy maceration?
(Insufficient acid treatment results in easy handling but difficult maceration; too severe acid treatment results in difficult
handling but easy maceration)
Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
STUDENT ACTIVITY GUIDE
Introduction
You are going to stain root tips and examine them for signs of cells dividing by mitosis. The chromosomes inside the nuclei of
such cells condense and become visible. You should know what condensed chromosomes look like and how they move
about inside a cell when undergoing mitosis.
Equipment and materials
Materials required by each student/group:
gloves and eye protection
compound microscope ( x100 - x400 magnification)
small beaker of 1 M hydrochloric acid (2 will be required if plant material is being investigated)
small beaker of water and dropper
microscope slides
coverslips
fine forceps
dissecting needle
scissors
soft tissue paper
ruler
fine thread
dropping bottle of lactopropionic orcein AND/OR (see below) dropping bottle of toluidine blue
garlic clove with suitable roots AND/OR (see below) hyacinth bulb with suitable roots
Materials to be shared:
water bath at 60°C
marker pen
timer
dropping bottle of 50% glycerol
dropping bottle of 70% ethanol
lens tissue
CARE! Wear gloves and eye protection whilst carrying out this experiment. Avoid skin contact with the stain(s) and avoid
breathing in the fumes of the stain, lactopropionic orcein, if used.
Unit: Cell and Molecular Biology (AH): Structure, function and growth of prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
Instructions
Either two types of roots OR two different stains will have been prepared. Find out what is available.
1.
One or two days before staining the root tips, remove the plant material carefully from the water and blot dry gently.
Use a permanent marker pen to mark a small dot about 2 mm from the end of some root tips. Replace the plant
carefully in the water.
2.
After one to two days, remove the plant material and use the thread and ruler to measure how much the root tips have
grown since marked.
3.
Preheat about 10 cm of 1 M hydrochloric acid in a small beaker to 60°C using a waterbath. Meanwhile, use a lens
tissue and alcohol to clean microscope slides and coverslips.
4.
Using scissors remove the last 2 mm from several young vigorously growing root tips. Place them in the preheated
acid and return to the waterbath for 4-5 minutes.
5.
Gently transfer each root tip to a clean microscope slide containing a large drop of water.
6.
Gently blot dry with a piece of soft tissue. It is important to remove as much water as possible.
7.
Using a dissection needle, thoroughly macerate the root tip and spread over an area equivalent to the size of a 5p
coin. (alternatively you could place another microscope slide at right angles to the original slide to form a cross, and
squash the tip between the two slides. This method will provide two samples for staining.)
8.
You are now ready to apply the stain.
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If using toluidine blue - add one drop to the macerated root tip and IMMEDIATELY cover with a coverslip, invert the
slide and blot firmly several times on a wad of tissues.
If using lactopropionic orcein - add one drop to the macerated root tip and leave for 3-4 minutes. To speed up
absorption of the stain, warm the slide gently by holding it 30-40 cm above a yellow bunsen flame (if your hand
becomes uncomfortable you are heating the slide too much). Cover with a coverslip, invert the slide and blot firmly
several times on a wad of tissues.
9.
View under a microscope, x40 - x100 magnification initially. Scan the slide to locate the region of mitosis.
10.
View this area at a higher magnification (x400 should be sufficient) and count:
(i) the total number of cells in the microscope field
(ii) the number of cells with condensed chromosomes which are going through any of the four stages of mitosis. You
will have to decide where your cut-off point is when considering if cells in prophase and telophase contain condensed
chromosomes (consult textbooks).
11.
Repeat steps 9 and 10 for the various microscope slides prepared. If you want to prevent the slides from drying out,
mount them in 50% glycerol.
12.
Calculate the mitotic index for each slide examined (the mitotic index is the fraction or percentage of cells containing
condensed chromosomes).
13.
Draw a table with suitable headings summarising your results.
14.
Compare your results with those of other groups.