• 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

• 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. Note.
  
• 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 0ºC 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

• 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.

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 1M 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 60ºC
marker pen
timer
dropping bottle of 50% glycerol
dropping bottle of 70% ethanol
lens tissue

Preparation of materials

• 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.
  
• Toluidine blue is harmful if swallowed. Prepare a 0.5% solution in a citrate/phosphate buffer at pH4 (20 cm³ 0.1 M citric acid + 10 cm³ 0.2 M disodium hydrogen phosphate + 8 cm³ distilled water).

  Fixing the roots

• This stage is required only if suitable roots are available but they are to be stained at a later date.
  
• Mix 6 cm³ absolute alcohol with 2 cm³ 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 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. Mounting bulbs for root production is most easily done by cutting small holes in a polystyrene ceiling tile and pushing the garlic cloves part way through these holes. Then float the tile in a tray of water. This is safer than pushing the clove into the top of a boiling tube as the combination of water uptake by the clove and water loss by evaporation can result in the developing roots drying up.

4. 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)

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)

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 60ºC
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.

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 cm³ of I M hydrochloric acid in a small beaker to 60ºC 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.
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.