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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 60 °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
● eye protection
● garlic clove with growing root tip
● glass slide and coverslip
● scissors
● water bath at 60 °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

DIAGRAM

figure A Section of a root tip showing meristem with dividing cells

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.

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.
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. Mitosis is part of the cell cycle. It is a form of nuclear division, followed by cytokinesis – division of the cytoplasm/rest of the cell.
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.
LEARNING TIPS
● 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.