2: CELLS - RP2

Question 1

RP2: Preparation of stained squashes of cells from plant root tips; set up and use of an optical microscope to identify the stages of mitosis in these stained squashes and calculation of a mitotic index - method

Answer 1

1. Cut off about 5 mm from several root tips using fine scissors. Choose root tips that are white and have a firm, rounded end; tips that are turning brown will give poor results.
2. Stand the beaker on a bench mat before adding approximately 10ml of hydrochloric acid (5mol dm-3)
3. Place the root tip you have cut into the acid and leave for 15 minutes
4. Set up your microscope while you are waiting
5. After 15 minutes put the root tips in a watch glass containing approximately 5 cm3 cold water. Leave the root tips for 4–5 minutes, and then dry them on filter paper. Take care – the root tips will be very fragile.
6. Transfer one of the root tips to a clean microscope slide.
7. Gently break up the root tip with a mounted needle (this is called maceration). Add one small drop of toluidine blue and leave to stain for 2 minutes.
8. Cover with a coverslip and blot firmly with several layers of tissue or filter paper. Press gently to spread the root tip, or tap gently on the coverslip with the end of a pencil.
9. View under the microscope (x400 magnification) and look for cells with visible chromosomes. If cells are overlapping, squash the slide again between two wads of filter paper. Avoid lateral movement of the coverslip.
10. Look for regularly shaped, actively dividing cells. DNA stains dark blue with toluidine blue stain so you should be able to see blue groups of chromosomes against a paler background.
11. If your preparation is not very successful, repeat with some of the other root tips from step 3. Try to adjust your procedure to remedy the problem; for example, if your cells are over- or understained, adjust the time they are left in the stain.

Question 2

How do you calculate mitotic index?

Answer 2

(no. of cells in mitosis/total no. of cells) x 100

Question 3

Why do is the root tip left in the acid?

Answer 3

to break down the cell wall so the root tip can be squashed more easily