Flash link:  https://vlab.amrita.edu/repo/BIOTECH/CEL/MITOSIS%20in%20ONION%20ROOT%20tips/index.swf

Materials required

 

1. Onion plant with root
2. Feulgen stain
3. 1 N HCl
4. Scissors
5. Forceps
6. Razor blade
7. Pasture pipette
8. 1.5 ml microfuge tubes
9. Dissection probe with wooden back
10. Microscopic slides and cover slips
11. Water bath
12. Light Microscope

Procedure

 

1. Take the onion plant with newly sprouted roots and cut two root tips using scissors and transfer them into a plastic microfuge tube.
2. Fill 2/3 of the tube with 1N HCl using a dropper.
3. Place the tube in a 60°C water bath and incubate the tube for 12- 15 minutes.
4. Remove the tube from the water bath after the incubation.
5. Discard the HCl from the tube using a Pasture pipette to the running tap water.
6. Add some drops of distilled water into the tube and rinse the root. Then remove the water from the microfuge tube using the Pasture  pipette. (Rinse the roots at least three times).
7. After the washing step add 2-3 drops of Feulgen stain into the tube with root tips and incubate the roots for 12-15 minutes. (During the incubation, the very tip of the root will begin to turn red as the DNA stains the numerous small actively dividing cells at the time).
8. After the incubation remove the stain using a Pasture  pipette.
9. Again rinse the root tips with distilled water. (Rinse the roots at least three times).
10. Transfer a root from the tube to the centre of the microscopic slide and add a drop of water over it.
11. Take a razor blade and cut most of the unstained part of the root.
12. Cover the root tip with a cover slip and then carefully push down on the cover slide with the wooden end of a dissecting probe. (Push hard, but do not twist or push the cover slide sideways). The root tip should spread out to a diameter  of about 0.5- 1cm.
13. Observe it under a compound microscope in 10x objective. Scan and narrow down to a region containing dividing cells and switch to 40x for a better view.

 

An Alternative Procedure

 

1. Cut the tip 5 to 8 mm from the tip of the freshly sprouted root.  Discard the rest of the root.
2. Place the cut tip on a clean microscope slide.
3. Add 2-3 drops of acetocarmine stain to the slide.
4. Warm the slide gently over the alcohol lamp for about one minute. ( Do not allow the slide to get hot to the touch; you don't want to cook either your fingers or the root.  Do not let the root dry out).
5. Cover the slide with a cover slip or lens paper.
6. Squash the slide with your thumb using a firm and even pressure. (Avoid squashing with such force that the cover slip breaks or slides).
7. Observe it under a compound microscope in 10x objective. Scan and narrow down to a region containing dividing cells and switch to 40x for a better view.