Root Tip Mitosis Lab x184f

Year 12 – Diploma Biology

Mitosis in Onion Root Tip Cells Introduction: The root tip is the area of a root that contains meristematic cells. These cells never specialize; rather, their function throughout the life of the plant is to produce cells by mitosis. These new cells will then specialize and become various tissues in the root. In this lab, you will prepare a slide of the meristematic cells in the root tip of a germinating garlic bulb. You should be able to identify cells in each stage of mitosis as well as cells in interphase. Materials: onion roots fine scalpel paper towel metric ruler forceps test tube transfer pipette warm water bath (50-60°c) small beaker

microscope slide and cover slip lens paper microscope aceto-orcein stain watch glass stirring rod test tube holder acetic alcohol fixative (3 : 1 absolute alcohol : glacial acetic acid) 5 ml 1M HCl

SAFETY: You must wear goggles until step 14. Both fixative and stain are strongly acidic! Procedure: This is all very tricky. Useful results are hard to obtain. Read through this procedure completely before starting anything. Then work carefully, trying to understand why you are doing what you are doing. 1. Pull off 2 roots from an onion bulb, which has been growing in water for a few days. Be sure to remove the ENTIRE roots. 2. Using a fine scalpel, cut off a 2cm long segment of each root tip. Be sure to make clean, straight cuts so you can distinguish between the cut end and the tip. KEEP THE 2cm LONG TIPS! Throw out the excess roots. Caution: handle fine scalpel carefully. 3. Place the two root tips in the bottom of a test tube. You may use a stirring rod to gently push them to the bottom. 4. Add just enough fixative solution to the test tube to cover the root tips. Caution: the solution is very acidic…wear safety goggles. Use forceps to handle “fixed” root tips from now on. 5. To help the fixative solution penetrate the cells, place your test tube in a test tube holder in a warm water bath at 50 degrees C. 6. After 6 minutes, take your test tube out of the water bath. Carefully pour the fixative and the root tips into the watch glass. 7. Wash the root tips gently in cold water. 8. Transfer the root tips to 1M hydrochloric acid in a small beaker and leave in the water bath for 5 minutes. TAKE CARE – THE ROOT TIPS WILL BE VERY FRAGILE NOW! 9. Wash the root tips again in cold water. 10. Place two clean microscope slides side by side on a clean paper towel. Very gently pick up the root tips with forceps and place one on each slide. Be sure to grab onto the cut end of the root with your forceps, NOT the tip! Once the root tips are on the slides, pour out your fixative with the cold water running. 11. Using a fine scalpel, cut off the very tip of the root so that only about 3-4 mm of the tip end is left (the root tips are the part you want to keep, toss out the other part). 12. Using the fine scalpel, chop up the 3-4mm root tip into tiny pieces. Make sure all the pieces remain on the slide! 13. Immediately add 1 or 2 drops of aceto-orcein stain. This stain gives colour to certain cell structures, including the chromosomes. Allow the piece of root tip to stay in the stain for 3 minutes. Do not let the preparation dry up. If it appears to be doing so, add another drop of the stain. 14. Place a clean cover slip over the pieces of root tip. 15. Fold the paper towel several times so it is the same shape as the slide but slightly larger. Then make a final fold in the paper bringing the ends together. Place the paper on the table and insert the part of the slide where the cover slip is located into the final fold of the paper towel. This forms a “sandwich” with several layers of paper under the slide and several layers on top of the slide. Make sure you know what you are doing here – ask if you are unsure. Diploma Biology 2014/2015/2016 Cell Biology PBL

1

16. With the “sandwich” resting flat on the table, press down vertically with your thumb on the upper layer of paper. This pressure will further spread the cells and flatten them. Be careful to apply pressure without twisting, so that the cover slip is not moved or broken. Carefully remove the slide from the paper towel. You have now made a slide of mitosis using the “squash preparation” technique. 17. You may now remove your goggles. Examine the slide under the microscope using the 10X objective (100X total power). Move the slide so that you can scan the entire area under the cover ship. Look for cells containing nuclei that appear to be made of distinct rod like structures (chromosomes). Such cells were undergoing mitosis at the time the garlic roots were cut and placed in the preservative solution. 18. Locate an area where the cells are in various stages of mitosis. Switch to high power (40x objective) and examine this area carefully. If necessary, adjust the diaphragm of your microscope to increase the clarity of the image. 19. Locate a cell in interphase and in each stage of mitosis.

Questions and Records 1. The cellulose walls of plant cells are held together by compounds in the middle lamella. Treatment with hydrochloric acid breaks these down. Why is this helpful in your preparation? 2. Which part, or parts, of the cells have taken up the stain? Are there cells present, in which the chromosomes are not condensed? If yes, why is this? 3. Identify cells in the following stages of mitosis: interphase, prophase, metaphase, anaphase and telophase. Draw outline sketches of a cell to illustrate each stage. 4. In a given field of view, count the number of cells at each of the five stages of mitosis. Record this in a suitable table. Present the results in the form of a pie chart. 5. Calculate the mitotic index. number of cells containing chromosomes Mitotic index = total number of cells in field of view Using these calculations, make some conclusions about the length of time for each stage of mitosis. Diploma Biology 2014/2015/2016 Cell Biology PBL

2