Idea Transcript
BIL 151 - Mechanisms of Mitosis PART II. Practicing Procedures and Developing a Research Project By Dana Krempels, PhD, Alesia Sharber, Linda White, Yunqiu Wang, PhD, Kathryn Tosney, PhD
In this project, you and your teammates will predict the outcome of treating a rapidly dividing tissue (onion root tip) with a substance that either promotes mitosis or inhibits mitosis. The unknown in this system is exactly how each substance either promotes or inhibits mitosis. For guidance in understanding the thought process you should be using in this project, be sure to read the Example Experiment linked to the online syllabus in this session’s slot. Your team should use this type of approach to analyze a particular structure or phenomenon that is affected by either trifluralin or indole-3-butyric acid (choose only one for your project) during mitosis.
I. Laboratory Equipment and Techniques To become good at examining cells in the act of mitosis will require that you attain some new skills that require practice and patience. Today you will learn how to do a chromosome squash, and learn to recognize and quantify cellular structures and events in a properly stained slide.
A. Materials Your team will have available the following equipment and materials. compound microscope dissecting microscope microscope slides cover slips 1 M hydrochloric acid (HCL) in 30mL dropper bottle 0.5% aqueous toluidine blue stain in 30mL dropper bottle one 250mL beaker labeled “DI Water” with watch glass lid one squeeze bottle with DI (deionized) water razor blade forceps (fine and large tip) dissecting needle probe scissors plastic squeeze pipets six 1.5mL microcentrifuge tubes in a rack one Petri dish one booklet bibulous paper (for absorbing liquid from slides) one box Kimwipes labeling tape Sharpie marker supply of live onions one 400mL beaker labeled “Liquid Waste” for all waste liquids
B. Methods Onion bulbs will sprout roots if they are placed in water for several days (Figure 1a). The bulbs should be placed in water about four days before the cell observations are to be made. Note that onion root tip cell cycle is about 24 hours. This means that it may take
approximately 24 hours of incubation with any particular reagent before one can expect to see any effect on mitotic cells. Side note: Individual batches of onion bulbs may respond quite differently to conditions suitable for root growth. Many onions obtained commercially have been treated to prevent sprouting, and will produce few roots. For this reason, we have done “test sprouting” to be sure the variety of onion we have chosen is viable. Green onions (scallions) are not treated to prevent sprouting, and sprout readily. Therefore, we are using them instead of bulb onions (Figure 1b). Organically grown onions are also not treated to prevent sprouting, and may yield better results than less expensive, conventionally grown onions. We give you this information only because we know that some day you may wish to perform fabulous onion root tip experiments on your own, and we just want you to know the perils you might face. You do not have to bring in your own onions to lab this week.
Figure 1a. Sprouting a yellow onion bulb in a glass of water.
Figure 1b. Sprouting green onions (scallions)
CAUTION! IN THE NEXT SECTION, YOU WILL BE GUIDED THROUGH A PROCEDURE FOR CHROMOSOME STAINING. BECAUSE SOME OF THE STAINS WILL PERMANENTLY MAR CLOTHING, BE SURE TO EITHER WEAR CLOTHES YOU DON’T MIND GETTING STAINED, OR BRING AN APRON OR OTHER COVERALL TO PROTECT YOUR FASHIONABLE NPIs (Nakedity Prevention Items; a.k.a. clothing).
C. Chromosome Squash and Toluidine Blue Staining Procedure To examine the mitotic process in the cells of the onion root tip, you must soften the root so the cells can be separated and flattened, thus making it possible to see the chromosomes, nuclei, spindles, and other cell parts. This procedure will help you perform a successful chromosome squash. You will probably need to practice it several times before getting a good squash, so every group member should set up several squashes (about 10 minutes apart) so that you can get enough practice to become a Root Squashing Pro! Make sure that all of your materials (beakers, plants, microscope slides) are properly labeled before you start this procedure. (When you examine root tips that have been treated with a compound next week, all microscope slides should be labeled appropriately as treatment or control and with
the treatment start-time.) Your team may have 10-20 slides out at any time, so it is critically important to label everything properly!
Before you begin, make sure all the microscope slides you plan to use are extremely clean. To properly clean a microscope slide, place three drops of 95% ethanol on the slide and wipe well with a Kimwipe. Do this on both sides of the slide, and repeat, as necessary, until the slide is very shiny and clean. If you fail to perform this important step, you will not be able to see structures on your slide with any clarity. Because some of the reagents you will be using are a bit caustic, WEAR THE NITRILE GLOVES PROVIDED AND YOUR OWN SAFETY GLASSES WHILE YOU PERFORM THE CHROMOSOME SQUASH. Do not waste gloves! One pair per student, or bring your own gloves if you plan to use more than one pair. 1. Snap an entire healthy root from an onion. The root tip is the most delicate part of the root and it desiccates very easily. You will need healthy, living cells, so keep your onion root wet at all times! Do not leave onion roots out of the water or lying on the lab bench. 2. Place the root on your labeled slide. Using the dissecting scope, identify the root tip. In plants, mitotic division occurs in the meristem cells, stem cells that can differentiate into any other type of cell. The apical (i.e., located at the apex, or tip) meristem is about one millimeter from the apparent tip of the root (the root cap) (Figure 2). If you cut off too much of the root, you will see long, rectangular cells in your squash. These cells are no longer undergoing mitosis, so you should not use or count them. Carefully cut off just the meristem region of the root tip with a sharp razor blade, and use that for your squash.
Figure 2a. Onion root tip anatomy. Only the cells at the very tip of the root (Zone of Cell Division) are undergoing mitosis. These are visually distinct in a fresh root tip, appearing more round or square than the elongated cells in the Zone of Elongation above it. (Campbell, 2005)
Figure 2b. Root tip of corn (Zea mays). Note the clear appearance of the root cap. Just above it is the apical meristem and the Zone of Cell Division. The darker, longitudinal lines above the cell division zone mark the newly formed vascular cambium.
3. Place just the root tip containing the apical meristem into a 1.5 ml microcentrifuge tube (Figure 3)
with your fine-tipped forceps. (Handle fine-tipped forceps with care. The tips are fragile, and will bend or break if you drop the forceps or handle them roughly.)
Figure 3. Microcentrifuge tube and needle-tip dissection probe.
4. Fill the centrifuge tube halfway with 1M HCl from the dropper bottle onto your root tip in the tube. This step will soften the connection between the cells. ***Use caution as HCl is a strong acid. 5. Close the tube and place in a hot 60°C waterbath for exactly 8 minutes (leaving the tip in the hot acid too long results in a soggy mass of cells that will disintegrate when you rinse). 6. Remove the tube carefully from the hot bath. To remove the 1M HCl, fill the tube with deionized (DI) water and remove it with a plastic squeeze pipet. Place all removed waste water into your 400mL beaker labeled "WASTE SOLUTIONS". Rinse a total of three times. 7. Add 2 drops of 0.5% toluidine blue to stain the root tip in the tube. Incubate at room temperature for 5 minutes, gently flicking the tube with the flat of your fingernail about once a minute to distribute the stain. Make sure the root tip stays in the stain. 8. Rinse off the excess toluidine blue as you did for the HCl: fill the tube with DI water and then remove with the plastic squeeze pipet. Repeat a total of three times, always placing the waste solution into the “WASTE SOLUTIONS” beaker. You should be able to see your blue root tip clearly by the time you have removed the last bit of rinse water. 9. Remove almost all of the last rinse, then use the dissecting probe (Figure 3) to gently push the root tip onto a clean, labeled slide. 10. Add one drop of DI water to the stained root tip, and then place a coverslip on top of it. You should now have "sandwich" of slide/root tip in water/coverslip. 11. Place a sheet of bibulous paper (from the booklet supplied on your tray) over the coverslip and GENTLY PRESS STRAIGHT DOWN onto the coverslip with root tip underneath. Be careful not to break the coverslip, or you’ll have to start over. DO NOT PLACE YOUR SLIDE INSIDE THE BIBULOUS PAPER BOOKLET! Please keep the pages clean and uncontaminated for your future slide preps. 12. Remove the bibulous paper and place the slide on the compound microscope to observe your stained root tip. ALWAYS BEGIN YOUR MICROSCOPE OBSERVATIONS ON LOW POWER. Find your root tip cells in the viewing field on low power, and then focus until the view is clear. Then swivel the objective to the next higher objective, and focus again. Do this until you are properly focused with the 40X objective, which you will need to use to see nuclear material clearly.
13. Examine your squash. You should be able to see cells in various stages of mitosis. (Figure 4)
Figure 4a. Allium root tip cells undergoing mitosis (acetocarmine stain). http://upload.wikimedia.org/wikipedia/comm ons/d/d3/Onion_root_mitosis.jpg
Figure 4b. Your preparation will probably look something like this. Yellow arrows indicate cells in various stages of mitosis. (preparation and photo courtesy of Linda White)
D. Data Collection The cells you should count will be round or square and flattened into a single cell layer. (Do not count long, rectangular cells, as these are no longer undergoing mitosis.) The darkened nuclei of mitotic cells will be large and appear to take up most of the space inside the cell. Ask your instructor to look at your slide if you’re not sure you have a good squash. At the highest magnification (400x), you will be able to identify mitotic cells. Look at an area that is properly squashed and count all of the cells you can see (around 50-200 cells). From among those, count how many cells—each—are in (1) interphase, (2) prophase, (3) metaphase, (4) anaphase, and (5) telophase. Record these numbers and repeat for 3-5 fields of view to obtain a good sample from your onion (about 250-600 cells total). Adding the number of actively dividing cells and dividing by the total number of cells you observed will give you the mitotic index for that root. Note that you are “sampling” multiple fields of view on one slide, but for the purposes of your experiment and statistical analysis, one sample = all the cells counted in one root from one onion AVOID PSEUDOREPLICATION! Do not take multiple roots from the same onion and do not count multiple fields of view as separate experimental samples. All samples from a single individual onion are part of the same treatment, and it would be invalid to count them as separate samples. All the cells counted from a particular onion plant should be considered one sample. 1. Count the number of cells you can identify in each stage of mitosis. When your team does its original project, you will use data like these for statistical analysis, to determine whether there is a difference between your treatment and control samples. 2. For comparison, you may wish to examine a commercially prepared slide of an onion root tip (available from your instructor). Observe the slide using low and high power. 3. When you are completely finished with your slide preparations, place them in the Broken Glass Disposal Container at the front of the lab room. Teams leaving slides
or other materials at their station will be docked 5 points.
D. Data Analysis If you were to perform the colchicine experiment described in the example project, you would prepare a report and presentation in which you might consider the following questions, and perhaps others. This should give you some ideas your team can apply to your own experiment. 1. Can you locate the various stages of mitosis – prophase, metaphase, anaphase, and telophase? 2. What are the mitotic indices of your treatment and control groups? 3. In what stage are most of the cells? (IMPORTANT: It is possible to calculate indices for each phase of mitosis by counting the number of cells in a particular phase of mitosis and dividing it by the total number of mitotic cells. This might help you determine whether treated onion root tips have been affected in a particular stage of mitosis.) 4. Do you observe any polyploid cells (i.e., those with multiple chromosome sets)? If so, what might be the significance of this finding? 5. What mitotic stages are most prevalent? (Next week, when you count cells in your experimentally treated roots, consider whether this is different between treatment and control onions.) 6. Are any mitotic stages completely lacking? (Next week, if you see any mitotic stages missing, you should be able to explain any difference between treatment and control.)
II. Your Team Research Project In your literature search, you will explore the effects of either the mitotic promoter indole-3-butyric acid or the mitotic inhibitor trifluralin on the structures or mechanisms of mitosis. The literature information should serve as an observation about something in this area. This observation should inspire you to suggest an overall hypothesis about what you should see in onion root tips treated with a either trifluralin or indole-3-butyric acid. From this hypothesis, you should be able to generate opposing statistical (null and alternative) hypotheses. The chemical compounds we have chosen for this experiment have been selected for their relative safety, but it will be up to your team to look up the Material Safety Data Sheet (MSDS) for these compounds and determine their mechanism of action, so that you can make a logical prediction. (Google: MSDS [name of compound] and you will find the necessary information.) Given what these compounds do to mitotic cells, do you have any idea why they might be potentially toxic to you and other vertebrates? In the next lab session, you and your team meet to discuss the findings of your literature search, and decide whether to examine the effects of either trifluralin or indole-3-butyric acid on growing onion root tips. Once you have decided, your team will complete a Mitosis Project Planning Worksheet (this is linked to the syllabus in the next session’s slot) to submit to your lab instructor. You will spend the following lab session comparing treatment and control onion root tips to test your hypotheses.
Literature Cited Reece, J.B., L.A. Urry, M. L. Cain, S.A. Wasserman, P.V. Minorsky, R.B. Jackson, N.A. Campbell. 2005. Biology. Boston: Benjamin Cummings.