BIL 151 -‐ Mechanisms of Mitosis

BIL 151 -­‐ Mechanisms of Mitosis Part 3. Collecting Data – Review of Procedures By Dana Krempels, PhD, Alesia Sharber, Yunqiu Wang, PhD, and Kathryn Tosney, PhD Quantifying Mitotic Cells Remember that the chromosome squash procedure requires practice and patience. Today you will actually perform the chromosome squash on treatment and control onions, and should be able to recognize and quantify the stages of mitosis in each. A. Equipment Your lab instructor will provide you with root tips from the appropriate treatment and control specimens that were prepared by our hard-­‐working Lab Manager as per the specifications of your team’s research protocol (as corrected by your lab instructor). PLEASE DO NOT HANDLE ANY OF THE ONIONS IN THE LAB! THESE HAVE BEEN CAREFULLY PREPARED FOR EACH LAB RESEARCH TEAM, AND NO ONE SHOULD BE TOUCHING THEM OR HANDLING THEM EXCEPT THE GRADUATE LABORATORY INSTRUCTOR. As before, your team will have available the following equipment and materials available. compound microscope dissecting microscope microscope slides cover slips acetocarmine stain in dropper bottle 1 M hydrochloric acid (HCL) in dropper bottle razor blades dissecting needles labeling tape Sharpie markers beakers supply of live onions (control and treatment) B. Model Organisms: Treatment and Control Recall that individual batches of onions may respond quite differently to conditions suitable for root growth. We are using green onions (scallions; Figure 1), and each individual onion plant is a single sample/replicate (why?). Remember that multiple roots from the same onion plant are NOT true replicates. To obtain a statistically meaningful sample, you must count cells from at least 10 different individual control and 10 different treatment onions. To get your samples, first LABEL EACH OF YOUR SLIDES METICULOUSLY with the onion’s individual ID# (e.g., C1, T1, etc.), date, team name, etc. Only properly labeled slides will be graced with an onion root tip from your lab instructor, who will be doing the cutting. Figure 1. Sprouting green onions (scallions) C. Chromosome Squash Procedure – You’ve done this before 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! Before you begin you should make sure that all of your materials (beakers, plants, microscope slides) are properly labeled. Microscope slides should be labeled as treatment or control and with the treatment start-­‐
time. Your team may have 10-­‐20 slides out at any time, so it is important to label everything properly! 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, which 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 meristematic 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 a drop or two of HCl on the root tip. The HCl will soften the connections between the cell walls and allow the root to be flattened into a single cell layer. 4. After 10-­‐15 minutes, place a Kimwipe on the edge of your HCl puddle to wick away as much of the liquid as possible. 5. Place a drop or two of acetocarmine stain on the root tip. Let the root tip sit in the stain for 15-­‐20 minutes. You may occasionally prod the root tip with a dissecting pin. The iron in the pin can help darken the stain, but may make the stain too dark if you prod too much. Do not let the acetocarmine completely dry up. ((Caution! Acetocarmine is caustic and corrosive! Handle with care, and flush well with clean water if you get any on your skin or clothing. Acetocarmine may stain skin and permanently stain clothing.) Acetocarmine will allow you to clearly see mitotic chromosomes. (Figure 3) Figure 3. Allium root tip cells undergoing mitotic division, stained with
acetocarmine stain.
(http://upload.wikimedia.org/wikipedia/commons/d/d3/Onion_root_mitosis.jpg)
6. Now it’s time to squash your root. Place a clean plastic coverslip over your root. Over the coverslip, place a Kimwipe to soak up excess stain that leaks out and to protect your coverslip from fingerprints. 7. Push down firmly with your thumb for a few seconds. 8. Place the slide on the platform of your compound microscope. 9. Starting at the lowest objective, focus on your slide. Examine your squash. You should be able to see cells in various stages of mitosis. (Figure 4) Figure 4. A well-­‐executed squash performed in lab by a BIL 151 student. The elongated (non-­‐mitotic) cells in the upper left quadrant should not be counted, as they are no longer undergoing mitosis. The cells should be round or square and flattened into a single cell layer. The darkened nuclei of these 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 whether the cells are undergoing mitosis. 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 are in interphase, prophase, metaphase, anaphase, and telophase. Record these numbers and repeat for 3-­‐5 fields of view to obtain a good sample of your slide (250-­‐600 cells total). 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. Because those are all from a single individual onion plant, it would be invalid to count them as separate samples. All the cells counted from a particular onion plant should be considered one sample. 10. Count the number of cells you can identify in each stage of mitosis. You will analyze these data to determine whether there is a difference between your treatment and control samples. D. Calculations Adding the number of mitotic cells and dividing by the total number of cells you observed will give you the mitotic index for that root. In any given sample: mitotic Index (MI) = # of cells in active mitosis total # of cells counted If appropriate to your project, you should identify the particular stage of mitosis, and calculate an index for that particular phase of mitosis. For example, if you are trying to determine whether treated onions are unable to enter metaphase, you could calculate a metaphase index or a prophase index for your samples. For example, a prophase index could be calculated as: prophase index (MIprophase) = # of cells in prophase total # of mitotic cells If your team will be reporting mitotic phase indices, be sure to also report an overall mitotic index, as well, and note whether there is a significant difference between your treatment and control indices. Consider… 1. What are the (various) mitotic indices of your treatment and control groups? 2. Do you observe any polyploid cells (i.e., those with multiple chromosome sets)? Significance? 3. What mitotic stages are most prevalent? Does this differ between treatment and control? 4. Are any mitotic stages completely absent? Again, you will eventually need to explain this, and any difference between treatment and control. Remember that you will be calculating a mitotic index (and/or mitotic phase indices) for at least 10 treatment and 10 control onions. The indices are your raw data that will be statistically analyzed using the student’s t test in the penultimate stage of your project.