Lab 3: Testing Hypotheses about Mitosis Why do cells divide? Lab today focuses on cellular division, also known as cellular reproduction. To become more familiar with why cells divide, the types of cell division, and how cell division occurs, please visit this website (you may need to type in the links if they do not work when you click on them) from the University of Florida (http://plaza.ufl.edu/alallen/pgl/modules/rio/stingarees/module/index.html). Next, visit the Cells Alive website (http://www.cellsalive.com/toc_cellbio.htm), where you have a chance to view simulations for (1) Cell Models, (2) Cell Cycle, and (3) Mitosis. Please take notes on the cycles, noting chromosome arrangement as you watch them. Cellular reproduction begins with cell growth during the G 1 (or "gap" 1) phase, followed by the replication of DNA during the S ("synthesis") phase of the cell cycle. Growth continues during the G 2 ("gap" 2) phase. The cell cycle is completed when the process of mitosis (the M phase) results in two identical "daughter cells". Mitosis is extremely accurate (in a biological sense) and ensures that each of the two daughter cells produced gets a genetically identical set of chromosomes copied from the parent cell. What organisms are best for studying cell division? All somatic cells in living organisms undergo mitosis as a way of replicating and growing. Some parts of plants, like the onion root tip we are using today, are ideal models for observing mitosis. Tips of plant roots and shoots contain regions, called meristems, which undergo rapid and continuous growth. Because rates of cell division are quite high, many meristem cells will be in various stages of mitosis at any given time. In root tips, meristem cells are rapidly undergoing mitosis as the root grows deeper into the soil. Snap shots of root tips allow scientists to measure rates of mitosis and investigate other questions about cell division. Longitudinal section of an onion root tip. (Image from Elzinga et al. 2007.) What you will do Today, you will use the compound microscope to observe onion root tip cells and test hypotheses about mitosis. You will apply simple statistical tests to make inferences based on your hypotheses. Additionally, you will continue to refine your skills in using microscopes, making slide preparations, and data analysis. Laboratory Objectives As a result of participating in this laboratory activity, you will: 1. Develop a hypothesis 2. Design and conduct experiments 3. Analyze data using statistics 4. Draw conclusions based on gathered data 5. Use appropriate terminology when discussing mitosis and integrate your knowledge of mitosis into the cell cycle 6. Model the process of mitosis 7. Make stained slides to observe mitosis and see phases of mitosis 1
Methods Part 1. Observing and Recording the Various Mitotic Phases in the Onion Root Tip Spend time becoming familiar with what each phase of mitosis looks like in onion root tip cells. 1. Create your own stained slide (methods in Appendix B) 2. Draw what you see in your own stained slide (use Appendix A to help determine what you see). 3. Using the professionally stained slide, sample 5 cells, sketching the (a) location of the chromosomes and (b) the arrangement of the chromosomes in the cell. Include sketches in the Data and Field Notes section. 4. Check your ability to identify stages of mitosis by identifying the stages of the 5 cells sketched above. Part 2. Cell Cycle How long does a cell spend in each phase of mitosis? Determine how long a cell spends in each phase of mitosis (Interphase (which includes G1, S, G2), Prophase, Metaphase, Anaphase, and Telophase). Complete this project with your lab group, and clearly write up your investigation in your lab guidebook. Parts 1 3 should be included in the data and field notes section. Part 4 should be in your data analysis and results section. Part 5 should be included in the conclusions section. 1. Develop a hypothesis for the percentage of time a cell spends in each phase of the cell cycle. 2. Make a prediction about your results that is based on your hypothesis. For example, if you were given 100 onion cells, and your alternative hypothesis is assumed to be true, how many of those 100 cells would be found in each of the stages of the cell cycle (that is, interphase, prophase, metaphase, anaphase and telophase)? These are your expected values based on your hypothesis. 3. Develop a method to test your hypothesis using professionally stained slides (these are easier for observing chromosomes). Record your method such that others could replicate your experiment. Collect your data. Construct a table to organize your raw data. Be sure to include appropriate titles and labels. Use the professionally prepared slides of onion root tips to sample the cells using the method you developed. 4. Analyze your results. Conduct a Chi squared statistical test on your collected data (see Appendix C). Organize your results as a pie chart. Don t forget to include a title and other appropriate labels. 5. Draw conclusions. Discuss and interpret what your results mean, including your statistical tests. Be sure to go back to your hypothesis to make a clear link between your hypothesis and your results. 2
Post Lab. Arrange your data, analyses and conclusions using the following headers and respective items under each heading. View the post lab rubric posted on the lab website for information about the evaluation of these items. 1. Raw Data, Field Notes and Observations (3 pts) a. Clearly labeled sketches of your own stained slide b. Clearly labeled sketches of 5 cells (from the professionally stained slide) with the location and arrangement of the chromosomes clearly marked, and the stages of mitosis identified. c. Record your hypothesis, prediction and method (#1 3). d. Construct a table to organize your raw data. Include titles and labels. 2. Data Analysis and Results (3 pts) a. Include all your work (i.e., calculations) for calculating Chi square. b. Organize your data into a pie chart. Include labels and figure legends. 3. Conclusions (3 pts) a. Discuss and interpret what your results mean, including results of your statistical test. 4. Reflection (3 pts) a. What is the purpose of mitosis? b. What might happen if your cells did not undergo mitosis? c. Create a model of mitosis. Include the following in your model: a. Three chromosomes (n=3) and distinguish between them in some fashion (e.g. color) b. Label each phase c. Begin your model with interphase (G1), where chromosomes are unduplicated, and carry the model through telophase and cytokinesis. 3
Appendix A. Cells in Mitosis Photographs In prophase, the chromatin has begun to gather together and form into chromosomes. Notice especially nuclear size and density. A cell in interphase has a nuclear membrane, but the one in the prophase cell has broken down. Prophase At metaphase, the chromosomes have begun to congregate in the center of the cell, between the poles, in an area called the equator. The identical chromosomes are being prepared for migration to the opposite sides of the cell toward the poles. Metaphase Anaphase is characterized by the migration of chromosomes toward the poles. Each set of chromosomes that you see in the center of the slide is an exact duplicate of the other. The two cells that will eventually form from this separation are thus duplicates, each with the same amount and quality of DNA. Anaphase During telophase, cytokinesis begins and a new cell wall forms at what was once the equator. In plant cells the cell wall forms in the center of the cell and is built outward until it reaches the periphery. Once the periphery is reached, the cell has completed the period and G 1 begins. Telophase 4
Appendix B. Preparing an Onion Root Tip Slide 1. Your instructor will hydrolyze your lab s onion root tips for 4 minutes in a vial of 1 M HCl. 2. Using forceps, transfer a root tip from the vial of HCl to a cup with Carnoy fixative for 4 minutes, then transfer the root tip to a clean slide. 3. With a razor blade, cut off the terminal 2 3 mm. This terminal portion is the part of the root tip with the most active cell division. Use it in step 4. 4. Add a few drops of toluidene blue to the root tip on your slide for 2 minutes. Be very careful with this stain as it is practically indelible on hands and clothes. Toluidene blue stains the chromosomes to form a blue purple color. Wait 2 minutes, then remove excess stain from the slide by blotting around the root tip with a paper towel. Do not touch the root tip. 5. After blotting, add a few drops of water and gently cover the root tip with a cover slip, being careful to avoid trapping air bubbles under the slip. Using the eraser end of a pencil, carefully push down on the coverslip where the tip is to squash the tissue. Make sure you are applying vertical pressure with the pencil eraser and steadily increasing the pressure in a firm, continuous movement. It will take a great deal of pressure to spread the cells into a single layer and to squash the cells, breaking the cell walls. Be very careful not to break the slide! The cells need to be in a single layer.* *If the cells are not sufficiently separated to permit viewing of each, squash the preparation again. 6. View slide and make observations. 5
Appendix C. Chi Square Statistic Today, we will be using statistics to test hypotheses, through the Chi Square (χ 2 ) Test Statistic. This test compares actual (observed) and predicted (expected) outcomes of an experiment. For each type of outcome (in our case, the phases in mitosis) in the experiment, there are both observed and expected numbers of occurrences. The Chi square statistic is used in this test:. Statisticians have determined the probability distribution of the Chi square statistic. You will use Table 1 for the Chi square statistic to analyze your results. This table will let you decide if the difference between observed and expected values is small enough to be due to chance (meaning the hypothesis may be true) or is too large to be due to chance (meaning the hypothesis produces a poor prediction of your results and is unlikely to be true). Table 1. Chi Square Probability Table Degrees of freedom Reject the hypothesis if Χ 2 is greater than: 1 3.841 2 5.991 3 7.815 4 9.488 5 11.070 Table 2. A simple way to manage the Chi Squared statistic calculations Stage of Observed (O) # Expected (E) # (O E) (O E) 2 (O E) 2 /E Mitosis Interphase Prophase Metaphase Anaphase Telophase SUM this column to get Chi Square In Table 2, the observed numbers will come from the data you collect during lab while the expected numbers will be derived from your hypotheses. To determine the degrees of freedom (df) you simply take the number of phases in mitosis (or rows in the table) and subtract 1. In our case, df = 5 1 = 4. Now use Table 1 to look at the row for the appropriate degrees of freedom (in our case, 4) to get a Chi Squared probability value. Compare this number to the value you obtained in Table 2 and determine whether or not the hypothesis you were testing holds true. If the number you calculated is greater than the number you pulled from Table 1, you fail to accept (i.e. reject) the hypothesis you were testing. However, if the value you calculated for Chi Squared is smaller than the number in Table 1, you will not reject the hypothesis you tested. Works Cited Elzinga, C., S. Michaud Lawrence, L. M. Leege, M. K. Heidemann, and D. O. Straney. 2007. Biological Sciences 110: Laboratory Manual. 12 th Edition. Plymouth, MI: Hayden McNeil Pub. 6