Exploring Mitosis Lab IMPORTANT LEARNING OBJECTIVES INTRODUCTION cell cycle interphase division (mitosis)

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1 Exploring Mitosis Lab Today s lab will give you a chance to get an up-close view of mitosis, the process by which eukaryotic cells divide to generate identical daughter cells. We re using garlic (A. sativium) root tip cells because garlic bulbs grow rapidly, and therefore the root tips of growing plants contain many actively dividing cells. A few days prior to the experiment you ll carry out today, two of the garlic bulbs you planted last week were treated with a chemical (Chemical X) that has been observed to alter the rate of mitosis in some cell types. Your goal is to determine whether Chemical X speeds up, slows down, or has no effect on mitosis in garlic root tip cells. IMPORTANT: In addition to reading this handout, you should review your notes from our discussion of mitosis in class. You also need to review dissecting and compound microscopes, which were described in your Paramecium Lab handout. LEARNING OBJECTIVES Improve your understanding of mitosis. Learn basic cell staining and slide preparation techniques and gain additional practice using microscopes. Practice developing hypotheses and predictions. Be able to apply data analysis skills developed in prior labs and to draw conclusions based on the results of your experiment. INTRODUCTION Cell Division Cell division is a carefully controlled process both when it occurs and the set of steps that ensure each daughter cell gets one complete copy of the genetic information. In eukaryotic cells (e.g. plant, animal, fungi), the cell cycle is the ordered series of events from when a cell is produced by division to when it divides. As shown in Figure 1 below, the cell cycle is divided into two main parts, interphase, and division (mitosis). During interphase, cells grow, produce additional molecules and organelles, and replicate their DNA. Cells spend most of their time in interphase. Actual division is a small part of a cell s life. The amount of time it takes for a cell to progress through the cell cycle varies HUGELY between cells. Some cells are produced and then never divide, while other cells divide rapidly. For example, most cells that transmit nervous system signals (neurons) never divide. Human skin cells, however, divide about once a day. As you learned, mitosis is the process of cell division by which eukaryotic cells divide to produce two genetically identical daughter cells. It involves a series of steps that must occur in a specific order to ensure that each daughter cell ends up with one, and only one, copy of every chromosome. The very first step in mitosis involves condensing the snarled mass of replicated DNA in a cell s nucleus into discrete, organized packages. At this point, each chromosome consists of a pair of sister chromatids identical DNA molecules generated by replication. Looking at the images in Figure 2 (see next page), you can see that the chromosomes remain condensed until after division is complete. Therefore, condensed chromosomes are a key indicator that a cell is in the process of mitosis. 1

2 Visualizing and Analyzing Cell Division To visualize the cells in your garlic bulb s roots, you ll cut off the very tip of the root and treat the cells with a fixative. A fixative is a chemical that fixes, or stops, the cells in whatever phase of the cell cycle they are undergoing at that moment. As noted above, condensed chromosomes are a sign that a cell is in the process of dividing, and this is what you ll use to identify mitotic phase cells. To determine whether a cell s chromosomes are condensed, you need to be able to see the chromosomes! After you fix your cells, you ll use a dye called Carbol-Fuchsin to stain the DNA (it will turn a dark red). Once the cells are fixed and stained, your goal is to prepare a microscope slide with a single layer of cells. This will allow you to see individual cells, and to count the number of cells that were undergoing mitosis when you harvested your root tip and fixed the cells. As described in the overview of this lab, one of the two garlic bulbs you planted was treated with a chemical that has been observed to alter the rate of mitosis in some types of cells, Chemical X. The other garlic bulb is your control. To determine if, and if so how, Chemical X affects the rate of garlic root tip cell mitosis, you need to determine the mitotic index for your control and treated root tips. The mitotic index is the ratio of the number of cells in mitosis to the total number of cells, and is calculated using the formula shown below. It s basically just the proportion of mitotic cells (cells undergoing mitosis) in a sample. IMPORTANT: You are using the mitotic index to assess the rate of mitosis how quickly or slowly a cell progresses through mitosis. Think about what you would expect to observe for your treated cells in comparison to the control cells if Chemical X made cells go through mitosis faster, more slowly, or did not alter the rate. Chemical X has NOT been observed to prevent cells from entering mitosis or to cause cells to start mitosis. After you collect your data, you ll apply your knowledge of averages, standard deviations, and t-tests to assess whether the chemical has a significant effect on the rate of mitosis in garlic bulb root tips, and if so, to draw a conclusion regarding whether the chemical increases or decreases the rate of mitosis. Again, note that Chemical X has NOT been found to prevent mitosis or cause cells to undergo mitosis. If the chemical inhibited mitosis, your garlic bulb roots would not grow. This is VERY important to keep in mind when analyzing your data! mitotic index = number of cells in mitosis total number of cells 2

3 LAB OUTLINE Below is a general outline for today s lab. The specific procedures you need for each part are described following the outline. The Exploring Mitosis Questions due next week will be provided for you in lab. You will work with a partner, but each person must turn in their OWN work. I. Quiz To prepare for this quiz, read this handout carefully focusing on the overall purpose of the experiment, and the main steps of the procedure (e.g. how many slides will you prepare, what data you are collecting, etc.). You should also review your notes from class on mitosis, and the microscope descriptions provided in the Paramecium lab handout. II. Lab Discussion: Developing a Hypothesis & Making Predictions III. Harvest Roots and Fix Cells Follow the appropriate procedure to collect tips from the garlic bulbs you planted last week and treat them with a chemical that will fix (stop) the cells in whatever stage of division they happen to be in when you cut off the tips. IV. Prepare Slides (aka Squashes ) Follow the appropriate procedure to stain the root tip cells you just fixed so you can see the DNA, and to spread the mass of tissue out into a thin layer, by squashing the tissue with a coverslip, so that you can view individual cells. It takes some patience, practice, and luck to create a good slide. You may (and likely will) need to repeat this process several times before you get a good slide. IMPORTANT: You need to make ONE good slide from a control tip and ONE good slide from a root tip that was treated with Chemical X. V. Collect and Analyze Data Use the procedures provided to collect and analyze your data. PROCEDURES Root Harvesting & Cell Fixation IMPORTANT: You MUST wear gloves and lab safety glasses (both provided in lab) while preparing your slides. You may remove your safety glasses and gloves when using the microscope. 1) Find your garlic bulbs, and bring them to your work space. Remove your control garlic bulbs from their cup and place them on the petri dish labeled control (if your dishes aren t labeled, label them). Carefully pour the liquid in the cup into the liquids waste container. Keep this cup; you will use it as a waste container later. Put the toothpick in the Laboratory Waste box. 2) Repeat the step above for your chemically-treated bulbs, putting the bulbs in the petri dish labeled treated. Dispose of the liquid, and put the toothpick and cup in the Laboratory Waste box. 3) Starting with your control bulbs, use dissecting scissors to cut approximately one centimeter off the ends of 6 to 8 of the sprouted roots. Put the root ends in a 1.5 ml microcentrifuge tube labeled control. Repeat for your chemically-treated bulb, putting the root ends in the microcentrifuge tube labeled treated. 4) Add 0.5 ml of Fixative (acetic acid/hydrochloric acid) to each tube. WARNING: This solution is caustic and can burn your skin! Make sure that you are wearing gloves and lab safety glasses when handling. 5) Place the tubes in a floating rack, and put the rack in the hot water bath (50 C) for 6 minutes. After the incubation period, transfer your tubes to a rack on your bench. 3

4 Slide Preparation IMPORTANT NOTES: - You need to make ONE good slide using a tip from a control bulb and ONE good slide using a tip from a chemically-treated bulb. - Be careful with the used kimwipes since they will have strong acids and dyes on them. - You want to squash the cells into a thin layer, NOT smear them by moving the cover slip side to side. - It will almost certainly take you multiple attempts to prepare good slides. Be PATIENT! - Make sure you have safety glasses AND gloves on before starting this procedure. 1) Clean a slide with 70% ethanol, and dry with a kimwipe. Label the slide on the very edge of one of the short sides C for control. Get a beaker, and fill it half way with tap water. You ll use this to rinse your tweezers. 2) Remove a control tip from the tube using a pair of tweezers, and place it on the slide. Rinse the acids off the tips of the tweezers in your beaker of water. 3) Using a razor blade, CAREFULLY cut away and save the first 1 to 2 millimeters of the root tip (the rounded end). You can use a dissecting scope to help you cut off the tip. Discard the remainder of the root end in the cups you kept earlier. IMPORTANT: Be careful using the razor blades and put them back in the container when you are done! 4) Place one drop of Carbol-Fuchsin stain on the root tip, and allow it to soak for 2 minutes. During this time, use the dissecting probes to soften the root tip tissue by lightly poking and working it apart. Be gentle! Being careful not to wipe away the root tissue (cells), blot away the excess stain using a kimwipe. 5) Carefully place a cover slip over the root tissue. Put your thumb squarely on the coverslip (GLOVES ON!), and push directly down using moderate pressure. Do NOT slide the coverslip from side to side! That will just smear the cells. Holding the edge of the cover slip with a finger can help keep it steady. Another method is to use the eraser end of a pencil to press gently on the coverslip. If liquid leaks out of the coverslip, blot carefully with a kimwipe. 6) Observe your squash (slide) using the compound microscope at low power (40X 100X). Do NOT touch the course focus knob again after leaving 40X. Search carefully for regions of the slide where you can clearly see a single layer of individual cells. This is important because it is challenging to understand what you see when you are looking through several layers of cells it s all a big blur! 7) When you identify a region of the squash where individual cells are clear, switch the objective to 400X and scan your slide for cells in stages of mitosis. A good slide will have lots of cells visible in various stages of mitosis, and lots of cells squashed into single cell layers. If you can identify about 5 fields of view (the area you see inside the view of the microscope at 400X, or camera at no higher than 50% zoom) that are clear enough to determine if cells are in interphase versus mitosis (uncondensed vs. condensed chromosomes), it is a good slide for analysis. 8) If you can t find regions of the slide with individual cells, your squash was unsuccessful. Try re-squashing by pressing on the coverslip again. If that fails, return to step #1 and make a new slide using one of your other root ends. Now that you know how to make a slide, you can make 2-3 slides at the same time. 9) Once you have one good slide for your control garlic bulb, repeat the process to prepare one good slide for your chemically-treated bulb. Label this slide T for treated. 4

5 Data Collection As described in the introduction, the mitotic index is the ratio of the number of cells in mitosis to the total number of cells. For the purposes of your experiment, you will take photos and make counts of cells within several fields of view of your microscope at 400X. Again, the field of view (FOV) is everything that you can see inside the view of the microscope, or camera at no higher than 50% zoom. For every FOV you select for observation, you will count the total number of mitotic cells and the total number of interphase cells (the sum of the two is the total number of cells). When you analyze your data, you will compare the mitotic index of untreated garlic root tips to chemically-treated garlic root tips to determine if, and if so how, Chemical X alters the rate of mitosis in garlic root tip cells. Using the camera to view your samples should make data collection easier, and will allow both partners to participate at the same time. Your lab TA will review how to use the cameras, and directions will also be provided in lab. NOTE: If you have issues with the cameras, you can do your counts looking through the microscope. 1) In the space provided under Data Collection (in the lab questions handed out in lab), make TWO tables to collect your data one for your control garlic bulb cells and one for your chemically-treated garlic bulb cells. You need rows for five FOV (see below) and four columns to record the number of cells in mitosis, the number of interphase cells, the total number of cells, and the mitotic index. Make sure you label your tables as control or chemically-treated samples. 2) Starting with your control garlic bulb slide, find a good FOV where you can clearly determine whether cells are in interphase vs. mitosis. Use the camera to capture the image (remember, no higher than 50% zoom). Save the file to the DESKTOP as control 1. Again, you can choose to do your counts looking through the scope. 3) Count all the cells that you can identify as being in mitosis, and record the number in your table (this maybe a very small number!). Count all the interphase cells, and record that data in the table. You do NOT need to calculate the total number of cells or mitotic index now. You ll do that later using Excel. 4) Repeat the step above for four additional FOV (five total), taking and saving photos of each. Make sure that your FOV do NOT overlap so you don t double count cells. The easiest way to do this is to pick a cell, and then move the field of view in a particular direction until the cell just barely disappears. If you keep moving the field of view in the same direction, you ll avoid double counting cells. 5) Use the same process to count five FOV (and take photos!) for your chemically-treated garlic blub slide, recording the data in the appropriate table. 6) After you have completed your counts, clean ALL your slides well in the sink using SOAP and WATER (NOT ethanol!). Put the slides in the tray provided to dry. Dispose of the cover slips, the cups with your discarded root tips, and your garlic bulbs in the Laboratory Waste box. Rinse your petri dishes with water and dry them with a paper towel. 5

6 Data Analysis To assess whether Chemical X altered the rate of mitosis in garlic root tip cells, you need to determine the average mitotic index and standard deviation (SD) for each of your conditions, and use a t-test to compare your control and chemically-treated samples to obtain a p-value. You can then use your p-value and the predictions you developed as a group at the beginning of lab to draw conclusions from your data. Use the table provided under Data Analysis in your lab questions document to record the results of your data analysis. 1) Open Excel. Using the knowledge you ve developed in previous labs, create tables and enter your data (number of mitotic cells and number of interphase cells) for your control and chemically-treated garlic bulb root tip cells into the spreadsheet. SAVE your file to the desktop (name the file whatever you want). 2) Use Excel to calculate the total number of cells and mitotic index for each FOV for each condition. Record the values in the table in your lab handout. 3) Use the Excel formulas below to calculate the average mitotic index and standard deviation for each condition. Record the values in the table provided under Data Analysis. Note that cell range in the formulas refers to the range of cells that contain your data (e.g. B3:B7). =AVERAGE(cell range) =STDEV(cell range) 4) Use the Excel formula =T.TEST(cell range,2,3) to compare your two conditions and obtain a p-value. IMPORTANT: For your cell range, you need to select ALL the Excel cells that contain mitotic index values for BOTH conditions, and NOT the averages. If you don t remember how to do this, please ask your lab TA! Record the p-value in your lab handout. 5) Open Word (you can save and close your Excel file). Save the Word file to the desktop. 6) Pick your BEST control root tip cell photo and your BEST chemically-treated root tip cell photo. In Word, got to the Insert Tab and click on Picture. Browse to the desktop and find your favorite control root tip cell image. Click on the file name and then click Insert. Resize your photo if you want. Label the image Control. SAVE YOUR FILE. NOTE: If you were unable to take photos, get images from another group or ask your lab TA. 7) Repeat the step above for your favorite chemically-treated root tip cell photo, labeling the image Chemically- Treated. SAVE YOUR FILE. Make sure that your data tables and Excel calculations are complete, and you have a Word document with two images. Send your WORD FILE to yourself by . You are welcome to send the other images and/or the Excel file too if you want. DELETE ALL FILES YOU CREATED FROM THE LAB COMPUTER. Before next lab, complete the remaining questions in the Exploring Mitosis Lab Questions handout. Your assignment is due at the beginning of lab next week. Remember each person must turn in their OWN work! REFERENCES Bonner, JM. (2010) A Scenario-Based Study of Root Tip Mitosis. 31: 36-49, in Tested Studies for Laboratory Teaching (K.L. Clase, Editor). 6