Lab 6, Part 1: Mitosis & Cytokinesis

Transcription

1 Biology 211, NSCC Lab 6, Part 1: Mitosis & Cytokinesis OBJECTIVES To observe the stages of mitosis in prepared slides of whitefish blastula and onion root tips. To gain a better understanding of the process of mitosis in plant and animal cells. Name, identify, and describe the events occurring during the phases of the cell cycle Relate the process of DNA replication to the process of mitosis Describe the structure of a chromosome during the cell cycle Describe the functions of centrioles, centromeres, and spindle fibers in mitosis Compare the process of cytokinesis in animal and plant cells Identify the stages of mitosis in living tissues from the onion root tip, and animal cells (from prepared slides) INTRODUCTION Cell division can be broken down into two processes, mitosis and cytokinesis. Mitosis is the process that leads to the equitable distribution of genetic material into the two nuclei of the two daughter cells. Cytokinesis ("cell movement") is the process that leads to the equitable distribution of cytoplasm to the two daughter cells. In short, mitosis is the division of the nucleus, and cytokinesis is the division of the cytoplasm. In this lab you will study these two processes. You will prepare slides of onion root tips and examine prepared slides of whitefish blastulae, which have also been stained to highlight the nuclei. A blastula is a developmental stage in many animals. It occurs shortly after fertilization and the formation of the zygote. It is the ball of cells stage of development. A QUICK REVIEW OF CELL DIVISION The genetic information of plants, animals and other eukaryotic organisms resides in several (or many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46 chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate their DNA before dividing. During DNA replication, the two strands of the DNA double helix separate, and for each original strand a new complementary strand is produced, yielding two identical DNA molecules. DNA replication yields an identical pair of DNA molecules (called sister chromatids) attached at a region called the centromere. DNA replication in eukaryotes is followed by the process called mitosis which assures that each daughter cell receives one copy of each of the replicated chromosomes. During the process of mitosis, the chromosomes pass through several stages known as prophase, metaphase, anaphase and telophase. The actual division of the cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages, particular events occur that contribute to the orderly distribution of the replicated chromosomes prior to cytokinesis. The stages of mitosis Prophase. During prophase, the chromosomes supercoil and the fibers of the spindle apparatus begin to form between centrosomes located at the pole of the cells. The nuclear membrane also disintegrates at this time, freeing the chromosomes into the surrounding cytoplasm. Prometaphase. During prometaphase, some of the fibers attach to the centromere of each pair of sister chromatids and they begin to move toward the center of the cell. It is difficult to determine the difference between prophase and prometaphase, so for this lab we will combine the two phases as prophase. Metaphase. At metaphase the chromosomes have come to rest along the center plane of the cell, called the metaphase plate.

2 Anaphase. During anaphase, the centromeres split and the sister chromatids begin to migrate toward the opposite poles of the cell. Telophase. During telophase, the chromosomes at either end of the cell begin to cluster together, which facilitates the formation of a new nuclear membrane. This also is when cytokinesis occurs, leading to two separate cells. One way to identify that telophase has begun in plants is by looking for the formation of the cell plate, the new cell wall forming between the two cells. PRELAB Before coming to class, review the chapter on mitosis in your biology book, read over this lab (parts 1 and 2), and answer the following questions. You may find it helpful to bring your textbook to lab! 1. Why would we look at onion root tips (as opposed to perhaps a leaf or stem) and whitefish blastula (as opposed to a fin or gill for example) to study the phases of mitosis? 2. In what other sorts of tissues would you expect to find mitosis occurring? 3. What function does meiosis serve in Lillium anthers? In what types of animal tissues would you expect to observe meiosis occurring? 4. When observing a specimen on a compound scope using the 4X objective lens, the specimen is magnified times. Part 1A: Mitosis in Onion Root Tips Why use onion roots for viewing mitosis? The roots are easy to grow in large numbers. The cells at the tip of the roots are actively dividing, and thus many cells will be in stages of mitosis. The tips can be prepared in a way that allows them to be flattened on microscopes slide ( squashed ) so that the chromosomes of individual cells can be observed. The chromosomes can be stained to make them more easily observable. Regions of Onion Root Tips There are three cellular regions near the tip of an onion root. 1. The root cap contains cells that cover and protect the underlying growth region as the root pushed through the soil. 2. The region of cell division (or meristem) is where cells are actively dividing but not increasing significantly in size. 3. In the region of cell elongation, cell are increasing in size, but not dividing. Viewing Chromosomes Chromosomes generally are not visible as distinct entities in nondividing cells, since the DNA is uncoiled, but the process of mitosis is facilitated by supercoiling of the chromosomes into a highly compacted form. Supercoiled chromosomes can be visualized in cells, particularly if they are treated with a DNA-specific stain, such as the Feulgen stain. MATERIALS (per group) safety goggles ruler pipette (1) gloves scissors discard flask (small glass) micro-tube (1) & styrofoam holder forceps water in dropper bottles onion root tip (1-2) dissecting probe Fuelgen stain (1/bench) MATERIALS (classroom station) HCL & pipettes large discard container Heat block slides cover slips

3 PROCEDURES You will work in groups of two for this lab exercise. Each group of two will be assigned either an onion that was growing in water or one growing in rooting hormone. The onions we will be using have been previously treated with a fixative to stabilize the cells. Important: We will be using HCL, so you will need to wear goggles and gloves while preparing the slides. Softening the roots (so that they later can be spread on a microscope slide). 1. Mark the micro-tube with your initials. 2. Using scissors, cut off one entire root from the onion (either A or B as assigned). Be sure to know which end is the root tip and which end is the cut end. 3. Next cut the root so you have a piece approximately 1cm long that includes the root tip. Using forceps, transfer the root tip into a plastic micro-tube. 4. Fill the tube about 2/3 full with 1M HCl, using a pipette. *** Caution: Work with the HCl carefully, it is a strong acid. *** 5. Place the tube in a heat block set to 60 o C, and allow the roots to incubate for 12 minutes. 6. After the 12 minute incubation period, remove the tube from the heat block. Removing HCL & rinsing roots Important: During these steps, do not put HCL, Feulgen stain or the water rinses down the sink! Use the discard flask. 7. Using a plastic squeeze pipet, carefully remove the HCl from the micro-tube and transfer it to the discard flask. 8. Add water from the dropper bottle to the micro-tube to rinse the root tip, and then use the pipette to remove the rinsing water and transfer it to the discard flask. Repeat 2 more times. 9. After removing the water from the third rinse, you are now ready to stain the root tip. Staining the chromosomes 10. Add enough Feulgen stain from the dropper bottle to cover the root in the micro-tube. *** Caution: the Feulgen stain will strongly stain skin and clothing. *** 11. Return the micro-tube to the heat block and incubate the roots in the stain for 12 minutes. During this time the very tip of the root will begin to turn red as the DNA stains the numerous small actively dividing cells at the tip. Removing the Feulgen stain & rinsing roots 12. Using a plastic squeeze pipet, carefully remove the Feulgen stain from the micro-tube and transfer it to the discard flask. 13. Add water from the dropper bottle to the micro-tube to rinse the root tip, then use the pipette to remove the rinsing water and transfer it to the discard flask. Repeat 2 more times. 14. After removing the water from the third rinse, you are now ready to prepare the slide. Preparing the slide 15. Transfer the root to the center of a clean microscope slide and add a drop of water. 16. Using a razor blade cut off most of the unstained part of the root, and discard it. Note if entire sample is stained, do not cut off anything. 17. Cover the root tip with a cover slip, and then carefully push down on the cover slide with the wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide sideways the goal is to flatten the root, not twist it. The root tip should spread out to a diameter about cm.

4 Observations of onion root tip slide. Scan the microscope under the 10x objective. Look for the region that has large nuclei relative to the size of the cell; among these cells will be found cells displaying stages of mitosis. Examples are shown in the figure to the right. Switch to the 40X objective to make closer observations. Since prophase and prometaphase are difficult to distinguish, classify all these cells as prophase. Find representatives of each stage (prophase, metaphase, anaphase, telophase) and make a drawing of each in your lab notebook. Be sure to note your total magnification and label the key structures in your sketches. Part 1B: Estimating the Relative Length of Each State of Mitosis. For this procedure, we will use permanently mounted slides of onion roots. These slides are prepared by slicing the roots into thin sections, mounting them on microscope slides, staining, and then mounting under a cover slip. While making your observations, consider the relative number of cells actually involved in mitosis. Cells that are actively dividing but not yet in mitosis are said to be in interphase, during which time the DNA is copied and the cell is otherwise preparing for replication. Some root cells may also have ceased dividing and be only increasing in size, whereas others have reached their final, mature size and function and are said to be in the G0 stage. Procedure: 1. Using the 10X objective, find the region of active cell division. 2. Switch to the 40X objective and begin observations at the lower end of this region. Students should then take turns as observer and recorder. The observer should call out the stage of mitosis of each cell to be tallied by the recorder in a results table in your lab notebook. Switch roles for a second slide. Since prophase and prometaphase are difficult to distinguish, classify these cells as prophase. Only count as prophase the cells that contain distinctly visible chromosomes. 3. Systematically scan the root tip, tallying each cell in a state of mitosis, taking care to not record the same cell twice. Tally the stages of 20 mitotic cells. Switch roles, allowing your partner to also identify 20 stages. 4. Add your data to the class data sheet, and record the class totals in your lab notebook. Part 2: Mitosis in animal cells Materials: Prepared slide: Cross sections of whitefish blastula Procedures 1. Obtain a prepared slide of cross sections of whitefish blastula and observe using the 10x objective. Look for cells undergoing each of the stages. 2. Switch to the 40X objective to make closer observations. Find representatives of each stage (prophase, metaphase, anaphase, telophase) and make a drawing of each in your lab notebook. Mitosis Cleanup: Pour group discard flasks into large discard flask. Then rinse the group discard flasks in the sink and return to the cart. Discard micro-tube and pipettes in garbage. Put cover slips in broken glass jar. Rinse slide, wipe dry with a tissue and return to the slide box. Please take care to make sure the slides are placed in the correct box and properly aligned!

5 Return all other equipment to the carts in the front of the room. Return whitefish slides neatly to the correct box. Put microscope away correctly unless you are continuing on to Part 3. Part 3: Meiosis OBJECTIVES: List and explain the principal events of the stages of meiosis. Define and explain the following terms: diploid, haploid, homologous chromosomes, alleles, synapsis, tetrad Explain and understand the difference between the first and second meiotic divisions List and explain the similarities and differences between meiosis and mitosis INTRODUCTION Meiosis consists of two nuclear divisions (meiosis I and meiosis II) and results in the production of four daughter nuclei, each of which contains only half the number of chromosomes (and half the amount of DNA) characteristic of the parental cells. During meiotic reduction of the chromosome number to half, however, chromosomes are not just divided into two sets at random. In diploid organisms, chromosomes occur in matched pairs called homologous chromosomes. These are identical in size, shape, location of their centromeres, and types of genes present. One member of each homologous pair is contributed by the male parent and one is contributed by the female parent during the process of sexual reproduction. Meiosis provides as precise a mechanism as possible for separating these homologous chromosomes so that daughter cells carry one member, or homologue, of each chromosomal pair. PROCEDURES Using the slides of Lilium anthers and ovules, observe and draw the phases of meiosis. Due to the coordination of the stages of meiosis, you will not be able to see all of the phases. Identify and draw as many as you can. Sketch and label each phase in your sketchbook. As always, label structures in your drawings and note the total magnification! When finished, please return all prepared slides correctly and put away your microscope. Remember that the microscopes should always be put away with the stage at its lowest setting, the 4X objective in place, and the cord tightly wrapped to prevent accidents! POSTLAB- Answer the following questions on a separate sheet of paper. These typed questions are due 1 week from the completion of your lab. 1. How does the arrangement of chromosomes differ when comparing metaphase of meiosis I and mitosis? 2. What happens to the sister chromatids during anaphase of meiosis I? 3. Compare the amount and arrangement of genetic material in each cell following telophase I of meiosis and telophase of mitosis? 4. How does metaphase II differ from metaphase I? 5. How does metaphase II compare to metaphase of mitosis? 6. How many cells were formed due to the process of meiosis? How many cells were formed during the process of mitosis? 7. List three major differences between meiosis and mitosis. 8. Use the class data to calculate the percentage of cells in each stage. (The relative time span of each stage is equivalent to the percentage of cells found in that stage.) Which stage is the longest? Which is the slowest? Why might this be?