Pope Sheldon Lab section L19 11-1-07 Mitosis and Chromosomes Introduction: Mitosis is a necessary function for all cells except for a certain few such as nerve cells. Mitosis is the splitting of one cell and creating two. There are four basic phases to mitosis which consist of prophase, metaphase, anaphase, and telophase. Before mitosis happens a cell starts in interphase. The gap 1 phase is where a cell spends most of its life. This is the growth phase and where the cell prepares DNA for replication. The Synthesis phase is where the cell replicates the DNA, duplicates the centriole, and synthesizes steroids. The gap 2 stage prepares the cell for mitosis. The basic function of mitosis is the splitting of chromosomes. Chromosomes are made of a material called chromatin. Chromatin is composed of DNA, as well as histones and other proteins called non-histones. During mitosis chromosomes become vivid and can be seen if they have been stained. In Prophase the spindle fibers attach to the centromeres of each chromosome, and then the spindle fibers line the chromosomes up. By metaphase the chromosomes are all aligned n middle of the cell. The nuclear membrane has disappeared allowing this to happen. Anaphase is next and the chromosomes which were duplicated in S phase are now separated and pulled towards the poles of the cell. The last step is telophase where the nuclear membrane starts to reform and a cleavage furrow is created. (Biology Lab Manual 2007) These are the steps of mitosis for a animal cell. A plant cell has the same effects except instead of spindle fibers from the centrioles, a plant cell uses meristems. Also in a plant cell there is not cleavage furrow created, instead the cell forms a cell plate which is where the new cell is formed.
This lab is meant to familiarize me with mitosis and it's certain steps. The experiment consists of using beads to show the S phase in interphase through anaphase. This shows what exactly happens to chromosomes during mitosis. The second part of the lab is to actually see different points of mitosis by observing the chromosomes in plant cells. Materials and Methods: Part I: Pop beads Magnetic strips. Using the beads, show the replication of chromosomes as would be seen in S phase for four chromosomes. Use the magnets to indicate centromeres. After completing S phase, jump to metaphase and separate the chromosome pairs by force. Let the chromosomes drag which shows the actual effect of the spindle fibers. Anaphase is next where the beads are separated and dragged toward opposite side of the cell. Notice that the chromosome "arms" drag behind them creating a V shape. Part II: Fava Bean root tips Forceps Microscope slides Cover slips Dissecting needle (preferably flattened) Pencil Hot 65C water bath 0.5N HCL solution Acetocarmine solution Rafts and milopette tubes Ice water bath Razor blades Using forceps, carefully place a root tip in a tube of hot 0.5N HCL. Place the tube back into the 65C water bath. Keep track of time and allow the root tip to stew for only 1 minute. Put the root tip now into an ice cold bath. Only allow this root tip to sit for 5-6 minutes. If it sits in the ice water for too long, it will absorb too much water. Take a root tip and place it on a slide without a cover slip, blot away water with paper towel. Do not touch the root tip with paper towel. Add a small drop of acetocarmine solution to the tip of the root. Don't let the root tip dry out. Use a razor blade to sever the tip from the rest of the root. The tip is small and to make sure you get it, uses a dissecting microscope. Leave the severed root tip in the stain while throwing away the rest of the root. Using the flattened end of the razor blade or tool, squash the root tip in the stain so it is broken into a few small pieces, make sure the root tip stays in the stain.
Place a cover slip over the solution, hold down one side and use a pencil eraser to push down gently on the cover slip, this will spread the cells out. Add more stain to the if slide there are air bubbles. Place a piece of paper towel on the cover slip and use a thumb to press on the cover slip for a few seconds, do not jostle the cover slip, and press straight down. Observe the slide at 400X magnification. Find a good cell to draw for each of the four phases of mitosis. Observe the other two slides, one with a plant cell plate, and the other is a fish cell in telophase. Results: The cells were hard to see due to the use of 400 times magnification, but they were still able to be seen performing mitosis. Prophase could be seen as just a cell with chromosomes inside. This is represented in figure 1. Figure 2 is the example of metaphase where the chromosomes are all lined up in middle of the cell, even though it was too small to see, the spindle fibers are attached to the centromeres of the chromosomes. The cells viewed that were performing anaphase could be seen pulling the chromatids to each side of the root cell in figure 3. The final phase of mitosis can be seen in figure 4, the plant cell is creating a cell plate, and the one in figure 4 is near the final stages of cell plate formation. On the reverse side of the observations sheet are figures 5 and 6. Figure 5 is the representation of animal cells performing cytokinesis. In the drawing, you can see the cytoplasm being sqeezed into two different cells. Also in this slide different stages of mitosis could be observed. Figure 6 is the illustration of a cell plate being formed in an onion root tip cell. This is basically the same as the root tip cells from the fava bean. Discussion: For the group that I worked with the lab was a little bumpy due to the cause of the root tip being lost, but in the end we ended up borrowing a slide with a well prepared root tip from another group. The cells could be seen due to staining, and they were performing mitosis at 400X
magnification, but since we were not able to use 1,000X it was a little bit harder to distinguish between structures in animal cells and plant cells. In plant cells there is no centriole but the meristem which is spindle fibers from the cytoskeleton. Also in the plant cells the cell plate is formed instead of the cytokinesis like in figure 5. This is because plant cells have cell walls which cannot easily be dissolved to perform cytokinesis. This lab definitely helped visualize the steps in mitosis and see what happens up close in a microscope. Cells perform mitosis to grow, and some plants grow at alarming rates, this must mean that mitosis is a generally fast process for plant cells. Animals grow slowly which must mean mitosis in animal cells is slowly performed. Giant kelp is grown underwater and can grow at rates of up to 3 feet per day. The speed of mitosis must be incredible. Some animals grow fast but not quite as fast as that. The parts of plant cells like cell plate and meristems may be a quicker way to perform mitosis. Questions: 2. If I wanted to isolate chromosomes I would extract them from the cell during anaphase because they are being drawn to one side of the cell. They are also separate because in mitosis the chromosomes separate which makes it easy to isolate chromosomes. 3. Edwards Syndrome is the genetic disease where chromosome number 18 is missing, the key traits of this disease is paralysis and mental retardation. Down Syndrome occurs in Chromosome 21 and the major effects Down Syndrome inflicts are mental retardation and eye problems. CriDu-Chat Syndrome is the effect of chromosome number 5 missing (also called 5p- syndrome). The visible traits of this disease are low muscle tones, problems eating food, drooling, and hearing impairments. (Genetics Education Center, University of Kansas Medical Center 19952007)
Bibliography: Minocha, S. C., Biology Laboratory Manual, Kendall/Hunt Publishing Co. (2005,2007); Bio 411 Genetics Education Center, University of Kansas Medical Center (1995-2007). Genetic disorders, Support Groups for Chromosomal Condition. Retrieved on 11-5-07 from http://www.kumc.edu/gec/support/