Structural Organization of Living Cells

Chapter 3 Structural Organization of Living Cells Osiris Boutros and Susan Noblit Boutros Department of Biology University of Pittsburgh Bradford Campus Bradford, Pennsylvania 16701 Dr. Osiris Boutros is Associate Professor of Biology at the Univer- sity of Pittsburgh Bradford Campus. His background includes de- grees from Cairo University, Florida State University and the University of Pittsburgh. Although his research interests are in the field of plant physiology, specifically dealing with the isolation, identification and mode of action of plant growth regulators, his teaching responsibilities include introductory biology, cellular and molecular biology, environmental biology and human anatomy and physiology. Dr. Susan Noblit Boutros is also an Associate Professor of Biology at the University of Pittsburgh Bradford Campus. She is a graduate of Dickinson College and the University of Pittsburgh. Her research interests are in cytogenetics and cytology. Her teaching responsibilities include introductory biology, cytology, human genetics and human development. 34 Living Cells Introduction Students tend to visualize cells as static entities based on their experience with textbook photographs and microscope slides. By focusing on living eukaryotic cells, this laboratory demonstrates that cells are dynamic units and that movement and change are intrinsic properties of life. Most of the unique features of the eukaryotic cell are visible in the light microscope. A number of cell organelles are readily observed and can be identified with a fair degree of confidence. In addition to examining and identifying cell organelles and cell structures, students are introduced to the techniques and concepts of microscopy and cytochemistry. Living cells other than heavily pigmented plant cells are best visualized with a phase microscope. Phase microscopy gives extraordinary detail to cells by taking advantage of the small differences in refractive indices and thick- nesses of various cell parts. Pleomorphic changes of mitochondria, cyclosis, and ciliar movement are spectacularly demonstrated. The exercise was designed for second-year biology majors. The material can be modified for freshman-level courses by excluding some stains and reducing the number of specimens examined. The instructor might also elim- inate the student use of the phase and polarizing microscope and retain these portions as demonstrations only. Times involved: Instructor: 4 hours reagent preparation 2 hours purchase of fresh materials Student: 6-8 hours (two laboratory sessions) Student Materials Background Information The cells of all higher plants and animals are eukaryotic cells character- ized by membrane-bounded nuclei and various membrane-constructed organelles which are lacking in prokaryotic cells. Most of the unique features of the eukaryotic cells are visible in the light microscope. Certain cell organelles can be identified with certainty in unstained prep- arations. Chloroplasts are easily visualized with the light microscope without stains or other special preparation. Other organelles are not so easily identified, and for observing these a variety of techniques are used. Chemicals that stain living cells are known as vital stains. Organelles such as vacuoles can be made visible with a vital stain because of their functional tendency to take in water- soluble materials for storage. An aqueous solution of neutral red becomes concentrated in the vacuoles, making them visible. Use of the vital stain Janus Living Cells 35 green B takes advantage of the functional activity of another cell organelle. The mitochondrion is the site of major reactions of biological oxidation and the electron transport system. When aqueous solutions of the blue-oxidized Janus green B diffuse into the cell, it is reduced in the cytoplasm to colorless compound and is locally re-oxidized in the mitochondria to the blue form. Procedures I. Observations of unstained cells A. Cell Surface Cell Membrane Examine one or both of the following preferably with a phase microscope: amoeba or buccal cells. To examine the amoeba, place a drop of culture, taken from the bottom of the dish, on a clean slide. Place a piece of broken cover slip beside the drop and add a cover slip. Scan the slide with the microscope while reducing the light level slightly. When an amoeba is found, watch it for a time to see the membrane motion and changes that occur in the membrane and cytoplasm as the amoeba moves. Buccal cells are prepared by gently scraping the inside of the mouth with the flat end of a clean toothpick. Place the cells in a drop of 0.9% saline solution on a glass slide. Add a cover slip. How does the cell membrane differ from that of the amoeba? The nucleus and mitochondria should be visible. I I Primary Cell Wall I 11 With fine forceps remove a piece of epidermis from the purple under- I3 side of a leaf of Rhoeo discolor and place the tissue in a drop of water. '1 Add a cover slip and observe with the microscope. Place a few drops of 10% sucrose at the edge of the cover slip and observe as the solution diffuses through the preparation. As the cell plasmolyzes, the cell wall will become distinguishable from the cell-membrane-bounded proto- I plast. If Rhoeo is not available, onion inner bulb scale epidermal peel I I may be substituted, but the onion tissue does not have the advantage I of the cytoplasmic pigment found in Rhoeo. I I Secondary Cell Wall I I Make a thin section of Coleus stem and mount on a microscope slide. I Under the microscope look at the vascular tissue to find cells with I secondary thickenings of the cell wall. Note that Coleus also has some I cells with cytoplasmic pigment. I I Cilia I Ciliated protozoans generally move too fast for easy observation of I I cilia, so slow the organism by placing a drop of 1.5% methyl cellulose I I together with a drop of protozoan culture on a microscope slide. Add I a cover slip before viewing with the microscope. 36 Living Cells B. The Cell Matrix and its Movement With fine forceps remove a stamen from a flower of either Trades- cantia or Rhoeo discolor, place it in a drop of water on a slide, and add a cover slip. Streaming cytoplasmic strands should be visible in the cells of the stamina1 hairs. Observe with a phase microscope if available. C. Cell Organelles Nucleus The nucleus is readily observed in onion bulb scale epidermis or in buccal cells. Use the slide of buccal cells prepared earlier in this exercise or prepare a slide of onion in the following manner. Cut a small triangle in the side of an onion bulb, pressing deeply enough to penetrate two or three layers of bulb scales. Remove the triangle and peel the epidermis from the inner bulb scale (see Figures 3.1 and 3.2). Place the epidermis in water on a glass slide. Add a cover slip. View the preparation with the phase microscope. Figure 3.1. Diagram showing a cell from onion inner bulb scale epidermis as seen with phase optics. Active cytoplasmic streaming will be noted in specimens. Prominent features include the nucleus (n), nucleoli, cell wall (w), numerous droplets (d), spherosomes (s), mitochondria and vesicles (e) tentatively identified as smooth endoplasmic reticulum. Living Cells 37 Figure 3.2. Cell organelles as seen with the phase microscope. At top: onion epidermal cell with numerous mitochondria (m). A large vacuole in the cell restricts the cytoplasm to the space between the cell wall and the vacuole. Area to the right above the nucleus contains vesicular elements suggestive of smooth endoplasmic reticulum (3). Bottom left: onion epidermal cell with large droplet structure (d) associated with mitochondria and vesicular structures. Bottom right: chromoplasts (c) of red pepper epidermis. Note distinctive cell wall (w). 38 Living Cells Mitochondria The onion bulb scale epidermal is one of the easiest cells in which to observe mitochondria. An epidermal peel of celery may also be used. Do the mitochondria move or change shape? Chloroplasts Examine one or more of the following: Elodea, spinach petiole epidermal peel, Euglena or Spirogyra. Prepare the spinach petiole epidermal peel by stripping a piece' of epidermis from the stem or petiole of a piece of spinach. Mount in water. Chromoplasts Examine one or more of the following mounted in water: carrot, tomato, red pepper. Prepare the carrot by making as thin a section as possible and mounting the section on a slide in water. With tomato and red pepper, either pulp or epidermis can be examined (see Figure 3.2). Endoplasmic reticulum Examine the onion bulb epidermal peel with a phase microscope. You should see a number of different structures. Can you distinguish between the nucleus, mitochondria, vacuole, and smooth endoplasmic reticulum (Fig. 3.1 )? D. Cell Communication Make thin cross-sections of one or both of the following, mount in water, and look for evidence of plasmodesmata: boiled date pits, co- tyledons of growing seedlings. E. Cell inclusions Make a thin section of one of the following or mount a whole leaf of Lemna frond (duck weed): rhubarb stem, wax begonia petiole, Aloe leaf. Look for crystalline inclusions. Use polarizing lenses if they are available. 11. Cytochemical Identification of Cell Organelles and Molecules A. Identi5cation of Vacuoles in Plant Cells with Neutral Red Place a few drops of 1% neutral red on a depression plate. Remove a root from an onion bulb and place the tip of the root in the neutral red. Crush the root gently with the rounded tip of a glass rod. Allow the root tip to remain in the solution 1G15 minutes. Carefully remove the root tip and mount it on a glass slide with a cover slip and a drop of the neutral red solution.

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