Lab #6: Cellular Transport Mechanisms Lab

Transcription

1 Lab #6: Cellular Transport Mechanisms Lab OVERVIEW One of the major functions of the plasma membrane is to regulate the movement of substances into and out of the cell. This process is essential in maintaining the homeostatic state of the cell. If you recall, the plasma membrane is composed primarily of a phospholipid bilayer and specialized proteins. The unique structure of the plasma membrane allows it to be selectively permeable to certain substances. The permeability of the membrane is regulated by several variables, including size of the molecule, polarity of the molecule, and external conditions such as concentration, temperature, and pressure. For molecules to enter or exit a cell, they must overcome the concentration gradient, which involves the movement of molecules from regions of higher concentration to regions of lower concentration. Living systems have two primary mechanisms for moving substances in and out of the cell passive and active transport. In passive transport the cell uses no energy (ATP) as essential substances are moved across the plasma membrane. Examples of molecules moved by the various means of passive transport are oxygen, water, and glucose. The most fundamental means of passive transport is diffusion, the random movement of molecules from regions of higher concentration to regions of lower concentration. This random movement is also known as Brownian motion. A state of equilibrium is attained when an equal distribution of molecules exists throughout the system. The movement of water across the plasma membrane in living systems is called osmosis. More specifically, osmosis is the movement of water down its concentration gradient (from regions of higher water concentration to regions of lower water concentration) through a selectively permeable membrane. However, when determining the movement of water in regard to solute concentration, osmosis will occur in the direction of higher solute concentration from regions of lower solute concentration. Other mechanisms of passive transport include facilitated diffusion and filtration. In facilitated diffusion, carrier proteins along the cell membrane are required to ferry specific molecules such as glucose across the membrane into the cell. Filtration involves hydrostatic pressure (water pressure) forcing molecules through a cell membrane. Filtration is an essential mechanism that takes place in the kidneys in the formation of urine. In active transport, energy in the form of ATP is required for the movement of substances against the concentration gradient. Biological pumps essential in this process are the following: 1. The proton or hydrogen pump is necessary to maintain the normal ph of the stomach. 2. The calcium pump is important in nerve and muscle function. 3. The sodium-potassium pump is integral in cellular metabolism. Macromolecules such as polypeptides and polysaccharides are too large to traverse the cell membrane by either passive or pump systems.. Instead, they must be transported into the 1

2 cell by endocytosis and out of the cell by exocytosis. Both endocytosis and exocytosis employ the formation of vesicles in transporting substances. In endocytosis, solid substances can be engulfed by a cell through phagocytosis. Everyday examples in biological systems include the ingestion of food particles by an amoeba and the engulfing of foreign particles by a macrophage. Another example of endocytosis is pinocytosis, or cell drinking, in which liquid droplets that may contain salts and other molecules are taken into the cell through vesicle formation. Specialized cells in the roots of plants use pinocytosis to ingest a variety of nutrients. DIFFUSION The instructor will demonstrate diffusion and how temperature affects the rate of diffusion at the front bench. Materials: 1. Two 500mL beakers 2. Distilled water 3. Potassium permanganate (KMnO 4 ) crystals 4. Hot plate Procedures: 1. The instructor will place a few KMnO 4 crystals in a beaker with room temperature water and a few KMnO 4 crystals in a beaker with hot water. 2. Observe the rate of diffusion of the purple KMnO 4 crystals. Question: Was the rate of diffusion faster in room temperature water or hot water? Why? 2

3 MEMBRANE PERMEABILITY In this activity, dialysis tubing will serve as the selectively permeable membrane. After making a bag with the dialysis tubing and filling it with colorless cornstarch solution, the bag will be immersed in a beaker containing an iodine solution that is caramel in color. Movement of the iodine molecules across the membrane can be detected by a change in the cornstarch solution to a purplish-brown color. Materials: ml beaker cm dialysis tubing ml graduated cylinder 7. String 3. Cornstarch solution 8. Timing device 4. Iodine solution 9. Paper towels 5. Water Procedures: 1. Obtain glassware and needed accessory materials. 2. Measure and cut a 15-cm length of dialysis tubing. 3. Place the tubing in water until it becomes soft and pliable. 4. Using string, form a bag with the dialysis tubing closing one end tightly. 5. Fill the dialysis tubing bag halfway with the cornstarch solution. 6. Using string, tie off the top of the bag. Ensure that both ends of the dialysis tubing bag do not have any leakages. 7. Immerse the bag containing the cornstarch solution into a beaker containing 200mL of iodine solution and record the time and the color of the solution. Question: What is the color of the iodine solution? Question: What is the color of the cornstarch solution? 8. Leave the apparatus undisturbed for 15 minutes. 9. Remove the bag from the solution and place it on a paper towel. 10. Observe the color changes in the dialysis bag and the beaker. Question: What color changes did you observe in the bag and in the beaker? Questions: Explain the color changes in the beaker and in the bag. 3

4 OSMOSIS Recall that osmosis is the diffusion of water across a selectively permeable membrane, and it occurs from regions of higher water concentration to regions of lower water concentration. A typical solution consists of two components the solvent as the dissolving medium and the solute as the substance dissolved in the solvent. In a saltwater solution, the water serves as a solvent and the salt as the solute. Tonicity refers to the concentration of solute in the solvent: 1. Hypotonic solution: there is a lower concentration of solute relative to the inside of the cell. If a cell such as a red blood cell or a potato cell is placed in a hypotonic solution, water will rush into the cell in attempt to reach a state of equilibrium. An ideal hypotonic solution is distilled water, because it is devoid of solutes. As the cell begins to fill with solvent, the cell will swell, and perhaps burst. The bursting of cells in a hypotonic solution is called cytolysis. 2. Hypertonic solution: there is a higher concentration of solute relative to the inside of the cell. If a cell such as a red blood cell or a potato cell is placed in a hypertonic solution, water will be drawn out of the cell into the outside solution in an attempt to reach a state of equilibrium. This is called crenation in red blood cells and plasmolysis in plant cells. 3. Isotonic solution: there is the same concentration of solute outside and inside of the cell. Normally, many cells such as red blood cells exist in an isotonic state in plasma. In plants, the swelling of cells placed in a hypotonic solutions results in turgor pressure. The framework cell wall protects the cell from bursting. Turgor pressure keeps the plant erect. If the turgor pressure is lost in a plant, the plant will wilt. Just think of the plants in your yard on a hot summer s day. Materials: 1. Potato strips 8. Paper towels 2. 3 test tubes 9. Forceps 3. Test tube rack 10. Scalpel 4. Sharpie 11. Microscope slides and coverslips 5. Water 12. Microscope 6. Distilled water 7. 10% NaCl solution Procedures: 1. Obtain the needed equipment and bring it to your lab station. 2. With a marker, label the tubes Control, Hypotonic Solution, and Hypertonic Solution. 3. Cut three strips of potato, each about 4 cm in length and 1-cm wide. 4. Place each strip in a labeled test tube. 5. Add tap water to the test tube labeled Control, totally immersing the potato strip. 6. Add 10% NaCl to the test tube labeled Hypertonic Solution, totally immersing the potato strip. 4

5 7. Add distilled water to the test tube labeled Hypotonic Solution, totally immersing the potato strip. 8. Let the solutions sit quietly for 30 minutes before making your observations. 9. Remove the potato strips and place them on a paper towel. Record your observations 10. Using a scalpel, carefully cut a thin piece of tissue from each strip. 11. Prepare a wet mount of each strip. 12. Using the microscope, observe each slide and record your results. 13. Clean your lab station. Control Hypotonic Solution Hypertonic Solution Question: Which strip was most limp? Why? Question: Which strip was most stiff? Why? 5

6 PLASMOLYSIS IN PLANT CELLS Plant cells are surrounded by a rigid cell wall composed primarily of the glucose polymer, cellulose. Many plant cells have a large central vacuole surrounded by the selectively permeable vacuolar membrane. Normally, the solute concentration within the cell s central vacuole is greater than that of the external environment. Consequently, water moves into the cell, creating turgor pressure, which pushes the cytoplasm and plasma membrane against the cell wall. Such cells are said to be turgid. Many non-woody plants (like beans and peas) rely on turgor pressure to maintain their rigidity and erect stance. In this experiment, you will explore the effects of external solute concentration on the structure of plant cells. Materials: 1. Compound microscope 2. A pair of forceps 3. Petri dish 4. Microscope slides and coverslips 5. Elodea leaves 6. Bottle of ddh 2 O 7. Bottle of 20% NaCl 8. Paper Towel 6

7 Procedures: 1. With a pair of forceps, obtain two healthy Elodea leaves and place in a petri dish with ddh 2 O until you are ready to use the leaves. Preparation of Elodea in ddh 2 O 2. Place one of the leaves on a microscope slide and add one drop of ddh 2 O and place a coverslip on top of the leaf. Place a paper towel on top of the slide and tap around the coverslip to extract excess water. 3. View the Elodea leaf in ddh 2 O through a compound microscope. Focus the slide with the 4X low-power objective. Once it is in focus, add a drop of oil immersion on top of the coverslip and switch the objective to the 100X-oil immersion lens. 4. Observe the cells and make note of how the structure of the cells appear. The cells under this condition are considered normal. You will use this to compare with the 20% NaCl condition. Preparation of Elodea in 20% NaCl 5. Place the other leaf on a microscope slide and add one drop of 20% NaCl and place a coverslip on top of the leaf. Place a paper towel on top of the slide and tap around the coverslip to extract excess water. 6. View the Elodea leaf in 20% NaCl through a compound microscope. Focus the slide with the 4X low-power objective. Once it is in focus, add a drop of oil immersion on top of the coverslip and switch the objective to the 100X-oil immersion lens. 7. Observe the cells and make note of how the structure of the cells appear. After a few minutes, the cell will lose water (Why?). This process is known as plasmolysis. Additional Review Questions 1. What is tonicity? 2. Is pure water considered hypotonic or hypertonic to the cytoplasm of the cell? 3. Is 20% NaCl considered to be hypotonic or hypertonic to the cytoplasm of the cell? 7

8 4. Suppose two different glucose solutions are separated by a membrane that is permeable to water but not to glucose. The glucose solution on side A has a concentration of 4.0g/mL. The glucose solution on side B has a concentration of 0.4g/L. State whether or not osmosis will occur and, if it will, in which direction. 5. Before the invention of refrigerators, pioneers preserved meat by salting it. Explain how meat can be preserved by this procedure. (Hint: think about what salting the meat would do to cells of decomposer organisms, such as bacteria and fungi). 6. Explain why a sailor set adrift cannot drink seawater. 8