MEMBRANE TRANSPORT
Also refer to Sherwood p. 49-69
1) To demonstrate the principles that govern transport of materials across cell membranes.
2) Explain the following terms:diffusion, osmosis (hypertonic, hypotonic, isotonic, solute and solvent), facilitated diffusion and active transport.
3) Understand osmotic principles and the relationship of osmotic pressure to hydrostatic pressure.
4) Apply the principles of osmosis to biological systems by observation of red blood cells:
a. define hemolysisb. define hemolysis time.
In order for cells to survive, they must procure an energy source from their surroundings. Cells extract energy by oxidative processes, and in so doing produce waste products which must not be allowed to accumulate in the cells. Materials can get into and out of cells by means such as: 1) diffusion; 2) osmosis; 3) facilitated diffusion; 4) active transport; 5) phagocytosis; 6) pinocytosis; and 7) filtration. The first three processes above use a difference in concentration (concentration gradient) on the two sides of the cell membrane to drive transport.
Diffusion is defined as the net random movement of particles from an area of higher concentration to an area of lower concentration. As long as temperature is above absolute zero, molecules will move randomly (i.e. they have kinetic energy) favoring movement toward an area of lower concentration.
Osmosis is simply a specialized type of diffusion with two restrictions: 1) diffusing particles are limited to solvent molecules (usually water); and 2) these solvent molecules must pass through a selectively permeable membrane. Even though osmosis is concerned with movement of solvent, concentration is typically expressed in terms of amount of dissolved substance (solute), rather than the amount of dissolving substance (solvent). However, since two objects cannot occupy the same space at the same time, as solute concentration increases, solvent "concentration" will diminish. To determine the direction of solvent movement during osmosis, two systems of solutions must be compared. This relative solute concentration is called tonicity. If both systems have the same solute concentration, they are isotonic to each other and will have the same solvent concentration. In contrast, if one system has a higher solute concentration than the other, the one with the greater amount of solute is hypertonicto the other and the system with the lower solute concentration is hypotonic to system with higher solute concentration. Conversely, the hypertonic solution will have a lower water concentration, so the solvent will move by osmosis from the hypotonic to the hypertonic solution if the two systems are separated by a membrane. NOTE: hypertonic and hypotonic are relative terms. A cell placed in pure water is said to be hypertonic to the solution or you may say that the cell is placed in a hypotonic solution. It is incorrect to say "the solution is hypertonic" without referencing what it is hypertonic to.
Facilitated (carrier mediated) diffusion is simply diffusion in which particles are physically transported across a membrane by a carrier (usually a protein). The interaction of a particle with the carrier protein is highly specific, meaning the carrier will only assist a certain particle across the membrane and will not bind with and move other types. Since there are a limited number of carrier molecules which transport a particular particle through a cell membrane, carrier proteins can be saturated (full) with particles waiting to move across the membrane. Thus, unlike regular diffusion, transport rate will increase as particle concentration gradient increases only to the point where carrier availability can keep up with movement. Eventually transport rate will slow and reach a plateau.
Active transport is similar to facilitated diffusion except that energy is required. Active transport is used to move particles against a concentration gradient, or from lower concentrations to higher concentrations.
Filtration is the movement of particles due to an applied force. For example, blood pressure can force fluids through capillary walls.
Phagocytosis (cell eating) is a method used by only a few cell types to internalize large multimolecular particles. This technique is primarily used by white blood cells which play an important role in the body’s defense system.
Pinocytosis: (cell drinking) is a method in which a small amount of extracellular fluid is brought into the cell in small pouch-like vesicles. This process is performed by most cells of the body as a way to bring extracellular fluid into the cell as well as retrieve extra plasma membrane that has been added to the cell surface during exocytosis (Sherwood).
In this series of lab activities, you will learn about basic laws that govern the ability of solutes and solvents (in this case, water) to move in biological systems. The movement of particles in biological systems is dictated in part by physical laws (moving from higher concentrations to lower concentrations), by physical barriers (cellular membranes), and physical portals (carrier proteins). We will examine simple diffusion, osmosis, and finally observe diffusion and osmosis in a biological context by observing the behavior of red blood cells which have been placed in two types of solutions of varying concentrations.
A. Diffusion. This activity will be done by the instructor
1. Place 1 ml of 0.01 M eosin (non-colloidal) solution in a test tube about 2/3 full of 2% agar-agar
2. Into a second similar tube place 1 ml of 0.01 M blue dextran
3. The instructor will measure the distance(cm) the dyes travel daily for one week. Results will be discussed in lab next week.
B. Osmosis.
See Figure below
1. Soak a piece of cellophane dialysis tubing about 20 cm long in tap water until it becomes pliable. Securely tie a knot in one end of the tube.
2. Fill the dialysis bag about 2/3 full of 1 M sucrose which has been colored with methylene blue for easy viewing.
3. Break or cut a rubber band. Immerse one end of a long capillary tube into the sucrose solution. Securely attach the bag to the tube by wrapping the stretched rubber band around the free end of the bag where the tube dips into the sucrose. Wrap the band down the bag until the solution is forced a few cm up the tube. Tie the free ends of the rubber band.
4. Immerse the bag in a beaker of deionized water and secure the tube with a burette or similar clamp. Immediately mark the level of the fluid. Use this mark for your "0" time reading.
5. Measure the total height of the solution in the tube at10 min intervals for the remainder of the period or until fluid flows from the top of the tube.
6. When the experiment is complete, remove the sucrose/dialysis tubing bag and clean the capillary tube by pulling water through the tube using the vacum aspirator located in the lab hood. After cleaning, return the capillary tube to the stand at your lab station.
C. Cell Permeability.
1. Add 2.0 ml (approximately 1 dropperful) of the following solutions to a series of test tubes:
These solutions cover the range of hypotonic to hypertonic solutions relative to the red blood cell.
Tube # NaCl (M) Tube # Glucose(M) 1 0.05 6 0.05 2 0.08 7 0.08 3 0.15 8 0.15 4 0.30 9 0.30 5 1.00 10 1.00
2. Determine the time it takes for red blood cells to hemolyze when placed in varying concentrations of NaCl (tubes 1-5) or glucose (tubes 6-10).Add a drop of mammalian blood to the test tube you are testing. Immedicately after vortexing place the test tude in front of the paper and stop timing when you can read through the tube to the print. Record the time. This may be demonstrated by your instructor. When you can clearly see letters through the solutions, hemolysis is complete.
3. Repeat with each tube in turn. If a solution does not clear within about 2 minutes, hemolysis did not occur for that solution (at least with these concentrations).
D. Microscope observations:
Slides will be prepared that show red blood cells in isotonic solution and hypertonic solution. Observe the shape of the cells in each solution and be prepared to sketch and describe their appearance, and provide an explanation of why the cells now look the way they do.
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