Lab 4: Diffusion and Osmosis Introduction The cell membrane encloses the contents of all cells, organelles and many cytoplasmic inclusions, and regulates what gets in and out. This is called selective permeability. Selective permeability is necessary for the maintenance of life processes. Diffusion Diffusion is the movement of particles from where there are many such particles to where there are few; in more scientific language, we say that diffusion is the movement of particles from areas of high concentration to areas of low concentration. Given a cell membrane, molecules will try to diffuse from the side of the membrane where the molecules in question enjoy a high concentration to the other side where the concentration is lower. This movement depends, of course, on whether or not the molecules are able to physically pass through the membrane. Molecules may be too large to pass through the tiny channels that exist in membranes; if the cell needs the components of these molecules, the molecules have to be disassembled (digested) into smaller units that can then pass through Kinetic Energy Molecules are constantly in motion. This motion, fueled by the energy absorbed from the environment, is called kinetic energy. Kinetic energy increases as the temperature of a system increases, and decreases as the temperature of a system decreases. Kinetic energy is the force behind diffusion. Osmosis If a cell is placed into a solution that has a higher water concentration than the cell, water tends to flow into the cell. Similarly, if a cell is place into a solution that has a lower water concentration than the cell, water tends to flow out of the cell. In the first case, we say that the solution in which the cell is placed is hypotonic, or having a lower solute concentration than the cell. In the second case, we say that the solution is hypertonic, since it has a higher solute concentration than the cell. We describe a third situation, where the cell and solution have the same water concentrations, as being isotonic. In isotonic solutions, water still flows from one system into another, only it occurs both ways, equally. The passage of water across a membrane from an area of lower solute concentration (higher water concentration) to higher solute concentration (lower water concentration) is called osmosis. Dialysis tubing is composed of a membrane synthesized of cellulose. Since it contains pores of a certain size, it allows molecules of certain size through, and those that are larger cannot get through; hence, it is semipermeable. Besides being used in medicine, dialysis tubing has many experimental applications, as we shall see in this lab. 4.1
Figure 4.1: Direction of water flow into or out of a cell placed in hypotonic, hypertonic and isotonic solutions. cell cell cell hypotonic solution Osmotic Potential hypertonic solution isotonic solution Osmotic potential,, is a measurement of the tendency of water to flow from one place to another, across a semipermeable membrane. It is a relative value. If a membrane-bound, aqueous system with a particle concentration of 0.5 M is placed into an aqueous system with a particle concentration of 1.0 M, we say that the membrane-bound system has a high water potential because water would tend to flow, via osmosis, from the membrane-bound system into the system in which it was placed. Similarly, if we placed the same membrane-bound, aqueous system in an aqueous system with a particle concentration of 0.25 M, then we d say that the membrane-bound system has a low water potential because water would tend to flow into it from the surrounding environment. There would, of course, be zero water potential if both systems were isotonic. The effect of the force of osmotic potential on individual cells largely depends on whether or not the cell possesses a mechanism to withstand that force. Plant cells, which generally have low water potential because they are surrounded by hypotonic environments, absorb water until their cell wall prevents further osmosis. At this point they are said to be turgid. This is what happens when a stalk of celery crisps up when you place it in water, and is the preferred state of most plants. Animal cells don t have cell walls and, indeed, if you place most animals cells in a markedly hypotonic environment, such as DW, water rushes into the cell and the cell explodes. This is call lysis. Erythrocytes that are placed in hypotonic environments and explode are said to have undergone hemolysis. Placing plant or animal cells in hypertonic environments increases the water potential of the cell, causing water to leave the cell. As water leaves, the cell shrivels; animal cells undergo crenation or pruning, whereas the membrane of plant cells shrinks back from the cell wall in a process called plasmolysis. Crenation and plasmolysis are generally fatal to the cell. Placing plant cells into an environment where the water potential is equal results in limp, flaccid plants; animal cells are normally in this kind of isotonic environment. Lugol s Iodine and Benedict s Tests Lugol s iodine turns dark blue/purple in the presence of amylose. Benedict s reagent, when mixed with monosaccharides and some disaccharides, turns orange/red when heated. These biochemical tests will be used in this lab. 4.2
Laboratory Objectives After mastery of this laboratory, including doing the assigned readings and required laboratory work, the student should be able to: 1. Define and discuss the relationship between temperature, kinetic energy and diffusion, and what Brownian motion has to do with kinetic energy. 2. Define and apply the terms hypotonic, hypertonic and isotonic. 3. Explain and demonstrate how solute concentration affects rates of osmosis. 4. Explain and demonstrate a technique to test whether or not glucose, iodine and amylose can pass through semipermeable membranes. 5. Describe what happens when cells with cell walls and cells without cell walls are placed in hypotonic, hypertonic and isotonic solutions, and describe and demonstrate experiments testing these observations. 6. Define and apply the terms, osmotic potential, turgid, lysis, hemolysis, crenation, plasmolysis, flaccid. 7. Describe and demonstrate the use of Lugol s iodine and Benedict s reagent tests. Materials and Methods Kinetic Energy of Molecules 1. Obtain a compound microscope as per the protocol you learned in Lab 3. Also obtain a standard microscope slide, cover slip, dropper bottle of water, and a needle probe. 2. Place a droplet of water on the microscope slide. Making sure the needle probe is dry, touch the probe into a tiny bit (about the size of a typed period) of powdered carmine located at the demo table. Mix the carmine sample into the droplet of water. Lower the cover slip onto the droplet of water and observe the carmine under the microscope. Use high power but don t use oil immersion. Carefully observe the behavior of the carmine particles. Describe this behavior in your lab report! Osmosis 1. Obtain five, 250 ml beakers per lab table you may be sharing these beakers with another lab group. They should be labeled 1 through 5 with plastic tape. 2. Into beakers 1 through 4 place about 200 ml of DW and into beaker 5 place about 200 ml of 60% sucrose solution. 3. Each lab group should obtain 5 lengths of dialysis tubing approximately 13 cm in length and 10 dialysis tube clips. If dialysis tube clips are not available, small paper clips will suffice. 4. Fold over about 1 cm of the end of each dialysis tube then fold it over again and clip securely 4.3
closed with a tube clip (or paper clip). The other end of each tube should be left open. 5. Soak the dialysis bags in DW for a minute or so, then fill the bags with 10 ml of the following solutions, using calibrated pipettes. DO NOT REMOVE THE PIPETTES FROM THE STOCK SOLUTIONS and DO NOT MIX UP THE PIPETTES! The pipettes at the demo table in the stock solutions are calibrated to deliver 1 ml. a. Fill bag 1 with 10 ml DW b. Fill bag 2 with 10 ml 20% sucrose solution c. Fill bag 3 with 10 ml 40% sucrose solution d. Fill bag 4 with 10 ml 60% sucrose solution e. Fill bag 5 with 10 ml DW 6. Squeeze the bottom of each bag as you fill it and clamp/clip it closed to eliminate any air trapped in the bag. 7. Weigh each bag as accurately as possible and record in your lab report. 8. You re going to weigh each bag every 15 minutes to determine changes in weight over time. At time zero, place each bag in the corresponding beaker (bag 1 goes into beaker 1, bag 2 goes into beaker 2, etc.). 9. Approximately every 15 minutes for about an hour, take the bags from the beakers, carefully pat dry with paper towels to remove excess fluid, and weigh as accurately as possible, recording the mass. After weighing, IMMEDIATELY return the bag to its corresponding beaker! DO NOT keep the bag out of a beaker while waiting to use a scale; it s okay to be a minute or two off of your time! 10. When finished, dispose of the fluids down the sink and the solids in the trash. Rinse the glassware very well and pick up any spilled sucrose solutions with lots of water to prevent ants from coming! Passive Transport of Molecules Through a Semipermeable Membrane 1. Place about 200 ml DW in a 250 ml beaker. Add just enough Lugol s iodine solution to distinctly color the water, but don t add so much that you can t clearly see through the solution; record the color in your lab report. 2. Obtain a 13 cm section of dialysis tubing. Fold over one end twice and clamp as before. 3. Soak the dialysis tubing in DW for about a minute. 4. Open the unclamped end and add 5 ml each of 30% glucose and 10% amylose solutions. 5. Hand close the end of the bag and shake well to mix. 6. Record the color of the contents of the bag in your lab report. 4.4
7. Rinse the outside of the dialysis bag in DW. 8. Place the dialysis bag into the beaker with the aqueous Lugol s iodine solution so that the unclosed end of the dialysis bag hangs over the edge; be careful not to allow the contents of the bag to spill out or mix with the Lugol s iodine solution! To secure the bag, if needed, wrap a rubber band around the beaker and tuck the untied end of the dialysis bag under it. 9. After 30 minutes, remove the dialysis bag from the solution and note the color of its contents, as well as the color of the solution in the beaker; enter these data in your lab report. 10. Start a boiling water bath using a hot plate and 600 ml beaker ½ full of tap water. 11. Obtain four test tubes, a test tube stand, test tube clamp, grease pencil and two disposable pipettes. Label the test tubes 1, 2, amylose + iodine and glucose + Benedict s. These last two test tubes will be positive controls for the Lugol s iodine and Benedict s tests. 12. Using a disposable pipette, into test tube #1, place 1 ml of solution from the dialysis tube. Using another disposable pipette, into test tube #2, place 1 ml of solution from the beaker. Toss the disposable pipettes into the trash. From the stock solutions, place 1 ml of amylose into the amylose + iodine test tube and 1 ml of glucose solution into the glucose + Benedict s test tube. 13. Into test tubes #1, #2 and the one labeled glucose + Benedict s, place 1 ml of Benedict s reagent. Into the test tube labeled amylose + Lugol s place 1 ml of amylose and 2 drops of Lugol s iodine solution. Note the color in Results and set this test tube aside. 14. Place test tubes #1, #2 and the one labeled glucose + Benedict s into the boiling water bath for 5 minutes. After 5 minutes, using a test tube clamp, remove the test tubes from the water bath and let them cool in a test tube rack for 10 minutes. Note the resultant color of both tubes and the positive reference, and record these data in your lab report. 15. When finished with the hot plate setup, ask to see if anyone else needs it; if not, turn the hot plate off, unplug it, and carefully set the hot water aside (or pour down the sink) using your beaker tongs. If the hot plate is on, add water to the beaker as needed! Biological Effects of Osmosis in Cells with Cell Walls 1. Obtain a compound microscope and standard microscope slide and cover slip. Make a wet mount using a small, young Elodea leaflet and a drop of DW; apply the cover slip. 2. Observe the Elodea under 430x; remember to first find the Elodea with 40x, increase the power to 100x, then up to 430x. Note the general shape and condition of the contents of the plant cells and record these observations in your lab report. Also make a sketch (not detailed drawing) of a representative cell showing this condition. Do your drawing as you observe your cell; you may NOT draw the cell later at home from memory!!! 4.5
3. While watching your representative cell, imbibe through one to several drops of hypertonic solution (5% NaCl) by placing the flat edge of a small piece (~ 3 cm x 3 cm) of paper towel on one edge of the coverslip and dispensing the solution directly on the edge of the coverslip on the opposite side. Note the general shape and condition of the contents of the plant cells and write this in Results. Again, make a sketch of your representative cell showing this condition and label your cell as being in a hypertonic environment! Do your drawing as you observe your cell; you may NOT draw the cell later from memory!!! Note: You do NOT need to calculate size rules for Elodea as we completed that task in the last lab! 4. While you are watching your representative cell, imbibe through one to several drops of hypotonic solution (DW again). Note the general shape and condition of the contents of the plant cells and write this in Results. Once again, draw your representative cell showing this condition and label the cell as being in a hypotonic environment. Draw an arrow from your first drawing to your second to your third to indicate the progression of drawings. Do your drawing as you observe your cell; you may NOT draw the cell later from memory! 5. Have your instructor initial your three drawings for credit. Biological Effects of Osmosis in Cells without Cell Walls 1. Obtain three standard microscope slides, three cover slips, three pieces of cellotape about 2 cm in length and a fine-point Sharpie. Also obtain gloves, as we ll be working with animal blood. 2. Cover the ends of the slides with cellotape and write on the tape, with the Sharpie, iso on one slide, hyper on the second slide, and hypo on the third. Lab safety advisory: Biohazard! We will be working with animal blood that may carry zoonotic pathogens, animal diseases that can be picked up by humans. Wear disposable gloves, and dispose of any materials that may have come in contact with the blood in the appropriate biohazard containers. If you come in contact with animal blood, immediately wash the area in question with soap and water. 3. Onto each slide place a tiny droplet (about 1 mm in diameter) of fresh sheep blood. If you get too much blood, wipe off the excess with a small square of paper towel. 4. Onto the slide marked iso, directly on top of the blood sample, place a full drop of isotonic solution (normal saline, 0.9% NaCl), and gently apply a cover slip. 5. Onto the slide marked hyper, directly on top of the blood sample, place a full drop of hypertonic solution (5% NaCl), and gently apply a cover slip. 6. Onto the slide marked hypo, directly on top of the blood sample, place a full drop of hypotonic solution (DW, 0% NaCl), and gently apply a cover slip. 7. Observe each treatment under the microscope, starting at lowest power and ending up at 4.6
400x (or 1000x) total magnification. Describe what you see in your lab report. 8. Dispose of all materials suspected of being contaminated with blood in the designated biohazard containers. Make sure you cleanup your work station, clean all equipment you used and put it back, and help in general to keep the lab clean and in order! 4.7
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Biol 160 Lab 4: Diffusion and Osmosis Prelab (5 points) Name: Date: Lab Section: ~Complete this prelab before coming to lab; it is due at the beginning of lab! 1. In your own words, define selective permeability. 2. Explain why can t all particles passively diffuse across cell membranes? 3. What is the energy that powers diffusion? 4. Draw an arrow showing the direction of net water flow across the membrane in each of the examples below! [Hint: Water flows toward the highest solute (salt) concentration!] c) Outside cell, 10% NaCl b) Outside cell, 0.9% NaCl a) Outside cell, 0.1% NaCl Inside cell: 0.9% NaCl Inside cell: 0.9% NaCl Inside cell: 0.9% NaCl 5. Plants do well living in environments with high osmotic potential. How do plants withstand the force of high osmotic potential? 4.9
6. What biohazard will we be working with in this lab? 7. What safety precautions will we be making with regards to the biohazard described above? 4.10
Biol 160 Lab 4: Diffusion and Osmosis Lab Report (20 points) Name: Date: Lab Section: Results Kinetic Energy of Molecules 1. Describe the behavior of the Carmine particles under the microscope! Osmosis 2. Table 4.1: Mass of dialysis bags over time. Time (min.) Mass of Bags (grams) Bag 1 Bag 2 Bag 3 Bag 4 Bag 5 0 Subtract the initial weight at time zero of each bag from subsequent weights and record these data in Table 4.2. 4.11
3. Table 4.2: Change in mass of dialysis bags over time. Time (min.) Mass Change of Bags (grams) Bag 1 Bag 2 Bag 3 Bag 4 Bag 5 0 Passive Transport of Molecules Through a Semipermeable Membrane 4. Table 4.3: Experimental results testing ability of glucose, iodine and amylose to passively transport across the membrane of dialysis tubing. Dialysis Bag Initial Contents Color Final Color Benedict s Test Color Beaker 5. Table 4.4: Positive references for Lugol s iodine and Benedict s test. Reagents Color of Positive Test Lugol s iodine + amylose Benedict s reagent + glucose + heat 4.12
Biological Effects of Osmosis in Cells With Cell Walls 6. Table 4.4: General shape and condition of the contents of Elodea cells in successively hypotonic (original), hypertonic and hypotonic solution. Tonicity [NaCl} General Shape & Condition hypotonic 0% original hypertonic 5% hypotonic at end 0% 7. Figure 4.2: Drawings of Elodea cells in successively hypotonic (original), hypertonic and hypotonic solutions. (Size rules not required!) Biological Effects of Osmosis in Cells Without Cell Walls Instructor s Signature: 8. Table 4.5: General shape and condition of the contents of blood cells in isotonic, hypertonic and hypotonic solution. Tonicity [NaCl] General Shape & Condition isotonic 0.9% hypertonic 5% hypotonic 0% 4.13
Discussion Kinetic Energy of Molecules 9. Why was the motion of Carmine particles under the microscope random? Osmosis 10. Which bag had the greatest change in mass? Why? 11. Which bag had the least change in mass? Why? 12. Can water pass freely through the membrane of a dialysis tube? What evidence is there supporting your answer? 13. Can sucrose pass freely through the membrane of a dialysis tube? What evidence is there supporting your answer? Passive Transport of Molecules Through a Semipermeable Membrane 14. Was there evidence of amylose being able to diffuse through the dialysis tubing membrane? What was the evidence? 4.14
15. Was there any evidence of glucose being able to diffuse through the dialysis tubing membrane? What was the evidence? 16. Based on this evidence, do you think that the sucrose in the osmosis exercise was able to diffuse across the membrane? What evidence supports your answer? Biological Effects of Osmosis in Cells with Cell Walls 17. Explain what happened when you placed Elodea cells in isotonic, hypertonic and hypotonic solutions, and why what you observed happened! 18. Why does a wilted plant crisp up when you water it? 19. Why do you think plants need to be kept in hypotonic solutions? 4.15
Biological Effects of Osmosis in Cells without Cell Walls 20. Explain what happened when you placed sheep erythrocytes in isotonic, hypertonic and hypotonic solutions. Why did this happen? 21. The fluid portion of blood is called plasma. Based on this experiment, suggest one important function of plasma. 22. Compare this experiment with the previous one involving Elodea cells and suggest an advantage to plants of having a cell wall! 4.16