Sixteenth Edition January 2010, Lab 4 Page 59 LABORATORY 4. GENERATING AND TESTING HYPOTHESES ABOUT WATER FLOW ACROSS CELL MEMBRANES PREPARATION 1. Reminder of the Process of Conjecture & Refutation Figure 4.1 Recall the process that we have been developing.
Sixteenth Edition January 2010, Lab 4 Page 60 2. What is Diffusion? According to the Laws of Thermodynamics: Law 1. Matter/energy cannot be created or destroyed, Law 2. Matter/energy tends to disorder, to become random, to entropy. In this exercise, we will be concerned with the second Law, that matter/energy tends to become disorganized. In terms of heat energy, we think of high temperature (a lot of energy - heat - in a contained area) as being more organized energy than low temperature. Since the energy in a high temperature body is organized, and since it has a tendency to become disordered or disorganized, the heat in this body will dissipate, it will flow out to colder areas. If you place a cold body next to it, the heat will rapidly pass into the cold body until both are the same temperature; or until the heat energy is completely randomized throughout the system. Another consequence of the Second Law of Thermodynamics is that energy transformations are never 100% efficient. Some energy is always lost (to entropy), usually in the form of heat. For example, when we convert electrical energy to light energy in electric lamps, the conversion is generally < 25% efficient. This is why light bulbs are so hot. You know that a bath of hot water, or a cup of hot coffee, will cool down. This cooling is a consequence of the second law of thermodynamics; the heat in the hot water is becoming randomized throughout the room. *2.1. How do you get the hot water for your coffee in the first place? The answer is that you heat it. The heating system (gas flame, electricity, whatever) must be at a higher temperature than the temperature at which water boils. *2.2. Why? *2.3. What, then, can we say about the temperature of the heating system in relation to the water? Now, since the second law of thermodynamics applies both to energy and matter, we can apply the same principle; matter tends to disorganize or become random also. *2.4. If you place a small drop of red dye in an aquarium of water, what happens?
Sixteenth Edition January 2010, Lab 4 Page 61 *2.5. If someone walked into the room wearing strong perfume, how would you know? *2.6. Explain these examples in terms of the second law of thermodynamics. Since air and water are fluids, their molecules are in constant motion. The movement of molecules from areas of high concentration (the drop of dye, the perfume, the hamburger) to areas of low concentration is called diffusion. Notice that, as the second law of thermodynamics predicts, the molecules tend to become random, disordered, disorganized; they flow down their concentration gradient from high to low concentration. 3. Diffusion and Solutions? Now, let us consider water and materials dissolved in water. The water is the solvent. The stuff dissolved in water is the solute. Consider Figure 4.2. Figure 4.2. Water (= solvent) and stuff (= solute) concentrations in two beakers. Beaker A Beaker B *3.1. Beaker A contains 100 ml of solution (= water + stuff). 80% is water. What percentage is stuff (solute)? *3.2. Beaker B contains 100 ml of solution, but this time 90% is water. What percentage is solute (stuff)?
Sixteenth Edition January 2010, Lab 4 Page 62 *3.3. Which beaker contains the higher water concentration? *3.4. Which beaker contains the higher solute concentration? *3.5. Which beaker contains the lower water concentration? *3.6. Which beaker contains the lower solute concentration? Suppose, now, we placed these two beakers next to one another, and removed the glass between them as depicted in Figure 4.3. Figure 4.3. The Same Two Beakers as in Figure 4.1 Have Been Placed Next to One Another, and the Glass Removed A B *3.7 According to the second law of thermodynamics, what would happen to the solute (stuff) and solvent (water) in the left and right regions? Water would flow down its concentration gradient from a high concentration in region B (90%) to a lower concentration in region A (80%) until the concentrations are the same in both regions (at approximately 85%). Meanwhile, the solute (= stuff) would flow from its high concentration in region A (20%) to its lower concentration in region B (10%) until solute concentrations are equal at 15% in the two regions.
Sixteenth Edition January 2010, Lab 4 Page 63 4. How is osmosis different? Now, suppose we place a membrane between the initial beakers A and B (Figure 4.3), and then remove the lower portion of the glass separating them. *4.1 If this membrane were completely permeable to water (= solvent) and stuff (= solute), what would happen? Since the membrane is completely permeable, the same flow of solute and solvent would happen as we just discussed without the membrane (see above). But, suppose now, the membrane is not completely permeable, but instead is selectively permeable (Figure 4.4); it only lets water molecules through but prevents the passage of solute molecules. Figure 4.4. The same two beakers as in Figure 4.1 have been placed next to one another, with the lower one-half of glass replaced by a selectively permeable membrane A B *4.2 What would happen to the amounts of solute and solvent in A & B? *4.3. Now what would happen to the solute concentrations either side of the selectively permeable membrane (Figure 4.4)? Since the concentration gradient of water molecules is still from higher in region B to lower in region A, water would flow from region B to region A exactly as before, down its concentration gradient.
Sixteenth Edition January 2010, Lab 4 Page 64 *4.4. Using the information given above and in relation to the concentration of solute and water either side, make a generalization about the direction of flow of water across a semipermeable membrane. We have seen that water will flow across a selectively permeable membrane from a region of high water (= low solute) concentration to a region of low water (= high solute) concentration. Consider the four conditions identified in Figure 4.5. Figure 4.5. Four beakers in which a cell, enclosed by a selectively permeable membrane, is placed in environments with different solute concentrations. A B C D Solute concentration in: -environment 10 % 5 % 1 % 20 % -cell 10 % 10 % 10 % 10 % *4.5. What do you predict will happen in each system, and why? A. B. C. D.
Sixteenth Edition January 2010, Lab 4 Page 65 *4.6. What difference do you expect (and why) between B and C in terms of (i) amount of osmosis? (ii) rate of osmosis? A Veggie Example: In class, you will undertake an exercise in which you review three culture dishes each containing a carrot and celery stalk and labeled imaginatively A, B, and C. Working in groups, you will develop a tentative explanation for the pattern that you see in which you explain in terms of osmosis for each dish: I) What the initial relative water concentrations of the cells and environment were. II) What the initial relative solute concentrations of the cells and environment were. III) What has been happening that resulted in the current appearance of the veggies. You will write a group analysis, discuss this in class, and then modify your answer to be submitted for points. 5. How Does Osmosis Occur in Red Blood Cells? Now, suppose we start with a new system. This time we have a cell which contains solutes at a concentration of 0.897% (Figure 4.6). The cell, a red blood cell (erythrocyte), is enclosed in a selectively permeable cell membrane. The membrane allows water to pass through, but not solutes. You will now generate four HYPOTHESES about the direction of water flow across red blood cell membranes in relation to the relative water concentrations on either side. Figure 4.6. Red blood cells (0.897 % solute concentration) in an environment with 0.897% solute concentration Make one hypothesis about the direction of water flow across the blood cell membrane in each of the following situations: DO NOT IDENTIFY WHAT YOU THINK WILL HAPPEN TO THE CELLS, ONLY WHICH WAY YOU THINK WATER WILL FLOW...AND EXPLAIN WHY YOU THINK THAT REFERRING TO THE PROCESS OF OSMOSIS.
Sixteenth Edition January 2010, Lab 4 Page 66 *5.1 If the red blood cells were placed in a beaker containing less than 0.897% solute. *5.2 If the red blood cells were to be placed in a beaker containing 0.897% solute. *5.3 If the red blood cells were placed in a beaker containing more than 0.897% solute. *5.4 If the red blood cells were placed in a beaker containing 0% solute. 6. What PREDICTIONS can you make from these 4 hypotheses about the shape of the cells? When a red blood cell loses water, it shrivels up (Figure 4.7A). Instead of looking like an aspirin tablet, it wrinkles and shrinks (crenates). When a red blood cell takes in water it swells, and looks much more rounded. If the red blood cell takes in a lot of water, it bursts (hemolysis), releasing the red pigment (hemoglobin) into solution as the cell membrane explodes (Figure 4.7B). Figure 4.7. Red blood cells (0.897 % solute conc.) in environments with higher solute concentration (A) and lower solute concentration (B).
Sixteenth Edition January 2010, Lab 4 Page 67 Suppose you have been provided with cow blood and four beakers of water containing various concentrations of salt (salt is the solute and water the solvent). The first beaker contains less than (< ) 0.897% salt, the second contains 0.897% salt, the third contains more than (> ) 0.897% salt, and the fourth contains 0% salt (distilled water). Now, PREDICT what will happen to the red blood cells (will they shrink, swell, stay the same, burst) if some of the cow blood is added to each of the four beakers and EXPLAIN why you think that (in relation to osmosis). Write your predictions and explanations in Table 4.1. Each treatment is one value for the manipulated variable. In this case the manipulated variable is the solute concentration in the environment surrounding the red blood cells. This variable has 4 values. Remember, the red blood cells contain 0.897% salt. Table 4.1. Predictions About the Effect for the Shape of Red Blood Cells of Different Solute Concentration in the Cells' Environment (from Table 4.1; p 81). ENVIRONMENT PREDICTION EXPLANATION *6.1 slightly < 0.897% salt *6.2 0.897% salt *6.3 slightly > 0.897% salt *6.4 distilled water (0% salt) * 6.5 In the above predictions what is the manipulated variable? *6.6 In the above predictions what is the responding variable? 7. How Do We Use a Microscope? Before working with a microscope, it is necessary to know how to use it. Even if you are absolutely convinced you know all you need to know about microscopes read Appendix I on microscopy. It will help you answer the following questions. *7.1 What is the difference between the objective and ocular?
Sixteenth Edition January 2010, Lab 4 Page 68 *7.2 When carrying a microscope, what should you do? *7.3 Before setting the microscope up to look at a slide, what should you do? *7.4 If you wish to use the high power objective, how do you get there? *7.5 When finished using the microscope, what do you do? 1. What are the Laws of Thermodynamics? PROBLEM SET 2. What does the Second Law of Thermodynamics tell us about energy and matter? 3. Suppose cells have a 10% solute concentration. They are surrounded by a selectively permeable membrane. Some of these cells are placed in each of the following environments. In terms of osmosis, predict what will happen to the solute (stuff) and solvent (water) concentrations (inside and outside the cells) in each environment AND explain why you think that. A. Environment contains less than 10% solute. B. Environment contains 10% solute. C. Environment contains more than 10% solute.
Sixteenth Edition January 2010, Lab 4 Page 69 LABORATORY EXERCISES Microscope Test of Hypotheses. Practice first with the simple slide of the letter ' e' *1.1 What is the relationship between the direction you move the slide, and the appearance of movement through the lens? *1.2 Now look at mammalian blood cells and onion root tip cells under low and high power, and compare the cell sizes. What is the difference between the size and shape of the central onion root cells and those of the mammal blood? Make sure you can focus and view under both low (approx 100X) and high (app 400X) power. *1.3 Adjust the amount of light passing through the diaphragm by rotating the adjustor; identify how this influences your ability to see the objects. Are all cells better seen at the same light intensity? Explain. Investigation of your water flow hypotheses using the microscope In the Introduction you developed some hypotheses and predictions about water flow across selectively permeable membranes in relation to solute concentrations (Your Hypotheses *4.1 - *4.4) and made predictions about the shape of red blood cells in environments with different solute concentrations (Your Predictions *5.1 -*5.4). These were based on information about osmosis and cell membranes. In the following study you will test these hypotheses and the consequent predictions with a microscope, by looking at cells in the 4 situations and comparing the results to what you predicted. As a consequence of this test you will re-evaluate your hypotheses. This process is numbers 2-6 below. Work in groups as directed by your instructor.
Sixteenth Edition January 2010, Lab 4 Page 70 In the Introduction, you made four hypotheses (*4.1 - *4.4) about the movement of water into and out of red blood cells in environments with different solute concentrations. These are the hypotheses that you will be testing in this exercise. 2. The Predictions These four hypotheses (*4.1 - *4.4) led to four predictions about the response of red blood cells to environments with different solute concentrations (*5.1 - *5.4 in Table 4.1). Reconsider your predictions about what will happen to the blood cells in relation to the following precise environmental solute concentrations and re-write them here. Your explanation MUST refer to osmosis (water flow across cell membranes) in red blood cells. 1. 0.6% solute in the environment. 2. 0.897% solute in the environment. 3. 5.0% solute in the environment. 4. 0.0% solute in the environment Now, you will test these four predictions. 3. The Tests Test Instructions i. Obtain 4 test tubes in a test tube rack and label them 1-4 using grease pencils or masking tape provided. ii. In the laboratory you will find four flasks containing the four different solute concentrations, one appropriate to each tube 1-4, as follows (watch the decimal point): 1: 0.4% - 0.6% salt 2: 0.897% salt 3: 5.0% salt 4: distilled water
Sixteenth Edition January 2010, Lab 4 Page 71 Into the 4 numbered test tubes place 10 ml of the appropriately numbered salt solution - keep the numbers consistent so you do not become confused. iii. In the ice chest you will find a flask of beef blood. This has been treated with sodium heparin to prevent it from clotting. But keep it in the ice chest. iv. Into each of your 4 test tubes place 8 drops of beef blood. v. Agitate on the mixer. vi Set up four microscopes (one for each test slide) on the same bench, and check them by setting up and viewing a prepared slide of blood. vii. Prepare one wet slide mount of blood from each test tube, labeling the slides with the appropriate numbers. Use a very small drop of the solution; otherwise you will have too many cells to see them individually. viii. Examine the slides under low and then high power and compare/contrast them with your predicted outcomes (Predictions 1-4 above). ix. Compare with the prepared slide of mammalian red blood cells if you are in doubt. REMEMBER - WHEN USING THE MICROSCOPE - always start and finish viewing under low power - never use the course focus with the high power lens - do NOT get the lower lens (objective) wet x. Record results below and discuss them with colleagues and the instructor. xi. Throw away solutions, wash all equipment and store in designated place. 4. What are the results? Return to YOUR predictions above. Clearly, YOUR results must be relevant to YOUR predictions with reference to swelling, shrinking, staying the same or bursting. *4.1 What happened to the red blood cells in: Test tube 1 Test tube 2 Test tube 3 Test tube 4
Sixteenth Edition January 2010, Lab 4 Page 72 *4.2 If any of the results were different from your predictions, note that here, and state the difference: Test Tube 1 Test Tube 2 Test Tube 3 Test Tube 4 *4.3 What do your results suggest to you about your predictions regarding the outcome for the blood cells (say more than ' correct' or ' incorrect,' identify the difference)? Prediction 1 Prediction 2 Prediction 3 Prediction 4 5. What do we do with test results? If we stop here, we have wasted our time; the purpose of a test is not just to get results, we are testing an opinion we have. So, it' s time to re-evaluate the Universe, to draw inferences/conclusions about the hypotheses you came up with initially - in this case, regarding the movement of water across a cell membrane. *5.1 What do your results suggest about the HYPOTHESES that YOU made concerning water movement through the membrane of red blood cells in (again, you must say more than "I was correct,' or ' I was wrong,' you must state clearly what you now think): 1. An environment with lower solute concentration than the cell? 2. An environment with similar solute concentration to the cell? 3. An environment with higher solute concentration than the cell? 4. An environment with no solute concentration?