HHMI-TRINITY SCIENCE ACADEMY # 3 BIOLOGY MODULE Achieving Balance. Effects of water, salt and sugar in your blood cells. PLEASE NOTE: It is VERY IMPORTANT for students to understand WHY they are doing each of these experiments. Please make sure that they are writing a hypothesis for every question/experiment and more importantly, that they are documenting their results! This is CRITICAL to developing their final thoughts towards a conclusion! For all the experiments today: we will be using erythrocytes from sheep. The erythrocyte (or red blood cell, RBC) is the largest membrane bound compartment found in animals. These cells are also the unique home of the protein hemoglobin, and the presence of a traceable molecule like hemoglobin has made these cells a model system for studying transport of molecules across membranes. Movements of molecules across membranes can be difficult to measure directly, but whenever molecules move, water moves to maintain an osmotic equilibrium. These bulk water movements will change the volume of any closed membrane compartment, including RBCs, and may eventually cause them to lyse due to internal pressure (distilled water experiment) or shrivel due to water molecules that leave the cells (salt experiment). In some instances, the property of the cell membranes change, as they become “sticky” causing cell aggregations called clots (sugar experiment). Thus, we can use sheep RBCs to ask questions about finding the right balance necessary for our RBCs to stay healthy. We will investigate questions concerning the amount of water we drink, how much sugar should we have in our diet as well as how much salt should we ingest every day. Basic Lab Materials: Distilled water Centrifuge tubes Micropipettes Sheep blood Spectrophotometer Microscope slides Cover slips Salt solutions Sugar solutions Microscope Instructor Guidance: How much water does my body need? For this particular module, the students will be divided into two groups with similar tasks (as described below) and they will then compare their observations to determine if drinking too much water can be detrimental to your health and if Gatorade (salt balanced water) is effective in keeping the salt balance in our bodies. 1 I - Morning Session Initiate discussion by asking how much water they drink in a day. Do you drink enough water everyday? What happens if you don’t drink enough? What happens if you drink too much? Do you drink a lot of water when you exercise? Do you think that excess of water can harm your cells in some way? Why do athletes drink Gatorade? Please have the students write their predictions/hypotheses in their lab books prior to each set of experiments. Water Balance Our body has a balanced method of processing and excreting fluids, either through your breath, urine and bowel movements or perspiration. Lack of water can lead to dehydration, a condition that occurs when you don't have enough water in your body to carry out its normal functions. Even mild dehydration can drain your energy and make you tired because water flushes toxins out of vital organs and carries nutrients to your cells. Therefore, your recommended water intake is the "8 x 8 rule" — drink eight 8ounce glasses of water a day (about 1.9 liters). Too much water is also not very good to your body. Over-diluting mineral levels in the blood plasma through excessive consumption of water contributes to what is called an osmotic shift of the fluid inside and outside the cells. This process causes swelling of the cells, often in the central nervous system, including the brain. This may result in water intoxication (also called hyper hydration). Athletes avoid water intoxication by drinking water with electrolytes added so that the balance of the body is maintained. Today’s experiment we will assess how easily we can disturb this balance by using red blood cells from sheep. We will also investigate if Gatorade is effective in keeping the salt balance, therefore keeping the RBCs healthy. General procedures Explain to the students that we can determine if too much water affects our cells by using erythrocytes from sheep. When erythrocytes burst, they release hemoglobin and we can measure the amount of lysis by quantifying the amount of hemoglobin you have in solution using a spectrophotometer. First Group - Three groups of 4 students – 12 total 1. Obtain 200 μl of washed sheep RBCs in 0.15 M NaCl 0.9% saline. 2. Prepare one microcentrifuge tube with 1.5 mls of 0.15 M NaCl, and another with 1.5 mls of distilled water. 3. Add 50 µl of RBC stock to each tube and mix thoroughly. 4. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at 7500 rpm for 6 minutes. 2 5. Record and interpret your results. Second Group - Three groups of 4 students – 12 total 1. Obtain 200 μul of washed sheep RBCs in 0.9% saline. 2. Prepare one microcentrifuge tube with 1.5 mls of 0.15 M NaCl, and another with 1.5 mls of Gatorade. 3. Add 50 µl of RBC stock to each tube and mix thoroughly. 4. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at 7500 rpm for 6 minutes. 5. Record and interpret your results. ** The groups discuss the results. Is there anything else that you could test here? What about different “Gatorades”? If they decide to try different Gatorades, colored ones, we need to adjust the calibration of the spectrophotometer for the appropriate wavelength. II - Afternoon Session Instructor Guidance: What happens to your cells when your diet has too much salt or too much sugar? Initiate questions about how much sugar should be eaten daily. How much salt should you have in your diet per day? Where do you find a lot of sugar? Where do you find a lot of salt? What happens when you have too much salt and sugar in your blood? First Group - Three groups of 4 students – 12 total How much sugar should you eat? What happens if you have too much sugar in your diet? The American Diabetes Association (ADA) criteria for diagnosing diabetes are met when your fasting blood glucose level is 126 mg/dL (7.0 mmol/L) or higher. Symptoms of diabetes include increased thirst and frequent urination (especially at night), unexplained increase in appetite, unexplained weight loss, fatigue, blurred vision, and tingling or numbness in the hands or feet. 1. Obtain 200 μl of washed sheep RBCs in 0.9% saline. 2. Prepare one microcentrifuge tube with 1.5 mls of 0.9% saline, and another with 1.5 mls of 4 mg/ml glucose solution. 3. Add 50 µl of RBC stock to each tube and mix thoroughly. 4. Take 25 µl of the RBC in glucose solution and put in a microscope slide and observe the cells under the microscope. What are you going to use as a control for comparison? Describe what you see. Can you reverse the process? What should you do to reverse the shrinking of the cells? Test it by adding increasing amounts of distilled water to 3 the 4 mg/ml glucose solution. 5. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at 7500 rpm for 6 minutes. 6. Read the amount of hemoglobin in the supernatant with the spectrophotometer. 7. Record and interpret your results. ** The groups discuss the results. Is there anything else that you could test here? What about increasing even more the amount of sugar? Would you see more cell aggregation? Is it possible to quantify the amount of aggregation? Thus, we can provide the students with 25%glucose solution and the students can add increasing amounts to the original 4 mg/ml glucose solution and observe if more cell aggregation will take place or if the cells will start shriveling. Have them speculate as to how these sorts of problems might contribute to the symptoms observed in diabetes. Second Group - Three groups of 4 students – 12 total Why do we need salt? Why is too much salt bad? How much salt should we eat? Salt is a commonly occurring mineral, the technical name of which is sodium chloride. Sodium helps to maintain the concentration of body fluids at correct levels. It also plays a central role in the transmission of electrical impulses in the nerves, and helps cells to take up nutrients. In adults, when levels of sodium are too high, the body retains too much water and the volume of bodily fluids increases. Many scientists are studying if salt is linked to high blood pressure, or hypertension, which in turn is linked to a greater risk of coronary heart disease and stroke. With high levels of fluid circulating through the brain there is a greater chance that weaknesses in the brain's blood vessels are exposed, and that they may burst, causing a stroke. Similarly, a greater volume of fluid passing through the heart can place additional strain on the organ, increasing the possibility of coronary disease. Thus, how much salt should you include in your diet? 1. Obtain 200 μl of washed sheep RBCs in 0.9% saline. 2. Prepare one microcentrifuge tube with 1.5 mls of 0.9% saline, and another with 1.5 mls of 2% sodium chloride (table salt) solution. 3. Add 50 µl of RBC stock to each tube and mix thoroughly. 4. Take 25 µl of the RBC in salt solution and put in a microscope slide and observe the cells under the microscope. What are you going to use as a control for comparison? Describe what you see. Can you reverse the process? What should you do to reverse the shrinking of the cells? Test it by adding increasing amounts of distilled water to the sodium chloride solution. 5. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at 4 7500 rpm for 6 minutes. 6. Read the amount of hemoglobin in the supernatant with the spectrophotometer. 7. Record and interpret your results. ** The groups discuss the results. Is there anything else that you could test here? Can you reverse the process? What should you do to reverse the shrinking of the cells? Test it by adding increasing amounts of distilled water to the sodium chloride solution. What about increasing even more the amount of salt? Would you see more cell lysis? We can provide the students with a 10% salt solution and the students can add increasing amounts to the original 2% sodium chloride solution and observe if more cells shrivel or if the cells start to lyse. 5