How To Test For Tonicity

Transcription

1 Pierce College Putman/Biol 241 Name: Lab 03 SGOs: The Cell: Transport Mechanisms (10 points) Pierce College Student Outcome: Lab Outcome 2: Perform a variety of cell physiology experiments; predict results of tonicity tests that utilize erythrocytes and varying concentrations of saline solutions; describe the differences between simple diffusion, facilitated diffusion, osmosis and filtration. By completing and understanding this lab, you should be able to do the following: 1. Observe the diffusion of a liquid through a colloid and explain what is happening. Give the relationship between molecular weight and the rate of diffusion. 2. Observe the diffusion of dye through water and explain the comparative rate of diffusion of dye through agar and through water. 3. Explain the results of an osmometer demonstration. 4. Observe and explain the results of placing a red blood cell in an isotonic, hypotonic and hypertonic solution. 5. State whether newsprint can be read through test tubes containing 0%, 0.9% and 5% saline solution plus blood, and why, based on the results of SGO 4 above! Exercise 3.1: Diffusion of a Liquid Through a Colloid. Introduction and Methods: In this experiment, you will determine which has a higher rate of diffusion through the solid gel, agar: KMnO 4 (potassium permanganate) or methylene blue. For reference, KMnO 4 has a mass of g/mole and methylene blue has a mass of g/mole. Which do you think? Record your hypothesis in the lab report (end of lab). Make the hypothesis simple and do not include a reason for your answer. A hypothesis such as, Methylene blue will have a higher rate of diffusion than KMnO 4, would be a good hypothesis. I think that (or) I believe that methylene blue will have a higher rate of diffusion than KMnO 4 because it s a protein stain and sticks to agar, would be a bad hypothesis; the I think that and I believe that are irrelevant, and the statement after because would require an additional experiment. Enter your hypothesis before moving on with the experiment! You will not be penalized for an incorrect hypothesis! A little on the scientific method. In science, we construct hypotheses to be the best possible explanations for natural phenomena. We then construct experiments that always must have, as one possible outcome, the falsification of the hypothesis. We run the experiment. If the results do not support the hypothesis, then we say the hypothesis has been falsified or disproved and we either modify it or throw it out completely. If the results of an experiment support the experiment, we say the results support the hypothesis, or the results suggest the hypothesis is the most likely explanation; we don t use the word prove because the next experiment must have, as one outcome, the possible falsification of the hypothesis, even if the falsification of the hypothesis is highly unlikely. To write that a hypothesis has been proven is contrary to the scientific method as this implies that further experiments cannot falsify the hypothesis; you must always have, as one possible outcome, the experimental falsification of the hypothesis. Putman/Pierce College Biol 241 Lab 03/ /Page 1 of 9

2 So, to test your hypothesis, work in groups of two. From the supply table obtain M K a petri dish containing agar and a soda straw. Make two wells in the agar. To do this, about 1/3 the way across the Petri dish, plunge the straw straight down into the agar, twist, and bring it straight out. Repeat another third the way across so that you have two wells as in the figure to the right. From the supply table, obtain a grease pencil or permanent marker and mark on one the plastic on one side of the dish an M for methylene blue and on the other side K for KMnO 4, so you can remember which is which. You will also need a 15 cm ruler. From the supply table, obtain a dropper bottle of methylene blue and one of KMnO 4. Careful: Don t get KMnO 4 in your mouth or eyes as it is toxic! First, note how the drops come out of the dropper bottles; you are going to fill the M well with methylene blue and the K well with KMnO 4 and you don t want any spillage. Carefully fill each well with the corresponding reagent; note the time from a clock this is your time zero. Enter the time in the lab report. Determine what time it will be when 60 minutes will have elapsed and enter this time in your lab report. While you are waiting for the reagents to diffuse through the agar, move on the the other exercises in this lab. At the end of 60 minutes, measure the distance each reagent has diffused from the edge of the well, in millimeters, and record these values in your lab report. Then calculate the rate as distance, in mm, divided by time, in minutes, and enter this value for each in the table in the lab report. Answer the questions in the lab report. Exercise 3.2: Diffusion of Dye Through Water. Introduction and Methods: Kinetic energy is heat, the energy of motion. Diffusion is the random movement of particles from high concentration to low concentration, from an ordered state to a disordered state, as powered by kinetic energy. Diffusion moves materials from high to low concentrations through the aqueous media of the body, inside cells, in extracellular fluids, in the lymph, cerebrospinal fluid, plasma, etc. At the demo table will be a large Petri plate containing water. It will be positioned on a couple of plastic rulers so that the 0 mm mark will be in the center of the plate. Be careful not to bump or breathe on the setup! When the class is ready, the instructor will ask for a time keeper. A few crystals of KMnO 4 will be placed at time 0 on the 0 mm mark. At 5 minutes, the time keeper will say time and another student will read the distance the reagent diffused through the water. Record this value in your lab report, calculate the rate of diffusion of KMnO 4 through the water by dividing the distance traveled by 5 minutes, and answer the questions. Putman/Pierce College Biol 241 Lab 03/ /Page 2 of 9

3 Exercise 3.3: Diffusion Through a Semipermeable Membrane Introduction and Methods: Osmosis is the diffusion of water through a semipermeable membrane. An osmometer is a device used to measure pressure resulting from osmosis. At the demo table, there will be an osmometer already setup. The osmometer will consist of a thistle funnel attached to dialysis tubing (dt) containing saturated sucrose (ss), immersed in distilled water (dw). Dialysis tubing is a semipermeable membrane that allows water through but not sucrose. Your instructor will have recorded time zero at the zero mark on the osmometer. During lab, observe the osmometer, note the height of the column, in mm, and the time, in minutes it took the column of fluid to reach that height; record these values in your lab report and calculate the rate at which the column of fluid is moving. Then answer the questions. dw ss dt Exercise 3.4: Microscopic Observation of Cells in Solutions of Different Tonicity. Introduction and Methods: For this lab, we will be using blood. Take appropriate precautions: Wear gloves. When finished, any materials that touched blood should go in the biohazard containers. Your instructor will give you further instructions regarding this. Obtain a microscope slide, three toothpicks, three coverslips and a permanent marker or grease pencil. Mark a dot in the upper right-hand corner of the microscope slide. At the supply table will be aqueous (water) solutions of 0%, 5% and 0.9% saline. Follow these directions very carefully: Place a single drop of 0% saline on the microscope slide as indicated to the right, followed by a droplet of blood from a toothpick dipped 2 to 3 mm in a blood tube. Gently mix the blood into the aqueous solution, then dispose of the toothpick in biohazard. Add a coverslip on the specimen. 0% 5% 0.9% Place a single drop of 5% saline on the microscope slide as above, mix, dispose of the toothpick in the biohazard and add a coverslip. Last, place a single drop of 0.9% saline on the microscope slide as above, mix, dispose of the toothpick in the biohazard and add a coverslip. Note: 0.9% saline is normal saline and is isoosmotic to blood plasma. Once you have made your slide, first observe the erythrocytes at 0.9% treatment. Focus on lowest power first, then at 100x, then finally at 400x. Describe the shape and condition of the cells in the lab report. Using your mechanical stage, move your slide over and examine the erythrocytes in the 5% treatment. Describe the shape and condition of the cells in the lab report. Finally, without refocusing, using your mechanical stage, move the slide over to examine the erythrocytes in the 0% treatment. Describe the shape and condition of the cells in the lab report. Putman/Pierce College Biol 241 Lab 03/ /Page 3 of 9

4 Again, if the erythrocytes were focused at the 5% treatment, you will not need to refocus at the 0% treatment. Answer the questions provided. Exercise 3.5: Macroscopic Observation of Cells in Solutions of Different tonicity. Introduction and Methods: At the demo table, you will find three test tubes: Test tube 0% will contain two drops of blood mixed in 2 ml of 0% saline solution (distilled water), test tube 0.9% will contain two drops of blood mixed in 2 ml of 0.9% saline solution and test tube 5% will contain two drops of blood mixed in 2 ml of 5% saline. Also at the table will be a scrap of newsprint. You will hold the newsprint behind each of the test tubes and observe if you can read the newsprint through the blood solution. Before you go to the demo table, construct a hypothesis stating which solution you will be able to read newsprint through and which you will not be able to read newsprint through. A good hypothesis might be: "I will be able to read newsprint through the 5% saline-blood solution but not through the 0.9% and 0% saline-blood solutions." Important: Do not include any possible reasons explaining your projected results! A bad hypothesis would be: "I will be able to read through the 5% saline-blood solution because the blood cells settle to the bottom, but I will not be able to read through the 0.9% and 0% saline-blood solutions because the blood cells coagulate and remain in solution." You leave explanations for the discussion portion of your lab! Please make sure the lab is in order before you leave! Putman/Pierce College Biol 241 Lab 03/ /Page 4 of 9

5 Biol 241 Name: Lab 3 Report: Microscopy (10 points) Date: Lab Section: Exercise 3.1: Diffusion of a Liquid Through a Colloid. Hypothesis: Results: Reagent: Time Time Elapsed (min) Distance Traveled (mm) Rate: Distance (mm)/time (min) 0 min 0 mm 60 min Reagent: Time Time Elapsed (min) Distance Traveled (mm) Rate: Distance (mm)/time (min) 0 min 0 mm 60 min Discussion: 1. Which had a higher rate of diffusion through the agar, methylene blue or KMnO 4? Why? 2. Did your results disprove your hypothesis or support your hypothesis? Putman/Pierce College Biol 241 Lab 03/ /Page 5 of 9

6 3. Even if your results supported your hypothesis, why can t we write that the results of an experiment proved the hypothesis? Exercise 3.2: Diffusion of Dye Through Water. Results: Distance KMnO4 traveled through water (mm) in 5 min Rate of diffusion of KMnO 4 through water: mm/min Discussion: 1. What was the rate of diffusion of KMnO 4 through water? 2. What would increase this rate? 3. Give an example of how or where diffusion might move a substance in a biological system? Putman/Pierce College Biol 241 Lab 03/ /Page 6 of 9

7 Exercise 3.3: Diffusion Through a Semipermeable Membrane. Results: Time (min) Height of Column (mm) Osmotic rate, mm/min Discussion: 1. Why did the column of water rise? Explain fully! 2. What was the energy source powering the rise in the column of water? Exercise 3.4: Microscopic Observation of Cells in Solutions of Different Tonicity. Results: Treatment: 0.9% 5% 0% Shape/condition of erythrocytes: Discussion: 1. In which treatment (if any) did water move out of the cells? What was the evidence for this? Putman/Pierce College Biol 241 Lab 03/ /Page 7 of 9

8 2. In the treatment you mentioned in #1, why did water move out of the cells? 3. In which treatment (if any) did water move into the cells? What was the evidence for this? 4. In the treatment you mentioned in #3, why did water move into the cells? Exercise 3.5: Macroscopic Observation of Cells in Solutions of Different Tonicity. Results Write your hypothesis here before beginning this part of the lab: Results: Follow the methods above and record your results here: 2 drops blood + % saline Can read newsprint through solution: Yes or No 0% 0.9% 5% Putman/Pierce College Biol 241 Lab 03/ /Page 8 of 9

9 Discussion: Based on your previous microscopic observations of what happened to red blood cells when they were placed in 0%, 0.9% and 5% saline solutions, answer the following questions: 1. Through which percent saline solution(s) could you read newsprint? Why? 2. Through which percent saline solution(s) could you not read newsprint? Why? How did osmosis affect these blood cells? 3. Did your results support or disprove your hypothesis? 4. If your results supported your hypothesis, why can't we write that your results "proved" your hypothesis? Putman/Pierce College Biol 241 Lab 03/ /Page 9 of 9

10 Putman/Pierce College Biol 241 Lab 03/ /Page 10 of 9