The Effect of Alcohol on Biological Membranes

Transcription

1 TEACHER INFORMATION The Effect of Alcohol on Biological Membranes Experiment 1. The student pages with complete instructions for using a Colorimeter or Spectrometer with LabQuest App and Logger Pro (computers) can be found on the CD that accompanies this book. Instructions for using a Colorimeter with EasyData (calculators) or DataPro (Palm handhelds) can also be found on the CD (EasyData and DataPro do not support the use of Spectrometers). See Appendix A for more information. 2. There are two models of Vernier Colorimeters. The first model (rectangular shape) has three wavelength settings, and the newest model (a rounded shape) has four wavelength settings. The 470 nm wavelength of either model is used in this experiment. The newer model is an auto-id sensor and supports automatic calibration (pressing the CAL button on the Colorimeter with a blank cuvette in the slot). If you have an older model Colorimeter, see for calibration information. 3. Isopropyl alcohol may be used in place of n-propanol. If rubbing alcohol is used, remember that it is already diluted to 70% or so (see the container label). You will need to recalculate the volumes used for this alcohol. Use 95% ethanol and absolute methanol. 4. Obtain large, red beets from a local store. Students will cut these into small 0.5 cm 0.5 cm 0.5 cm pieces. To save class time, you may want to cut a 0.5 cm 0.5 cm 12 cm section for each group. They can slice 0.5 cm sections from this. The end pieces should be discarded, since they will dry out and behave differently than freshly cut surfaces. 5. Beets stain hands and clothing. Lab aprons and gloves should be worn to prevent staining. 6. The cuvette must have at least 2 ml of solution in order to get a valid absorbance reading. If students fill the cuvette as described in the procedure, they will have sufficient volume. 7. Since there is some variation in the amount of light absorbed by the cuvette if it is rotated 180, you should use a water-proof marker to make a reference mark on the top edge of one of the clear sides of all cuvettes. Students are reminded in the procedure to align this mark with the white reference mark to the right of the cuvette slot on the colorimeter. 8. The use of a single cuvette in the procedure is to eliminate errors introduced by slight variations in the absorbance of different plastic cuvettes. If one cuvette is used throughout the experiment by a student group, this variable is eliminated. 9. We recommend the use of cotton swabs to dry a cuvette after water or a solution has been emptied from it. The cotton swab will remove any remaining droplets in the cuvette. 10. This experiment gives you a good opportunity to discuss the relationship between percent transmittance and absorbance. You can also discuss the mathematical relationship between absorbance and percent transmittance, as represented by the formula: A = log(100 / %T) Biology with Vernier 8-1 T

2 Experiment 8 SAMPLE RESULTS Absorbance Trial Concentration (%) Methanol Ethanol 1-Propanol Absorbance readings in different concentrations of alcohols ANSWERS TO QUESTIONS 1. Propanol damaged membranes more at low concentrations. At 20% alcohol, propanol caused three times the pigment to be released into the solution, compared to the other two alcohols. 2. Propanol seems to affect membranes the most, as there is more pigment (cellular damage) at any concentration of alcohol. 3. Student answers may vary. In most experiments, the percentage of methanol, ethanol, and 1-propanol that is most damaging is 60%. 4. Answers may vary. CHALLENGE QUESTION 1. The larger the molecule, the greater the extent of membrane damage. Propanol (a threecarbon molecule) is larger than ethanol (a two-carbon molecule) and methanol (a one-carbon molecule). Lipids can be composed of hydrocarbon chains, which are non-polar. As the alcohol increases in length, it becomes less polar than smaller alcohols, and are able to mix with lipids to a greater extent. The OH branch of the alcohol can mix with water in all three molecules. The longer alcohols can mix very effectively with both lipids and water. This tends to disrupt the membrane, much as detergents and soaps do. For this reason, larger alcohols might cause more extensive membrane damage. 8-2 T Biology with Vernier

3 Computer The Effect of Alcohol on Biological Membranes The primary objective of this experiment is to determine the stress that various alcohols have on biological membranes. Membranes within cells are composed mainly of lipids and proteins and often serve to help maintain order within a cell by containing cellular materials. Different membranes have a variety of specific functions. One type of membrane-bound vacuole found in plant cells, the tonoplast, is quite large and usually contains water. In beet plants, this membrane-bound vacuole also contains a watersoluble red pigment, betacyanin, that gives the beet its characteristic color. Since the pigment is water soluble and not lipid soluble, it remains in the vacuole when the cells are healthy. If the integrity of a membrane is disrupted, however, the contents of the vacuole will spill out into the surrounding environment. This usually means the cell is dead. In this experiment, you will test the effect of three different alcohols (methanol, ethanol, and 1-propanol) on membranes. Ethanol is found in alcoholic beverages. Methanol, sometimes referred to as wood alcohol, can cause blindness and death. Propanol is fatal if consumed. One possible reason why they are so dangerous to living organisms is that they might damage cellular membranes. Methanol, ethanol, and 1-propanol are very similar alcohols, differing by the number of carbon and hydrogen atoms within the molecule. Methanol, CH 3 OH, is the smallest, ethanol, CH 3 CH 2 OH, is intermediate in size, and 1-propanol, CH 3 CH 2 CH 2 OH, is the largest of the three molecules. If beet membranes are damaged, the red pigment will leak out into the surrounding environment. The intensity of color in the environment should be proportional to the amount of cellular damage sustained by the beet. To measure the color intensity, you will be using a Colorimeter or Spectrometer. In this device, blue light from the LED light source will pass through the solution and strike a photocell. The alcohol solutions used in this experiment are clear. If the beet pigment leaks into the solution, it will color the solution red. A higher concentration of colored solution absorbs more light and transmits less light than a solution of lower concentration. The device monitors the light received by the photocell as either an absorbance or a percent transmittance value. You are to prepare five solutions of differing alcohol concentrations (0%, 10%, 20%, 30%, and 40%) for each of the three alcohols. A small piece of beet is placed in each solution. After ten minutes, each alcohol solution is transferred to a cuvette that is placed into the Colorimeter or Spectrometer. The amount of light that penetrates the solution and strikes the photocell is used to compute the absorbance of each solution. The absorbance is directly related to the amount of red pigment in the solution. By plotting the percent alcohol vs. the amount of pigment (that is, the absorbance), you can assess the amount of damage various alcohols cause to cell membranes. OBJECTIVES In this experiment, you will Use a Colorimeter or Spectrometer to measure the color intensity of beet pigment in alcohol solutions. Test the effect of three different alcohols on membranes. Test the effect of different alcohol concentrations on membranes. Biology with Vernier 8-1

4 Computer 8 MATERIALS computer cotton swabs Vernier computer interface* forceps Logger Pro knife Colorimeter or Spectrometer lab apron four graduated Beral pipets microplate, 24-well 10 ml 1-propanol one pair gloves 10 ml ethanol ruler (cm) 10 ml methanol tap water three mm test tubes with rack timer or stopwatch 100 ml beaker tissues (preferably lint-free) beet root toothpick * Not necessary if using a Spectrometer. PROCEDURE 1. Obtain and wear goggles, an apron, and gloves. CAUTION: The compounds used in this experiment are flammable and poisonous. Avoid inhaling vapors. Avoid contacting them with your skin or clothing. Be sure there are no open flames in the lab during this experiment. Notify your teacher immediately if an accident occurs. 2. Obtain the following materials: a. Place about 10 ml of methanol in a medium sized test tube. Label this tube M. b. Place about 10 ml of ethanol in a medium sized test tube. Label this tube E. c. Place about 10 ml of 1-propanol in a medium sized test tube. Label this tube P. d. Place about 30 ml of tap water in a small beaker. 3. Prepare five methanol solutions (0%, 10%, 20%, 30% and 40%). Using Beral pipets, add the number of drops of water specified in Table 1 to each of five wells. Use a different Beral pipet to add alcohol to each of five wells in the microwell plate. See Table 1 to determine the number of drops of alcohol to add to each well. Table 1 Well number H 2 O drops Alcohol drops Concentration of alcohol (%) Biology with Vernier

5 The Effect of Alcohol on Biological Membranes 4. Clean the pipet used to transfer alcohol. To do this, wipe the outside clean and empty it of liquid. Draw up a little ethanol into the pipette and use the liquid to rinse the inside of the pipette. Discard the ethanol. 5. Prepare five ethanol solutions. To do so, repeat Step 3, substituting ethanol for methanol. Place each solution in the second row of wells. See Figure Prepare five 1-propanol solutions. To do so, clean your pipette and repeat Step 3, substituting 1-propanol for methanol. Place each solution in the third row of wells. See Figure Now, obtain a piece of beet from your instructor. Cut 15 squares, each 0.5 cm X 0.5 cm X 0.5 cm in size. They should easily fit into a microwell without being wedged in. While cutting the beet, be sure: There are no ragged edges. No piece has any of the outer skin on it. All of the pieces are the same size. The pieces do not dry out. 8. Rinse the beet pieces several times using a small amount of water. Immediately drain off the water. This will wash off any pigment released during the cutting process. Figure 1 9. Set the timer to 10 minutes and begin timing. Use forceps to place a piece of beet into each of 15 wells, as shown in Figure 1. Stir the beet in the alcohol solution once every minute with a toothpick. Be careful not to puncture or damage the beet. While one team member is performing this step, another team member should proceed to Step Prepare a blank by filling a cuvette 3/4 full with distilled water. To correctly use a cuvette, remember: Wipe the outside of each cuvette with a lint-free tissue. Handle cuvettes only by the top edge of the ribbed sides. Dislodge any bubbles by gently tapping the cuvette on a hard surface. Always position the cuvette so the light passes through the clear sides. Spectrometer Users Only (Colorimeter users proceed to the Colorimeter section) 11. Calibrate the Spectrometer. a. Use a USB cable to connect the Spectrometer to your computer. Choose New from the File menu. Biology with Vernier 8-3

6 Computer 8 b. To calibrate the Spectrometer, place the blank cuvette into the cuvette slot of the Spectrometer, and choose Calibrate Spectrometer from the Experiment menu. c. The calibration dialog box will display the message: Waiting 90 seconds for lamp to warm up. After 90 seconds, the message will change to Warmup complete. Click Finish Calibration and then click. 12. Prepare the solutions for data collection. a. After the 10-minute period is complete, remove the beet pieces from the wells. Remove them in the same order that they were placed into the wells. Discard the beet pieces and retain the colored solutions b. Transfer all of the 0% methanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the Spectrometer. 13. Determine the optimum wavelength for creating the standard curve. a. Click. A full spectrum graph of the solution will be displayed. Note that one area of the graph contains a peak absorbance. Click to complete the analysis. b. To select a wavelength for analysis, click the Configure Spectrometer Data Collection icon,, in the toolbar. c. Select Absorbance vs. Concentration (under the Collection Mode). The wavelength of maximum absorbance (λ max) will be selected. (It should be close to 540 nm.) Click. Leave the cuvette in the Spectrometer. d. Proceed to Step 14. Colorimeter Users Only 11. Connect the Colorimeter to the computer interface and prepare the computer for data collection by opening the file 08 Alcohol and Membranes from the Biology with Vernier folder of Logger Pro. 12. Calibrate the Colorimeter. a. Open the Colorimeter lid. b. Holding the blank cuvette by the upper edges, place it in the cuvette slot of the Colorimeter. Close the lid. c. Press the < or > button on the Colorimeter to select a wavelength of 470 nm (Blue) for this experiment. Note: If your Colorimeter has a knob to select the wavelength instead of arrow buttons, ask your instructor for calibration information. d. Press the CAL button until the red LED begins to flash, then release. When the LED stops flashing, the calibration is complete. 13. Prepare the solutions and cuvette for data collection. a. After the 10-minute period is complete, remove the beet pieces from the wells. Remove them in the same order that they were placed into the wells. Discard the beet pieces and retain the colored solutions. b. Empty the water from the cuvette. Use a cotton swab to dry the cuvette after the water has been emptied from it c. Transfer all of the 0% methanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the Colorimeter. Close the lid. Proceed to Step Biology with Vernier

7 Both Colorimeter and Spectrometer Users The Effect of Alcohol on Biological Membranes 14. You are now ready to collect absorbance data for the alcohol solutions. a. Click. b. Wait for the absorbance value displayed in the meter to stabilize. c. Click, enter 0 in the edit box and then press ENTER. The data pair you just collected should now be plotted on the graph. 15. Discard the cuvette contents into your waste beaker. Remove all of the solution from the cuvette. Use a cotton swab to dry the cuvette. Fill the cuvette with the 10% methanol solution from Well 2 using a Beral pipet. Wipe the outside with a tissue and place it in the device (close the lid if using a Colorimeter). Wait for the absorbance value displayed in the meter to stabilize. Click, enter 10 in the edit box and then press ENTER. 16. Repeat Step 15, using the solutions in Wells 3, 4, and 5. When you have finished with all of the methanol solutions click. 17. In the data table, record the absorbance and concentration data pairs listed in the data table. 18. Store the data for the methanol solutions by choosing Store Latest Run from the Experiment menu. 19. Repeat Steps 14 18, measuring the five ethanol solutions. 20. Repeat Steps 14 17, measuring the five propanol solutions. 21. To print a graph of concentration vs. absorbance showing the data for all three alcohols: a. Label all three curves by choosing Text Annotation from the Insert menu, and typing Ethanol (or Methanol, or 1-Propanol ) in the edit box. Then drag each box to a position near its respective curve. Adjust the placement of the arrow head. b. Print a copy of the graph, with all three data sets displayed. Enter your name(s) and the number of copies of the graph you want. c. Use your graph to answer the discussion questions at the end of this experiment. DATA Absorbance Trial Concentration (%) Methanol Ethanol 1-Propanol Biology with Vernier 8-5

8 Computer 8 QUESTIONS 1. Which alcohol damaged the beet at the lowest concentrations? How did you determine this? 2. Which of the three alcohols seems to affect membranes the most? How did you come to this conclusion? 3. At what percentage of alcohol is the cellular damage highest for methanol? ethanol? 1-propanol? CHALLENGE QUESTION 1. What is the relationship between the size of the alcohol molecule and the extent of membrane damage? Hypothesize why this might be so. 8-6 Biology with Vernier

9 Calculator Effect of Alcohol on Biological Membranes The primary objective of this experiment is to determine the stress that various alcohols have on biological membranes. Membranes of cells are composed mainly of lipids and proteins and serve to maintain cellular integrity by containing cellular materials. Different membranes have a variety of specific functions. One type of membrane-bound vacuole found in plant cells, the tonoplast, is quite large and usually contains water. In beet plants, this membrane-bound vacuole also contains a watersoluble red pigment, betacyanin, that gives the beet its characteristic color. Since the pigment is water soluble and not lipid soluble, it remains in the vacuole when the cells are healthy. If the integrity of a membrane is disrupted, however, the contents of the vacuole will spill out into the surrounding environment. This usually means the cell that made the vacuole is dead. In this experiment, you will test the effect of three different alcohols (methanol, ethanol, and 1-propanol) on membranes. Ethanol is found in alcoholic beverages. Methanol, sometimes referred to as wood alcohol, can cause blindness and death. Propanol is fatal if consumed. One possible reason why alcohols are so dangerous to living organisms is that they might damage cellular membranes. Methanol, ethanol, and 1-propanol are very similar alcohols, differing by the number of carbon and hydrogen atoms within the molecule. Methanol, CH 3 OH, is the smallest; ethanol, CH 3 CH 2 OH, is intermediate in size; and 1-propanol, CH 3 CH 2 CH 2 OH, is the largest of the three molecules. If beet membranes are damaged, the red pigment will leak out into the surrounding environment. The intensity of color in the environment should be proportional to the amount of cellular damage sustained by the beet. To measure the color intensity, you will be using the Colorimeter shown in Figure 1. In this device, blue light from the LED light source will pass through the solution and strike a photocell. The alcohol solutions used in this experiment are initially colorless. If the beet pigment leaks into the solution, it will color the solution red. A higher concentration of colored solution absorbs more light and transmits less light than a solution of lower concentration. The Colorimeter monitors the light received by the photocell as either an absorbance or a percentage transmittance value. Figure 1 You are to prepare five solutions of differing alcohol concentrations (0%, 10%, 20%, 30%, and 40%) for each of the three alcohols. A small piece of beet is placed in each solution. After ten minutes, each alcohol solution is transferred to a small, rectangular cuvette that is placed into the Colorimeter. The amount of light that penetrates the solution and strikes the photocell is used to compute the absorbance of each solution. The absorbance is directly related to the amount of red pigment in the solution. By plotting the percent alcohol vs. the amount of pigment (that is, the absorbance), you can assess the amount of damage various alcohols cause to cell membranes. Biology with Vernier 8-1

10 Calculator 8 OBJECTIVES In this experiment you will, Test the effect of three different alcohols on membranes. Use a colorimeter to measure the color intensity of beet pigment in alcohol solutions. Compare the effects of different concentrations of alcohol solutions on membranes. MATERIALS TI-83 Plus or TI-84 Plus graphing calculator cotton swabs EasyData application forceps data-collection interface knife Vernier Colorimeter lab apron graph paper (or Logger Pro ) microwell plate, 24 well two 2 ml pipets or 2 Beral pipets one pair gloves 10 ml 1-propanol pipet pump or pipet bulb 10 ml ethanol ruler (cm) 10 ml methanol tap water three mm test tubes with rack timer or stopwatch 100 ml beaker tissues (preferably lint-free) beet root toothpick PROCEDURE 1. Obtain and wear goggles, an apron, and gloves. CAUTION: The compounds used in this experiment are flammable and poisonous. Avoid inhaling vapors. Avoid contacting them with your skin or clothing. Be sure there are no open flames in the lab during this experiment. Notify your teacher immediately if an accident occurs. 2. Turn on the calculator. Connect the Colorimeter, data-collection interface, and calculator. 3. Obtain the following materials: Place about 10 ml of methanol in a medium sized test tube. Label this tube M. Place about 10 ml of ethanol in a medium sized test tube. Label this tube E. Place about 10 ml of 1-propanol in a medium sized test tube. Label this tube P. Place about 30 ml of tap water in a small beaker. 4. Prepare five methanol solutions (0%, 10%, 20%, 30% and 40%). Using Beral pipets, add the number of drops of water specified in Table 1 to each of five wells in the microwell plate. 5. Use a different Beral pipet to add alcohol to each of five wells in the microwell plate. See Table 1 to determine the number of drops of alcohol to add to each well. 8-2 Biology with Vernier

11 Effect of Alcohol on Biological Membranes Table 1 Well H 2 O Alcohol Concentration number drops drops of alcohol (%) Clean the pipet used to transfer alcohol. To do this, wipe the outside clean and empty it of liquid. Draw up a little ethanol into the pipette and use the liquid to rinse the inside of the pipette. Discard the ethanol. 7. Prepare five ethanol solutions. To do so, repeat Steps 4 and 5, substituting ethanol for methanol. Place each solution in the second row of wells. See Figure Prepare five 1-propanol solutions. To do so, clean your pipette and repeat Steps 4 and 5, substituting 1-propanol for methanol. Place each solution in the third row of wells. See Figure Now, obtain a piece of beet from your instructor. Cut 15 squares, each 0.5 cm X 0.5 cm X 0.5 cm in size. They should easily fit into a microwell without being wedged in. While cutting the beet, be sure: There are no ragged edges. No piece has any of the outer skin on it. All of the pieces are the same size. The pieces do not dry out. 10. Rinse the beet pieces several times using a small amount of water. Immediately drain off the water. This will wash off any pigment released during the cutting process. Figure Set the timer to 10 minutes and begin timing. Use forceps to place a piece of beet into each of 15 wells, as shown in Figure 2. Stir the beet in the alcohol solution once every minute with a toothpick. Be careful not to puncture or damage the beet. Biology with Vernier 8-3

12 Calculator While one team member is performing Step 11, another team member should calibrate the Colorimeter. Prepare a blank by filling an empty cuvette ¾ full with distilled water. Seal the cuvette with a lid. To correctly use a Colorimeter cuvette, remember: All cuvettes should be wiped clean and dry on the outside with a tissue. Handle cuvettes only by the top edge of the ribbed sides. All solutions should be free of bubbles. Always position the cuvette with its reference mark facing toward the white reference mark at the right of the cuvette slot on the Colorimeter. 13. Calibrate the Colorimeter and set up EasyData for data collection. a. Start the EasyData application, if it is not already running. b. Select from the Main screen, and then select New to reset the application. c. Select from the Main screen, and then select Events with Entry. d. Place the blank in the cuvette slot of the Colorimeter and close the lid. e. Set the wavelength on the Colorimeter to 470 nm (Blue). Then calibrate by pressing the CAL button on the Colorimeter. 14. When the timer set to 10 minutes goes off, remove the beet pieces from the wells. Remove them in the same order that they were placed in the well. Discard the beet pieces and retain the colored solutions. 15. Select from the Main screen. 16. You are now ready to collect absorbance data for the alcohol solutions. a. Transfer all of the 0% methanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the Colorimeter. After closing the lid, wait for the absorbance value displayed on the calculator screen to stabilize and select. b. Enter the concentration of alcohol from Well 1 and select. The absorbance and concentration values have now been saved. c. Discard the cuvette contents into your waste beaker. Remove all of the solution from the cuvette. Use a cotton swab to dry the cuvette. 17. Repeat Step 16, using the solutions in Wells 2, 3, 4, and When all solutions have been measured, select to stop data collection. Using the graph, record your data in Table Store the data so that it can be used later. To do this: a. Select to return to the Main screen. b. Select, and then select Store Run. c. Select to store your latest data and overwrite the data in Lists 3 and 4 (L3 and L4). 20. Repeat Steps 15 19, substituting the ethanol solutions in place of the methanol. Note: After selecting to begin data collection, select to start collecting data. Your stored data will not be overwritten. 21. Repeat Steps 15 18, substituting the propanol solutions in place of the methanol. 8-4 Biology with Vernier

13 Effect of Alcohol on Biological Membranes 22. Graph all three runs of data on a single graph. To do this: a. Select and then select L2, L3 and L4 vs L1. b. All three runs should now be displayed on the same graph. The flashing cursor will be on the plot of 1-propanol, each point of ethanol is plotted with a square, and each point of methanol is plotted with a plus sign. c. Examine the data points along the displayed curve of L2 (1-propanol) vs. L1. As you move the cursor right or left, the concentration (X) and absorbance (Y) values of each data point are displayed above the graph. Record the absorbance values in your data table (round to the nearest 0.001). d. Press to switch the cursor to the curve of L3 (ethanol) vs. L1. Examine the data points along the curve. Record the absorbance values (round to the nearest 0.001). e. Press to switch the cursor to the curve of L4 (methanol) vs. L1. Examine the data points along the curve. Record the absorbance values (round to the nearest 0.001). f. Select to return to the Main screen. 23. (Optional) Transfer your data to a computer for later printing per your teacher s instructions. DATA Table 2 Absorbance Trial Concentration (%) Methanol Ethanol 1-Propanol QUESTIONS 1. Which alcohol damaged the beet at the lowest concentrations? How did you determine this? 2. Which of the three alcohols seems to affect membranes the most? How did you come to this conclusion? 3. At what percentage of alcohol is the cellular damage highest for methanol? ethanol? 1-propanol? CHALLENGE QUESTION 1. What is the relationship between the size of the alcohol molecule and the extent of membrane damage? Hypothesize why this might be so. Biology with Vernier 8-5

14 Calculator Biology with Vernier

15 LabQuest Effect of Alcohol on Biological Membranes The primary objective of this experiment is to determine the stress that various alcohols have on biological membranes. Membranes of cells are composed mainly of lipids and proteins and serve to maintain cellular integrity by containing cellular materials. Different membranes have a variety of specific functions. One type of membrane-bound vacuole found in plant cells, the tonoplast, is quite large and usually contains water. In beet plants, this membrane-bound vacuole also contains a watersoluble red pigment, betacyanin, that gives the beet its characteristic color. Since the pigment is water soluble and not lipid soluble, it remains in the vacuole when the cells are healthy. If the integrity of a membrane is disrupted, however, the contents of the vacuole will spill out into the surrounding environment. This usually means the cell that made the vacuole is dead. In this experiment, you will test the effect of three different alcohols (methanol, ethanol, and 1-propanol) on membranes. Ethanol is found in alcoholic beverages. Methanol, sometimes referred to as wood alcohol, can cause blindness and death. Propanol is fatal if consumed. One possible reason why alcohols are so dangerous to living organisms is that they might damage cellular membranes. Methanol, ethanol, and 1-propanol are very similar alcohols, differing by the number of carbon and hydrogen atoms within the molecule. Methanol, CH 3 OH, is the smallest; ethanol, CH 3 CH 2 OH, is intermediate in size; and 1-propanol, CH 3 CH 2 CH 2 OH, is the largest of the three molecules. If beet membranes are damaged, the red pigment will leak out into the surrounding environment. The intensity of color in the environment should be proportional to the amount of cellular damage sustained by the beet. To measure the color intensity, you will be using a Colorimeter or Spectrometer. In this device, light from the LED light source will pass through the solution and strike a photocell. The alcohol solutions used in this experiment are initially colorless. If the beet pigment leaks into the solution, it will color the solution red. A higher concentration of colored solution absorbs more light and transmits less light than a solution of lower concentration. The device monitors the light received by the photocell as either an absorbance or a percentage transmittance value. You are to prepare five solutions of differing alcohol concentrations (0%, 10%, 20%, 30%, and 40%) for each of the three alcohols. A small piece of beet is placed in each solution. After ten minutes, each alcohol solution is transferred to a cuvette that is placed into the Colorimeter or Spectrometer. The amount of light that penetrates the solution and strikes the photocell is used to compute the absorbance of each solution. The absorbance is directly related to the amount of red pigment in the solution. By plotting the percent alcohol vs. the amount of pigment (that is, the absorbance), you can assess the amount of damage various alcohols cause to cell membranes. OBJECTIVES In this experiment you will Test the effect of three different alcohols on membranes. Use a Colorimeter or Spectrometer to measure the color intensity of beet pigment in alcohol solutions. Compare the effects of different concentrations of alcohol solutions on membranes. Biology with Vernier 8-1

16 LabQuest 8 MATERIALS LabQuest cotton swabs LabQuest App forceps Vernier Colorimeter or Spectrometer knife graph paper or Logger Pro lab apron two 2 ml pipets or 2 Beral pipets microwell plate, 24-well 10 ml 1-propanol toothpick 10 ml ethanol one pair gloves 10 ml methanol ruler (cm) three 18 X 150 mm test tubes with rack tap water 100 ml beaker timer or stopwatch pipet pump or pipet bulb tissues (preferably lint-free) beet root PROCEDURE 1. Obtain and wear goggles, an apron, and gloves. CAUTION: The compounds used in this experiment are flammable and poisonous. Avoid inhaling vapors. Avoid contacting them with your skin or clothing. Be sure there are no open flames in the lab during this experiment. Notify your teacher immediately if an accident occurs. 2. Obtain the following materials: Place about 10 ml of methanol in a medium sized test tube. Label this tube M. Place about 10 ml of ethanol in a medium sized test tube. Label this tube E. Place about 10 ml of 1-propanol in a medium sized test tube. Label this tube P. Place about 30 ml of tap water in a small beaker. 3. Prepare five methanol solutions (0%, 10%, 20%, 30% and 40%). Using Beral pipets, add the number of drops of water specified in Table 1 to each of five wells in the microwell plate. 4. Use a different Beral pipet to add alcohol to each of five wells in the microwell plate. See Table 1 to determine the number of drops of alcohol to add to each well. Table 1 Well number H 2 O (drops) Alcohol (drops) Concentration of alcohol (%) Clean the pipet used to transfer alcohol. To do this, wipe the outside clean and empty it of liquid. Draw up a little ethanol into the pipette and use the liquid to rinse the inside of the pipette. Discard the ethanol. 6. Prepare five ethanol solutions. To do so, repeat Steps 3 and 4, substituting ethanol for methanol. Place each solution in the second row of wells. See Figure Biology with Vernier

17 Effect of Alcohol on Biological Membranes 7. Prepare five 1-propanol solutions. To do so, clean your pipette and repeat Steps 3 and 4, substituting 1-propanol for methanol. Place each solution in the third row of wells. See Figure Now, obtain a piece of beet from your instructor. Cut 15 squares, each 0.5 cm X 0.5 cm X 0.5 cm in size. They should easily fit into a microwell without being wedged in. While cutting the beet, be sure: There are no ragged edges. No piece has any of the outer skin on it. All of the pieces are the same size. The pieces do not dry out. 9. Rinse the beet pieces several times using a small amount of water. Immediately drain off the water. This will wash off any pigment released during the cutting process. Figure Set the timer to 10 minutes and begin timing. Use forceps to place a piece of beet into each of 15 wells, as shown in Figure 2. Stir the beet in the alcohol solution once every minute with a toothpick. Be careful not to puncture or damage the beet. While one person is performing this step, another team member should proceed to Step Prepare a blank by filling an empty cuvette 3/4 full with distilled water. Seal the cuvette with a lid. To correctly use a cuvette, remember: Wipe the outside of each cuvette with a lint-free tissue. Handle cuvettes only by the top edge of the ribbed sides. Dislodge any bubbles by gently tapping the cuvette on a hard surface. Always position the cuvette so the light passes through the clear sides. Spectrometer Users Only (Colorimeter users proceed to the Colorimeter section) 12. Calibrate the Spectrometer. a. Connect the Spectrometer to LabQuest and choose New from the File menu. b. Choose Calibrate from the Sensors menu. c. When the warm-up period is complete, place the blank in the Spectrometer. Make sure to align the cuvette so that the clear sides are facing the light source of the Spectrometer. d. Tap Finish Calibration, and then select OK. 13. Prepare the solutions and cuvette for use. Biology with Vernier 8-3

18 LabQuest 8 a. When the timer set to 10 minutes goes off, remove the beet pieces from the wells. Remove them in the same order in which they were placed in the well. Discard the beet pieces and retain the colored solutions. b. Empty the blank. Transfer all of the 0% methanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the Spectrometer 14. Determine the optimal wavelength for creating the standard curve. a. Start data collection. A full spectrum graph of the solution will be displayed. Stop data collection. b. Choose an absorbance peak in the green region of the spectrum by tapping the graph (or use the or keys on LabQuest). The wavelength should be close to 540 nm. 15. Set up the data-collection mode. a. Tap the Meter tab. On the Meter screen, tap Mode. Change the mode to Events with Entry. b. Enter the Name (Concentration) and Units (%). c. Select OK. Proceed to Step 16. Colorimeter Users Only 12. Connect the Colorimeter to LabQuest and choose New from the File menu. If you have an older sensor that does not auto-id, manually set up the sensor. 13. Calibrate the Colorimeter. a. Place the blank in the cuvette slot of the Colorimeter and close the lid. b. Press the < or > button on the Colorimeter to select a wavelength of 470 nm (Blue) for this experiment. Note: If your Colorimeter has a knob to select the wavelength instead of arrow buttons, ask your instructor for calibration information. c. Press the CAL button until the red LED begins to flash, then release. When the LED stops flashing, calibration is complete. 14. Set up the data-collection mode. a. On the Meter screen, tap Mode. Change the data-collection mode to Events with Entry. b. Enter the Entry Label (Conc) and Units (%). c. Select OK. 15. Prepare the solutions and cuvette for use. a. When the timer set to 10 minutes goes off, remove the beet pieces from the wells. Remove them in the same order in which they were placed in the well. Discard the beet pieces and retain the colored solutions. b. Empty the blank. Transfer all of the 0% methanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the Colorimeter. Close the lid. Proceed to Step Biology with Vernier

19 Effect of Alcohol on Biological Membranes Both Colorimeter and Spectrometer Users 16. Start data collection. 17. You are now ready to collect absorbance data for the alcohol solutions. a. Wait for the absorbance value to stabilize and tap Keep. b. Enter the concentration of alcohol from Well 1. Select OK. The absorbance and concentration values have now been saved. 18. Collect the next data point. a. Discard the cuvette contents into your waste beaker. Remove all of the solution from the cuvette. Use a cotton swab to dry the cuvette. b. Transfer all of the methanol solution from Well 2 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the device (close the lid if using a Colorimeter). c. Wait for the absorbance value to stabilize and tap Keep. d. Enter the concentration of alcohol from Well 2. Select OK. The absorbance and concentration values have now been saved. 19. Repeat Step 18 using the solutions in Wells 3, 4, and Stop data collection to view a graph of absorbance vs. concentration. To examine the data pairs on the displayed graph, select any data point. 21. Store the data from the first run by tapping the File Cabinet icon. 22. Discard the solution in the cuvette and transfer all of the ethanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the device (close the lid if using a Colorimeter). Repeat Steps 16 21, substituting the ethanol solutions in place of the methanol. 23. Discard the solution in the cuvette and transfer all of the propanol solution from Well 1 into the cuvette using a Beral pipet. Wipe the outside with a tissue and place it in the device (close the lid if using a Colorimeter). Repeat Steps 16 20, substituting the propanol solutions in place of the methanol. 24. Graph all three runs of data on a single graph. a. Select Run 3, and select All Runs. All three runs will now be displayed on the same graph axes. b. As you tap the data points, the absorbance and concentration values of each data point, for all three runs, are displayed to the right of the graph. c. Record the absorbance values for all three runs in your data table (round to the nearest 0.001). 25. (Optional) Transfer your data to a computer for later printing per your teacher s instructions. Biology with Vernier 8-5

20 LabQuest 8 DATA Table 2 Absorbance Trial Concentration (%) Methanol Ethanol 1-Propanol QUESTIONS 1. Which alcohol damaged the beet at the lowest concentrations? How did you determine this? 2. Which of the three alcohols seems to affect membranes the most? How did you come to this conclusion? 3. At what percentage of alcohol is the cellular damage highest for methanol? ethanol? 1-propanol? CHALLENGE QUESTION 1. What is the relationship between the size of the alcohol molecule and the extent of membrane damage? Hypothesize why this might be so. 8-6 Biology with Vernier