Cellular Respiration Inquiry Introduction: Respiration refers to two different but related processes: one is the active acquisition of gaseous oxygen by an organism and the other is the release of energy from organic compounds within a cell. The latter process, a chemical oxidation, is more commonly referred to as cellular respiration. During this laboratory investigation you will measure the rate of cellular respiration under different conditions. You will experimentally answer the following three questions: Does cellular respiration occur in living pea seeds? Does cellular respiration occur at the same rate for germinating and dormant pea seeds? Does cellular respiration occur at the same rate for pea seeds at 10 C and 25 C? You will also answer a fourth question of your own design, investigating the effect of another variable of your choosing on the rate of cellular respiration. The chemical equation for the complete oxidation of glucose is shown below. Note that oxygen is required for this energy-releasing process to occur. C6H12O6 + 6 O2 6 CO2 + 6 H2O + 686 calories of energy The chemical formula gives some clues as to how you might study the rate of cellular respiration. You could measure the consumption of oxygen (i.e., how many molecules of oxygen are consumed when 1 molecule of glucose is oxidized?), the production of carbon dioxide (i.e., how many molecules of carbon dioxide are produced when 1 molecule of glucose is oxidized?), or the consumption of oxygen and the release of carbon dioxide (i.e., how many molecules of carbon dioxide are produced for every molecule of oxygen consumed?). In this laboratory exercise, you will measure the relative volume of oxygen consumed by germinating and nongerminating pea seeds at two different temperatures over time. In order to measure the volume of oxygen consumed over time you must eliminate all the carbon dioxide produced by the reaction. In this experiment, the carbon dioxide will be removed by reacting with potassium hydroxide (KOH) to form solid potassium carbonate (K2CO3). CO2 + 2 KOH K2CO3 + H2O Therefore, if the temperature and volume in the experimental apparatus remain constant, any gas volume change will be due to the amount of oxygen consumed. A control apparatus will be used to detect any experimental results due to atmospheric pressure change or temperature change. Materials Plastic pan Absorbent cotton Ice Pasteur pipette Thermometer KOH solution (15%) 100 ml graduated cylinder Nonabsorbent cotton Germinating peas Masking tape Dormant peas Respirometer weights Plastic beads Graph paper Test tubes (25 200 mm) Aprons Stopper pipette apparatus Black plastic Procedure Part A. Preparations 1. Before coming to class on the day of the experiment, propose and record hypotheses for each of the problems being studied in this laboratory investigation. Be sure to include your rationale for
Cellular Respiration Inquiry Page 2 each hypothesis. After studying the apparatus and procedures, write predictions for each experiment. 2. Working with your group, develop a fourth experimental question to be investigated. Make sure to write your hypothesis and rationale for your fourth question before coming to class on the day of the experiment. Examples of student inquiries that have been conducted include (but are NOT limited to): Do sliced peas respire at a different rate than whole peas? Do boiled/frozen peas respire at a different rate than unboiled/unfrozen peas? Do peas treated with acid/base/salt solution respire at a different rate than untreated peas? You are encouraged to develop your own experimental question for this investigation. 3. Both a room temperature bath and a 10 C bath should be set up immediately in plastic pans to allow time to adjust each temperature. Add ice to attain the 10 C bath. (If your teacher assigns different groups to only one temperature, you need only set up only one bath.) Add a piece of black plastic to the bottom of each pan. Part B. Determining Pea Volume 1. While the baths are equilibrating, obtain a 100 ml graduated cylinder and fill it with 50 ml of tap water. Drop in 25 germination peas and determine their volume by measuring the amount of water that was displaced. Record the volume of the 25 germinating peas, then remove them and place them on a paper towel. They will be used in Respirometer 1a. 2. Refill the graduated cylinder with 50 ml of water. Drop 25 dormant peas into the graduated cylinder and then add enough plastic beads to attain a volume equal to that of the germinating peas. Remove the peas and beads and place them on a paper towel. They will be used in Respirometer 2a. 3. Refill the graduated cylinder with 50 ml of water. Determine how many plastic beads alone are required to attain a volume equal to the germinating peas. Remove the beads and place them on a paper towel. They will be used in Respirometer 3a. 4. Finally, use the same procedure to create your 4a Respirometer mixture of peas and beads. 5. If you are investigating both temperatures, Repeat Procedures 1 4 above to prepare a second set of germinating peas, dormant peas and beads, and beads only for use in Respirometers 1b, 2b, and 3b respectively. Table 1. Experimental protocol Treatment Apparatus 1a Apparatus 2a Apparatus 3a Apparatus 4a Room Temperature Germinating seeds Dry Seeds + Beads Beads Experimental Peas/Beads Treatment Apparatus 1b Apparatus 2b Apparatus 3b Apparatus 4b 10 C Germinating seeds Dry Seeds + Beads Beads Experimental Peas/Beads Part C. Setting up the Apparatus 1. To assemble the respirometers, obtain 8 test tubes (4 if your class is dividing experimental treatments) and 8 (3) stoppers with attached pipette. Place a small wad of absorbent cotton in the bottom of each test tube and, using a 1-ml pipette, saturate the cotton with 15% KOH solution. Make sure that the respirometer tubes are dry on the inside; do not get KOH on the sides of the respirometer. Place nonabsorbent cotton on top of the KOH-soaked absorbent cotton. 2. Label the respirometer tubes with appropriate numbers and then place one set of germinating peas, dormant peas and beads, and beads in Tubes 1a, 2a, and 3a respectively. (Place the second set of germinating peas, dormant peas and beads, and beads in tubes 1b, 2b, and 3b respectively if you are doing both temperature experiments.) Insert the stoppers fitted with calibrated pipettes.
Cellular Respiration Inquiry Page 3 3. Make a sling of masking tape attached to each side of the water baths to hold the pipettes out of water during an equilibration period of 5 minutes. Place the respirometers in the water bath. (See Figure 1 below.) Attach weights to the respirometers according to your teacher's directions. Respirometers 1a, 2a, 3a, & 4a should rest in the room temperature water bath and Respirometers 1b, 2b, 3b, & 4b should rest in the 10 C water bath. Figure 1: Respirometer Diagrams (experimental not shown) Part D. Collecting Data 1. After an equilibration period of 5 minutes, immerse all respirometers entirely in their water baths. Water will enter the pipettes for a short distance and then stop. (If the water continues to move into a pipette, check for leaks in the respirometer.) Working swiftly, arrange the pipettes so that they can be read through the water at the beginning of the experiment. They should not be shifted during the experiment. Keep your hands out of the water bath after the experiment has started. Make sure that a constant temperature is maintained. Figure 2. Respirometers in the water bath (experimental not shown) 2. Allow the respirometers to equilibrate for 30 seconds and then record, to the nearest 0.01 ml, the initial position of water in each pipette (time = 0). Check the temperature in both baths. Record the changes in the water's position in each pipette every 5 minutes for 20 minutes. Data can be recorded in a table similar to Table 2 below. Table 2. Measurement of oxygen consumption by germinating and dormant pea seeds at room temperature (~25 C) and at 10 C using volumetric methods (Note: Experimental Data Not Shown).
Cellular Respiration Inquiry Page 4 Tem p. ( C) Beads Alone Germinating Peas Dormant Peas Experimental Time (min.) (ml) (ml) (ml) (ml) Diff.* Corr. Diff.* Diff.* Corrected Diff.* Corrected 0 0 0 0 0 0 0 5 25 10 15 20 0 0 0 0 0 0 0 5 10 10 15 20 3. Share data with other class teams. 4. On the same set of axes, graph the results for germinating peas, dormant peas, at both room temperature and 10 C, along with your experimental set up (at both temperatures if possible). Be sure to put the independent and dependent variables on the correct axes. From the graphs, determine the rate of oxygen consumption of germinating and dormant peas at each temperature. In Conclusion: 1. Restate all of your hypotheses and discuss the results of your experiments as relates to the support or refutation of those hypotheses. 2. Do you have reason to doubt your data in any of your experiments? Provide any explanation as to why or why not. 3. Would you expect a difference in cellular respiration rate between germinating and dormant pea seeds? Explain your reasoning. 4. Would you expect a difference in cellular respiration rate between pea seeds at different temperatures? Explain your reasoning. 5. Explain any differences that you see in the rate of cellular respiration in your fourth tube (the experimental tube) as compared to tubes 2 and 3. Propose explanations as to why these differences, or lack of differences are demonstrated. 6. Don t forget to include discussion of modifiying the experiment, and further avenues of investigation. Analysis Questions (following Conclusion): 1. Why was it necessary to equalize the pea volumes in each respirometer? 2. Why were plastic beads used to equalize the dormant pea volumes instead of using more peas? 3. Why was it important not to leave potassium hydroxide (KOH) on the test tube sides? 4. Why was it necessary to absorb all of the carbon dioxide produced by the reaction? 5. What function did Respirometers 3 and 6 serve? 6. Students often get pressure changes in Respirometers 3 and 6. Would you predict such a change for inert plastic beads? What could cause this change?
Cellular Respiration Inquiry Page 5 Teacher Notes: Materials: 1. 15% KOH 37.5 g KOH brought to 250 ml with distilled water 2. Pea Seeds 25 germinating pea seeds / team; same for nongerminating (~ 100 peas at 30 ml mark on a 50 ml beaker) [Order info: one pound of peas / year is more than sufficient] 3. Respirometer weights Use water-filled specimen dishes with equilibrated water Suggestions: 1. Must germinate peas a few days in advance (soak overnight; then roll in moist paper towels for the next night in 26 C incubator) 2. Can set up room temperature baths the night before to equilibrate 3. Get ice from the cafeteria 4. Divide class into teams of 25 C or 10 C experiment; one half on one side of the room, the other on the other half. 5. Remind students that if they need to turn the apparatus under water to better read the pipette, only touch the pipette, not the test tube. Touching the test tube will change the temperature of the apparatus and, thus, the pressure in the pipette. 6. Use extra long forceps to clean cotton out of tubes Results: 1. See APB Lab Manual Notes: 1. Written by Fred Brown, AP Biology Teacher and APB Consultant [FredBrown3@aol.com] 2. Last revised: 6/06