Lab 5- Cellular Respiration

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1 Lab 5- Cellular Respiration Background: Many cellular processes require energy. Aerobic cellular respiration supplies energy by the oxidation of glucose. This is a complex process involving a number of enzymemediated reactions; however we can summarize the process in terms of input and output products with a very simple equation: C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O + energy You will use a respirometer to measure the rate of respiration of germinating and nongerminating pea seeds at two different temperatures. The respirometer consists of a vial that contains the peas and a volume of air. The mouth of the vial is sealed with a rubber 1-hole stopper that has a pipette inserted in it. The respirometer is submerged in water. If the peas are respiring, they will use oxygen and release carbon dioxide. Since 1 mole of carbon dioxide is released for each mole of oxygen consumed, there is no change in the volume of gas in the respirometer. (Avogadro s Law: At constant temperature and pressure, 1 mole of any gas has the same volume as 1 mole of any other gas.) You will alter this equilibrium by placing a solution of potassium hydroxide (KOH) in the vial. Potassium hydroxide reacts with carbon dioxide to form potassium carbonate, which is a solid. CO 2 + KOH K 2 CO 3 + H 2 O Since the carbon dioxide produced is removed by reaction with potassium hydroxide, as oxygen is used by cellular respiration, the volume of gas in the respirometer will decrease. As the volume of gas decreases, water will move into the pipet. You will use this decrease of volume, as read from the scale printed on the pipet, as a measure of the rate of cellular respiration. PV = nrt is the formula for the inert gas law where P = pressure of the gas, V = volume of the gas, n = number of molecules of gas, R = gas constant, and T = temperature of the gas in degrees K. This law implies several important things about gases. If temperature and pressure are kept constant then the volume of the gas is directly proportional to the number of molecules of the gas. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume. If the temperature changes and the number of gas molecules is kept constant, then either pressure or volume or both will change in direct proportion to the temperature. Purposes: - Measure the consumption of oxygen by respiring seeds (germinating vs. non germinating seeds on respiration rate) - Compare respiration rates at two different temperatures (room temperature vs. cold water on respiration rate) Hypothesis 1 (germination): Hypothesis 2 (temperature): IV: DV: Control: Experimental groups (list): # Total Trials (at both temps): Constants:

2 Materials: - Room Temp. Water bath - Cold Temp. Water bath - Container of ice - Germinating peas - Non-germinating peas - Glass beads - Respirometers - Graduated tube - Absorbent cotton balls - Nonabsorbent cotton - Dropping pipettes - Forceps - Thermometers - Stopwatch - 15% potassium hydroxide (KOH) solution Procedure: 1. Prepare a room-temperature bath (approx. 25 o C) and a cold water bath (approx. 10 o C). 2. Add ice cubes to the cold water bath until the desired temperature of 10 o C is obtained. 3. Put a sheet of white paper lying flat in the water bath. The paper will help provide a contrast when reading the pipette. 4. Fill a 100 ml graduated cylinder with 50 ml of water. 5. Add 25 germinating peas and determine the amount of water that is displaced. 6. Record this volume of the 25 germinating peas, then remove the peas and place them on a paper towel. They will be used for respirometer Next, refill the graduated cylinder with 50 ml of water and add 25 non-germinating peas to it. 8. Add glass beads to the graduated cylinder until the volume is equivalent to that of the expanded germinating peas. Remove the beads and peas and place on a paper towel. They will be used in respirometer Now, refill the graduated cylinder with 50 ml of water. Determine how many glass beads would be required to attain a volume that is equivalent to that of the germinating peas. Remove the beads. They will be used in respirometer Then repeat the procedures used above (steps 3-8) to prepare a second set of germinating peas, dry peas and beads, and beads to be used in respirometers 4, 5, and Wear gloves before assembling the six respirometers: 12. Obtain 6 glass vials (3 for room temp and 3 for colder temps), each with an attached stopper and pipette. 13. Place a small wad of absorbent cotton in the bottom of each vial, using tweezers. 14. Using a plastic pipette, saturate the cotton with 15 % KOH. Be sure not to get the KOH on the sides of the respirometer. 15. Place a small wad of nonabsorbent cotton on top of the KOH-soaked absorbent cotton, using tweezers. 16. Repeat these steps (steps 10-14) to make the other respirometers. It is important to use about the same amount of cotton and KOH in each vial.

3 17. Place the first set of germinating peas, dry peas and beads, and beads alone in vials 1, 2, and 3, respectively. 18. Place the second set of germinating peas, dry peas and beads, and glass beads in vials 4, 5, and 6, respectively. 19. Insert the stoppers in each vial with the proper glass pipette. There should be a washer on each of the pipettes to be used as a weight. (See Figure 5.1) Table 1 of your setup: Respirometer Temperature (Record Contents Initial Temperature) o C 1 Room= Germinating Seeds 2 Room= Dry Seeds + Glass Beads 3 Room= Glass Beads 4 Cold= Germinating Seeds 5 Cold= Dry Seeds + Glass Beads 6 Cold= Glass Beads 20. Make a sling using masking tape and attach it to each side of the water baths to hold the pipettes out of the water during the equilibration period of 10 minutes. Vials 1, 2, and 3 should be in the bath containing water at room temperature. Vials 4, 5, and 6 should be in the bath containing water that is 10 o C. (See Figure 5.2) 21. It is difficult to see the water/air interface when taking readings from the pipettes. To help with this, you may want to touch a drop of food coloring to the tips of the pipettes just before immersing them. 22. After the equilibration period, immerse all six respirometers into the water completely. Water will enter the pipette for a short distance and stop. If the water does not stop, there is a leak. Make sure the pipettes are facing a direction from where you can read them. The vials should not be shifted during the experiment and your hands should not be placed in the water during the experiment. 23. Allow the respirometers to equilibrate for three more minutes and then record the initial water reading in each pipette at time Check the temperature in both baths and record the data. 25. Every five minutes for 20 minutes take readings of the water s position in each pipette, and record.

4 26. For your calculations in the data table: = V at time 0 V at current reading = (for Respirometer 1 or Respirometer 2) - of Respirometer 3 Lab 5-Data Table 2: Measurement of O2 Consumption by Soaked and Dry Pea Seeds at Room Temperature Using Volumetric Methods Temperature ( o C) Time (Min) Respirometer 1 Germinating Peas Respirometer 2 Dry Peas + Beads Respirometer 3 Beads Only Table 3: Measurement of O2 Consumption by Soaked and Dry Pea Seeds at 10 C Using Volumetric Methods Respirometer 1 Germinating Peas Respirometer 2 Dry Peas + Beads Respirometer 3 Beads Only Temperature ( o C) Time (Min) Graph respiration rate of all six variables on one graph (include key)

5 Lab 5- Analysis of Results 1. What are the sources of error in this experiment? 2. Describe (What?) and explain (Why?) the relationship between the amount of oxygen consumed and time. 3. Explain the effects of germination (versus non-germination) on pea seed respiration. 4. Why is it necessary to correct the readings from the peas with the readings from the beads? 5. What is the purpose of KOH in this experiment? 6. In this experiment, you measured the change in volume of the gas inside the respirometers. The general gas law describes the state of a gas under given conditions: PV = nrt is the formula for the inert gas law or V = nrt/p (since you are measuring changes in volume. P = pressure of the gas V = volume of the gas n = kmoles (number of molecules) of gas R = universal gas constant [8314 joules/ (kmole) (K)] T = temperature of the gas in degrees K Using the general gas law and your experience in this lab, give the variables that had to be controlled for your data to be valid. State the controls used for each variable and any means used to correct for the influence of a variable(s).

6 7. Which of the respirometers (1, 2, or 3) serves as a negative control? Explain your answer. 8. From your graph, calculate the rate of oxygen consumption for each treatment: a. Germinating seeds at room temperature = ml/min b. Germinating seeds at colder temperature = ml/min c. dry seeds at room temperature = ml/min d. dry seeds at colder temperature = ml/min 9. Using your graph and data tables, summarize your findings, comparing results from respirometers 1 and 2, and results obtained at room temperature vs. results at the colder temperatures. Speculate as to the cause (s) of any differences between the treatments. 10. If you used the same experimental design to compare the rates of respiration of a 35g mammal at 10 o C, what results would you expect? Explain your reasoning. 11. If respiration in a small mammal were studied at both room temperature (21oC) and 10 o C, what results would you predict? Explain your reasoning.

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