Lab 5 Cell Respiration

Lab 5 Cellular Respiration OVERVIEW In this experiment, you will be working with Alaskan Peas. Some of these will be dormant (dry and inactive) and some will be germinating (active). A seed contains an embryo plant and a food supply surrounded by a seed coat. When the necessary conditions are met, germination occurs and the rate of cellular respiration greatly increases. In this lab you will: • • • measure oxygen consumption during germination, measure the change in gas volume in respirometers containing either germinating or nongerminating pea seeds, and measure the rate of respiration of these peas at two different temperatures. OBJECTIVES Before doing this lab you should understand: • respiration, dormancy, and germination; • how a respirometer works in terms of the gas laws; • the general processes of metabolism in living organisms; and • how the rate of cellular respiration relates to the amount of activity in a cell. After doing this lab you should be able to: • calculate the rate of cell respiration from experimental data; • relate gas production to respiration rate; • test the rate of cellular respiration in germinating versus nongerminated seeds in a controlled experiment; and • test the effect of temperature on the rate of cell respiration in germinating versus nongerminated seeds in a controlled experiment. INTRODUCTION Aerobic cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria within each cell. In simpler terms, sugar is broken down and made into ATP. Cellular respiration involves a series of enzyme-mediated reactions. The equation below shows the complete oxidation of glucose. Oxygen is required for this energy-releasing process to occur. C6H1206 + 602 → 6 CO2 + 6 H2O + 686 kilocalories of energy/mole of glucose oxidized (ATP!!) By studying the equation above, you will notice there are three ways cellular respiration could be measured. One could measure the 1. Consumption of O2 (How many moles of 02 are consumed in cellular respiration?) 2. Production of CO2 (How many moles of CO2 are produced in cellular respiration?) 3. Release of energy during cellular respiration In this experiment the relative volume of 02 consumed by germinating and nongerminated (dry) peas at two different temperatures will be measured. Background Information In order to understand the apparatus in this experiment and how it works, a general knowledge of gas laws is important. The laws are summarized in a general gas law that states: PV=nRT where P is the pressure of the gas, V is the volume of the gas, n is the number of molecules of gas, R is the gas constant (its value is fixed), and T is the temperature of the gas (in K). This law implies the following important concepts about gases: 1. If the temperature and pressure are kept constant, then the volume of the gas is directly proportional to the number of molecules of the gas. 2. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas present. 3. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume. 4. If the temperature changes and the number of gas molecules is kept constant, then either the pressure or volume (or both) will change in direct proportion to the temperature. It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure. In this experiment the CO2 produced during cellular respiration will be removed by potassium hydroxide (KOH) and will form solid potassium carbonate (K2CO3) according to the following reaction: CO2 + 2 KOH → K2 CO3 + H2O Since the CO2 is being removed, the change in the volume of gas in the respirometer will be directly related to the amount of oxygen consumed. This will be measured by watching the bubble move through the pipette part of the respirometer. In the experimental apparatus shown in Figures 1.1 and 1.2, if water temperature and volume remain constant, the water will move toward the region of lower pressure. During respiration, oxygen will be consumed. Its volume will be reduced, because the CO2 produced is being converted to a solid. The net result is a decrease in gas volume within the tube and a related decrease in pressure in the tube. The vial with glass beads alone will permit detection of any changes in volume due to atmospheric pressure changes or temperature changes. The two variables being tested in this experiment are activity of peas (germinating vs. nongerminated) and temperature (room temperature vs. cold). We are going to examine how each of these effect the rate of respiration. We will be measuring the rate of respiration by determining how much oxygen the peas are consuming. Since the CO2 gas that is produced as a waste will be converted into a solid (by KOH), we can measure how much oxygen is being used by watching the air inside the pipette moving into the respirometer. We will have two setups, one in room temperature water, and one in cold water. Each setup will have 3 respirometers: one with germinating peas, one with dry peas and glass beads (to equal the volume of the germinating peas), and one with glass beads only. The one with glass beads only will be our control and if we see any changes in that one we will know it is due to a change in pressure and that change will be applied to our other respirometers. The amount of 02 consumed will be measured over a period of time. Six respirometers should be set up as follows: Respirometer Temperature Contents l Room Germinating Seeds 2 Room Dry Seeds + Beads 3 Room Beads 4 10°C Germinating Seeds 5 10°C Dry Seeds + Beads 6 10°C Beads PreLab Questions 1. What is the difference between a germinating and a nongerminating pea seed? Which do you expect to use more energy? 2. Name 3 ways that you could measure cellular respiration. 3. How will we measure cellular respiration in our lab? 4. What is the equation for cellular respiration? 5. Which respirometer do you expect (and at what temperature) to consume the most oxygen and why? Procedure 1. Both a room-temperature bath (by convention, 25°C) and a 10°C bath should be set up immediately to allow time for the temperature of each to adjust. Add ice to attain 10°C. Use the masking tape to create a sling that the tips of the pipettes can rest on while they are acclimating. See the teacher example. 2. Respirometer 1: Obtain a 100-mL graduated cylinder and fill it with 50 mL of H2O. Drop 25 germinating peas in the graduated cylinder and determine the amount of water that was displaced (which is equivalent to the volume of the peas). Record the volume of the 25 germinating peas. Remove these peas and place them on a paper towel. They will be used in respirometer 1. Make sure you label your paper towel with Respirometer #1 so you can keep all your peas organized. Make a note of this volume for use in the next several steps…record below in your lab notebook: Volume of Germinating Peas = ________________ mL Setup #1 3. Respirometer 2: Refill the graduated cylinder with 50 mL of H2O. Drop 25 dried peas (not germinating) into the graduated cylinder and then add enough glass beads to attain the same volume that the germinating peas had. Remove these peas and beads and place them on a paper towel. They will be used in respirometer 2. 4. Respirometer 3: Refill the graduated cylinder with 50 mL of H2O. Determine how many glass beads would be required to attain a volume equivalent to that of the germinating peas. Remove these beads and place them on a paper towel. They will be used in respirometer 3. 5. Repeat Steps 2-4 to prepare a SECOND SET of germinating peas, dry peas plus beads, and beads for use in respirometers 4, 5, and 6, respectively. Volume of Germinating Peas = ________________ mL Setup #2 6. To assemble the six respirometers, obtain six vials, each with an attached stopper and pipette. Place a small piece of absorbent cotton in the bottom of each vial and, using a dropper; moisten the cotton with 15% KOH. Count the number of drops you are using because it needs to be the same for each setup. The cotton should be moist, but there should NOT be excess KOH at the bottom of the respirometer. Make sure that the respirometer vials are dry on the inside→ Do not get KOH on the sides of the respirometer. Place a small wad of nonabsorbent cotton on top of the KOH-soaked absorbent cotton so that the peas do NOT come into contact with the KOH (Figure 1.1). Do not use too much cotton because all the peas need to fit in the respirometer, so there needs to be enough room. Figure 1.1: Assembled Respirometers 7. Place the first set of geminating peas, dry peas plus beads, and beads in vials 1, 2, and 3, respectively. Place the second set of germinating peas, dry peas plus beads, and beads in vials 4, 5, and 6, respectively. Insert the stopper with the calibrated pipette. 8. Once all 6 of your respirometers are fully assembled, confirm that the temperatures of your two water baths are correct (room temperature- about 25°C and 10°C). Place the respirometers in the water baths with the tips of the pipettes up on the slings. Vials 1, 2, and 3 should rest in the room-temperature water bath (approximately 25°C) and vials 4, 5, and 6 should rest in the 10°C water bath (see Figure 1.2). THE TIPS OF THE PIPETTES SHOULD NOT BE SUBMERGED IN WATER YET. Let the respirometers sit and acclimate for 7 minutes. Make sure they are turned so the numbers on the pipette are facing up so you can read it. While they are acclimating, you can clean up your lab station. Figure 1.2: Respirometers Equilibrating in the Water Bath 9. After the equilibration period of seven minutes, put a drop of food coloring in each pipette. Just put the food color container right next to the tip of the pipette and a single drop should go in when it touches → no need to squirt it in. 10. Next, immerse all six respirometers entirely in their water baths by breaking the sling. Make sure they are turned so that the numbers on the pipette are facing up. You want to be able to read them without touching anything. The setup should not be disturbed. 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. Make sure that a constant temperature is maintained. 11. Allow the respirometers to equilibrate for three more minutes 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 and record it in Data Tables 1.1 and 1.2. Every 5 minutes for 20 minutes, take readings of the water's position in each pipette and record the data in your data tables. You will be filling in the column “Reading at Time X” and after we are done the experiment, we will fill in the rest of the table. Data Tables Table 1.1: Room Temperature Water Bath Temperature of Water = _______°C Beads Alone Time (min) 0 5 10 15 20 Reading at time X Diff. Germinating Peas Reading at time X Diff. Correct Diff. Dry Peas & Beads Reading at time X Diff. Correct Diff. Table 1.2: Cold Water Bath Temperature of Water = _______°C Beads Alone Reading at time X Time (min) Diff. Germinating Peas Reading at time X Diff. Correct Diff. Dry Peas & Beads Reading at time X Diff. Correct Diff. 0 5 10 15 20 Analysis Calculations: In order to calculate the difference in Data Tables 1.1 and 1.2, use the following equation: Difference = (initial reading at time 0) – (reading at time X) In order to calculate the corrected difference in Data Tables 1.1 and 1.2, use the following equation: Corrected Difference = Difference of Germinating Peas – Difference of Beads Alone OR = Difference of Dry Peas/Beads - Difference of Beads Alone Do these calculations and fill in your data tables. You do not need to show your work. SAMPLE DATA → for your reference only to check to see how to do calculations Time 0 5 10 Beads Alone Reading at Diff X .78 .79 -.01 .82 -.04 Germinating Peas Reading at Diff Corrected X Diff .72 .65 +.07 +.08 .62 +.10 +.14 Dry Peas & Beads Reading at Diff Corrected X Diff .72 .81 -.09 -.08 .83 -.11 -.07 To get the difference, subtract from the initial → Ex → .78 - .79 = -.01 .72 - .65 = +.07 Notice at the second time point, you still use the INITIAL→ Ex → .78 - .82 = -.04 .72 - .62 = +.10 To get the corrected difference, take the difference and subtract the difference of the beads alone→ Ex → .07 – (-.01) = +.08 -.09 – (-.01) = -.08 *This step corrects for any changes in atmospheric pressure Analysis Questions: 1. Make a graph of time (minutes) vs. mL of O2 consumed. Be sure to graph the results from the corrected difference column for the germinating peas and the dry peas at both room temperature and at 10°C. Make a best fit line for each data set. (There should be 4 lines on the graph. Provide a key.) Include a title and label your axis correctly. Make this NEAT. **This question does not have to be copied into your lab notebook. For #1 just write “see graph”. 2. Looking at your graph, describe and explain the relationship between the amount of 02 consumed and time. 3. Which respirometer contained the peas that did the most cellular respiration? Why do you think this is? 4. In order to calculate rate, use ∆y/∆x. Look at your graph and use the data from there. Remember to use your corrected difference in your calculations (you used corrected difference in your graph, so if you use your graph that should be fine). Rather than copying this question into your lab notebook, just complete the following table (or cut it out and paste it in): Condition Germinating Peas/ 10°C Germinating Peas/25°C Dry Peas/10°C Dry Peas/25°C Show Calculation Here Rate (mL O2/minute) 5. Why is it necessary to correct the readings from the peas with the readings from the beads? 6. Explain the effect of germination on pea seed respiration. 7. What is the purpose of KOH in this experiment? 8. Why did the vial have to be completely sealed around the stopper? 9. Explain why water moved into the respirometers' pipettes. 10. If you used the same experimental design to compare the rates of respiration of a 25g reptile and a 25g mammal at 10°C, what results would you expect? Explain your reasoning. 11. If respiration in a small mammal were studied at both room temperature (21°C) and 10°C, what results would you predict? Explain your reasoning. 12. Design an experiment to examine the rates of cellular respiration with peas that have been germinating for different lengths of time: 0, 24, 48, and 72 hours. What results would you expect? Why?