ࡱ> ` Wjbjb +ddO%PPPPPPPp8,Dpp "BBB  ,dRr7-P7^7PPBBdPBPB dhPPPP aPP ^M-<&z0G((4PppppLab Activity No. 4 Cellular Respiration General Biology Lab A. Objectives: Upon completion of this lab activity, you should be able to: 1. Define/explain the following terms: aerobic respiration, anaerobic metabolism, second law of thermodynamics, germination, sodium hydroxide, pH indicator, and phenolphthalein. 2. Write a balanced chemical equation for aerobic respiration and explain it. 3. Describe the apparatuses used to collect data for aerobic respiration by seeds, mice, and humans and explain the physiological rationale for these apparatuses. 4. Interpret collected and presented data and draw appropriate conclusions and be able to support/refute the conclusions using lecture and/or text information. 5. Correctly present the collected data in a graphical manner. 6. Critically evaluate the experiment designs and suggest specific improvements. 7. Use appropriate and specific terminology as part of all explanations. B. Introduction: The oxidation of organic molecules (e.g. sugars) releases energy that is stored in its chemical (covalent) bonds. Organisms are able to capture some of this energy in the form of ATP, which can be used to do cellular work. This capturing of chemical energy occurs in a number of steps, with each step under the control of a specific enzyme (Review figures 7.5 (p. 129), 7.8 (p. 133) and, Fig. 7.9 (p. 134). The most energy-efficient form of the oxidation of organic molecules is termed aerobic (oxygen-requiring) respiration. The process by which glucose is oxidized to CO2, H2O, and energy can be represented by the following chemical equation: 36 ADP + 36 Pi + 6 O2 + C6H12O6 ---> 6 CO2 + 6 H2O + 36 ATP This process yields a net of 36 ATP molecules for each glucose molecule oxidized. Approximately 40% of the released energy is captured to make this ATP, while the rest is lost as heat (Recall the second law of thermodynamics). Anaerobic (non-oxygen requiring) metabolism produces a net of only 2 molecules of ATP per glucose, so the way to maximize ATP production is to carry out aerobic respiration . One byproduct of aerobic respiration is carbon dioxide (CO2). For example, CO2 generated by human cells diffuses into the blood stream where it is taken to the lungs. Note from the previous chemical equation that the amount of CO2 generated is equal to the amount of oxygen consumed (assuming that the source of energy is glucose). Therefore, if a metabolically active plant or animal is confined in a closed chamber and the CO2 generated is removed from the chamber, the volume of air in the chamber will decrease in proportion to the amount of O2 used for cellular respiration. C. Plant Cellular Respiration Rate Experiment: Influence of Temperature and Germination Status The plant respiration experiment measures the utilization of oxygen by seeds in a special container. The class will investigate the effects of temperature on aerobic respiration in germinating and non-germinating seeds. Each group will test all four treatments: (1) Approximately 10 germinating seeds at 3 to 5 C (ice-water bath) (2) Approximately 10 germinating seeds at room temperature (22-24 C) (3) Approximately 10 germinating seeds at 45oC (warm-water bath) (4) Approximately 20 non-germinating seeds at room temperature (22-24 C) Hypothesis: Write a hypothesis for this experiment. Think about the conditions under which chemical reactions can take place when formulating your hypothesis.    Materials: Germinated pea seeds Non-germinated pea seeds Four respiration chambers Blue dye in dropper bottle Sodium hydroxide Balance Ice Hot water bath Two 600 ml beakers with large rubber stoppers with holes 1. Place approximately 10 germinating seeds in each of three weighing pans. Use forceps to select the seeds and be careful not to damage them during the transfer. Do not select seeds with a substantial amount of mold on them. 2. Place approximately 20 non-germinating seeds in a weighing pan. 3. Tare the scale with an empty pan and record only the weight of the seeds. Record these weights in the appropriate data table in your report. 4. Place each sample into an individually labeled test tube (respiration chambers). Make certain you record the respective test tube number/temperature condition into which the seeds were placed. 5. Place approximately 3/4 of one cotton ball over the seeds in the tube and use a spatula to gently push the cotton down the test tube (See the diagram presented in class). The cotton should not be packed so tightly as to interfere with gas flow. The depth of the cotton layer should be no more than 1/2 inch (approximately 1.5 cm). The cotton keeps the sodium hydroxide (which will be added in the next step) from damaging the seeds. 6. Using a spatula, add enough sodium hydroxide to create a 1/2 inch layer over the cotton. Caution: Do not touch the sodium hydroxide. If it does come into contact with your skin, wash your hands with soap and water. The sodium hydroxide will function to remove the expired carbon dioxide from the tube. 7. Firmly insert the rubber stopper with the attached graduated pipette into each of the four test tubes. Note the measurement scale on the graduated tube. 8. Prepare a 600 ml beaker, with a one-hole rubber stopper at the bottom, containing an ice water solution (approximately 3-5 C). Prepare a second beaker, with a two-hole rubber stopper at the bottom, containing room temperature water (approximately 22-24 C). Beakers with the warm water (approximately 45 C) are located in the water bath in the back of the room. Leave these beakers in the water bath until directed to remove them. 9. Position the respiration tubes into the rubber stoppers in their respective beakers. Make certain the graduated pipettes are approximately horizontal to the surface of the table. 10. Allow all respiration tubes to equilibrate to the experimental temperature for 15 minutes. Leave the 45 C tube and beaker in the water bath during this 15 minutes. The other two beakers should be at your table. 11. During the next 15 minutes, someone should practice adding the colored water to the ends of the pipettes. 12. Remove the beaker from the water bath, place it into a foam insulator (to reduce heat loss) and take it back to your table. Using a dropper, immediately add colored water (enough to give a band approximately 1/8 in. diameter) to the end of all four of the graduated pipettes (This will be demonstrated). Make certain that approximately the same volume of dye is in all four graduated pipettes. Record the starting time in the appropriate data table in the report. 13. Continue the experiment until the dye band in any one of the four tubes has moved up to the 0 mark on the graduated pipette, which represents 1 ml of oxygen consumed. 14. Record the time and location (with respect to the gradients on the pipette) of the dye in all four tubes at the end of the experiment in the appropriate data table in the report. By comparing the starting position of the dye with the final position, you can determine the volume of oxygen used during the experiment. The time value (# Min. Run) for all four tubes should be the same, assuming that the dye was added at approximately the same time. You can use the data collected to determine the volume of oxygen consumed per minute per gram (ml/min./g). Complete these calculations in the data table in your report. 15. At the conclusion of the experiment, remove the stoppers from the test tubes and rinse the colored water out of the graduated pipettes. Discard the cotton in the trash can. Discard the used sodium hydroxide in designated containers. Remove the seeds and return them to their proper places (Do NOT throw them in the trash can!). Rinse the test tubes and the pipettes with distilled water and return them to the rack on your table. Make certain you wash your hands with soap and water if you handled the sodium hydroxide. Data Tables: 1. Oxygen Consumption Data for Seeds (Germinating and Non-germinating) Exposed to Three Different Temperatures (2 pts. for accurate completion) Temperature (C)Seed ConditionSeed Weight (grams)Vol. of Air Consumed (ml)No. Min. RunO2/min.O2/min/gram 22-24  Non-Germ. 3-5 Germinating 22-24 Germinating 45 Germinating Time Started: ________ Time Completed: ________ Total Time (min.): _______ Note: To convert seconds to minutes, divide by 60. For example 140 seconds is 2.3 minutes (140/60 = 2.3) 2. Class Oxygen Consumption Data (ml O2/min./g) for Seeds (Germinating and Non-germinating) Exposed to Three Different Temperatures Group No.Non-Germinating (22-24 C)Germinating (3-5 C)Germinating (22-24 C)Germinating (45 C) 1 2 3 4 5 6 AVERAGE Graph of Plant Cellular Respiration Data: Create a bar graph that shows the class averages for the four, temperature/germination status combinations. Remember to use a key/legend.  Questions: 1. What did you conclude from this experiment? Did you accept or reject your hypothesis? 2. Reflect on the design of this experiment. Come up with two specific changes you would make if starting over again. Make certain you explain how the changes will result in an improved design. First Improvement:___________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ Second Improvement:_________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ D. Human Cellular Respiration Experiment This activity will assess the effects of activity level on human cellular respiration. Two persons from each group will act as experimental subjects. Do not participate in this simulation if you have any health condition that could be problematic during periods of physical activity. A by-product of aerobic respiration is carbon dioxide (CO2). Thus, the release of CO2 in our exhaled breath should be an indicator of the amount of oxygen (O2) consumed. We will assay the amount of CO2 in exhaled air by exploiting the fact that CO2, when mixed with water, forms carbonic acid, in the manner shown by the following chemical reaction: CO2 + H2O HCO3- + H+ Phenolphthalein is a pH indicator--it is clear in acid solutions (pH < 7) and pink at neutral (pH = 7) or alkaline solutions (pH > 7). Thus if a person exhales into water that has some phenolphthalein, the solution should be clear. If we now add a base, sodium hydroxide (NaOH) to the solution, we can restore neutral pH, and the solution should turn pink. By adding NaOH one drop at a time, we can obtain a quantitative (i.e. numerical) estimate of the amount of CO2 produced by a person. We can then use this rationale to examine the effects of exercise on the rate of aerobic respiration. Materials: Distilled water Phenolphthalein Three, 250 ml beakers Straws 0.4% NaOH 1. Designate two students in each group as the experimental subjects. If possible, select one person who exercises (e.g. aerobics, jogging, or biking) at least 3x per week and one person who does not exercise on a regular basis. Do not select any student to participate in this activity if they have any health condition that could be problematic during periods of physical activity. While preparing the beakers, have the two students sit quietly. 2. Place 50 ml of distilled water in a provided 250 ml beaker. Add 4 drops of phenolphthalein to the water and gently swirl to mix. i. If the water turns pink, assume there is little if any CO2 dissolved in the water. Proceed to step 3. ii. If the water does not turn pink, CO2 must be present. To begin the experiment, we must remove this CO2 from our assay. Add 0.4% NaOH to the solution, one drop at a time, gently swirling the solution between each drop. Continue to add NaOH until the solution turns pink and stays pink for at least one minute. It will help to put a sheet of white paper behind the bottle to more accurately evaluate the color. iii. This beaker will act as a color control (bottle #1) for both students in your group. 3. Repeat step 2 for two other beakers, one for each of the experimental students (beakers #2 and #3). Attain the same pink color in both of these beakers as was in beaker #1. 4. Make certain that the experimental students have been sitting quietly for at least 5 minutes before proceeding. The data that you collect from this portion of the experiment will define the cellular respiration rate at rest. 5. After this time, place the end of a soda straw into each of the other beakers (1 straw for beaker 2 and one straw for beaker 3). Each of the experimental students should take one of the beaker and steadily blow air (Do NOT suck up liquid in the straw!) from their lungs through the straw and into the water for 10 seconds. 6. Add NaOH, one drop at a time (swirling between drops), until the original pinkish color is restored (compared to beaker #1, the color control). Record the number of NaOH drops required to obtain the color change in the appropriate data table as the respiration rate at rest. Remember that the number of drops is an indication of the CO2 output from the experimental students. ****Note: These beakers are already ready for the next experiment, so just have each student use their beaker and straw over again. 7. Next, have both students exercise at a "vigorous pace" by running as fast as possible in place for 3 minutes. Immediately repeat steps 5 and 6. Record the number of NaOH drops as the cellular respiration rate with "vigorous exercise". 8. Add your data into the class data table on the board and then transfer the results to the appropriate data table in your report. Data Tables: 1. Group Data for the Number of Drops of NaOH Required to Neutralize the Carbon Dioxide, Exhaled After Three Different Levels of Activity Subject No. Fitness Level Activity Level Tested No. Drops of NaOH Required 1 Regular Exercise Resting  1 Regular Exercise Vigorous 2 Infrequent Exercise Resting 2 Infrequent Exercise Vigorous 2. Class Data for the Number of Drops of NaOH Required to Neutralize the Carbon Dioxide Exhaled, After Three Different Levels of Activity Group No. Regular Exercise Subjects Resting Vigorous Infrequent Exercise Subjects Resting Vigorous 1 2 3 4 5 6 AVERAGE Questions: 1. An experiment was conducted to evaluate the effects of activity level on human respiration rate. A total of 100 males were selected to participate in the study. Fifty of the test subjects were classified as athletic (i.e. participated in 30 minutes of aerobic activity at least 4 times per week) and the remaining 50 were classified as sedentary (i.e. no participation in a regular exercise program). The carbon dioxide content of each subjects exhaled air was then determined at rest and after 5 minutes of intense exercise. Below is a summary of the collected data: Average Carbon Dioxide Levels in Air Exhaled by Athletes and Non-Athletes At Rest and After Five Minutes of Intense Exercise Physical Status at Time of Testing: Athletic Classification:  At Rest Athletes Non-Athletes After Exercise Athletes Non-AthletesCarbon Dioxide Level in Exhaled Air 15 14 28 10 Provide a physiological explanation for average carbon dioxide level differences between the athletes and the non-athletes (i.e. Why the athletes had a HIGHER average carbon dioxide level in the exhaled air after 5 minutes of intense exercise versus at rest and the non-athletes had a LOWER average value). ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ E. Review Questions 1. What is a fundamental distinguishing feature between aerobic and anaerobic metabolism? 2. In what organelle does the majority of aerobic respiration occur? 3. What is the second law of thermodynamics? Is it relevant to aerobic respirations efficiency? 4. What is the function of the sodium hydroxide in the seed respiration experiment? How is this function critical to the success of data collection? 5. Prior to conducting the seed respiration rate assay, what was your hypothesis regarding the influence of temperature and germination status on this process? Write your response in the space provided below. Justify your response with details from the reading and/or lecture. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 6. Explain, referring to the data table, how you calculated oxygen consumption for the seeds. Use the units (ml oxygen/minute/gram) as part of your explanation. 7. Explain, using the class data, the influence of temperature and germination status on seed respiration rate. 8. Present a completed bar graph of the class data (i.e. NOT just your individual groups data) from the seed respiration rate experiment. Explain the procedures used to construct it. 9. In the human respiration rate assay, why did pink solution turn clear as you blew through the straw? Why did a clear solution turn pink as you added sodium hydroxide? 10. Prior to conducting the human respiration rate assay, what was your hypothesis regarding the influence of exercise status (i.e. frequent versus infrequent) on the amount of carbon dioxide produced at rest and after vigorous exercise? Write your response in the space provided below. Justify your response with details from the reading and/or lecture. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______ 11. Samples from some people require a lower number of drops of NaOH after vigorous exercise than after mild exercise. How can this be explained?     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