Survey of Biology

Instructor: Steven Clark            Clark Community College    Email: sclark@clark.edu

Lab Activity No. 4

Cellular Respiration

Survey of 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 methods used to collect aerobic respiration data for seeds and humans and explain the physiological rationale for these methods.

4.  Interpret collected and presented data, 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), 7.7 (p. 132),  and Fig. 7.8 (p. 133) in your text. 

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 (Remember 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 organism 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 and weighing pan
• Ice
• Hot water bath
• Two 600 ml beakers with large rubber stoppers with holes

Procedure:

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.  Record the weights of the pans and the seeds.  Subtract the approximate weight of the pan (1.41 g) to determine the weight of each of the seed samples.  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. Place a large rubber stopper with a hole in it into the bottom of each of two 600 ml beakers. NOTE: Large rubber stoppers should not be placed into any beaker that is smaller than 600 ml. If they are stuffed into smaller beakers, they cannot be removed without breaking the beaker! Fill one of the 600 ml beakers with ice and a little cold water to make an ice water solution that is approximately 3-5 C. Fill the second 600 ml beaker, with warm water from the bath and leave it in the water bath until you are ready to begin data collection. 

9. Place one of the respiration chambers with the germinated seeds in the test tube rack that is in the warm water bath. Place a second respiration chamber with the germinated seeds in the beaker with the ice water solution at your desk. The remaining two respiration chambers can be placed in a test tube rack at your desk. Allow all four respiration chambers to “equilibrate” to the experimental temperature for 15 minutes.  After 15 minutes, put the respiration chamber that was in the warm water bath into the 600 ml beaker that is in the bath and bring them to your desk. Place the beaker into a foam rubber insulating jacket to help it to reduce heat loss while the experiment is running.

10.  Stand the bottom ends of the two respiration chambers in the beakers over the holes in the rubber stoppers to help hold them in an upright position.  Use some tape stretched across the top of the beakers and test tube rack to hold each of the test tubes upright so the graduated pipettes are sitting horizontal to the surface of the table.

11. Using an eye dropper, immediately add colored water (enough to give a band approximately 1/8 in. long) 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.

12.  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.

13.  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.

14.  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 trashcan.  Discard the used sodium hydroxide in designated containers.  Remove the seeds and return them to their proper places (Do NOT throw them in the trashcan!).  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)

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

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.