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Cellular Respiration Cellular Respiration Inquiry

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					                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 X 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
  AP Biology Lab:                          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
                                                                                                            Experimental
       Room Temperature                Germinating seeds              Dry Seeds + Beads         Beads
                                                                                                            Peas/Beads

             Treatment                    Apparatus 1b                   Apparatus 2b        Apparatus 3b   Apparatus 4b
                                                                                                            Experimental
                10 C                   Germinating seeds             Dry Seeds + Beads          Beads
                                                                                                            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.


Adapted from a college board lab activity by Fred Brown, 2006, Modified by D Knuffke, 2011
  AP Biology Lab:                          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).


Adapted from a college board lab activity by Fred Brown, 2006, Modified by D Knuffke, 2011
  AP Biology Lab:                          Cellular Respiration Inquiry                                                     Page 4



                       Beads Alone                 Germinating Peas                         Dormant Peas               Experimental
 Tem                  Reading                 Reading                                Reading                      Reading
            Time
  p.                    (ml)                    (ml)                                   (ml)                         (ml)     Diff.*   Corr.
           (min.)              Diff.*                   Diff.* Corrected                       Diff.* Corrected
 (°C)                 at time                 at time                                at time                      at time
                         X                       X                                      X                            X
              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?




Adapted from a college board lab activity by Fred Brown, 2006, Modified by D Knuffke, 2011

				
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