AP Biology Lab #5: Cell Respiration OVERVIEW: In this experiment you will work with seeds that are living but dormant. 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:

1) Measure oxygen consumption during germination. 2) Measure the change in gas volume in respirometers containing either germinating or nongerminating

pea seeds. 3) Measure the rate of respiration of these peas at two different temperatures.

OBJECTIVES: Before doing this lab you should understand:

A) Respiration, dormancy, and germination. B) How a respirometers works in terms of the gas laws. C) The general processes of metabolism in living organisms. D) How the rate of cellular respiration relates to the amount of activity in a cell.

After doing this lab you should be able to:

A) Calculate the rate of cell respiration form experimental data. B) Relate gas production to respiration rate. C) Test the ate of cellular respiration in germinating versus nongerminating seeds in a controlled

experiment. D) Test the effect of temperature on the rate of cell respiration in germinating versus nongerminating

seeds in a controlled experiment. 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.

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 kilocalories of energy/mole glucose oxidized

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 (dry) pea seeds at two different temperatures over time. A number of physical laws relating to gases are important to the understanding of how the

apparatus that you will use in this exercise works. The laws are summarized in the 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, and T is the temperature of the gas (in K)

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.

In the experimental apparatus below, 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 amount of O2 consumed will be measures over a period of time. Six respirometers should be

set up as follows: Table 1

Respirometer Temperature Contents 1 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

PROCEDURE: Both a room temperature bath and a 10°C bath will be set up in plastic pans. 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.) Part A. 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 1.

2. Refill the graduated cylinder with 50 ml of water. Drop 25 dormant (dry) 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 2.

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 3.

4. If you are testing for both temperature conditions, repeat Procedures 1�3 above to prepare a second set of germinating peas, dormant peas and beads, and beads only for use in Respirometers 4, 5, and 6 respectively.

Part B. Setting up the Apparatus 1. To assemble the respirometers, obtain 6 test tubes (3 if your class is dividing experimental

treatments) and 6 (3) stoppers with attached pipette. Place a small wad of absorbent cotton in the bottom of each test tube and, using a Pasteur 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,

dry peas and beads, and beads in Tubes 1, 2, and 3 respectively. (Place the second set of germinating peas, dry peas and beads, and beads in tubes 4, 5, and 6 respectively if you are doing both temperature experiments.) Insert the stoppers fitted with calibrated pipettes.

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 7 minutes. Place the respirometers in the water bath. (See Figure 1 below.) Attach weights to the respirometers according to your teacher's directions. Respirometers 1, 2, and 3 should rest in the room temperature water bath and Respirometers 4, 5, and 6 should rest in the 10�C water bath.

Part C. Collecting Data 1. After an equilibration period of 7 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.

2. Allow the respirometers to equilibrate for 3 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. 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 1 below.

Table 2. Measurement of oxygen consumption by germinating and dry pea seeds at room temperature (25° C) and at 10°C using volumetric methods.

Beads alone

Germinating Peas

Dry Peas and Beads

TEMP (oC)

TIME (min.)

reading @ time x

change

reading @ time x

change

corrected change

reading @ time x

change

corrected change

initial - 0

0 - 5

5 - 10

10 - 15

15 - 20

initial - 0

0 - 5

5 - 10

10 - 15

15 - 20

3. Share data with other class teams. 4. Graph the results for germinating peas and dry peas at both room temperature and 10°C. Place time

in minutes on the x-axis and ml of O2 consumed on the y-axis. From the graphs, determine the rate of oxygen consumption of germinating and dry peas at each temperature.

PROCEDURE (CALCULATORS): 1. Connect the plastic tubing to the valve on the Gas Pressure Sensor. If your sensors have blue plastic

valves on them, place the valve in the position shown in Figure 2.

2. Plug the first Gas Pressure Sensor into Channel 1 of the LabPro CBL interface. Plug the second Gas Pressure Sensor into Channel 2. Use the link cable to connect the TI Graphing Calculator to the interface. Firmly press in the cable ends.

3. Turn on the calculator and start the DATAMATE program. Press CLEAR to reset the program.

4. Set up the calculator and interface for two Gas Pressure Sensors.

a. If the calculator displays PRESS (KPA) in CH 1 and CH 2, proceed directly to Step 5. If it does not, continue with this step to set up your sensors manually.

b. Select SETUP from the main screen. c. Press to select CH 1. ENTER

d. Select PRESSURE from the SELECT SENSOR menu. e. Select the correct pressure sensor from the PRESSURE menu. f. Select the calibration listing for (KPA). g. Press once, then press ENTER to select CH2. h. Select PRESSURE from the SELECT SENSOR menu. i. Select the correct pressure sensor from the PRESSURE menu. j. Select the calibration listing for (KPA). k. Select OK to return to the main screen.

5. To test whether germinating peas undergo cellular respiration, you will need to

• Set up two water baths. • Prepare a respirometer for the germinating peas. • Prepare a second, control respirometer containing glass beads.

6. Set up two water baths, one at about 25°C and one at about 10°C. Obtain two 1-liter beakers and

place about 800 mL of water in each. Add ice to attain the 10°C water bath.

7. To be sure the volumes of air in all respirometers are equal, you will need to measure the volume of the twenty-five peas that will be in the experimental respirometer. The control respirometer must have an equal volume of glass beads (or other non-oxygen consuming material) to make the air volume equal to the respirometer with germinating peas. Similarly, glass beads will be used to account for any volume difference between the germinating and non-germinating peas.

8. Obtain three test tubes and label them “T1”, T2”, and “T3”.

9. Place a 3-cm wad of absorbent cotton in the bottom of each test tube. Using a dropper pipette, carefully add a sufficient amount of KOH to the cotton to completely saturate it. Do not put so much that liquid can easily run out of the tube. Note: Do not allow any of the KOH to touch the sides of the test tube. The sides should be completely dry, or the KOH may damage the peas. CAUTION: Potassium hydroxide solution is caustic. Avoid spilling it on your clothes or skin.

10. Prepare the test tube containing germinating peas (T1):

Cotton with KOH

Peas

Cotton non-absorbent

Figure 3

a. Add 50 mL of water to a 100-mL graduated cylinder. b. Place 25 germinating peas into the water. c. Measure the volume of the peas by water displacement. Record that

volume in Table 1. d. Gently remove the peas from the graduated cylinder and blot them dry with

a paper towel. e. Add a small wad of non-absorbent dry cotton to the bottom of the test tube

to prevent the peas from touching the KOH saturated cotton. f. Add these germinating peas to the respirometer labeled “T1”.

11. Prepare the test tube containing non-germinating peas (T2):

a. Refill the graduated cylinder with 50 mL of water. b. Place 25 non-germinating peas into the water. c. Measure the volume of the peas by water displacement. Record the

volume in Table 1. d. Add a sufficient number of glass beads to the non-germinating peas and water until they displace

exactly the same volume of water as the germinating peas. e. Gently remove the peas and glass beads from the graduated cylinder and dry them with a paper

towel. f. Add a small wad of dry non-absorbent cotton to the bottom of the test tube to prevent the peas

from touching the KOH saturated cotton. g. Add the non-germinating peas and glass beads to the respirometer labeled “T2”.

12. Prepare the test tube containing glass beads (T3):

a. Refill the graduated cylinder with 50 mL of water. b. Add a sufficient number of glass beads to the water until they displace exactly the same volume of

water as the germinating peas. c. Remove the glass beads from the graduated cylinder and dry them. d. Add a small wad of dry non-absorbent cotton to the bottom of the test tube to prevent the peas

from touching the KOH saturated cotton.

e. Add the glass beads to the respirometer labeled “T3”. Part I Germinating peas, room temperature

13. Insert a single-holed rubber-stopper into test tube T1 and T3. Note: Firmly twist the stopper for an airtight fit. Secure each test tube with a utility clamp and ring-stand as shown in Figure 1.

14. Arrange test tubes T1 and T3 in the warm water bath using the apparatus shown in Figure 1. Incubate the test tube for 10 minutes in the water bath. Be sure to keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove about as much water as you will be adding, or the beaker may overflow. Use a basting bulb to remove excess water. Record the resulting temperature of the water bath once incubation has finished in Table 2.

Note: Be sure the tubes are submerged to an equal depth, just up to the rubber stoppers. The temperature of the air in the tube must be constant for this experiment to work well.

Figure 4

15. When incubation has finished, connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 4.

16. Select START to begin data collection. Maintain the temperature of the water bath during the course of the experiment.

17. Data collection will end after 15 minutes. Monitor the pressure readings displayed on the calculator screen. If the pressure exceeds 130 kPa, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kPa.

18. When data collection has finished, an auto-scaled graph of pressure vs. time will be displayed on the calculator screen. As you move the cursor right or left, the time (X) and pressure (Y) values of each data point are displayed below the graph.

19. Press , then select QUIT from the main screen to exit the DATAMATE program. ENTER

20. To account for any pressure changes due to experimental error, it is necessary to create a new data column. This new column will consist of the experimental data subtracted from the control data.

83 Plus Calculators:

Press L3 2nd – L2 2nd STO L2, then press 2nd . The adjusted pressure is now stored in list L2.

ENTER

21. Start the DATAMATE program.

22. Perform a linear regression to calculate the rate of respiration.

a. Select ANALYZE from the main screen. b. Select CURVE FIT from the ANALYZE OPTIONS menu. c. Select LINEAR (CH 1 VS TIME) from the CURVE FIT menu. d. The linear-regression statistics for these two lists are displayed for the equation in the form:

Y=A∗X+B

e. Enter the absolute value of the slope, A, as the rate of respiration in Table 2. f. Press to view a graph of the data and the regression line. ENTER

g. Press to return to the ANALYZE menu. ENTER

h. Select RETURN TO MAIN SCREEN from the ANALYZE menu. Part II Non-germinating peas, room temperature

23. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber stopper from each test tube.

24. Repeat Steps 13 – 22, using test tubes T2 and T3.

Part III Germinating peas, cool temperatures 25. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber stopper from

each test tube.

26. Repeat Steps 13 – 22, using test tubes T1 and T3 in a cold water bath.

DATA

Table 1

Peas Volume (mL)

Germinating

Non-germinating

Table 2

Water bath Temperature (°C)

warm

cool

Table 3

Peas Rate of respiration (kPa/s)

Germinated, room temperature

Non-germinated, room temperature

Germinated, cool temperature

DISCUSSION: 1. Would you expect a difference in cellular respiration rate between germinating and dormant pea

seeds? Why/why not? 2. Would you expect a difference in cellular respiration rate between pea seeds at different

temperatures? Why/why not? 3. Why was it necessary to equalize the pea volumes in each respirometer? 4. Why were plastic beads used to equalize the pea volumes instead of using more peas? 5. Why was it important not to leave KOH on the test tube sides? 6. Why was it necessary to absorb all of the carbon dioxide produced by the reaction? 7. What function did Respirometers 3 and 6 serve? 8. Students often get pressure changes in Respirometers 3 and 6. Would you predict such a change for

inert plastic beads? What could be causing this change?