bwcbl11

Biology with CBL 11-1

Experiment

11Name ____________________________________ Date __________________

Cell RespirationCellular respiration refers to the process of converting the chemical energy of organicmolecules into a form immediately usable by organisms. Glucose may be oxidized completelyif sufficient oxygen is available, by the following equation:

C6H12O6 + 6O2(g) 6 H2O + 6 CO2(g) + energyAll organisms, including plants and animals, oxidize glucose for energy. Often, this energy isused to convert ADP and phosphate into ATP.

To measure the rate of cellular respiration, the pressure change due to the consumption ofoxygen by peas will be measured with a pressure sensor. It is not possible to directly measurepressure changes due to oxygen, since the pressure sensor measures the total pressure change.Carbon dioxide is produced as oxygen is consumed. The pressure due to CO2 might cancel outany change due to the consumption of oxygen. To eliminate this problem, a chemical will beadded that will selectively remove CO2. Potassium hydroxide, KOH, will chemically react withCO2 by the following equation:

2 KOH + CO2 K2CO3 + H2O

This will allow you to monitor pressure changes that are exclusively due to the consumption ofoxygen.

A respirometer is the system used to measure cellular respiration. Pressure changes in therespirometer are directly proportional to a change in the amount of gas in the respirometer,providing the volume and the temperature of the respirometer do not change. If you wish tocompare the consumption of oxygen in two different respirometers, as we will in thisexperiment, you must keep the volume and temperature of the air equal in each respirometer.

Both germinating and non-germinating peas will be tested. Additionally, cellular respiration ofgerminating peas at two different temperatures will be tested.

OBJECTIVESIn this experiment, you will use a CBL and gas pressure sensors to measure pressure changes. study the effect of temperature on cellular respiration. determine whether germinating peas respire. determine whether non-germinating peas respire. determine whether germinating peas respire differently than non-germinating peas. compare the rates of cellular respiration in germinating and non-germinating peas. compare the rates of cellular respiration in warm and cool peas.

to pressuresensor

11-2 Biology with CBL

Name ________________________________________________ Date __________________________

MATERIALSCBL System glass beadsTI Graphing Calculator ice2 Vernier Biology Gas Pressure Sensors non-absorbent cotton2 Vernier DIN adapter cables thermometer15% KOH in a dropper bottle test tube rack25 germinating peas timer with a second hand25 non-germinating peas three 28 X 150 mm test tubes100-mL graduated cylinder two rubber stopper assembliesabsorbent cotton two 1-L beakersforceps ringstand2 utility clamps Graphical Analysis (optional)

Figure 1

PROCEDURE1. Obtain and wear goggles.

2. Connect the CBL and calculator with the link cable using the port on each unit. Firmlypress the cable ends into each port.

3. Prepare the Biology Gas Pressure Sensors for data collection. Plug the gas pressure sensors into the adapter cable in Channel 1

and Channel 2 of the CBL. A rubber-stopper assembly has alreadybeen connected to each of the gas pressure sensors as shown in Figure1.

Check and make sure that the two-way valve connected to the rubber-stopper assembly is closed as shown in Figure 2. Keep the valveclosed at all times.

Open the gas pressure sensor valves so the test tubes are open tothe atmosphere. Align the blue handle with the arm that leads tothe gas pressure sensor, as shown in Figure 3. The handle of thevalve will always point to the arm that is closed off. In Figure 3, the pressure sensor isclosed off from the test tube and the atmosphere. Any gas generated in the test tube willbe vented into the air.

Figure 3

valve closed

Figure 2


Name ________________________________________________ Date __________________________

4. 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.

5. Set up two water baths, one at about 25C and one at about 10C. Obtain two 1-literbeakers and place about 800 mL of water in each. Add ice to attain the 10C water bath.

6. To be sure the volumes of air in all respirometers are equal, you will need to measure thevolume of the twenty-five peas that will be in the experimental respirometer. The controlrespirometer must have an equal volume of glass beads (or other non-oxygen consumingmaterial) 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 thegerminating and non-germinating peas.

7. Obtain three test tubes and label them T1, T2, and T3.

8. Place a small wad of absorbent cotton in the bottom of each test tube. Using a dropperpipette, carefully add a sufficient amount of KOH to the cotton to completely saturate it. Donot put so much that liquid can easily run out of the tube. Note: Do not allow any of theKOH to touch the sides of the test tube. The sides should be completely dry, or the KOHmay damage the peas. CAUTION: Potassium hydroxide solution is caustic. Avoid spillingit on your clothes or skin.

9. Prepare the test tube containing germinating peas (T1): Add 50 mL of water to a 100-mL graduated cylinder. Place 25 germinating peas into the water. Measure the volume of the peas by water displacement. Record that volume in Table 1. Gently remove the peas from the graduated cylinder and blot them dry with a paper

towel. 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. Add these germinating peas to the respirometer labeled T1.

10. Prepare the test tube containing non-germinating peas (T2): Refill the graduated cylinder with 50 mL of water. Place 25 non-germinating peas into the water. Measure the volume of the peas by water displacement. Record the volume in Table 1. 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. Gently remove the peas and glass beads from the graduated cylinder and dry them with a

paper towel. 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. Add the non-germinating peas and glass beads to the respirometer labeled T2.

11. Prepare the test tube containing glass beads (T3): Refill the graduated cylinder with 50 mL of water. Add a sufficient number of glass beads to the water until they displace exactly the same

volume of water as the germinating peas. Remove the glass beads from the graduated cylinder and dry them. 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. Add the glass beads to the respirometer labeled T3.


Name ________________________________________________ Date __________________________

12. Connect the pressure sensor in Channel 1 to test tube T1. Securely fit the rubber stopperassembly into test tube T1 to be sure there is no air leakage. Connect the pressure sensor inChannel 2 to test tube T3, the control.

Part I Germinating peas, room temperature13. Arrange test tubes T1 and T3 in the warm water bath using the apparatus shown in Figure

1. Allow them to equilibrate with the temperature of the water bath for 8-10 minutes. Besure the tubes are submerged to an equal depth, just up to the rubber stoppers. Record thefinal resulting temperature of the water bath in Table 2.

14. Turn on the CBL unit and the calculator. Start the CHEMBIO program and proceed to theMAIN MENU.

15. Set up the calculator and CBL for two biology gas pressure sensors. Select SET UP PROBES from the MAIN MENU. Enter 2 as the number of probes. Select MORE PROBES from the SELECT PROBE menu. Select BIO PRESSURE from the SELECT PROBE menu. Enter 1 as the channel number. Select USE STORED from the CALIBRATION menu. Select ATM from the PRESSURE UNITS menu. Select MORE PROBES from the SELECT PROBE menu. Select BIO PRESSURE from the SELECT PROBE menu. Enter 2 as the channel number. Select USE STORED from the CALIBRATION menu. Select ATM from the PRESSURE UNITS menu.

16. Set up the calculator and CBL for data collection. Select COLLECT DATA from the MAIN MENU. Select TIME GRAPH from the DATA COLLECTION menu. Enter 30 as the time between samples, in seconds. Enter 40 as the number of samples Press ENTER , then select USE TIME SETUP to continue. Enter 0.9 as the minimum pressure (Ymin). Enter 1.0 as the maximum pressure (Ymax). Enter 0.01 as the pressure increment (Yscl). When the test tubes have been in the water

bath for 8-10 minutes proceed to Step 17.

17. Close the air valves on the pressure sensors. Alignthe blue handle with the side stem, as shown inFigure 4.

18. Start measuring the pressures by pressing ENTER on the calculator. Monitor the temperatureof the water bath. The temperature should not change by more than a few C. To maintainwater bath temperature, add hot or cold water as needed. Be sure to remove an equalamount of water with a turkey baster or Beral pipet before you add water to the beaker, or itmay overflow.

19. After 20 minutes, the CBL will stop taking measurements.

20. Once data collection has stopped, SAMPLING will change to DONE on the CBL screen.A message will be displayed on the calculator indicating the data lists in which the timeand pressure data are stored.

Figure 4

to pressuresensor

Name ________________________________________________ Date __________________________


Name ________________________________________________ Date __________________________

21. Press ENTER to display a graph of pressure vs. time on the calculator screen. As you move thecursor right or left, the time (X) and pressure (Y) values of each data point are displayed belowthe graph.

22. Press ENTER on the calculator and a message will appear asking if you want to repeat theexperiment. Select NO to return to the main menu. Select QUIT from the MAIN MENU to exitthe CHEMBIO program.

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

TI-82 or TI-83 Calculators:Press 2nd L3

2nd L2 STO 2nd L4, then press ENTER . The adjusted pressure is nowstored in list L4.

TI-85 or TI-86 Calculators:Press

APPS

[LIST], then select . Press and then press ENTER .

TI-92 Calculators:Open up the Data/Matrix and subtract the experimental data from the control data. Press , select Data/Matrix Editor, then select Current. The Data/Matrix will open. The cursor can be moved from cell to cell using the

cursor pad. Move the curson to the column heading c4. Type c3-c2 and press ENTER . The adjusted pressure is now stored in c4.

24. Start the CHEMBIO program and proceed to the MAIN MENU.

25. The rate of respiration can be measured by examining the slope of the adjusted pressure vs.time plot. Perform a linear regression to calculate the slope: Select FIT CURVE from the MAIN MENU. Select LINEAR L1,L4 (c1,c4 on the TI-92) from the REGRESSION MENU. The linear-regression statistics for these two lists are displayed for the equation in the

form:Y=AX+B

Enter the slope, A, as the rate of oxygen consumption by germinating peas in Table 3.

26. Plot a graph of the data and the regression curve. Select SCALE TO DATA to view a graph of the data and the regression line. Press ENTER to return to the MAIN MENU.

Part II Non-germinating peas, room temperature27. Open the air valves on the pressure sensors as shown in Figure 3. Remove test tube T1 and

replace it with test tube T2. Be sure the stoppers are firmly in place for an airtight fit.

28. Repeat Steps 16 26, substituting test tube T2 for test tube T1.

Part III Germinating peas, cool temperatures29. Open the air valves on the pressure sensors as shown in Figure 3. Remove test tube T2 and

replace it with test tube T1. Be sure the stoppers are firmly in place for an airtight fit.


Name ________________________________________________ Date __________________________

30. Repeat Steps 16 26, using a cold water bath in place of warm water.

DATA

Table 3

Peas Rate of O2 consumption (atm/s)

Germinating, warm

Non-germinating, warm

Germinating, cool

QUESTIONS1. Do you have evidence that cellular respiration occurred in peas? Explain.2. What is the effect of germination on the rate of cellular respiration in peas?

3. What is the effect of temperature on the rate of cellular respiration in peas?4. What was the role of the control respirometer in each series of experiments?

5. Why do germinating peas undergo cellular respiration?

EXTENSIONS1. Compare the respiration rate among various types of seeds.

2. Compare the respiration rate among seeds that have germinated for different time periods, suchas 1, 3, and 5 days.

3. Compare the respiration rate among various types of small animals, such as insects orearthworms.

Table 1

Peas Volume (mL)

Germinating

Non-germinating

Table 2

Water bath Temperature (C)

warm

cool

Biology with CBL 11-1 T

Experiment

11TEACHER INFORMATION

Cell Respiration1. This experiment may take several 50-minute lab periods to complete. A good stopping

place might be at the end of Step 26. If Part III is completed during a second or third labperiod, you may want to cool the respirometers in the refrigerator prior to the class. Thiswill save a substantial amount of equilibration time for students.

2. Allow the seeds to germinate for three days prior to the experiment. Prior to the first day,soak them in water overnight. On subsequent days, roll them in a moist paper towel andplace the towel in a paper bag. Place the bag in a warm, dark place. Check each day to besure the towels remain very moist.

3. The night before the experiment, draw a sufficient quantity of water for the room-tempera-ture water baths. This will allow the water to equilibrate to room temperature.

4. To prepare the 15% KOH solution, add 75 grams of solid KOH to distilled water to make atotal volume of 500 mL. If the solution will be stored for an extended time, it will be best tostore it in a plastic container. Strong bases will damage glass containers. HAZARDALERT: Corrosive solid; skin burns are possible; much heat evolves when added to water;very dangerous to eyes; wear face and eye protection when using this substance. Weargloves. Hazard Code: BHazardous.The hazard information reference is: Flinn Scientific, Inc., Chemical & Biological Catalog/Reference Manual, 1997, P.O. Box 219, Batavia, IL 60510-0219. See Appendix E of thisbook, Biology with Computers, for more information.

5. The Biology Gas Pressure Sensor has a plastic 3-way valve. Align the blue handle with anyone of the three valve stems to close off the stem (the word off is imprinted on the bluehandle). To open the side valve stem to the atmosphere and allow air to enter or exit the testtube, the handle is aligned with the stem that enters the Biology Gas Pressure Sensor box.To confine air in the test tube for data collection, the handle is aligned with the side valvestem.

6. The Vernier Barometer sensor can also be used to perform this experiment. If you alreadyhave a Barometer and wish to do this activity, you will need to order the following partsfrom Vernier Software:Pressure Sensor Accessories Kit: order code: PS-ACC .................................................. $5.00Pressure Sensor Valve: order code: PSV ........................................................................ $2.00

7. All of the pressure valves, tubing, and connectors used in this experiment are included withVernier Biology Gas Pressure Sensors shipped after February 15, 1998. These accessoriesare also helpful when performing respiration/fermentation experiments such as Experi-ments 6, 12, 16, 23, and 24 in this manual.

11-2 T Biology with CBL

Experiment 11

8. If you purchased your biology gas pressure sensor at an earlier date, Vernier Software has aPressure Sensor Accessories Kit (PS-ACC, $5) that includes all of the parts shown here fordoing respiration/fermentation experiments (except the test tube). Using this kit allows foreasy assembly of a completely airtight system. The kit includes the following parts: two ribbed, tapered valve connectors inserted into a rubber stopper. two Luer-lock connectors connected to either end of a piece of plastic tubing. one two-way valve one 20-mL syringe

9. Connect the piece of plastic tubing to the shorter valve connector on the rubber stopperprior to the start of class (as shown in the figure on this page). Threaded Luer-lock connec-tors are attached to both ends of the plastic tubing. Connect one of these to the valve stemwith a 1/2 clockwise turn.

10. If you do not have the connectors and valves currently shipped with the Biology GasPressure Sensor or in the Pressure Sensor Accessories Kit, an alternative is to connect theplastic tubing to a piece of glass tubing inserted in a #5 one-hole rubber stopper. The shortpiece of glass tubing that is inserted into the rubber stopper can be drawn out to a taperedend in a burner flame and then fire polished. The plastic tubing supplied with the BiologyGas Pressure Sensor can then be easily connected to the tapered end of the glass tube, andthe stopper inserted into a 28 X 150-mm test tube for an airtight system.

11. The length of plastic tubing connecting the rubber stopper assemblies to each Biology GasPressure Sensor must be the same. Use the shortest length of tubing reasonable to minimizethe volume of the respirometer. Note: If pressure changes during data collection are toosmall, you may need to decrease the total gas volume in the system. Shortening the lengthof tubing used will help to decrease the volume.

Biology with CBL 11-3 T

Experiment 6

SAMPLE RESULTS

Germinating peas and glass Regression statistics for the germinating peas, along with abeeds at 22C plot of the regression line and pressure data

Table 1Temperature Rate of O2 consumption

Seeds (C) (atm/s)Germinating, warm 22 1.60 X 10-5

Non-germinating, warm 22 1.62 X 10-6

Germinating, cool 6 7.78 X 10-6

ANSWERS TO QUESTIONS1. Yes. The pressure change vs. time graph indicates that some gas is being removed at a con-

stant rate from the respirometer when germinating seeds are present.

2. Germination greatly accelerates the rate of cellular respiration. This reflects a higher rate ofmetabolic activity in germinating seeds. In most experiments, non-germinating seeds do notseem to be respiring. Occasionally, however, some respiration is detectable.

3. Warm temperatures increase the rate of respiration. This reflects a higher rate of metabolicactivity in warm germinating seeds than in cool seeds.

4. Gas in the control respirometer responded to temperature changes exactly as the experimentalrespirometer. By subtracting the control respirometers pressure readings from the experi-mental respirometers pressure readings, only the pressure change due to the removal ofoxygen gas by seeds is detected. Any pressure change due to temperature fluctuations iseliminated.

If one looks only at the graph of the experimental respirometer, it appears that both germinat-ing and non-germinating seeds respire. This would be an erroneous conclusion, as the controlrespirometers graph indicates a change as well. The control would not be necessary if thegraph indicated no change in pressure throughout the experiment.

5. It is necessary for germinating seeds to undergo cellular respiration in order to acquire theenergy they need for growth and development. Unlike their mature relatives, seeds do not yethave the necessary photosynthetic abilities needed to produce their own energy sources.

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