AP BIO Lab #5: Cellular Respiration

BACKGROUND
Aerobic cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria with in each cell in the presence of oxygen. Cellular respiration involves a series of enzyme-mediated reactions. All organisms, including plants and animals, oxidize glucose for energy. Often, this energy is used to convert ADP and phosphate into ATP. The equation below shows the complete oxidation of glucose.

C6H12O6 + 6 O2(g) --> 6 H2O + 6 CO2(g) + 686 kilocalories of energy/mole of glucose oxidized

 By studying the equation above, there are three ways cellular respiration could be measured. 1) Consumption of O2 (the number of moles of O2 consumed), 2) Production of CO2 (How many moles of CO2 are produced), 3) Release of energy during cellular respiration.  To measure the rate of cellular respiration in this experiment, the pressure change due to the consumption of oxygen by peas will be measured with a Gas Pressure Sensor. It is not possible to directly measure pressure changes due to oxygen, since the Gas Pressure Sensor measures the total pressure change. Carbon dioxide is produced as oxygen is consumed. The pressure due to CO2 might cancel out any change due to the consumption of oxygen. To eliminate this problem, a chemical will be added that will selectively remove CO2. Potassium hydroxide, KOH, will chemically react with CO2 by the equation below and will allow you to monitor pressure changes exclusively due to the consumption of oxygen:

2 KOH + CO2 --> K2CO3 + H2O

 A respirometer is the system used to measure cellular respiration. Volume changes in the respirometer are directly proportional to a change in the amount of gas in the respirometer, providing the pressure and the temperature of the respirometer do not change. This can be shown in the ideal gas law PV=nRT where P is the pressure of the gas, V is 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). If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of gas molecules present. If you wish to compare the consumption of oxygen in two different respirometers, as we will in this experiment, you must keep the volume, pressure and temperature of the air equal in each respirometer. If respiration occurs in the tubes, oxygen will be consumed. Its volume will be reduced because the CO2 produced is being converted to a solid. A vial with beads alone will permit detection of any changes in pressure due to atmosphere or  temperature changes.

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 (soaking in water) germination occurs and the rate of cellular respiration greatly increases. Both germinating and non-germinating peas will be tested. Additionally, cellular respiration of germinating peas at two different temperatures will be tested.

OBJECTIVES
 In this experiment, you will
- Measure gas consumption
- Study the effect of temperature on cell respiration.
- Determine whether germinating peas and non-germinating peas respire.
- Compare the rates of cell respiration in germinating and non-germinating peas

PURPOSE QUESTION

HYPOTHESIS

NULL HYPOTHESIS

DRAWING OF SET UP

 

 

 

 

PROCEDURE (LABLE VARIABLES)

1. To assemble a respirometer, obtain a vial, with an attached stopper and pipette. Then place a small wad of absorbent cotton in the bottom of the vial and, using a pipette, saturate the cotton with 15 % KOH. Be sure not to get the KOH on the sides of the respirometer. Then place a small wad of non-absorbent cotton on top of the KOH-soaked absorbent cotton. Repeat these steps to make the other respirometers you will use. It is important to use about the same amount of cotton and KOH in each vial.

2. Next, place the same volume of germinating peas, dry peas and beads and/or beads alone in vials.  Insert the stoppers in each vial with the proper pipette. Place a washer on each of the pipettes to be used as a weight.

3. Make a sling of masking tape attached to the side of a water bath. This will hold the ends of the pipettes out of the water during an equilibration period of 7 minutes. After 7 min, put all respirometers entirely into the water. Put a drop of food coloring at the end of the pipette as you put it under the water to help you see the water line easier.  A little water should enter the pipettes and then stop. If the water continues to enter the pipette, check for leaks in the respirometer.


4. Allow the respirometers to equilibrate for 3 more minutes and then record the initial position of the water in each pipette to the nearest 0.01mL (time 0).

DATA TABLE

 

ANALYSIS

1. This activity uses a number of controlled variables. What conditions must remain constant? Why?

2. Calculate the rate of oxygen consumption (cellular respiration) for the organism. Reminder: Rate= Δy/Δx (Show work!) and Record results in data table below.

Table 4: Rate of Cellular Respiration

Condition Rate of Cellular Respiration (mL/min)
   
   
   

3. Graph the data, independent variable on x-axis and dependent variable on y-axis. Be sure to include a title, labeled axis with units.

4. Do you have evidence that cellular respiration occurred in your organism? Explain.

5. Complete a Chi square analysis for your data using rates of respiration.

6. What was the role of the control respirometer (tube with beads alone)?

7. What is the purpose of the KOH in this experiment?

8. If you used the same experimental design to compare the rates of respiration of a 25 g reptile and a 25 g mammal at 10 °C, what results would you expect ? Explain your reasoning.

9. If respiration in a small mammal were studied at both room temperature and 10 °C, what results would you predict? Explain your reasoning.

 

CONCLUSION
 As usual from blue sheet