Cellular Respiration LAB
Micro Respirometer
Pea Seeds
Group Members:
Lab Station:
Standard: AP Big Idea #2
What factors affect the rate of cellular respiration in multicellular organisms?
Background:
Living organisms must metabolize compounds derived from food to produce energy for maintenance, growth
and reproduction. Cellular respiration is a process that produces
energy by metabolizing glucose in the presence of oxygen (O2) while
removing carbon dioxide (CO2), a waste product. The carbohydrate,
glucose, is the most basic stored energy source used by cells that
can be supplied directly or through the catabolism of other
carbohydrates, proteins and fats. However, in order for this energy
stored in glucose to be useful to us, it must be converted to
adenosine triphosphate (ATP), the common carrier of chemical
energy in the cell. All cells split glucose molecules to transfer the
energy to ATP through a process called cellular respiration. The
energy transfer occurs in two stages, glycolysis and respiration. In
the first stage, a small amount of ATP is produced when glucose is
broken down to pyruvate during glycolysis in the cellular cytosol.
When oxygen is present, pyruvate is used to generate ATP through
aerobic respiration. In eukaryotes aerobic respiration occurs in the
mitochondria and in prokaryotes it occurs at the cell membrane. In
the absence of oxygen, pyruvate is converted to either lactate or
ethanol and carbon dioxide in the cytosol through the less efficient process of anaerobic fermentation.
In eukaryotic cells, aerobic respiration occurs in the mitochondria, but in prokaryotic cells this occurs in the
cell membrane. ATP provides the energy used for synthetic reactions, active transport, and all cell processes.
The overall equation for the efficient breakdown of glucose in aerobic respiration can be represented in the
following way: C6H12O6 + 6O2 → 6CO2 + 6H2O + 36ATP (energy), where one mole of glucose can ultimately
produce 686 kcal of energy. Oxygen gas is consumed and carbon dioxide gas is produced at equal rates. In the
presence of potassium hydroxide (KOH), carbon dioxide will react with it to form a solid—potassium
carbonate. This reaction is represented in the following equation:CO2 + 2KOH → K2CO3 + H2O. By consuming
the carbon dioxide gas in this way, oxygen consumption during respiration can be measured, with a
respirometer or other gas pressure gauge, as a change in gas volume. An understanding of the gas laws is
important to the functioning of a respirometer.
The laws are summarized in the combined gas law of: PV = nRT.
P: pressure
R: gas constant
V: volume
T: temperature
n: number of molecules
Rearranging the equation into V=nRT/P, this implies that a change in temperature will cause a direct change in
volume. It is important to remember that gases and fluids flow from regions of high pressure to regions of low
pressure. This law summarizes the following important concepts about gases:
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Given a constant temperature and pressure, the volume of the gas is directly proportional to the number of
molecules of the gas.
Given a constant temperature and volume, the pressure of the gas changes in direct proportion to the number
of molecules of gas present.
Given a constant number of gas molecules and temperature, the pressure is inversely proportional to the
volume.
Given a change in temperature while the number of gas molecules remain constant, then either pressure or
volume, or both, will change in direct proportion to the temperature.
Gases and fluids flow from a high-pressure area to a low-pressure area.
If the given conditions of constant temperature and pressure are satisfied, the change in volume of gas in the
respirometer will be directly related to the amount of oxygen consumed and can be used to generate a rate of
respiration. The total volume of gas will be reduced because the CO2 produced is being converted to a solid.
Since it is being removed, the change in the volume of gas in the respirometer will be directly related to the
amount of oxygen consumed. The tube with beads acts as a control to detect any changes in volume due to
atmospheric pressure changes or temperature changes. When the tip of the respirometer is submerged under
water, no additional air will enter. As O2 is used up, the pressure of gases inside the respirometer decreases.
This causes water to enter the pipette. When the gas volume inside the vial decreases, the pressure of water
outside the vial forces water into the pipette. Because the amount of water that enters the pipette is directly
proportional to the amount of oxygen consumed by the peas, measuring the water volume in the pipette
allows you to measure the rate of respiration. Water in a pipette adheres to the side of the tube and forms a
curved surface called a meniscus. By common practice, all readings are made at the bottom of the meniscus.
In this experiment you will observe the rate of respiration in peas that are germinating at two different
temperatures: 10°C and room temperature (20-25°C). In addition, you will compare these rates to a nonmetabolizing control.
More Information on Germinating Peas
Seeds contain a plant embryo and its initial food supply protected by a seed coat. When warmth and moisture
conditions are favorable, germination, or sprouting, will begin. When you soak pea seeds for this laboratory,
germination begins. Enzymes begin using the stored food supply to generate ATP, and the rate of cellular
respiration accelerates. It is important to know that non-germinating (dry) seeds are not dead; they are
dormant and still perform a small amount of respiration.
Pre-lab Questions:
1. What is the purpose of a respirometer?
2. What happens to glucose if sufficient oxygen is available? (use the following terms: oxidize, pyruvate,
glycolysis, citric acid cycle, mitochondria, and ATP)
3. Describe the processes of anaerobic respiration to that of aerobic respiration. (include major details of
each step within each process)
4. What kinds of organisms perform aerobic respiration? Anaerobic respiration?
5. How does respiration differ between eukaryotes and prokaryotes?
6. How does respiration differ between plants and animals.
7. Where do gases enter and exit a plants cell?
8. What is ATP used for within a cell? (use specific examples discussed this year)
9. How would you compare the rate of respiration in germinating seeds to those that are dormant?
10. How would you compare the rate of respiration in germinating seeds to plants that have already
germinated?
11. How would you compare the rate of respiration in a 25g reptile and a 25g mammal at 10 degrees
Celsius?
12. How would you compare the rate of respiration in a 25g reptile and a 25g mammal at 25 degrees
Celsius?
Prepare your water baths: Fill the large black plastic trays with water until the water level is around 3cm to
the top. To one of your plastic trays add enough ice that there is a layer of ice cubes floating at the top of the
water (you do not need ice stacked and packed in the tray, just a layer). The trays should be set out the night
prior so that they start at room temperature, then add the ice at the beginning of the lab set-up. *Note that
the temperatures described in the procedure may be off so record your initial room temperature and the
initial temperature of the ice bath just prior to starting data collection.
Part 1 - Materials
2 large black plastic trays
4 vials with weights attached to the bottom
4 graduated pipets
4 stoppers
4 metal washers
2 thermometers
4 cotton balls
2 rayon balls (cut in half to make 4)
1 plastic pipet for KOH drops
1 100mL graduated cylinder
20 germinating peas
Beads (number depends on pea volume)
Part 1 – Procedure
***KOH is corrosive, goggles and aprons must be worn during the lab***
1. Fill a 100 mL graduated cylinder with 50 mL water. Add 10 germinating peas and take a reading of the
displaced water. This is the volume of the germinating peas. Record the volume in the table below.
Decant the water, remove the peas and place them on a paper
towel; pat the peas dry and set aside.
2. Refill the graduated cylinder with 50 mL water. Add beads
until the water level is the same as that of the germinating
peas. Be sure to get the water level as close as possible to that
of the germinating peas. If you go over, pour out the contents
of the graduated cylinder and start again. Record the volume
in the table below. Decant the water, remove the beads and
place them on a paper towel; pat the beads dry and set aside.
3. Obtain four vials with steel washers on the bottoms (to
prevent floating). Number the vials 1 @ 10 degrees C, 2 @ 10
degrees C, 1 @ 20 degrees C, 2 @ 20 degrees C. Place an
absorbent cotton ball in each of the vials and push each down
to the bottom using a pipet or pencil tip. Be sure to use the
absorbent cotton balls and NOT the non-absorbent rayon.
Without getting liquid on the sides of the respirometers, use a
pipet to add 1 mL 15% potassium hydroxide (KOH) to the
cotton.
4. Add a piece of non-absorbent rayon that is slightly smaller than that of the cotton ball and place it on
top of the KOH-soaked cotton. Do not tamp down this layer.
5. Add the germinating peas to vial 1 @ 10 degrees C and the acrylic beads to vial 2 @ 10 degrees C.
6. Repeat steps 2-5 for the 20 degree vials.
Table 1. Volume of Germinating peas and beads per vial.
Vial
Volume (mL)
Peas
Beads
o
Vial 1 @10 Celsius
Vial 2 @10o Celsius
Vial 1 @20o Celsius
Vial 2 @20o Celsius
7. Insert the non-tapered end of the graduated pipet into the wide end of a stopper so that the tapered
end of the pipet points away from the stopper and so that the pipet extends just beyond the bottom
of the stopper.
8. Firmly insert the stopper into the vial. The seal that has been created between the stopper and the vial
should be sufficient to prevent the pipet from
easily moving up and down in the stopper.
Add a small amount of petroleum jelly to the
area of the pipet that will be located in the
stopper hole to ensure a water tight seal.
9. Place a washer over the pipet tip and guide it
down the pipet until it rests on the stopper.
Repeat this entire step for the other vial as
well as the vials required for the second
water bath.
10. Place a thermometer in each water bath.
11. Place the vial-pipet apparatus so that the
pipet tips resting on the edge of the tray or a
piece of tape that crosses the tray (as seen in
the diagram). Allow the respirometers to
equilibrate for 10 minutes.
12. Add one drop of food coloring to the exposed
tip of each respirometer and wait one minute. Turn each of the respirometers so that the graduation
marks on the pipets are facing up.
13. Carefully shift the respirometers until the seed containers and the pipet tips with food coloring are
completely immersed in the water bath. Do not touch the respirometers once the experiment has
started! Let the respirometers equilibrate for another 5 minutes before proceeding. Lab Hints:
 The stopper must be firmly inserted for an airtight seal. Check that nothing blocks the pipette
opening.
 If water continues to move into the pipette when first immersing the respirometers, there is a
leak. Start over by drying the stopper and tube, then tape around the leaks with duct tape.
 Allow the respirometers to equilibrate for several minutes in their respective water baths. This
will minimize volume changes due to change in air temperature.
14. Read all of the respirometers to the nearest 0.01 mL and take the temperature of the water bath to
ensure temperature stability. Record the initial readings of volume (mL) and the temperature of the
water bath (°C) in Tables 2 and 3 below.
15. Take additional readings every 5 minutes for 25 minutes, and record the readings and temperature in
Tables 2 and 3.
16. When all of the readings have been taken, calculate the difference and the corrected difference for
each result and record each value in Tables 2 and 3.
17. Calculation equations:
*Difference = (initial reading at time 0) – (reading at time X)
*Corrected difference = (initial pea reading at time – pea seed reading at time X) – (initial bead reading at time 0 – bead reading a time X)
18. Graph your results from the corrected difference column in Table 2 for the germinating peas and
beads of each water bath. Plot the time in minutes.
Table 2. Measurement of oxygen consumption by peas at 10 oC.
Beads Reading
Reading at Time X
Calculated
*Difference
Germinated Peas Readings
Reading at Time X
Calculated
*Difference
Calculated
*Corrected
Difference
Time 0 min
Time 5 min
Time 10 min
Time 15 min
Time 20 min
Time 25 min
Table 3. Measurement of oxygen consumption by peas at 20 oC.
Beads Reading
Reading at Time X
Calculated
*Difference
Germinated Peas Readings
Reading at Time X
Calculated
*Difference
Calculated
*Corrected
Difference
Time 0 min
Time 5 min
Time 10 min
Time 15 min
Time 20 min
Time 25 min
Analysis Questions:
1. Graph your corrected data (the difference) for each vial in both the 10oC and 20oC water baths.
2. Graph your corrected difference data for the peas in both the 10 oC and 20oC water baths.
3. What accounts for the difference in oxygen consumption seen between the seeds in the 10oC and 20oC
water baths?
4. Provide examples from the experiment for each of the variables listed below:
a. Independent variable
b. Dependent variable
c. Constants
d. Control
5. Why do the beads seem to be using oxygen?
6. Why are the readings corrected using the beads?
7. What is the function of KOH in the experiment?
8. From the slope of the lines, determine the rate of oxygen consumption at 10 oC and 20oC for the
germinating pea seeds. Determine
Experimental
Calculations
Rate in mL
the slope of the lines over a middle
Conditions
O2/minute
section of each line by dividing the
difference in the volume reading by Germinating Seed
o
the difference in time. Volume (mL 10 C
Germinating Seed
O2 consumed) values are
20oC
determined from the line.
9. Compare the rate of oxygen consumption at 10oC and 20oC for the germinating pea seeds.
10. Explain why or why not you would predict the rates of respiration to change in peas that have been
germinating for different periods of time (example: 48 hours to 96 hours).
11. Write a hypothesis using the same experimental design to compare the rates of respiration in a mouse
at both room temperature and 10oC.