Name: Per.
Learning objectives:
1. Describe and explain the cellular respiration lab
2. Relate the overall equation of cellular respiration to the cellular respiration experiment.
3. Quantify oxygen consumption rates in germinating seeds under different conditions
Introduction
This investigation uses respirometry techniques to calculate the rate of oxygen consumption (cellular respiration) in germinating seeds. The consumption rate is quantified and graphed.
Background
Each individual cell is responsible for the energy exchanges necessary to sustain its ordered structure. Cells accomplish this task by breaking down nutrient molecules to generate ATP (adenosine triphosphate), which can then be used to run cellular processes that require energy. This process is called cellular respiration which requires nutrient molecules and oxygen. Carbon dioxide and water are products of the series of reactions involved in cellular respiration.
C
6
H
12
O
6
+ 6O
2
 6CO
2
+ 6H
2
O
There are several methods of indirectly measuring the rate of cellular respiration in organisms. One method involves monitoring changes in temperature; since the process of respiration is exergonic (produces heat). Another method is to measure either the oxygen consumption or the carbon dioxide production.
Respirometers are devices that measure these types of gas volume changes, and therefore provide information about the rate of cellular respiration.
During cellular respiration, two gases are changing in volume. Oxygen gas is being consumed by the respiring cells and carbon dioxide gas is diffusing out of the cells. The respirometer, therefore, has to be able to deal with two simultaneously changing gas volumes. This is accomplished by introducing potassium hydroxide into the device. KOH absorbs carbon dioxide, following this equation
CO
2
+ 2KOH  K
2
CO
3
+ H
2
O

Potassium carbonate ( K
2
CO
3
) is a solid precipitate. Any CO
2
produced is immediately converted from a gas to a solid and is therefore no longer governed by gas laws. This allows the respirometer to measure only one variable, the consumption of oxygen gas by living cells.
Materials:

4 weighted vials

4 cotton balls

4 rayon balls

2 mL premeasured disposable pipets

1 bottle 15% KOH (class stock bottle).

1 bottle red food coloring

metal washers

These seeds soaked in water overnight:

~30 peas

~30 beans

~30 wheat berries

~30 glass beads

1 large shallow tray for water

3 volumeters (rubber stopper with serological pipet inserted)

goggles

wooden sticks

masking tape

forceps

thermometer

timer
Procedure
Day 1
1. Soak 25 peas, 25 beans and 25 wheatberries in water overnight.
2. Discuss how you will determine an equal volume of each kind of seed plus the beads.
3. Read through the experiment and discuss how you will collect your data.
4. Construct a blank data table.
Day 2
5. Goggles and gloves should be worn at all time during this experiment. KOH is a strongly corrosive base. When done, following your lab ins tructor’s directions for disposal of the KOH saturated cotton and seeds.
6. Label the weighted vials # 1-4
7. Place one cotton ball in the bottom of each vial. Use the wooden stick or forceps to push the cotton to the bottom of the vial.

8. Partially open the KOH bottle. Do not remove the cap! Fill the disposable pipet with 1 mL of KOH and pour 1mL onto the cotton ball in each vial.
COVER the KOH!
9. Place a dry rayon ball on top of each saturated cotton ball to protect the organism.
10. Find the volume of 25 soaked peas. Drain the peas and then add them to the first vial.
11. Find the volume of 25 beans and equalize the volume to that of the 25 peas by adding glass beads. Drain the beans and add them (and any beads) to vial
#2. Do the same for the wheat berries for vial #3. Vial 4 should contain an equal volume of only beads.
12. Insert the volumeters into each vial, making a tight fit.
13. Fill the shallow plastic tray about 3/4 full 20 o C with water. Lay a piece of white paper along the bottom so you can read your respirometers. Lay a piece of masking tape across top of the tray about ¼ of the way down the length of the tray. This will support your tubes.
14. Place the respirometers into the room temperature water bath so that the pipets rest on the masking tape prop. Begin time for a total of 7 minutes – this is the equilibration period, where your respirometers will become the same temperature as the water.
15. While you are waiting, use a disposable pipet to put a drop of red food coloring into the tip of the serological pipet.

16. At the end of 7 minutes, slowly and carefully submerge each of the tubes entirely in the water bath. If you don’t do it slowly and carefully, the red dye will shoot out the tip.
17. At this point, check to make sure you can read the pipets. The air bubble should extend from the main chamber up the tube of the pipet. The pipet may need to be rotated so that you can see the numbers.
18. If your respirometers float, you may need to weight them down with washers.

19. Data table: As oxygen is consumed by cellular respiration, the red dye will move toward the chamber. You will be recording the position of the red dye in the pipet in mLs and also the change in volume in mLs every 5 minutes for 20 to 25 minutes for each of the four respirometers. Start collecting data at 0 minutes.
20. Remove the respirometers from the water. Dispose of the seeds according to directions given. Be sure to rinse the serological pipet carefully to remove all the dye. Return all equipment to your lab station clean and dry.
21. Graph the rate of oxygen consumption for each of the vials – on the same graph.
Analysis:
1. Identify a hypothesis being tested in this experiment. Indicate the variables and controls. Identify the experimental control and its purpose.
2. Why were the seeds soaked overnight?
3. What is the purpose of the KOH?
4. Explain why the water moved into the pipets causing the red dye to move.
5. Describe and explain the relationship between the amount of oxygen consumed and time.
6. Designing future experiments: Explain how changing some of the variables could change the rate of oxygen consumption. Give at least 3 examples with detailed explanations.
7. Calculate the RATE of oxygen consumption in each vial. Recall that rate =
ΔY/ΔX.
8. Why do the rates of oxygen consumption different from vial to vial?