Cellular Respiration Assay: Using respirometers
to detect O2 gas consumption by germinating peas
Adapted from the AP Biology Laboratory Manual – revised Nov 2014
Cellular respiration consists of dozens of enzyme-mediated reactions, but can be summarized:
C6H12O6 + 6 O2  6 CO2 + 6 H20 + 686 kcal heat energy
In this assay for cellular respiration, we quantify oxygen consumption by germinating seeds. The carbon dioxide
respired by the seeds’ cells reacts with potassium hydroxide (KOH) in the respirometer to form solid potassium
carbonate, K2CO3 (s).
Since the carbon dioxide gas is turned to a solid, any change in the gas volume inside the respirometers is directly
related to the amount of oxygen gas consumed by the germinating seeds. A vial containing inert glass beads (or rocks)
will allow us detect and control for changes in gas volume due to changes in atmospheric pressure or temperature in
the classroom.
MATERIALS
- Materials for 2 respirometers:
2 vials and stopper/pipets
2 wads of absorbent cotton
2 wads of nonabsorbent cotton
25 germinating seeds*
“Enough” beads/rocks (see step 2)
15% KOH solution
- In the classroom:
Newspaper for underneath pans
Large pans of water (filled day before)
Graduated cylinders
PROCEDURE
1.
Measure the volume of 25 germinating peas by the process of water displacement. Hint: Start with a 100 mL
graduate filled with a known amount of water, say 50 mL. Drop in the peas. Record the change in volume.
2.
After measuring their volume, pat the peas dry and set them aside until step # 8.
3.
Determine with water displacement how many glass beads (or rocks) are required to attain the equivalent volume.
4.
When finished, pat the beads/rocks dry and set them aside until step # 8.
5.
Obtain two clean vials and two stopper-and-pipet assemblies. Please handle the latter with care, only picking
them up by the stopper and never by the pipet. Holding by the pipet might cause the seal to break & the
respirometer to leak!
WEAR GOGGLES FOR STEPS 6-10 & CLEANUP OF RESPIROMETERS
6.
Place a small, approximately equal-sized wad of absorbent cotton in the bottom of each vial and saturate it with
15% KOH. Do not get corrosive KOH on the sides of the vials where it could come in contact with the peas.
7. Place a small approximately equal-sized wad of NONabsorbent cotton on top of the KOH-soaked cotton in each
vial.
8.
Place the germinating peas in one vial on top of the nonabsorbent cotton; place the beads (rocks) in the other vial.
9.
Insert the stopper/pipet assemblies with a gentle turning motion applied to the stoppers. See Fig. 1.
10. Make a sling of masking tape across a pan of water (on newspaper) to hold the pipets’ tips out of the water;
submerge the rest of the vial in the water. See Fig. 2. Wait 7 min. for equilibration. Place weights on the vials so
they don’t float.
11. After equilibration, immerse the two respirometers completely in the pan of water. Work swiftly to adjust their
*Note: to prepare germinating peas, we soak them in water for 6-8 h, then place them between sheets of moist paper towel for 1-2
days.
positions so that you can read the water lines in the pipets. You won’t be able to move the respirometers after this
step!
12. Wait 3 minutes, then record to the nearest 0.01 mL the position of the water line in each pipet at time 0.
13. Every 3 minutes take readings of the water line in the pipet of each respirometer. Record on the table (fig. 3)
provided
14. Clean up:
 Carefully dismantle respirometers.
 Discard the cotton & the KOH-contaminated peas. KEEP the beads!
 Rinse vials, stoppers & pipets.
At the conclusion of data collection, plot the corrected difference in the pea vial gas volume (mL) vs. time (min).
From
the trend line, calculate the slope. This is the peas’ respiration rate. Write it on your graph.
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