Procedure (water bath setup):
1. If you are doing two different temperatures or need to
separate your samples - then you would need to setup two
baths.
 You may have to use ice or hot water to maintain
the correct temperature.
2. Tape masking tape across the top width of the water bath
approximately ¼ of the way from one side.
Procedure (preparing your organisms):
Respirometer 1
3. Obtain a 50-mL graduated cylinder and fill it with 25 mL of H2O.
4. Drop in your organism and determine the amount of water that was
displaced.
 Organism will be either one worm or 25 germinating peas.
 If you choose ants or crickets - then they cannot be displaced
by water and you will have to estimate an equal volume.
 The amount of water displaced is equal to the volume of the
organism.
5. Record the volume of the organism(s) here
_______________________.
6. Remove the organism(s) and gently dry on a paper towel.
7. Set the organism(s) aside. The organism(s) will be used in respirometer 1.
Respirometer 2
8. Obtain a 50-mL graduated cylinder and fill it with 25 mL of H2O.
9. Drop glass beads into the graduated cylinder until you obtain the same volume
displacement as the organism(s) in Respirometer 1. This will be your control for "no
organism"
 If you are using Crickets or Ants - then you will need to just estimate how many
glass beads equals the mass of your organism.
Respirometers 3-4
These respirometers need to be set up according to your experiment. Repeat steps 3-9
accordingly. For example, if you are testing the effects of cellular respiration at different
temperatures, then respirometer 3 and 4 will be at the different temperature (#3 will be a
control without organism and #4 will be with organism). If you are looking at the size of
an organism, then you would have 2 differently sized organisms (each with a control, all
at same temperature). If you are looking at effect of light on germinating seeds, then one
of your setups (1-2) would have a lamp, while 3-4 would not. Make a subprocedure of
what you are doing to set up the conditions for respirometers 3-4:
10. a) _____________________________________________________________________
_____________________________________________________________________
b) _____________________________________________________________________
_____________________________________________________________________
c) _____________________________________________________________________
_____________________________________________________________________
1
Fill in the following table for your respirometer assignments:
Table I. Contents of Four Respirometers.
Respirometer Temperature
Contents
Volume
of
Contents
Other External
Condition
1
2
3
4
Procedure (respirometer setup):
11. Using tweezers, place one absorbent cotton ball at the bottom of the respirometer vial.
 When pipetting your KOH - be very careful to have it absorb
into the cotton ball. Do not allow it to touch the sides of the
glass vial.
12. Pipet 3 milliliters of 15% KOH onto the cotton balls.
13. Place one nonabsorbent cotton piece on top of the cotton ball.
 It is very important that the amount of cotton you add be the
same for each respirometer in order to be able to compare raw
volume change.
 The nonabsorbent cotton piece protects your organism from
touching the toxic KOH.
14. For each respirometer, place the sample being tested (i.e.: germinating
peas) or the control on top of the nonabsorbent cotton.
15. Add the rubber stopper/pipette to the vial. Make a tight seal.
Procedure (experimentation):
16. Slip two metal washers over the pipette to form a metal collar.
 The metal weight will help keep the respirometer submerged in the water to
maintain a constant pressure and to help equilibrate the respirometer faster.
17. Place the respirometers onto the overhanging tape in the water bath so that the vials are
submerged but the pipette is resting on the masking tape and the pipette tip is exposed to
air.
18. Let the respirometers equilibrate for five minutes.
 Equilibration is needed to make sure that respiratory rates don’t change due to a
change in temperature DURING the experiment.
19. After the five minutes, remove the masking tape sling across the top and fully submerge
each respirometer.
 Work quickly in steps 20-22 as the experiment data cannot be collected until the
entire setup is stable.
20. Check for any leaks in the pipettes and orient them so that the pipette can be easily read.
21. Be sure the glass vial is not floating. You may have to adjust the metal collars.
2
22. Allow three more minutes of equilibration and begin to record volumetric data. Make
sure the temperature of the water bath stays constant and record the volume for each
respirometer every five minutes for twenty minutes.
 Do NOT touch the water bath or respirometers or table until the experiment is
completed after this step.
23. Take your initial reading at then begin timing. Take readings each five minutes for at
least 20 minutes. Record your data in Table 2.
Results:
Table 2.
Condition
Time
(min)
Control (no organism)
Reading Difference
at Time X
0 (initial)
5
10
15
20
0 (initial)
5
10
15
20
3
Reading
at Time X
Organism
Difference
Corrected
Difference