Cellular Respiration
Carbon dioxide and water are two of the most common and energetically stable
compounds found in nature. Thus, energy is released when complex molecules that contain
carbon and hydrogen are chemically converted to carbon dioxide and water. The burning of
methane fuel (natural gas) is a dramatic example of this type of energy-yielding conversion:
1CH4 + 2O2  1CO2 + 2H2O + 213,000 calories (energy)
This is an oxidation reaction because oxygen combines with another substance (methane in this
example), with the release of energy during the reaction.
Cell respiration is the process by which organisms oxidize food items to CO2 and H2O.
Energy is released during this process. Cell respiration differs greatly from combustion,
however, in that the energy of respiration is released in a controlled fashion through a series of
small steps (chemical reactions). This sequential breakdown of food items permits some of the
energy from the food to be transferred to “high energy” compounds like ATP instead of being
entirely dissipated as heat. The ATP, in turn, is used to provide energy that is needed to drive
reactions in the cell.
The most common fuel for respiration in plants and animals is the six-carbon sugar
glucose, which is often available via the breakdown of other complex carbohydrates, such as
starch. Starch is the major storage form of glucose in plants. In cellular respiration glucose is
oxidized to carbon dioxide with the release of energy:
C6H12O6 + 6O2  6CO2 + 6 H2O + 674,000 calories (energy)
All those calories that are released are either transferred to other reactions to form ATP, or are
lost as heat.
For many reasons it would be useful if we could measure the rate of respiration. Fortunately,
there are several ways in which this can be performed in the laboratory:
Heat Production – not all the energy released in respiration is converted to ATP. The
remainder is given off as heat, which can be measured over time to give an estimate of
the rate of respiration. This source of heat, in fact, is what maintains your relatively high
body temperature. But, in plants, this loss of heat is largely just wasted energy that is
dispersed into the environment.
Liberation of Carbon Dioxide – the rate of respiration can be determined by measuring
the amount of CO2 given off over a period of time.
Oxygen Consumption – the rate at which oxygen is consumed is a generally accepted
index of the rate of respiration. This is the method we will utilize in lab, by the use of a
simple respirometer (see Figure below). The respirometer consists of a sealed chamber
attached to a calibrated tube. Actively respiring tissue is placed in the chamber along
with a smaller tube containing potassium hydroxide (KOH). A drop of water is then
placed into the top of the calibrated tube. As CO2 is produced by the tissue it is absorbed
by the KOH to form potassium carbonate. Thus, as oxygen is consumed by the respiring
tissue, the volume of air in the chamber and calibrated tube decreases because the CO2 in
the respired air is being absorbed. This decrease in volume will be observed by noting
the movement of the drop of water down the calibrated tube. By observing this volume
change per unit of time an estimate of the rate of oxygen consumption can be determined,
in ml of O2 per minute
The seeds of flowering plants are typically resistant structures in which embryonic plants
are enclosed. The outer layer of the seed is called the seed coat, which protects the embryo from
adverse environmental conditions. The seeds of most flowering plants are similar in that each
contains a seed coat, an embryo, and some form of food storage tissue. The food for the
development of the embryo is largely located in the tissue called endosperm. The cells of the
endosperm are rich in stored protein, and especially rich in starch.
Mature seeds have a low water content and the cells of the embryo are dormant. The
cells of the seed can be activated by changes in environmental conditions, particularly an
increase in the moisture content of the atmosphere and/or soil. This activation of the cells of the
seed is called germination, and it begins by the uptake of water (the imbibition phase) and
progresses to the point of protrusion of the embryonic root from the seed. In peas and barley,
which we will use today, germination requires 24-48 hours under ideal conditions.
OBJECTIVE: to measure the oxygen consumption in germinating peas and barley seeds at
different environmental temperatures.
PRE-LAB PREPARATIONS
Barley seeds were germinated 2-3 days in advance of lab by soaking mature seeds on wet
paper towels. Additional seeds were boiled, then cooled, to serve as non-respiring controls.
Three water baths were prepared in which your experiments will be conducted. The purpose of
the water baths is to maintain different temperatures for the experiment. Water baths will be
held at these temperatures:
Bath 1, 0-3 degrees C; (cold)
Bath 2, 10-13 degrees C; (cool)
Bath 3, 20-25 degrees C, and (room)
A number of respirometer parts have been assembled. Your group will be assigned specific
tissues and a particular water bath for incubating your seeds.
For this experiment you will work in groups of three. Each group will be assigned a Group
number. We will run a replicate of each tissue at each temperature. A control respirometer will
be assembled for each temperature, as well. Groups will be assigned a tissue type and a
temperature as follows:
Group 01
Barley at cold temperature #1
Group 02
Barley at cold temperature #2
Group 03
Barley at cool temperature #1
Group 04
Barley at cool temperature #2
Group 05
Barley at room temperature #1
Group 06
Barley at room temperature #2
PROCEDURES
01. Obtain the components for assembling a respirometer. Refer to the figure above.
02. Using a waterproof pen, mark a line 5 cm from the top of the 25 ml tube.
03. Fill the tube to this line with the seeds assigned to your group. The seeds should fill the
tube to the line, but not be packed into the tube. All groups should be consistent in how
they fill their tubes.
04. Fold a small piece of white tissue (about the size of a postage stamp) several times and
place it into the 1.5 ml tube.
05. Using a transfer pipet CAREFULLY add about 0.5 ml of KOH to the tube, allowing it to
soak into the paper towel. DO NOT allow any of this base to leak out of the tube. The
KOH is a caustic base and will damage clothes, furniture, and your skin (or the seeds).
Handle it carefully. If an accident occurs where KOH contacts your skin or clothes,
wash at once with water. Notify the instructor of any spills.
06. Gently lower the 1.5 ml tube with KOH into the large glass tube so that it rests on the
seeds. Be sure to keep the tubes upright at all times.
07. Place the stopper with the attached calibrated tube into the top of the large tube and push
down gently to form an air tight seal. Handle only the rubber stopper. Pressure on the
calibrated tube may break the airtight seal. Use gentle pressure only. Push too hard and
the tube may break, causing injury. Use caution.
08. If a control tube has not already been assembled, prepare a second tube to serve as a
control for your experiment. Obtain some of the boiled seeds and assemble a
respirometer following the same instructions above. N.B. Only the first lab group of the
09.
10.
11.
12.
day need prepare a control. Other groups during the same day may use the already
prepared control.
Place your assembled respirometers into the designated water bath, and allow at least ten
minutes for temperature equilibration. The respirometer must remain upright or just
slightly tilted in the water bath during the entire balance of this experiment. DO NOT
remove the respirometer from the water bath for the rest of the experiment
After equilibration a drop of water must be placed in the top portion of each calibrated
tube. This drop will allow you to monitor the oxygen consumed. Draw water into a
small pipet and place the tip of the pipet in the calibrated tube a few mm from the open
end of the tube. Gently squeeze the bulb to expel one drop of water. When done
properly, the drop should be held near the end of the tube by surface tension. If the drop
runs down the tube, draw it out with the pipet and try again. The drop should now be
near the first calibration line.
Wait 2-3 minutes more and record to the nearest 0.01 cc the position of the drop of water
in each tube. Record this as Time Zero. Take readings in this manner every three
minutes until at least eight readings have been taken. Record the data for your Group in
Table 1 below. Note that 1 cc of water = 1 ml
a. If your indicator drop moves the entire length of the calibrated tube it will be
necessary to start a second drop. Simply add a second drop to the tube at an
appropriate time interval and record the position and change in position for the
new drop for the balance of the experiment
Record the cumulative oxygen consumption for all groups in Table 2 below, then
calculate the mean change for seeds at each temperature. Use the data from Table 2 for
your analysis to be performed later.
a. You must allow for any change that occurred in the control tube. Any oxygen
consumption that was measured in the control must be SUBTRACTED from the
cumulative consumption of the experimental tube at the same temperature.
b. The control is non-respiring tissue and therefore, should not consume oxygen.
Any change in gas volume in the control respiromenter is due to changes in
temperature during the experiment.
c. From the table, determine the mean (average) oxygen consumption for replicate
tubes held at the same temperature, and record in the appropriate column of the
table
d. The graphs will display the data for mean cumulative oxygen consumption vs.
time for all temperatures for each seed type. Each group will graph the data for
all groups.
e. Table 3 will be oxygen consumption rates for each tissue. For this, use the total
mean cumulative oxygen consumed.
ASSIGNMENT: Each group will hand in a completed Table
2, Graphs, and Table 3
Table 1. Drop position, change in position over each time interval, and cumulative change in drop position for experimental and
control seeds
Seed type: __________________________
Time
(minutes)
0
3
6
9
12
15
18
21
24
27
30
Drop position
Control
Change since
last reading
Control
Cumulative
change in position
Control
Drop position
Experimental
Change since last
reading
Experimental
Cumulative change
in position
Experimental
Table 2. Cumulative change in oxygen consumption for respiring seeds.
NAMES:
YOUR GROUP # ______
SEED TYPE: __________________
Time Cumulative Cumulative
change
change
Group 1
Group 2
0
3
6
9
12
15
18
21
24
27
30
Mean
cumulative
change
(cold)
Cumulative
change
Group 3
TEMPERATURE: ______________
Cumulative
change
Group 4
Mean
cumulative
change
(cool)
Cumulative
change
Group 5
Cumulative
change
Group 6
Mean
cumulative
change
(room)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
Cumulative mean oxygen consumption (ml)
ANALYSIS
In the space below, graph the mean cumulative oxygen consumption vs. time for all three
temperatures. Label the different temperatures of incubation clearly. Use only data that has
been corrected for changes in the controls.
0
3
6
9
12 15 18 21 24
Time (minutes)
27 30
Finally, from your data, and the data from the other groups, determine the oxygen consumption
rates for each tissue at different temperatures.
Table 3. Oxygen consumption rates for peas and barley.
Group
#
Temperature
Total O2
Consumed
(ml)
Mean total O2
consumed
(ml)
Mean rate of O2
consumption
(ml/min)
1
2
3
4
5
6
What do you conclude about the effects of temperature on respiration?