This describes how to investigate the
rate of respiration of small organisms like woodlice (an
arthropod) by measuring their
rate of uptake of oxygen that is being used in the organisms metabolism.
A respirometer is a device
designed to measure the rate of consumption of oxygen by a living organism.
The rate of oxygen depletion in
the air e.g. cm3/min by gas volume or mm/min on some
scale is taken as a measure of the rate of respiration.
You can also use germinating peas or
germinating beans to investigate the effect of temperature on the rate of
their respiration. Germinating seeds need to respire to
provide energy from them to grow and develop into the plant.
The experimental set up - a
respirometer system
A
boiling tube is set up in a thermostated water bath to control the
temperature of the experiment - you should use a thermometer to
accurately monitor the temperature of the water. A syringe containing air and a manometer
are connected via glass tubing through a rubber bung to the boiling
tube. Together with the soda lime this set up is called a respirometer.
A manometer is a device to measure
pressures. A common simple manometer consists of a U shaped tube of
glass filled with some liquid - in this case coloured water NOT
poisonous mercury! A ruler scale is placed in between the arms of the U
tube so the difference in heights of the liquid can be measured in both arms.
The syringe is used to set the level
of the liquid in the manometer and refresh the air between experiments.
At the bottom of the boiling tube soda lime granules are placed to
absorb carbon dioxide given
out by the respiring woodlice. On top of the soda lime is a wad of
cotton wool to prevent contact with the woodlice - they would be harmed
by the strongly alkaline soda lime - an ethical point when using live
animals in experiments.
The live woodlice are carefully
placed on the cotton wool and the rest of the apparatus of the
respirometer is carefully assembled so that boiling tube is vertical in
the water bath.
Experimental notes
Note 1: The reduction in volume
shows a gas is being removed from air, and that gas is oxygen. There
would be no change in volume without the soda lime - the volume of CO2
formed would equal the volume of O2 used. But, since
the soda lime removes the carbon dioxide given out by the organism's
respiration, there is no confusion that the gas volume reduction is due
to the uptake of oxygen by respiration and the liquid in the manometer
moves towards respiring organism in the test tube.
Note 2: The right-hand limb of
the manometer can be connected to a 2nd control test tube set up in the
same way as the respiring organism test tube. This test tube is
connected to the manometer and a tap is fitted instead of the syringe. A
control test tube helps check that movement of the fluid in the
manometer is only caused by the respiration of the organism.
Note 3: Instead of soda lime
granules, you can use cotton wool soaked in a concentrated solution of
an alkali e.g. sodium hydroxide or potassium hydroxide.
Investigation procedure
(a) The water bath, and boiling tube
without woodlice in the boiling tube, are left for a short time until
the temperature of the water bath has settled down to your desired start
temperature e.g. 20oC.
(b) The woodlice are then quickly
placed in the boiling tube, connect everything up as shown in the
diagram.
(c) The syringe is used to equate the
liquid levels in the manometer, preferably to zero difference in height
(R1 = 0).
R1 doesn't have to be zero as
long as you carefully read and record readings in both limbs of the
manometer at the start and end of the respiration experiment.
(d) Let the woodlice respire for a
fixed time.
As they respire and use up oxygen, carbon dioxide is
produced, which is absorbed by the soda lime, decreasing the volume of
air.
(e) As the air volume decreases, it
temporarily reduces the pressure in the boiling tube containing
the respiring organism, therefore, to
maintain the constant external pressure ('fair test'), the liquid moves up the left limb of the
U tube manometer.
(f) After a set time, you read the
two levels to determine R2 (left reading - right reading in the U
tube).
The total distance moved by the
liquid = the difference between the two readings, R2 - R1, and this
gives you a relative measure of the rate of respiration.
(technically it doesn't matter if
R1 isn't zero at the start, as long as you subtract the initial
differential reading R1 from R2, you obtain the actual numerical change desired.)
(g) You then repeat (a) to (f) with
the same woodlice, at a higher temperatures going up 5o at
time up to 50oC.
You need to replenish the air in
the test tube and move the manometer liquid down and away from the
test tube.
The greater the rate of movement
of the coloured liquid in the thermometer, the greater the rate of
respiration
Further experimental notes:
Theoretically, because the
syringe is also calibrated, the syringe 'plunger' can be carefully
depressed to return the manometer liquid levels to their
original readings.
The volume of oxygen used =
final syringe volume - initial syringe volume (e.g. in cm3).
This allows you to calculate
the woodlice rate of respiration in cm3/min.
OR, you can just use the
manometer level readings as a relative measure of respiration
e.g. mm/min.
Ethical note:
The woodlice should not be
used at sufficiently high temperatures that they die.
The woodlice should not be
left in the respirometer too long so that they run out of oxygen
and die.
After the experiment they
should be released back into their natural outside habitat.
The woodlice should be
allowed to come into contact with the soda lime or any other
harmful chemical used to absorb the carbon dioxide.
Results, analysis and conclusions
As described above, the relative rate
of respiration is measured as the rate of oxygen consumption in mm/min
or cm3/min.
From your data table of results you can plot a graph of rate of respiration versus temperature.
You should find initially the rates
increases, passes through an optimum at ~35-40oC and then decreases.
steadily as the temperature goes higher.
This is typical of the behaviour
of enzyme controlled reactions - which includes the metabolic
chemistry of respiration.
Initially, as with any chemical
reaction, the rate increases with increase in temperature.
However, as the temperature rises the
enzymes become denatured and the rate of respiration will fall and the
organism will be harmed.
This is not acceptable - unethical,
so you should not raise the temperature too high and obtain just the
first half of the graph above.
However, there is no reason why you
cannot do higher temperature experiments with respiring seeds and see if
you can get the full graphical picture of rates of respiration versus
temperature.
