These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Feeding a growing planet: respiration and ripening project starter.
Potential areas for investigation
You can carry out your own practical investigations into the respiration rates of fruit and vegetables using the protocols given below. Topics might include
- How does the temperature of storage affect the rate of respiration?
- Does an infected fruit or vegetable have a higher respiratory rate than a healthy one?
- Does the skin affect the exchange of gases between a fruit or vegetable and the atmosphere?
- Does the rate of respiration vary during the ripening process?
This could feed into a broader research project considering how food waste can be reduced by understanding respiration in fruit and vegetables. Topics might include
- How can farmers in different parts of the world reduce post-harvest losses?
Protocol: Measuring the production of carbon dioxide (from respiration) with baryta water
Respiration in plant tissues gives rise to carbon dioxide as a waste product. This gas is absorbed by barium hydroxide solution, an alkaline solution known as baryta water. This is then titrated with an acid to find out how much carbon dioxide has accumulated in it. This test allows an estimation of the carbon dioxide output of the plant material to be made.
• Obtain two air-tight containers (such as a tupperware box, or jam-jar with lid) of an appropriate size for the plant material. Make sure that there is plenty of room for some air as well – the plant material requires oxygen for respiration!
• Place the solution of baryta water (barium hydroxide 0.025 M) in an open dish of some sort, near the bottom of each container. Remember that the carbon dioxide is heavier than air and will settle downwards.
• Place the plant material in one of the containers, and leave the other as a control. The baryta water must not be in direct contact with the plant material.
• Leave the containers for a few hours – or overnight. Then remove the dishes of baryta water from each container, and titrate them with 0.01 M hydrochloric acid, using phenolphtalein as an indicator:
• Carbon dioxide reacts with the barium hydroxide as follows:
Ba(OH)2 + CO2 ——> BaCO3 + H2O
• Remove any crust that forms on the baryta water and continue with the clear solution from underneath. Add a few drops of phenolphthalein indicator to a sample of baryta water which should become pink. Then add the acid carefully, and note the volume required to just turn the pink to colourless. Repeat this procedure with the second sample.
• The difference in the results of the titration between the control container, and the container with plant material, as determined by the volumes of acid needed, gives a measure of the carbon dioxide produced by the plant material.
Given that 1 cm3 of 0.01 M acid is equivalent to 0.22 mg of CO2, it should be possible to calculate the mass of CO2 produced by the plant material.
Protocol: Comparing differences in respiratory activity in tissue slices using TTC
Dehydrogenase enzymes have an important role in aerobic respiration. The technique described below depends upon the fact that these dehydrogenase enzymes can donate the hydrogen ions they remove from a respiratory substrate, to a colourless compound, causing it to change colour.
When the colourless chemical tetrazolium chloride (TTC) diffuses into actively respiring tissues it accepts electrons from the mitochondrial electron transport chain, reducing it to a pink compound, known as formazan. The accumulation of this pink compound stains the tissues red, and the intensity of the red colour is proportional to the rate of respiration in those tissues.
This test can be used with slices of fruits or vegetables, or imbibed seeds. The formation of the pink colour in the tissues allows the active and less active areas of the specimen to be distinguished.
Cut the material (with a razor or scalpel) and place face down in a watch glass or Petri dish, or other shallow container, containing some 1% TTC solution (1).
In a warm room, the most actively respiring tissues of the cut surface becomes coloured pink within 10 to 20 minutes, but longer periods of incubation may be needed if the respiratory rate is slow.
The staining is permanent and such slices can be kept in a refrigerator until the next lesson, or even dried out.
(1) Tetrazolium chloride is available as a powdered sodium salt from Philip Harris Ltd.