Practical: Investigating the Rate of Respiration (OCR A Level Biology)

• There are several different experimental methods that can be used to investigate the rate of anaerobic respiration and aerobic respiration in organisms
• Some methods involve the use of a redox indicator such as the experiment described below
• A redox indicator is a substance that changes colour when it is reduced or oxidised
• DCPIP and methylene blue are redox indicators
  • They are used to investigate the effects of temperature and substrate concentration on the rate of anaerobic respiration in yeast

• These dyes can be added to a suspension of living yeast cells as they don’t damage cells
• Yeast can respire both aerobically and anaerobically, in the first experiment it is their rate of anaerobic respiration that is being investigated

Using redox indicators to investigate anaerobic respiration in yeast

• Dehydrogenation happens regularly throughout the different stages of respiration
• The enzyme dehydrogenase catalyses the production of reduced NAD in glycolysis
• When DCPIP or methylene blue are present they take up hydrogens from the organic compounds and get reduced instead of NAD
• Both redox indicators undergo the same colour change when they are reduced
  • Blue → colourless

• The faster the rate of respiration, the faster the rate of hydrogen release and the faster the dyes get reduced and change colour
  • This means that the rate of colour change can correspond to the rate dehydrogenase would be working at and therefore, the rate of respiration in yeast

• The rate of respiration is inversely proportional to the time taken

Rate of respiration (sec^-1) = 1 / time (sec)

Apparatus

• Yeast
• Glucose solution
• Test tubes
• Stopwatch
• DCPIP
• Temperature-controlled water bath

Method - Temperature

• Add a set volume of yeast suspension to test tubes containing a certain concentration of glucose
• Put the test tube in a temperature-controlled water bath and leave for 5 minutes to ensure the water temperature is correct and not continuing to increase or decrease
• Add a set volume of DCPIP to the test tube and start the stopwatch immediately
• Stop the stopwatch when the solution becomes colourless or loses all blue colour
  • This is subjective and therefore the same person should be assigned this task for all repeat experiments

• Record the time taken for a colour change to occur once the dye is added
  • Repeat across a range of temperatures. For example, 30^oC, 35^oC, 40^oC, 45^oC

Methylene blue or DCPIP is added to a solution of anaerobically respiring yeast cells in a glucose solution. The rate at which the solution turns from blue to colourless gives the rate of dehydrogenase activity.

Controlling other variables

• It is important when investigating one variable to ensure that the other variables in the experiment are being controlled
  • Volume of dye added: if there is more dye molecules present then the time taken for the colour change to occur will be longer
  • Volume of yeast suspension: when more yeast cells are present the rate of respiration will be inflated
  • Type of substrate: yeast cells will respire different substrates at different rates
  • Concentration of substrate: if there is limited substrate in one tube then the respiration of those yeast cells will be limited
  • Temperature: an increase or decrease in temperature can affect the rate of respiration due to energy demands and kinetic energy changes. The temperature of the dye being added also needs to be considered
  • pH: a buffer solution can be used to control the pH level to ensure that no enzymes are denatured

Results

• A graph should be plotted of temperature against time
• As the temperature increases, the rate of respiration also increases so the time taken for the solution to go colourless reduces
  • This means hydrogens are released by the reactions more quickly, hence the DCPIP accepts electrons/hydrogens more quickly until all molecules of DCPIP are reduced. This means that it will take less time to turn from blue to colourless

Limitations

• This experiment is not measuring the rate of dehydrogenase activity directly (through measuring the rate of substrate use or product made) but is instead predicting what the rate would be by measuring the rate of electron transfer
• Distinguishing the end of the reaction and the colour change is subjective and therefore one person should be used to attempt to control this
  • Although the DCPIP and methylene blue undergo a colour change from blue to colourless it is important to remember that the yeast suspension in the test tube may have a slight colour (usually yellow). That means when the dye changes to colourless there may still be an overall yellow colour in the test tube

How to investigate the effect of other factors on the rate of anaerobic respiration in yeast

• The effect of substrate concentration can be investigated by adding different concentrations of a substrate to the suspension of yeast cells and recording the time taken for a colour change to occur once the dye is added
  • For example, 0.1% glucose, 0.5% glucose, 1.0% glucose

• The effect of different respiratory substrates can be investigated by adding different respiratory substrates such as lipids or proteins (at the same concentration) to the suspension of yeast cells and recording the time taken for a colour change to occur once the dye is added

Using a respirometer to investigate aerobic respiration

• Respirometers are used to measure and investigate the rate of oxygen consumption during aerobic respiration in organisms
• By adding the apparatus to a thermostatically controlled water bath the effect of temperature on the rate of respiration can be investigated
• The experiments usually involve organisms such as seeds or invertebrates

Respirometer set up with temperature-controlled water bath

Method

• Measure oxygen consumption: set up the respirometer and run the experiment with both tubes in a controlled temperature water bath. Use the manometer reading to calculate the change in gas volume within a given time, x cm³ min^-1
• Reset the apparatus: Allow air to reenter the tubes via the screw cap and reset the manometer fluid using the syringe. Change the temperature of the water bath and allow the tubes to acclimate, then close the screw clip to begin the experiment
• Run the experiment again: use the manometer reading to calculate the change in gas volume in a given time, y cm³ min^-1
• Repeat experiment several times at different temperatures

Calculations

• The volume of oxygen consumed (cm³ min^-1) can be worked out using the diameter of the capillary tube r (cm) and the distance moved by the manometer fluid h (cm) in a minute using the formula:

 πr²h

Analysis

• The rate of oxygen consumption (cm³ min^-1) is often taken as the rate of respiration for organisms
• The different volumes of oxygen consumed obtained for the different temperatures can be presented in table or graph form to show the effects of temperature

Graph showing the effect of temperature on the rate of respiration