RPQ 9 - investigating factors affecting respiration

• aerobic resp:

  1. put a known volume and concentration of substrate solution (e.g. glucose) in a test tube. add a known volume of buffer solution to keep the pH constant (choose the optimum pH for the yeast you are testing - usually 4-6)
  2. place the test tube in a water bath set to one of the temps being investigated leave it there for 10 min to allow the temp of the substrate to stabilise
  3. add a known mass of dried yeast (e.g. Saccharomyces cerevisiae) to the test tube and stir for 2 min
• 4. after the yeast has dissolved into the solution, put a bung with a tube attached to a gas syringe in the top of the test tube - gas syringe should be set to zero

  5. start a stop watch as soon as the bung has been put in the test tube

  6. as the yeast respire, theco2 formed will travel up the tube and into the gas syringe, used to measure the volume of co2 released
• 7. at reg intervals (e.g. every min) record the volume of CO2 that is present in the gas syringe - for a set amount of time e.g. 10 min

  8. a neg control experiment should also be set up at each temp - where no yeast is present - no co2 should be formed without the yeast

  9. repeat the experiment 3x at each temp - use data to calc the mean rate of CO2 production at each temp
• anaerobic:

  • set up the apparatus according to steps 1-3 of previous experiment
  • after yeast has dissolved into substrate solution, trickle some liquid paraffin down the inside of the test tube so that it settles on and completely covers the surface of the solution - this will stop oxygen getting in - force the yeast to respire anaerobically
  • put a bung with a tube attached to a gas syringe in the top of the test tube - gas syringe should be set to 0
  • perform steps 5-9 from previous experiment
• 1. set up the apparatus partially submerged in a water bath at 15 degrees to provide the optimum temp for the woodlice and therefore the optimum temp for the enzymes involved in their respiration
  2. the control tube is set up exactly the same way as the woodlouse tube except that the woodlice are substituted with glass beads of the same mass
  3. for 10 min the tap is left open and the syringe is removed to allow the apparatus to equilibrate (allow for any expansion that might cause the pressure to change inside) and the resp rate of the woodlice to stabilise in their new environ
• 4. when the 10 min is up, the tap is closed and the syringe is attached

  5. the syringe is used to reset the manometer, so that the ends of the fluid are at the same level on either side of the U and the reading from the volume scale on the syringe (cm3) is recorded

  6. as respiration occurs, the volume of air in the test tube containing woodlice will decrease due to the oxygen consumed during respiration (all the CO2 produced is absorbed by the potassium hydroxide)
• 7. the decrease in the volume of air will reduce the pressure in the test tube, causing the coloured fluid in the capillary tube of the manometer to move towards it

  8. after leaving the apparatus to run for a set period of time (e.g. 10 min) the syringe is used to reset the manometer and the reading on the syringe's volume scale is recorded again. the difference between this figure and the figure taken at the start of the experiment is the oxygen consumption for this time period - can use this to calculate the rate of respiration
• 9. to check the precision of the results, the experiment is repeated and a mean volume of O2 is calculated
• investigating factors affecting respiration in single-celled organisms:

  • yeast are a single celled organism that can be grown in a culture
  • can respire aerobically when plenty of oxygen is available
  • can respire anaerobically when oxygen isn't available
  • both aerobic and anaerobic respiration in yeast produce CO2
  • so rate of CO2 production gives an indication of the yeast's respiration rate
• • one way to measure CO2 production is by using a gas syringe to collect the CO2
• a buffer solution is able to resist changes in the pH when a small amount of acid or alkali is added - can get acidic buffers (pH less than 7) and alkaline buffers (pH more than 7)
• the yeast will only respire aerobically until the oxygen trapped in the tube is all used up - if want to run an experiment for more time or with more yeast or glucose - could use a conical flask that can trap more oxygen
• to calculate the rate of CO2 production, divide the total volume of CO2 produced at a particular temp by the number of minutes the apparatus was left for
• you can easily adapt these methods (aerobic and anaerobic) to investigate the effects of other variables, such as substrate concentration and the use of different respiratory substrates (e.g. sucrose) on the respiration rate

  • just remember to only change 1 variable at a time
  • all other variables that could affect results need to be controlled (kept constant) or results won't be valid
• to test that the gas produced is definitely CO2, connect the yeast and substrate solution to a test tube of limewater rather than a gas syringe. the limewater will turn cloudy in the presence of CO2
• there are other ways of measuring the respiration rate in yeast e.g. could use an indicator dye (e.g. methylene blue) and a colorimeter to measure the rate of aerobic resp (similar method to the one used in photosynth experiment) - the dye takes the place of electron acceptors in ox phos
• using a respirometer to measure oxygen consumption:

  • respirometers can be used to indicate the rate of aerobic resp by measuring the amount of oxygen consumed by an organism over a period of time
• • if working with woodlice - need to consider ethical issues - treat woodlice with respect and ensure they are not harmed or distressed unnecessarily
  • could use plant seeds or germinating peas instead
• wear eye protection and gloves when working with potassium hydroxide - make sure the woodlice do not come into contact with it
• oxygen consumption can also be calculated by recording the movement of the fluid in the manometer - read from the scale on the manometer itself