Required practical 9

• Describe how a respirometer can be used to measure the rate of

  aerobic respiration (by measuring oxygen uptake):

  1)Add set mass of single-celled organism eg. yeast to set volume / conc. of substrate eg. glucose

  2)Add a buffer to keep pH constant

  3)Add a set volume / conc. of a chemical that absorbs CO2 eg. sodium hydroxide

  4)Place in water bath at a set temperature and allow to equilibrate

  5)Measure distance moved by coloured liquid in a set time
• Explain why the liquid moves:

  ● Organisms aerobically respire so take in O2

  ● CO2 given out but absorbed by sodium hydroxide solution

  ● So volume of gas and pressure in container decrease

  ● Fluid in tube moves down pressure gradient towards organism
• Explain why the respirometer apparatus is left open for 10 minutes:

  ● Allow apparatus to equilibrate

  ● Allow for overall pressure expansion/change throughout

  ● Allow respiration rate of organisms to stabilise
• Explain why the apparatus must be airtight:

  ● Prevent air entering or leaving

  ● Would change volume and pressure, affecting movement of liquid
• Describe a more accurate way to measure volume of gas:

  ● Use a gas syringe
• Suggest a suitable control experiment and explain why it is necessary:?

  ● No organisms OR use inert objects OR use dead organisms AND all other conditions / apparatus / equipment the same

  ● To show that (respiring) organisms are causing liquid to move / taking up oxygen / causing the change in volume / pressure
• Describe how a respirometer can be used to measure the rate of

  anaerobic respiration (by measuring carbon dioxide release):

  ● Repeat experiment as above but remove chemical that absorbs CO2

  ● Make conditions anaerobic, for example:

  ○ Layer of oil / liquid paraffin above yeast to stop O2 diffusing in

  ○ Add a chemical that absorbs O2

  ○ Leave for an hour to allow O2 to be respired and used up
• Explain why the liquid moves:

  ● Yeast anaerobically respire so release CO2

  ● So volume of gas and pressure in container increase

  ● So fluid in capillary tube moves down a pressure gradient away from organism
• Explain why the apparatus is left for an hour after the culture has reached a constant temperature:

  ● Allow time for oxygen to be used / respired
• Describe how rate of respiration can be calculated:

  1)Calculate volume of O2 / CO2 consumed / released (calculate area of a cylinder)

  a)Calculate cross-sectional area of capillary tube using π r2

  b)Multiply by distance liquid has moved

  2)Divide by mass of organism and time taken

  3)Units - unit for volume per unit time per unit mass eg. cm3min -1g
• What are Redox Indicators?

  ● Redox indicators (eg. methylene blue) change colour when they accept electrons becoming reduced

  ● Redox indicators take up hydrogens and get reduced instead of NAD / FAD → modelling their reactions
• Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration:

  1)Add a set volume of organism eg. yeast and a set volume of respiratory substrate eg. glucose to tubes

  2)Add a buffer to keep pH constant

  3)Place in water bath at a set temperature and allow to equilibrate for 5 mins

  4)Add a set volume of methylene blue, shake for a set time (do not shake again)

  5)Record time taken for colour to disappear in tube

  6)Rate of respiration (s-1) = 1 / time (sec)
• Give two examples of variables that could be controlled:

  ● Volume of single-celled organism

  ● Volume / conc. / type of respiratory substrate

  ● Temperature (with a water bath)

  ● pH (with a buffer)

  ● Volume of redox indicator (only control)
• Why leave tubes in the water bath for 5 minutes?

  ● Allow for solutions to equilibrate and reach the same temperature as the water bath
• Suggest a suitable control experiment and explain why it is necessary:

  ● Add methylene blue to boiled / inactive / dead yeast (boiling denatures enzymes)

  ● All other conditions the same

  ● To show change is due to respiration in organisms
• Suggest and explain why you must not shake tubes containing methylene blue:?

  ● Shaking would mix solution with oxygen

  ● Which would oxidise methylene blue / cause it to lose its electrons

  ● So methylene blue would turn back to its original blue colour
• Suggest one source of error in using methylene blue. Explain how this can be reduced:

  ● Subjective as to determination of colour change / end point

  ● Compare results to a colour standard (one that has already changed) OR use a colorimeter for quantitative results