Required practical 9 - Investigation into the effect of a named variable on the rate of respiration of cultures of single-celled organisms

Question 1

Describe how a respirometer can be used to measure the rate of aerobic respiration (by measuring oxygen uptake)

Answer 1

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

Question 2

Explain why the liquid moves (4) - in aerobic respiration

Answer 2

● 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

Question 3

Explain why the respirometer apparatus is left open for 10 minutes. (1)

Answer 3

● Allow apparatus to equilibrate
● Allow for overall pressure expansion/change throughout
● Allow respiration rate of organisms to stabilise

Question 4

Explain why the apparatus must be airtight. (2)

Answer 4

● Prevent air entering or leaving
● Would change volume and pressure, affecting movement of liquid

Question 5

Describe a more accurate way to measure volume of gas. (1)

Answer 5

● Use a gas syringe

Question 6

Suggest a suitable control experiment and explain why it is necessary. (2)

Answer 6

● 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

Question 7

Describe how a respirometer can be used to measure the rate of anaerobic respiration (by measuring carbon dioxide release)

Answer 7

● 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

Question 8

Explain why the liquid moves. (3) - in anaerobic respiration

Answer 8

● 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

Question 9

Explain why the apparatus is left for an hour after the culture has reached
a constant temperature. (1)

Answer 9

● Allow time for oxygen to be used / respired

Question 10

Describe how rate of respiration can be calculated

Answer 10

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

Question 11

Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration

Answer 11

● 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

Question 12

Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration - method

Answer 12

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)

Question 13

Give two examples of variables that could be controlled. (2) (Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration)

Answer 13

● 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)

Question 14

Why leave tubes in the water bath for 5 minutes? (1) (Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration)

Answer 14

● Allow for solutions to equilibrate and reach the same temperature
as the water bath

Question 15

Suggest a suitable control experiment and explain why it
is necessary. (3) (Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration)

Answer 15

● 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

Question 16

Suggest and explain why you must not shake tubes
containing methylene blue. (3) (Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration)

Answer 16

● 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

Question 17

Suggest one source of error in using methylene blue. Explain
how this can be reduced. (2) (Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration)

Answer 17

● 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