rp9 Investigation into the effect of a named variable on the rate of respiration of cultures of single-celled organisms

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

respiration

Measures O2 uptake:

1. Add a set mass of single-celled organism e.g. yeast to a

set volume / concentration of substrate e.g. glucose

2. Add a buffer to keep pH constant

3. Add a chemical that absorbs CO2 e.g. 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 → take in O2

● CO2 given out but absorbed by sodium hydroxide solution

● So volume of gas and pressure in container decrease

● So fluid in capillary tube moves down a pressure gradient towards

organism

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

● 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

Describe how the rate of respiration can be calculated

1. Calculate volume of O2 consumed / CO2 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 volume per unit time per unit mass eg. cm3min-1g-1

formula

Respiratory Quotient (RQ): indicates which respiratory substrate is being broken down

volume of co2 produced/o2 consumsed

RATE OF RESPIRATION: pi x r² x h

mass x time taken for fluid to move

pi x r² = cross sectional area of the tube

h = distance fluid moved

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

respiration

Measures CO2 release:

● Repeat experiment as above but remove chemical that absorbs CO2

● Make conditions anaerobic, for example:

○ Layer of oil / liquid paraffin above yeast → 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 → 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

redox indicator

● 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

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

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

Give 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

Describe a control experiment

and why it would be done.

● 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

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