Core practical 15: yeast respiration with a redox indicator

The principle

Dehydrogenase enzymes remove hydrogen from respiratory substrates. Normally the hydrogen is taken up by NAD or FAD. A redox indicator can accept the hydrogen instead and changes colour when it is reduced:

• TTC (triphenyl tetrazolium chloride): colourless → red when reduced
• methylene blue: blue → colourless when reduced

The faster the yeast respires, the faster the colour changes.

Method

1. Make a suspension of yeast in glucose solution and leave it to become active.

2. Put a fixed volume of yeast suspension (for example 10 cm³) and, in a separate tube, a fixed volume of indicator (for example 1 cm³ TTC) in a water bath at the chosen temperature for 5 minutes to equilibrate.

3. Mix, start a stopwatch and time how long the colour change takes, judging against a colour standard.

4. Repeat at other temperatures (for example 15 to 35 °C), or with different substrates, three times each.

Control: yeast concentration and volume, glucose concentration, indicator volume, and the end point. A tube of boiled yeast shows the colour change needs living enzymes.

Results

Rate = 1 / time (s-1). Plot rate against temperature.

Limitations: judging the end point is subjective (a colorimeter improves this); methylene blue is reoxidised by oxygen from the air if shaken; yeast cells settle, so mix the suspension each time.

Worked example

The TTC in a tube of yeast turns red after 125 s. Calculate the rate.

1. Rate = 1 / time
2. = 1 / 125 = 0.008 s-1

Answer: 0.008 s-1 (8.0 x 10^-3 s-1).

Check yourself

1. Methylene blue in a yeast suspension goes colourless after 80 s. Calculate the rate in s-1.
   Show answer

   0.0125 s-1. 1 / 80 = 0.0125 s-1.

2. At 20 °C the colour change takes 300 s; at 30 °C it takes 120 s. How many times faster is the rate at 30 °C?
   Show answer

   2.5. 300 / 120 = 2.5 times faster.

3. What colour change shows TTC has been reduced?
   • Red to colourless
   • Colourless to red
   • Blue to colourless
   • Orange to blue-black
   Show answer

   Colourless to red. Reduced TTC forms a red compound.

4. Why does the indicator change colour in respiring yeast?
   • It reacts with carbon dioxide
   • It is broken down by amylase
   • It accepts hydrogen removed by dehydrogenase enzymes
   • It absorbs oxygen from the air
   Show answer

   It accepts hydrogen removed by dehydrogenase enzymes. It acts as an artificial hydrogen acceptor.

5. What is the purpose of a tube containing boiled yeast?
   • To show that living enzymes are needed for the colour change
   • To speed up the reaction
   • To provide extra glucose
   • To act as a colour standard for red
   Show answer

   To show that living enzymes are needed for the colour change. Boiling denatures enzymes, so no colour change should occur.

6. Why are the yeast and indicator left in the water bath for 5 minutes before mixing?
   • So the yeast dies
   • So the indicator changes colour
   • So the yeast and indicator reach the test temperature before mixing
   • So oxygen can escape
   Show answer

   So the yeast and indicator reach the test temperature before mixing. Equilibration makes sure the temperature is correct from the start.

7. Why should tubes containing methylene blue not be shaken during timing?
   • Oxygen from the air would reoxidise it back to blue
   • It would turn red
   • Shaking kills yeast
   • It would make the yeast respire anaerobically
   Show answer

   Oxygen from the air would reoxidise it back to blue. Reduced methylene blue reacts with oxygen and turns blue again.

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Sources

• Pearson Edexcel International Advanced Level in Biology specification (XBI11/YBI11, first teaching 2018)
• Physics & Maths Tutor: Edexcel IAL Biology CP15, Respiration in yeast

Last updated 7 November 2026. Spotted a mistake? Tell us.