Student worksheet

Activity 12: Investigating dehydrogenase activity in anaerobic respiration of yeast

Objective Equipment/materials
 To investigate the effects of living yeast cells on  Two 500 cm3 beakers for water baths
methylene blue  Three boiling tubes labelled A, B and C with rubber
bungs
 Boiling tube rack
 30 cm3 yeast and glucose solution
 10 cm3 syringe or pipette & filler
Safety
 Bunsen burner, tripod and gauze
 Supply of warm water
 Thermometer (–10 to 100 °C) × 1 °C
 Methylene blue solution
 1 cm3 pipette
 Stopwatch

Procedure Data/diagram
1. Set up a water bath of hot water using a large beaker Figure 12
and bring it to the boil over the Bunsen burner.
2. Label three boiling tubes A, B and C.
3. Stir the yeast solution provided and pipette 10 cm3 of
the yeast solution into each of the three boiling tubes.
4. Place tube C into the boiling water bath and heat for
five minutes, then remove and cool under a cold
running tap.
5. Whilst you are waiting, set up the second water bath to
be maintained at approximately 40 °C by using a
mixture of warm and tap water. Check the temperature
using a thermometer.
6. Add 1 cm3 of methylene blue to each of the tubes A, B
and C. Analysis of results
7. Place a rubber bung into each tube and gently shake to
mix to a uniform colour; place the tubes into the water
bath at 40 °C and start the stopwatch.
8. After 10 minutes, remove all the tubes and stand in a
rack. Observe tubes A, B and C and record the results.
9. Place tube B into the boiling water bath and leave for
five minutes, then remove and allow to cool. From the examiner
10. Shake tube A vigorously five times and note your  Methylene blue can be used to investigate redox
observations. reactions in living cells.
11. Shake tube B five times and record your observations.  Methylene blue is reduced to a colourless form but
will easily re-oxidise to its blue form.
 It is an artificial hydrogen acceptor.

Questions
1. Is the reaction enzyme controlled? Explain your answer.
2. Explain the observations you made on tubes A and C at step 8.
3. Explain your observations on tube A after shaking it vigorously.
4. How could you modify this experiment to test the effect of temperature on the activity of dehydrogenase? Give details
of what you would do.

Practical activities have been checked for health and safety advice by CLEAPSS. All users will need to review the © Pearson Education Ltd 2009
risk assessment information and may need to adapt it to local circumstances. This document may have been altered from the original 12/30
 Teacher worksheet

Activity 12: Investigating dehydrogenase activity in anaerobic respiration of yeast

Objective From the examiner

 To investigate the effects of living yeast cells on  Methylene blue can be used to investigate redox
methylene blue reactions in living cells.
 Methylene blue is reduced to a colourless form but
will easily re-oxidise to its blue form.
 It is an artificial hydrogen acceptor.
Safety

Answers
1. The reaction is enzyme controlled since boiling tube
Notes on procedure C prevents any reaction taking place. The
 A water bath of boiling water from a kettle could also be dehydrogenase enzymes in the yeast cells have
used but it will be difficult to keep the water hot enough been denatured.
for this part of the experiment, since tube C needs to 2. (a) In tube A, the release of hydrogen by the action
be in boiling water for a total of five minutes. of the dehydrogenase enzymes has reduced the
methylene blue to its colourless form.
 The second water bath at 40 °C is easier to set up
using a plastic/metal container and hot water from a (b) The denatured enzymes in tube C mean no
kettle, adjusted with cold water. The thermometer will hydrogen is released and the methylene blue is
be needed to check the correct temperature has been not reduced.
reached. 3. The methylene blue in tube A is re-oxidised after
shaking the tube vigorously and so the blue colour
 The yeast solution must be stirred before using as it
returns.
would have settled.
4. To test the effect of temperature on the activity of
 You may need to adjust the volume of methylene blue
dehydrogenase, students should be encouraged to
added in order to gain results in the time allocated.
change the temperature of the water bath used to
 Timing for removal from the water bath may need to be incubate the three tubes during the experiment.
reduced if the time is short in order to achieve the
 A temperature range of at least five independent
colour change. The correct period can be found via
variable values is required.
trials and may also produce information on the volume
of methylene blue required to be added in Step 6 of the  The range needs to be reflective of the fact that
procedure. yeast will normally survive in an optimum of 30
 Shaking tube A will cause the methylene blue to be re- °C.
oxidised.  It would therefore be sensible to include
temperatures from 0 °C to approximately 60 °C.
 Replicate data should be collected to give
reliable evidence.

Practical activities have been checked for health and safety advice by CLEAPSS. All users will need to review the © Pearson Education Ltd 2009
risk assessment information and may need to adapt it to local circumstances. This document may have been altered from the original 12/30
 Technician worksheet

Activity 12: Investigating dehydrogenase activity in anaerobic respiration of yeast

Objective Safety
 To investigate the effects of living yeast cells on  Methylene blue solid is harmful (the working solution
methylene blue is normally 1 % and is low hazard).

Requirements per student*/group of students Notes

3
Two 500 cm beakers for water baths Water baths can be shared between several students.

Three boiling tubes labelled A, B and C with rubber bungs

Boiling tube rack

30 cm3 yeast and glucose solution See notes below.

10 cm3 syringe or pipette and filler

Bunsen burner, tripod and gauze

Supply of warm water

Thermometer (-10 to 100 °C) × 1 °C

Methylene blue solution Use standard methylene blue solution as prepared for
staining animal cells in microscopy.

1 cm3 pipette

Stopwatch

Notes

Yeast and glucose solution:

 Prepare a yeast suspension using 4 g of dried yeast, 1 g of yeast extract and 100 cm3 of water. Mix well together.
 For every 20 cm3 of yeast suspension, add 20 cm3 of 5% glucose solution and allow fermenting to begin by incubating
at a temperature of 30 °C.
 The yeast and glucose need to be premixed and incubated, ready to use at the start of the lesson.

Practical activities have been checked for health and safety advice by CLEAPSS. All users will need to review the © Pearson Education Ltd 2009
risk assessment information and may need to adapt it to local circumstances. This document may have been altered from the original 12/30