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SB0023 Biology : Structure and Function Preparatory Centre for Science & Technology
2011/2012
REACTION OF YEAST CELLS WITH METHYLENE BLUE Introduction Yeast are simple organisms that can adapt to either aerobic or anaerobic conditions and a wide range of nutrients. In this experiment you will study the activity of two enzymes, lactic acid dehydrogenase and alcohol dehydrogenase, which both use NAD+ as the cellular oxidizing agent. You will use methylene blue as an artificial electron acceptor (oxidizing agent) in the reaction, which changes from blue to colorless as a result of its reduction in the enzyme reaction. Objectives: To observe enzyme activities in actively respiring yeast by observing colour changes in methylene blue dye as a substitute for NAD+ N AD+ in the yeast respiratory chain. Apparatus: Boiling tubes Water bath (380C-420 C) Measuring cylinder, 10 cm3 Pastuer pipettes Cork or rubber stopper Thermometer Material: Methylene blue 0.05% Yeast suspension (10%) 1% glucose (newly prepared) Method: 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11.
Label 3 boiling tubes with A, B, C. Fill each tube with 10 cm3 of o f yeast suspension Boil tube C for 5 minutes Add 1% glucose to tube A only. Add 10 drops of methylene blue into each of the tubes. Shake and let the colour evenly distributed Heat all tubes in the water bath with 380C-420 C for 15 minutes Observe colour changes in all tubes after 15 minutes Place tube B in boiling water for 5 minutes. Do not shake the tube Plug tube A with cork or rubber stopper. Press it with your thmb and shake the tube vigourously for about 10 times. Observe the colour changes. Remove the stopper and place tube A into the water bath again. Repeat step 8 using tube B and C. Observe the colour of suspension precipitated in each tube Record the times your observation (how long it takes for the blue colour to disappear)
SB0023 Biology : Structure and Function Preparatory Centre for Science & Technology
2011/2012
Results : Test Tube A B C
Contents Yeast+1% glucose Yeast Boiled yeast
Observation
Discussion 1.
Yeast are used to make beer and wine, producing alcohol in the process. What are the conditions needed for yeast to produce alcohol 2. Explain your result for test tube C. 3. Explain the effect of adding 1% glucose to test tube A. 4. Why methylene blue is used in this experiment? What is the substance in living cell that perform the same function as methylene blue. Conclusion
