Studies of yeast respiration with the oxygen electrode
Studies of yeast respiration with the oxygen electrode
Aims
You will measure compare the rates of respiration by yeast of sugars at different conditions using the oxygen electrode.
Good laboratory practice is essential: no eating or drinking; wear lab coats at all times; handle chemicals carefully and with gloves; read, understand and reflect about the experiment.
Introduction
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Under aerobic conditions sugars are respired to carbon dioxide and water via the glycolytic pathway and the citric acid cycle. The respiration of a yeast suspension is conveniently monitored by following the decline in the concentration of dissolved oxygen using an oxygen electrode. It is essential to starve the yeast suspension by bubbling a stream of air through it overnight to deplete endogenous reserves of glycogen, otherwise a high rate of respiration is observed before the substrate (eg. a sugar) is added. Even then, a small rate of respiration is usually observed due to metabolism of remaining endogenous reserves. It is usual practice to estimate this, the endogenous rate, and to apply a correction for it.
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Materials:
· 7.5 mL 0.05M K phosphate buffer pH 5.0, saturated with air at 30C by bubbling.
· 7.5 mL 0.05M K phosphate buffer pH 1.0, saturated with air at 30C by bubbling.
· 7.5 mL 0.05M K phosphate buffer pH 9.0, saturated with air at 30C by bubbling.
· 5 mL 1% (w/v) suspension of fresh yeast in the buffer above, starved by bubbling with air at 30C for at least 4 hours.
· Glucose (0.16M), Galactose (0.16M), Ethanol (0.16M), Sodium Azide (0.16M)
Procedure
1. Pipette 1500 L of air-saturated buffer into the reaction vessel of an oxygen electrode. Note the temperature of the water bath in which the buffer is immersed; you will need this for a subsequent calculation. If you do not have a thermometer, note the temperature as 30C.
2. Calibrate the electrode by turning the dial till it reaches 100. If the electrode reading fluctuates wildly or drifts steadily downwards, consult a member of staff.
3. Add 1000 μL of the 10 mg/mL yeast suspension to the reaction vessel, and start recording the data every 5 seconds.
5. When you reach 100 seconds, inject 25 L of 0.16 M glucose solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate until 200 seconds, at this time inject 25 L of 0.16 M sodium azide solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate the end of the run (360 seconds). Ensure that the needle enters the electrode chamber.
6. Pipette out the contents of the reaction chamber with a dropping pipette and wash out the reaction chamber three times with distilled water.
7. Determine respiration rates before and after the addition of 25 L of 0.16 M solution of galactose by repeating steps 1-3.
8. Pipette out the contents of the reaction chamber with a dropping pipette and wash out the reaction chamber three times with distilled water.
9. Pipette 1500 L of air-saturated buffer (pH 1) into the reaction vessel of an oxygen electrode. Note the temperature of the water bath in which the buffer is immersed; you will need this for a subsequent calculation. If you do not have a thermometer, note the temperature as 30C.
10. Calibrate the electrode by turning the dial till it reaches 100. If the electrode reading fluctuates wildly or drifts steadily downwards, consult a member of staff.
11. Add 1000 μL of the 10 mg/mL yeast suspension to the reaction vessel, and start recording the data every 5 seconds.
12. When you reach 100 seconds, inject 25 L of 0.16 M glucose solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate until 200 seconds, at this time inject 25 L of 0.16 M sodium azide solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate the end of the run (360 seconds). Ensure that the needle enters the electrode chamber.
13. Pipette out the contents of the reaction chamber with a dropping pipette and wash out the reaction chamber three times with distilled water.
14. Determine respiration rates before and after the addition of 25 L of 0.16 M solution of galactose by repeating steps 1-3.
15. Pipette out the contents of the reaction chamber with a dropping pipette and wash out the reaction chamber three times with distilled water.
16. Pipette 1500 L of air-saturated buffer (pH 1) into the reaction vessel of an oxygen electrode. Note the temperature of the water bath in which the buffer is immersed; you will need this for a subsequent calculation. If you do not have a thermometer, note the temperature as 30C.
17. Calibrate the electrode by turning the dial till it reaches 100. If the electrode reading fluctuates wildly or drifts steadily downwards, consult a member of staff.
18. Add 1000 μL of the 10 mg/mL yeast suspension to the reaction vessel, and start recording the data every 5 seconds.
19. When you reach 100 seconds, inject 25 L of 0.16 M glucose solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate until 200 seconds, at this time inject 25 L of 0.16 M sodium azide solution through the vent to the electrode chamber using a Hamilton syringe and allow the data to accumulate the end of the run (360 seconds). Ensure that the needle enters the electrode chamber.
20. Pipette out the contents of the reaction chamber with a dropping pipette and wash out the reaction chamber three times with distilled water.
21. Determine respiration rates before and after the addition of 25 L of 0.16 M solution of galactose by repeating steps 1-3.
seconds
Glucose pH 5
Galactose
pH 5
Glucose pH 1
Galactose
pH 1
Glucose pH 9
Galactose
pH 9
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