Yeast are eukaryotic microorganisms just like other organisms, they must respire in order to survive. Respiration can be defined as the controlled release of energy from organic compounds to form adenosine triphosphate (ATP). The type of respiration that occurs is called aerobic respiration. It occurs when glucose and oxygen are present. It can be summarized by the equation: .
Enzymes play a very significant part in respiration. During the different stages of respiration, enzymes that are often found in the matrix of the mitochondria are used to speed up reactions in the body by reducing a reaction’s activation energy, which means that less energy is required to start a reaction. Enzymes need to collide with their respective substrates in order to generate a reaction, increasing the temperature will give the enzymes and their substrates more energy, increasing the likelihood of collisions to occur, and successful collisions to happen, ultimately increasing the rate of respiration.
Triphenyl Tetrazolium Chloride (TTC) is a redox indicator often used in biochemical experiments to indicate cellular respiration. It is a white crystalline powder that can be dissolved in water, acetone and ethanol. TTC is colorless when oxidized and pink when reduced. TTC can be used to show the presence of active dehydrogenase enzymes by a change in color. TTC is heat and light unstable. During respiration, hydrogen ions or protons are released and picked up hydrogen acceptors, either NAD or FAD to form NADH+H+ or FADH respectively.
In this experiment, I will be find out how long does it take for yeast to dehydrogenases in different temperatures with the help of TTC. I was intrigued that TTC could change color. By using the kinetic theory and collision theory, it can be assumed that as the temperature increases, it would be expected that the time taken for the TTC to be reduced would decrease. They need to collide in order for the reactions to occur. Besides collision, they also require enough activation energy in order for the reaction to occur. It is more likely that molecules will have the sufficient activation energy at high temperature due to the increased kinetic energy in the molecules.
How does the temperature affect the time taken for the Triphenyl tetrazolium chloride to be reduced in actively respiring yeast cells?
Time Taken for indictor to reach endpoint (minutes) (possible errors ±1 seconds)
Temperatures 50˚C, 55˚C, 60˚C, 65˚C, 70˚C (possible errors ±0.5°C)
Measures to control the controlled variables
Effect on results
Actively respiring yeast suspension (ml of yeast and 4ml of 10% glucose diluted in water (6:4 ratio)) Syringe used to measure volume of yeast in order to get accurate and precise measurements so the volume in each test will as close as possible. Volume stated in method. Milliliters
If a bigger volume of Actively respiring yeast suspension was measured than what was stated, the time taken for the end point to be reached will be faster than it would be normally, because there are more respiring yeast cells present, reducing the time taken for the TTC to become reduced. Vice versa, if a smaller volume of Actively respiring yeast suspension was measured than what was stated, the time taken for the end point to be reached will be shorter because there are less respiring yeast cells present, increasing the time taken for the TTC to become reduced. Temperature in water bath
Water bath used to ensure that temperature remains as constant as possible. Degrees Celsius If one test is exposed to a higher temperature, the end-point might be reached faster as the enzymes have more kinetic energy. Alternatively, a lower temperature will make the time to reach the end-point slower.
Measures to control the controlled variables
Effect on results
Concentration of yeast
Yeast solution should be drawn from a single stock solution to prevent fluctuations in concentrations. %
If a bigger concentration of yeast was used than what was stated, the time taken for the end point to be reached will be faster than it would be normally, because there are more respiring yeast cells present which will increase rate of reaction, reducing the time taken for the TTC to become reduced. Vice versa, if a smaller concentration of yeast was used than what was stated, the time taken for the end point to be reached will be shorter because there are less respiring yeast cells present, increasing the time taken for the TTC to become reduced. Concentration of glucose solution
Glucose solution should be drawn from a single stock solution to prevent fluctuations in concentrations. %
If a bigger volume of glucose was measured than what was stated, the time taken for the end point to be reached will be faster than it would be normally, because there are more glucose molecules present, reducing the time taken for the TTC to become reduced. Vice versa, if a smaller volume of glucose was measured than what was stated, the time taken for the end point to be reached will be shorter because there are less glucose molecules present, increasing the time taken for the TTC to become reduced.
Measures to control the uncontrolled variables
Effect on results
Light sensitivity for TTC
Keep the TTC under shade
The solution will not or have a delay when turning pink, thus making the results inaccurate and unreliable Yeast concentration over time
Since yeast reproduces by binary fission, it will be hard to keep a constant concentration throughout the experiment. To reduce this effect, the experiment should be done as fast as possible and as close together as possible in order to reduce fluctuations in concentrations between tests. The results would be slightly in inaccurate.
i. Actively respiring yeast suspension (186 g of yeast and 124 g of 10% glucose diluted in water. This mixture should be prepared 2 hours in a beaker before the experiment is conducted) ii. Triphenyl Tetrazolium Chloride solution (31 cm3)
iii. Test tubes and rack
iv. Glass rods
v. Water baths at temperatures 50˚C, 55˚C, 60˚C, 65˚C, 70˚C vi. Thermometers
1. Set up water baths at 50˚C, 55˚C, 60˚C, 65˚C and 70˚C 2. Pipette 10 cm3 of yeast suspension into one test tube and 1 cm3 of TTC solution into another test tube and stand them both in the water bath. Leave for several minutes to the temperature of the water bath. 3. When the desired temperature is reached, mix the yeast suspension with the glass rod and TTC solution together and return the test tube to the water bath and start the stopwatch immediately. 4. Observe and note the time taken when the color reaches the endpoint (fig 1.) 5. Take qualitative observations (such as uncertainty of equipment and initial color) 6. Repeat this procedure 5 times for each temperature of water bath. (30 times in total) 7. Calculate the mean and standard deviation for the data in each temperature. 8. Plot data in a line graph, with temperature on the x-axis and average time taken to reach TTC’s end point on the y-axis.
As seen in the table above, the fastest mean time for the solution to reach the endpoint is 60˚C. The there is relatively low standard deviation for 50˚C, 55˚C, 60˚C and 70˚C except 65˚C. This suggests that the data does not have widely spread which is more reliable. However, the data for 65˚C has a high standard deviation, therefore suggests that the data has a wide spread which is less reliable. 70˚C has the same mean and no standard deviation because it took too long to reach the endpoint.
From the data above, we can see that as the temperature increased, the average time taken for the TTC to reach it’s endpoint inclined from 50˚C to 55˚C then gradually decline from 60˚C to 70˚C. It declines because yeast cells enzymes start to denature. The inclination can be explained through the kinetic theory. As the temperature increases, the enzyme and substrates have more kinetic energy. Thus, it will increase the chance of successful collisions occur as there is a higher chance that the enzyme and substrate both have the sufficient activation energy for the reaction to occur.
Firstly, there would have been less glucose available to the yeast than stated due to the fact that they respire when waiting for the mixture to be heated to the set temperature. When there are less glucose available, TTC may take longer to reach it’s point or may not reach the endpoint at all because of the lack of glucose. In order to improve this, the yeast and glucose should be heated separately and mixed together after it reaches the desired temperature.
The TTC’s endpoint could have been perceived differently. Since a colorimeter could not be used, it is difficult to be accurate about the same endpoint for each of the test. If the endpoint was perceived too early, the time recorded would have been shorter than the actual time. On the order hand, if the endpoint was perceived too late, a longer time would be recorded.
This may affect the results slightly as average times may be slight fluctuated from what it would be but not as significant to affect our conclusion, as there will be a general increase as the averages for each temperature are far from each other, so slight changes would not have a significant affect on the results. To improve this, this experiment should have more than one person to determine if the TTC has reached its endpoint. This will increase the reliability as it is not solely based on one opinion and reduces researcher bias. Instead of using TTC, there may be other forms of measurement, which may be less ambiguous, relation to rate of respiration could be used such as gas collection. Appendix 1