Membrane Transport of Neutral Red Dye in Saccharomyces cerevisiae
The purpose of the experiment was to determine which type of diffusion is used for membrane transportation in Saccharomyces cerevisiae. We took different concentrations of red dye to measure the absorbance rate the yeast cells took in. To compare our results, we had a controlled group of yeast growth medium with the concentrations of dye and we made a group of the same dye concentrations but with sodium azide mixed in the yeast growth medium. Our results showed that the control and +azide group both increased in absorbancy, although the +azide group had a higher absorbance rate. We concluded that yeast cells use facilitated transport. In our “Design Your Own” Experiment (DYO), we further supported our results by adding double the amount of glucose in the yeast growth medium. The added glucose would demonstrate a larger gap in the graph comparing the absorbance of +azide group and control. Our results from the DYO also showed an increase all groups but deemed inconclusive due to experimental errors.
The yeast species used was Saccharomyces cerevisiae cells. Their membrane allows certain things to pass in and out of the cell. Depending on what wants to come into the cell, a certain type of diffusion needs to be used: simple diffusion, facilitated diffusion, or active transport. Simple diffusion allows oxygen, carbon dioxide, and water in and out of the cell easily. Facilitated diffusion is divided into two subgroups that either use carrier proteins or channel proteins. Carrier proteins can move glucose across the membrane or they can use anion exchange proteins to create a balanced gradient of ions. Channel proteins use aquaporin to transport water rapidly. Active transport requires ATP to use a pump that takes sodium ions out of the cell and potassium ions in. Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) was used as an inhibitor. It is unclear how the yeast cells transport the dye so we mixed it with yeast growth medium (YGM) to make conclusions based on absorbance rate. The YGM was made of 56mM glucose and 20mM HEPES with a pH of 6.8. Glucose is used as an energy food source and will allow the red dye to be absorbed by the cells. To obtain our results, we made two experiments. Our first experiment was to determine the type of diffusion. The control group was a set of different concentrations of red dye with YGM. The +azide group had sodium azide mixed with the same YGM. From previous readings, we know that sodium azide is a poison. It blocks electron transfer that ultimately stops production of ATP. Therefore, since active transport requires ATP, the absorbance of +azide group could show a decrease if active transport is present. Comparing the control to +azide group will tell us the type of diffusion yeast cells use to transport the neutral red dye. We predicted that the yeast cells use facilitated transport since carrier proteins have a glucose transporter (GluT1). The absorbance would show an increase with a high concentration of dye to account for the large amount compared to a small amount. This type of diffusion would allow more dye in since the glucose is transported with it. Glucose is needed for the cell’s energy. Once we had concluded on the type of diffusion used, we created a second experiment (DYO). The purpose of our DYO was to further prove our conclusion by adding more glucose in the YGM, which would produce more ATP. More ATP would show a larger gap between +azide and control groups since +azide group cannot utilize the ATP. We hypothesized that by introducing a third experimental group, carrier proteins will be further supported by using a glucose rich yeast group to promote increased ATP production and thusly a lower absorbance than either azide or control groups. To record the absorbency in both trials, a microspectrophotometer was used to observe the wavelength for each