The uptake of neutral red dye in a yeast cell using different solutions
Every cell transports materials in and out throught something called a membrane. There are many different methods of transport in the cell Saccharomyces cerevisiae (Serrano, 1977) We want to know does adding higher concentrations of azide more effectively block dye transport? We tested the transport of dye in yeast cells with a metabolic inhibitor. When we did this we showed no difference in the absorbance between different azide solutions, and our control. From this we concluded that azide has no effect on the transport through a yeast cell membrane.
Every cell has a layer of protection called the cell membrane. This cell membrane has many functions. The Saccharomyces cerevisiae cell has a selectively permeabile membrane which means it allows certian materials to pass through its membrane more or less than others ( Campbell et al., 2008). In a certian process called active transport these cells need energy to move material across the membrane ( Campbell et al., 2008). This energy that is being used is called adenosine triphosphate or ATP (Campbell et al., 2008). The production of ATP, which helps cells maintain a cells pH, helps with the uptake of neutral red dye because neutral red dye cannot be absorbed if the pH of the cell is reduced (Repetto, 2008). Saccharomyces cerevisiae has two types of active transporters through the cell membrane: primary and secondary active transporters (Stambuk, 2000). Sodium azide is a metobolic inhibitor which means it prevents ATP from being produced (Rowan University, 2009). In a study of Echerichia coli it was found that ATP was inhibited more than 90% using sodium azide ( Noumi, 1987).
The study of how things pass through different cell membranes is very common. So, studying how neutral red passes through a yeast cell is something that seems plausable to look at. We want to understand how different things will pass through the the cell membrane of yeast. In a previous experiment we saw that using a 10% azide solution, and a control group with neutral red gave us the same results of absorbance with the exception of two errors at 0%, and 2%. (Figure 1). This was concluded by using standard deviation error bars to see where the azide and control treatments were similar in absorbance.
Since, it was still believed that azide, being a metobolic inhibitor, should block dye transport we wanted to find out how much of this azide would cause this to happen. In a study done by Rikhvanov et al. (2001) Saccharomyces cerevisiae was compleatly inhibited by sodium azide. These results helped us to form a new question for our next experiment. We wanted to know does adding higher concentrations of azide more effectivly block dye transport? We predicted that higher azide concentrations will inhibit the dye transport into the yeast cell. There is always a possibility that more azide will have no effect on the dye transport.
First, we calculated how much azide we needed to make our concentrations of 10%, 20%, and 30%. We next made a yeast suspension using yeast growth medium ( 56mM Glucose, 20mM HEPES, pH 6.8) and Saccharomyces cerevisiae. After making our azide concentrations we made four dye concentrations (0%, 0.5%, 1.25%, 2.5%) from a stock dye solution and yeast growth medium (YGM). Next, we took the yeast suspension and added it to fresh YGM, to make a more diluted yeast solution. We seperated four 15-mL centrifuge tubes and added some of this diluted yeast solution to each. In tube 1, we added fresh yeast growth medium. In tube 2, we added 10% sodium azide, tube 3 we added 20% sodium azide, and tube 4 we added 30% sodium azide. 16 microfuge tubes were obtained and labled in groups of 4 every four tubes contained our 4 different dye concentrations.
We then transfered the control (diluted yeast suspension), and our Azide concentrations into smaller microfuge tubes that contained the different dye concentrations (0%, 0.5%, 1.25%,2.5%) respectivly. We then allowed these microfuge tubes to sit for 30 minutes, so transport could take place. We next placed these tubes in a microcentrifuge on 5000 rpm’s for 2 minutes. Finally, we removed the liquid portion from these tubes, making sure not to disturb the pellets on the bottom, and then resuspended each tube with fresh YGM, we did this 3 times. We did one more resuspension before we transfered these solutions into a microtitrate plate. We used a microspectrophotometer at 520 nm to read the absorbances of our solutions.
Figure 2: Dye percents versus absorbance in a control, 10%, 20%, and 30% azide solutions. In this graph you can see the error bars (using standard dieviation) are overlaping at every point except at the outliner which was at 2.5% dye concentration with 20% azide. Note how all the solutions (control, 10% azide, 20% azide, and 30% azide) show similar aborbance with each concentration of dye.
The results that were obtained showed us that higher azide solutions did not inhibit the dye transport as we thought. Therefore, our alternate hypothesis was not supported. Although, azide is a meabolic inhibitor it did not show any effects during this experiment. The overlaps in the error bars suggest that there is no significant difference in the absorbance with different dye concentrations. The source of error in this experiment is the outliner that was previously mentioned. This could have been caused from several different things, most likely a pipetting error. In other experiments they found that the absorbance of neutral red dye into a yeast cell will increase the longer you leave the dye to sit with the cells (Repetto, 2008).
Since, the dye and the cells were only incubated for 30 minutes it is possible that the absorbance was low because of the minimal amount of time. If azide is known to inhibit transport why in this situation did it not? In an experiment conducted by Rikhvanov et al. (2001) they found that azide compleatly inhibited the respiration of Saccharomyces cerevisiae . They instead grew the Saccharomyces cerevisiae cells at 30*C. Possibly growing these cells using different temperature requirements is what allows the sodium azide to take effect.
2009. Uptake of neutral red dye by Saccharomyces cerevisiae in the presence of ametabolic inhibitor. Biological Sciences, Rowan University. pp 1-8. Campbell, N.A., Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V.,Jackson, R.B. 2008. Biology, 8th ed. Pearson Benjamin Cummings, San FranciscoCA, pp. 125-138. Repetto, G., del Peso, A., LZurita, J.2008. Neutral red uptake assay for the estimation ofcell viability/cytotoxicity. Nature Protocols. 3: 1125 – 1131. Rikhvanov, E.G., Varakina, N.N., Rusaleva, T.M., Rachenko, E.I., Voinkov, V.K. 2002.The effects of sodium azide on the thermotolerance of the yeasts Saccharomycescerevisiae and Candida albicans. Microbiology. 71(6): 662-665
Stambuk, B.U. 2000. A simple laboratory exercise illustrating active transportnext term inprevious termyeast cellsnext term. Biochemistry and Molecular Biology Education.28(6): 313-317. Serrano, R. 1977. Energy requirments for maltose transport in yeast. European Journal ofBiochemisrty. 80: 97-102
Noumi, T., Maeda, M., Futai, M. 1987. Mode of inhibition of sodium azide on H+-ATPaseof Escherichia coli. Department of organic chemistry and biochemistry, theinstitute of scientific and industrial research, osaka university. 213(2): 381-384
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