Biology lab assignment

Biology lab assignment

Abstract

Sodium azide is known to be metabolic inhibitor. There are experiments that have been done to demonstrate this effect. On the experiments in which this substance, sodium azide, was used where method of cellular transport used by saccharomyces cerevisiae (s. cerevisiae) was determined. The conclusion that was made from the experiment was that there was increase in the dye intake in the presence of sodium azide. The control experiment showed otherwise, that is decrease in uptake. From the suggestion point of view, the dye was able to diffuse freely into the cell. The other research with an aim of determining what may   affect the inhibiting effects of azide was conducted. In the experiment I made a decision to increase the concentration of glucose and observe what effect would have on the inhibiting factor of transport channels in s. cerevisiae. But from the results there was no change in the dyes uptake when the glucose concentration was increased.

Introduction

The experiment was performed to find out the uptake of neutral red dye by S. cerevisiae when metabolic inhibitor is present. From the results as shown in figure 1, the absorption rate increased in yeast cells when sodium azide was introduced. Since sodium azide is a metabolic inhibitor, it inclusion in the sample was anticipated to decrease the dye uptake. The opposite results were recorded where the dye uptake was increased. It is, in fact, the higher absorption rate was due to the introduction of the sodium azide in the experiment. Now the aim of the experiment was to find how this happened.  Getting different results made me think that S. cerevisiae, considering whether it is product uptake or secretion, involve two methods of cellular transportation.

Analyzing a similar experiment performed in the University of California where inhibitory effects of azide were tested. The results archived showed that azide (0.1-10millimolar) on the activity of the mitochondria fraction is a selective inhibitor of pH 9.0. Conclusion drawn from these experiment showed that the presence of sodium aside depleted the energy present in the mitochondria. The diffusion of across the cell were unaffected because the diffusion do not really rely on the energy to in order for it to take place. The other consideration was whether the increase in Adenosine triphosphate availability would result in an increase in the efficiency of active transport channels hindering the inhibitory effects of sodium azide. To check this required an experiment to find out the effects of increase of glucose concentration in an aim to promote glycolysis in releasing energy through which ATP is derived. From the results obtained by increasing the glucose decreased S. cerevisiae’s’ absorption of dye.

Methods

In performing the experiment I made use of Rowan (2010) methods to find out Cerevisiea uptake of neutral red dye in presence of a metabolic inhibitor. To ensure that the methods were correct, I made slight modification with an aim of obtaining the optimum results. Three separate solutions of glucose were made. These solution had different concentrations, that is, 5.6mM, 56mM and 560mM. In order to archive homogeneity, the preparation were vortex and then adjust the pH to 6.8.  To the three samples, I added 9ml of fresh yeast growth medium to suspension respectively. For further mixing I inverted the centrifuge tubes in order to produce the suspension and after which I separated the suspension for at least 12 minutes in order to rehydrate. After rehydration, each suspension were measured after which I put into centrifuge which is non-sterile.

Using different solution, I made three 1% stock of red dye.   To prepare the red neutral dye, the concentration obtained each yeast growth were used in the following ratios:  0%, 0.25 %, 0.75 and 1 %. After the dye was prepared, I took 200µl from each concentration and I added into the yeast growth medium preparations I took 200µl of 10 % Sodium azide and added to one set of samples from each suspension. I kept the remaining samples as a control. The next task I did was to vortex the   preparation and incubated for 30 minutes at room temperature after which I moved  the samples  into microfuge tubes and I  vortexed gently a leave the for approximately 30 minutes.

After the elapse of the 30 minutes I span the sample for approximately 2 minutes at 5000 rpm using a micro centrifuge machine. Using pipette I removed the supernatant and the cell resuspension followed with 1ml of fresh yeast growth medium and then I placed back the re-suspended samples in to the micro-centrifuge and spun again for 2 minutes at 5000 rpm. I then repeated this procedure for three washes. To the final resuspension 400 µl of yeast growth was added. I transferred 3-100µl of each suspension onto a microtiter plate and at read at 520 nm by the microspectrophotometer.

Results

Both Azide and the control group, figure 1 and 2 respectively were noted to yield similar concentrations. However, there is a possibility of an experimental error presented from the outliers that I observed where the data points were 3.386 and 2.285 (figure 2). The error is likely to have been caused by the abnormal transfer of the experimental samples into the microtitre plate’s wells. I noted that the standard deviation that was presented by the Azide groups 1.26. This results is an indication that the experimental results were not distributed normally. I therefore, omitted the outliers to affirm whether the distribution was really distributed abnormally. The results that came out of the omission became 0.769, suggesting that the distribution was normal.

The control groups presented similar results. Their relative standard deviation was noted to be 1.54, a figure that depicts abnormal distribution of results as well. Since there was a presentation abnormal distribution of results in both azide and the control groups, I found it useful to apply the KS-test comparative, cumulative test to compare both of them. In regard to increase in the concentration of glucose and that of dye, both azide and the control groups depicted similar groups.

While calculating the probability, there was no indication of much difference considering the data sets (p=0.96). When the compiled data is analyzed, there is an evidence that glucose had overawed the azide’s inhibition.

Figure 1: Graph of mean A 520 mm (read using microspectrophotomete) against the Actual dye (percentage).

The 0mM was used as a control experiment that determined the normalcy of the distribution of distribution of results.

Figure 2: Mean A 520 (read using microspectrophotomete) against the Actual dye (percentage).

I did this experiment as a self-design based on the previous experiment. In the previous experiment. The second part presents the results obtained from the initial experiment

Data collected from yeast samples in 5.6mM and 560mM glucose corresponded to our hypothesis. Yeast with 5.6mM glucose concentration had the highest absorbance of red neutral dye, 560mM came in middle, and 56mM came in the lowest, having the least absorbance of neutral red dye. From 0% dye concentration to 0.75% dye concentration, 5.6mM yeast given the control treatment had lower absorbance in comparison to its azide treated counterpart, but at 1% dye concentration control 5.6mM shows higher absorbance than azide.

Figure 3: Graph of Absorbance against dye concentration (Azide Group)

From 0% to 0.5% azide solutions containing 5.6mM and 560mM glucose concentrations had almost identical data points, but from 0.5% to 1% 560mM shows much lower absorbance than the 5.6mM solution. For azide vs. control at 560mM and 56mM control solutions always had lower absorbance than their azide counterpart as seen in figure 4.

When comparing absorbance of red neutral dye of all glucose concentration that were given the control treatment to the previous lab’s control, a large decline in 56mM can be seen. The 56mM of this experiment and control lab 1 were made using the same solution, and followed the same procedure.

Figure 4: Graph of Absorbance against Dye concentration (Control groups)

Comparing figure 1, 3 and figure 2, 4 respectively, the results in the former figures depicts smoothness in transition compared to those in the previous experiment (figure 3 and 4). This is because in the personal design, I endeavored to avoid any possible experimental error, thus, giving out a normal distribution of results which consequently enhanced smoothness in the results.

Discussion of the results

The objective of this experiment was to come up with the personal design upon the determination of the effects of azide in the glucose concentration. I rationalized that the active transport’s efficiency networks in cerevisae and that they depended upon the availability of the ATP. From this results, we noted that increasing the concentration of glucose is would consequently lead to more ATP being generated. However, by proving that there is a relationship between the variables used, then the final outcome would have indicated a decrease in the concentration of dye. When I noted that the results depicted no significant correlations based on the concentration of glucose and reducing the uptake of dye, then I had to reject the hypothesis that was given as an alternative.

However, the results indicated that there is a given amount of glucose whereby there is a permission of a higher concentration of glucose regardless of if there is sodium azide present or not. This is an indication that glucose can enhance decrease in metabolic functions and the activity of transport regardless of its inadequacy or excess of glucose. On the other hand, the transport activity and the metabolic functions are medium when the glucose concentration is medium as well.

In the previous experiment, I noted that Sodium azide behaves as a metabolic or mitochondrial inhibitor. Azide was found to inhibit the production of ATP by interfering with oxidative phosphorylation. Methods of transporting in a cell include diffusion or protein mediated active transport, and sometimes for large molecules endocytosis may be used. Movement of substances in and out of the cell is dictated by the gradient, the size of the molecule and the permeability of the membrane. Movement across the membrane occurs from high to low concentration in passive transport. A cell needs the assistance of ATP to go against the gradient from low to high concentration. A cell will always work to maintain a state of homeostasis, constantly importing and exporting to keep that balance. Since the neutral red dye is actively transported into living cells its import and export will depend on the cells energy availability.

Also in the new experiment, we made different solutions, where each solution had different glucose concentrations. Our solutions contained 5.6mM, 56mM, and 560mM concentrations of glucose. The hypothesis, which states that an increase in glucose concentration will allow anaerobic respiration to overcome the export inhibiting effects of azide on the yeast cell; is supported by solutions containing concentrations of 5.6mM and 560mM, as they have shown, that with an increase in glucose concentration the absorbance in red neutral red dye is decreased. This trend is seen in both the control and azide solutions containing 5.6mM and 560mM glucose concentrations. The only solution to show unpredicted trends was the solution containing 56mM, this solution was the only solution not to be made newly. Not only did it show random data points, but when compared to its control data from the previous lab it shows a large decline in absorption of the red neutral dye, as seen in figure 4.

In conclusion, the comparison of results from various experimental outcomes indicated that both the null and the alternative hypothesis are substantial to the experiment. This is based on the improvements and the repetitiveness that was adopted in observing the glucose concentration, sodium azide, and the uptake of dye as compared to the previous experiment. Therefore, it is worth concluding that there is no effect on the concentration of dye when the glucose concentration is altered.