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Dr. Tahamont
3 November 2010
cerevisiae. Findings showed that the uptake of neutral red dye increased in the
possibility that the dye was diffusing freely into the cell and becoming trapped
was done to examine the variables that may affect the inhibitory effects of azide.
introduction of neutral red dye and exposure to the metabolic inhibitor, sodium
azide. Results showed no significant increase in neutral red dye uptakes with
Introduction: Results of the experiment (Fig. 1), Uptake of Neutral Red Dye by S.
absorption rate in yeast cells in when sodium azide is introduced. Because sodium
azide is a metabolic inhibitor, it was hypothesized that its inclusion would lead to a
decrease in the dye uptake; however, results disproved the hypothesis since there
was an increase in dye concentration. The results showed that sodium azide did
have an influence on the dye concentrations, but the uncertainty of how remained.
The idea that S. cerevisiae may involve two methods of cellular transport, with
respect to either product uptake or secretion was considered. A similar study on the
inhibitory effects of azide at The University of California showed that azide (0.1-10
fraction (Gallagher and Leonard, 1982). This study adds to the evidence that
depletion of energy resulted from the presence of sodium azide, and a reduction in
the efficiency of active transport. However, the ability of the dye to enter the cell
was unaffected because diffusion does not require an energy source. This led us to
question whether or not an increase in ATP availability would increase the efficiency
release energy from which ATP is derived. This lead to the alternate hypothesis
that increased glucose will decrease the absorbance of dye uptake by S. cerevisiae.
The null hypothesis stated that no change in dye absorption will transpire under the
Figure. 1
(Rowan 2010) were used. Four separate 2.0% yeast suspensions were prepared
with 20mM HEPES and S. cerevisiae [Universal Foods, Milwaukee, WI], using glucose
concentrations in the amount of; 0.00mM, 56.0mM, 84.0mM, and 112.0mM. The
the pH to (6.8). Dilution of the samples was done by adding 9ml of fresh yeast
inverting the centrifuge tubes. The suspensions were then set aside for 12 minutes
suspension were measured out and put into non-sterile centrifuge tube. Each
concentration of fresh yeast growth mixture were used to prepare neutral red dye
at four concentrations; 0.00%, 0.25%, 1.25%, and 2.5%. Once dye was prepared,
200µl of each dye concentration were added to the yeast growth medium
preparations. 200 µl of 10% Sodium azide was then added to one set of samples
from each suspension and the remaining sets were used as a control. After
4 |Mitchell, J.L.
minutes. Following incubation, 200 µl of both azide exposed and control samples
were transferred into microfuge tubes, vortexed gently, and set aside for another
30 minutes. The samples were then spun at 5,000 rpm for 2 minutes in a
resuspension with 1ml of fresh yeast growth medium. The resuspended samples
were placed back into the microcentrifuge and spun at 5,000 rpm. This wash
procedure was repeated again, totaling three washes. The final resuspension
involved adding 400 µl of fresh yeast growth medium. Three 100 µl samples of each
suspension were transferred onto a microtitre plate and read at 520 nm by the
microspectrophotometer.
Results: In this study, both azide (fig.2) and control groups (fig.3) yielded similar
dye concentrations. Outliers observed (fig.2) at data points 3.397 and 3.295, can
be contributed to an experimental error during transfer of the sample into the wells
of the microtitre plate. Relative standard deviation of the azide groups is 1.27,
suggesting that the results are not normally distributed. However, when outliers are
omitted from the calculations, the relative standard deviation becomes .784,
indicating a normal distribution. Similar findings were found in the control group,
which have a relative standard deviation value of 1.45, also indicating that results
are not normally distributed. Since, both the azide and control groups presented
comparison of the azide and control group was useful. The two groups shared
Analysis of compiled data showed no evidence that glucose had overcome the
inhibition of azide.
Figure 2.
Figure 3.
6 |Mitchell, J.L.
principally dependent upon ATP availability. Therefore, the idea was adopted that
needed to carry out active transport. If these variables were proven to related, a
final result would show a decrease in dye concentration. After reviewing the data
reduced dye uptake, we had to reject the alternate hypothesis. However, results
showed that using 84mM of glucose permits the highest amount of dye secretion,
regardless if sodium azide is present or not. This suggests that both an inadequate
function and transport activity. I decided to look for other studies that demonstrated
study done on glucose concentration in growth medium and the metabolic activities
was also an indication that acidic pH levels are also product of excess glucose
suppresses enzyme function (Alm and Friedman, 1962). The pH was adjusted at
6.8 only once in our experiment. Subsequently, we had carried out pH-uncontrolled
between glucose, pH, and cellular permeability. They found that during
pH. They compared the permeability factor of these cells to those that were ph-
significant effect on PF. In fact, when glucose levels were maintained at .20M during
1978). An additional study that linked glucose and pH levels showed that metabolic
7.0g/L (Margaritis and Vogrinetz, 1983). Rather than experimental error, another
explanation should be considered regarding the outliers at data points 3.397 and
3.295 in the azide group, (Fig.2). Clearly, a relationship can be seen between the
high rise in dye concentration in accordance with both azide presence and high
indicated that the presence of azide suppresses enzyme function similar to that of
high glucose levels. Interestingly, azide does not directly interfere with metabolic
8 |Mitchell, J.L.
functions that require glucose, such as oxidation (Mills and Randall, 1957).
Therefore, it can be rationalized that in the presence of azide and glucose levels
above .20M, that metabolic functions are highly impeded. Additionally, I found that
ScADH3, is a gene that encodes for the mitochondrial protein responsible for
converting ethanol to acetaldehyde. Studies have proven that this gene can be
suppressed in the presence of glucose (Barrette et el., 1970). The null hypothesis
states that there is no effect on dye concentration with respect to increased glucose
not only the alternate hypothesis, but the null hypothesis as well.
contributions and academic input in developing the experimental protocol for, The
Furthermore, I owe infinite gratitude to Jason Sierberlich of Chickies and Petes, for
allowing me the extra time and therefore, opportunity to successfully construct this
lab report.
9 |Mitchell, J.L.
Bibliography
Alm, W. L., Friedman, M. E., 1962. Effect of glucose concentration in the growth
Kolmogorov-Smirnov Test. K-S data entry. [Internet]. [cited 8 Mar 2010]. Available
from http://www.physics.csbsju.edu/stats/KS-test.n.plot_form.html
Hydrodrogen. 8: 281-284.
10 | M i t c h e l l , J . L .
Mills, G. C., Randall, H.P. 1957. The protection of hemoglobin from oxidative
TX, pp.589-598.
6-12.
Mitchell 2010. The effects of increasing glucose in the presence of sodium azide on