1 |Mitchell, J.L.

Jaime Lee Mitchell

Bio-3T Lab Report (1)

Dr. Tahamont

3 November 2010

The Effects of Increasing Glucose in the Presence of Sodium Azide on the

Uptake of Neutral Red Dye in Saccharomyces cerevisiae

Abstract: In a previous experiment the metabolic inhibitor, sodium azide, was

used to help determine the method of cellular transport used by Saccharomyces

cerevisiae. Findings showed that the uptake of neutral red dye increased in the

presence of sodium azide as compared to the control. This suggested the

possibility that the dye was diffusing freely into the cell and becoming trapped

intracellularly due to inhibition of the active transport channels. Additional research

was done to examine the variables that may affect the inhibitory effects of azide.

We decided that changing glucose concentration could overcome inhibition of active

transport channels in S. cerevisiae. It was proposed that raising the concentrations

of glucose lead to an increase of ATP made available to the cell; therefore a

decrease in dye absorption would be observed due to increase in active transport

channels efficiency. The previous experiment was adjusted to include four

variations of yeast growth medium by integrating changes in glucose

concentrations. Microspectrophotometric readings were taken subsequent to the

introduction of neutral red dye and exposure to the metabolic inhibitor, sodium

azide. Results showed no significant increase in neutral red dye uptakes with

respect to the amount of glucose introduced. Therefore, the alternate hypothesis

was rejected and it is concluded that an increase in glucose has no effect

concentration of dye uptake by S. cerevisiae.

2 |Mitchell, J.L.

Introduction: Results of the experiment (Fig. 1), Uptake of Neutral Red Dye by S.

cerevisiae in the Presence of a Metabolic Inhibitor, indicate that there is a higher

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

millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial

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

of active transport channels thereby, overcoming the inhibitory effects of sodium

azide. To investigate this question, the experiment was developed to include an

increase in glucose concentrations. The intention was to promote glycolysis to

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

conditions of increased glucose.

3 |Mitchell, J.L.

Figure. 1

Methods and Materials: Similar methods derived the previous experiment,

Uptake of Neutral Red Dye by S. cerevisiae in the Presence of a Metabolic Inhibitor

(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

preparations were vortexed until homogeneity was achieved followed by adjusting

the pH to (6.8). Dilution of the samples was done by adding 9ml of fresh yeast

growth medium to each respective suspension. Further mixing was done by

inverting the centrifuge tubes. The suspensions were then set aside for 12 minutes

to completely rehydrate. Succeeding rehydration, two 1.8 ml samples of each yeast

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.

vortexing the preparations, they were incubated at room temperature for 30

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

microcentrifuge. Supernatant was removed with a pipette followed by cell

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

non-normality in distribution patterns; the KS-test comparative cumulative test for a

comparison of the azide and control group was useful. The two groups shared

similar results regarding increased glucose and dye concentration. Calculations of

probability showed no significant difference between the sets of data (P=0.95).

5 |Mitchell, J.L.

Analysis of compiled data showed no evidence that glucose had overcome the

inhibition of azide.

Figure 2.

Figure 3.
6 |Mitchell, J.L.

Discussion: The objective of this experiment was to determine the effects of

higher glucose concentration on inhibition caused by sodium azide. We

rationalized that efficiency of active transport channels in S. cerevisiae were

principally dependent upon ATP availability. Therefore, the idea was adopted that

an increase glucose concentration would lead to generation of more ATP, which is

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

and finding no significant correlations between higher glucose concentrations and

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

glucose concentration, as well as an excess, can cause a decrease in metabolic

function and transport activity. I decided to look for other studies that demonstrated

correlations between metabolic activity and glucose concentration. Results from a

study done on glucose concentration in growth medium and the metabolic activities

of L. mononcytogenes showed that an increase in glucose concentration led to a

7 |Mitchell, J.L.

decrease in metabolic activity, in addition to a decrease in catalase functions. There

was also an indication that acidic pH levels are also product of excess glucose

concentrations, an additional contributor that lowers metabolic activity and

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

fermentation, which indisputably led to a rise in pH value of the yeast cell

suspensions. Uncontrolled and controlled fermentation of B. cereus were performed

in a study at Johns Hopkins University. Their results indicated a valuable connection

between glucose, pH, and cellular permeability. They found that during

uncontrolled fermentation, depletion of glucose was observed, which led to a rise in

pH. They compared the permeability factor of these cells to those that were ph-

controlled and found that maintaining a pH of 7.0 with respect to glucose

concentration, is directly related to boosting the permeability factor (PF).

Furthermore, increasing the initial glucose concentration above .20M showed no

significant effect on PF. In fact, when glucose levels were maintained at .20M during

controlled fermentation, a 50% decrease in PF was observed (Silverman and Spira

1978). An additional study that linked glucose and pH levels showed that metabolic

functions are optimal when pH is maintained at (7.0) and glucose concentration is

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

glucose concentrations. Evidence presented in a study on hemoglobin catabolism

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

gene suppression in the mitochondria of S. cerevisiae is related to high glucose

concentrations. Seven genes are known to be directly affected by glucose.

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

concentration. After comparing results from our experiment to the findings of

several credible sources, relationships repeatedly observed between; glucose

concentration, sodium azide, and dye uptake. Therefore, it is acceptable to reject

not only the alternate hypothesis, but the null hypothesis as well.

Acknowledgments: I would like to thank my Biology 3T lab partner for their

contributions and academic input in developing the experimental protocol for, The

Effects of Increasing Glucose in the Presence of Sodium Azide on the Uptake of

Neutral Red Dye in Saccharomyces cerevisiae (Rowan 2010). Additionally, I would

like to express appreciation towards my colleagues at Rowan University for any

suggestions that aided in the formulation and completion of our experiment.

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

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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

Margaritis, A., Vogrinetz, J. 1983. The effect of glucose concentration and pH on

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10 | M i t c h e l l , J . L .

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Mitchell 2010. The effects of increasing glucose in the presence of sodium azide on

the uptake of neutral red dye in Saccharomyces cerevisiae. Biological

Sciences, Rowan University. pp. 14-17.

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