Giana Melfi

Bio 3T Lab #1 Report

Does neutral red dye transport as simple or facilitated diffusion?

Abstract
In our preliminary experiment, we concluded that azide had no effect on
active transport, when determining its type of transport using Saccharomyces
cerevisiae. Because of those results, we decided to study the absorption of neutral
red dye in the cell membrane. In this experiment, we removed azide and added
additional dye concentrations ranging from 0 to 2% to determine if neutral red dye
transports as simple or facilitated diffusion. We replicated our experiment by using
Sample A and Sample B, and performed the same steps on both. Our results from the
readings of a microspectrophotometer showed that the dye moved into the yeast
cell by facilitated diffusion.

Introduction
Membrane transport is a main function in cells. While active transport
requires cellular energy, passive transport does not, and will move material from an
area of high to low concentration. In the preliminary experiment, sodium azide was
used to observe what type of transport occurred using Saccharomyces cerevisiae and

a variation of dye concentrations. Based off the graph below (figure 1), the
difference between the absorbencies of the control and the absorbencies of the
azide showed little to no difference. This experiments result led us to two alternate
hypotheses. The hypotheses stated, Neutral red dye moves into yeast cells by
facilitated diffusion, and neutral red dye moves into the yeast cells by simple
diffusion. In an experiment performed at the Indian Institute of Science, they used
Saccharomyces cerevisiae and aluminum, and their results showed that aluminum
enters the cell, and over a period of time a state of equilibrium is reached due to
passive transport (Rao and Easwaran, 1997). This experiment shows similar results
as to what we found where you can clearly see the plateau or state of equilibrium in
our results in both the preliminary and current experiment.

Effect of Azide on the Absorbancy of

0.8

Mean A520 Azide

0.6
0.4
0.2
0
0

0.2

0.4

0.6

0.8

1.2

Figure 1: The graph shows the preliminary results of the mean absorbance
measured I nthe microspectrophotometer at 520 nm for both the control and
azide concentrations. Data are presented as means +/- one standard deviation.

Methods
We prepared a 2.0% yeast suspension adding Saccharomyces cerevisiae to
yeast growing medium, and vortexed it until it was homogeneous. After, we allowed
it to sit for 5 minutes and vortexed it again. This was repeated twice. Next, we
created a more dilute yeast cell suspension (0.2%), and had it sit for 12 minutes.
While waiting, a 2.0% dye stock was made in a microfuge tube in the
following concentrations: 0, 0.125, 0.250, 0.325, 0.5, 0.625, 0.75, 0.825, 1.0, 1.125,
1.250, 1.325, 1.5, 1.625, 1.75, 1.825, and 2.0. There were two replicates for each dye
concentration. Each microfuge tube was vortexed and 30 minutes were allowed for
dye transport to occur. The microfuge tubes centrifuged at 5,000 rpm for 2 minutes.
The pellets were washed and then resuspended into yeast growing medium
followed by gentle vortexing. These wash steps were repeated 3 times. The cells
were resuspended in yeast growing medium. Each sample was then placed into 3
wells of a microtitre plate, and measured in a microspectrophotometer for its
absorbances at 520 nm.

Results
In this experiment, we used replication by doing sample A and sample B. The
results seemed to follow the pattern of the last experiment, where the dye
concentration and absorbances slightly increased, and then plateaued. In our last
experiment we found there was no difference in azide and the control. The graph
below (figure 2) displays the results of the dye absorbance in the yeast cells of

sample A. The mean absorbance of 0.75% did not follow the pattern shown and had
a mean absorbance of 1.99 nm.

Dye Absorbance in Yeast Cells (Sample A)

Absorbance (nm)

4
3.5
3
2.5
2
1.5
1
0.5
0
0 0.1250.250.375 0.5 0.6250.750.875 1 1.1251.251.375 1.5 1.6251.751.875 2 2.125
Dye Concentration (%)

Figure 2: The graph shows the results of the mean absorbance measured
in the microspectrophotometer at 520 nm for each dye concentration in
sample A. Data are presented as means +/- one standard deviation.

Discussion
Our main goal of the experiment was to see based off of our preliminary
experiment if the dye would move into the yeast cell by either simple or facilitated
diffusion. Based off our results, we concluded that the dye moved into the yeast cell
by facilitated diffusion. The results seemed to follow the pattern of the last

experiment, where the dye concentration and absorbances slightly increased, and
then plateaued. There is a clear plateau in our sample, which indicates the carrier
molecule was involved. In sample B, the results were considered inconclusive due to
an experimental error when transferring the samples from the microfuge tube to
the microtitre plate. Although contradicting our results, a similar study was
experimented at the University College of Dublin, studying the effect of redox dyes
using Hydrogen, Potassium, and Sodium, and they found very strong evidence of
active transport (Conway and Jernan, 1954). These results could have been affected
by the size of hydrogen, potassium, and sodium ions, in comparison to ours. They
also believed that dyes that lower the potential lowered the excretion and
absorption of the ions that they used. In future experiments, I feel that other factors
should be considered. An experiment was performed which used Listeria
monocytogenes grown in the presence of 1.0% glucose, and their results showed as
the glucose concentration increased, metabolic activity and catalase functions
decreased (Alm and Friedman, 1962). They also indicated the pH level when acidic
lowers the metabolic activity and suppresses enzyme function. Our experiments pH
was measured at 6.8 in the beginning of the experiment. In a future experiment, the
pH can be considered more. In conclusion, we were able to accept our one alternate
hypothesis that the dye moves into the yeast cell by facilitated diffusion.

Acknowledgements
I would like to thank my Biology 3T lab group Jessica, Amanda and Celeste,
for all working together and coming up with a successful experiment. Also, we

would not have been able to do this experiment without Rowans Lab Prep team. I
also thank my manager Ann Sundstrom for allowing me to be flexible with my
schedule while going to school.
Works Cited

________ 2009. Uptake of neutral red dye by Saccharmyces cervisiae in the presence
of a metabolic inhibitor. Rowan University. pp. 1-7.

Cirillo, V. 1962. Mechanism of glucose transport across the yeast cell membrane. J.
Bacteriol. 84(3): 485-491.

Conway, E., Kernan, P. 1955. The effect of redox dyes on the active transport of
hydrogen, potassium, and sodium ions across the yeast cell membrane.
Biochemical journal. 61(1): 32-36.

Friedman, M., Alm, W. 1962. Effect of glucose concentration in the growth

Rao, K., Easwaran, K. 1997. Al-NMR studies of aluminum transport across yeast cell
membranes. Molecular and Cellular Biochemistry. 175(1-2): 59-63.