DETERMINING THE LETHAL CONCENTRATION OF 2, 4DINITROPHENOL AND ETHANOL ON THE GROWTH OF YEAST

CELLS
Prepared by: Isaac Castellano and Alexa Pudil
Prepared for: Dr. B. Mogen
Cell and Molecular Biology
Lab Section 11 & 12
Spring 2015
Abstract:
The main purpose of this experiment is to determine the minimum lethal concentration of 2, 4dinitrophenol and ethanol on common yeast cells, Saccharomyces cerevisiae (S. cerevisiae). The main
component is 2, 4- dinitrophenol, but the stock solution is too concentrated to what we prefer to start with.
The concentration of the stock solution of 2, 4- dinitrophenol is 250 mM, which is not soluble in water, so
we must dilute it with 95% ethanol. Our target is to determine if it is specifically the 2, 4- dinitrophenol,
or the ethanol control, that is inhibiting the cell growth. As one, or both, of these components are lethal to
S. cerevisiae at a specific concentration, we used multiple experiments and tests to determine the
concentration needed to inhibit S. cerevisiae. First, we created a standard curve for yeast cells by using a
hemocytometer, which is used to count the number of cells in a defined volume and the spectronic 20
(Spec 20) readings, measuring the ability of the yeast cell suspension to scatter light, in units of Optical
Density. Common yeast cells have an ideal wavelength between 625- 700 nm for absorption that creates
the minimal amount of interference to the yeast cell count. With the Spec 20 readings and the cell count,
we constructed a standard curve, which will help in the ability to count cells at a rapid rate. Next, we
observed how the toxicity of certain chemicals (2, 4- dinitrophenol and ethanol) affected the growth rates
of S. cerevisiae, and in turn, what concentration of these toxins that will inhibit, or terminate, the common
yeast cell growth. We concluded that the concentration between 1.8 mM of 2, 4- dinitrophenol and 5.4
mM of 2, 4- dinitrophenol inhibited yeast cell growth. To further the research, we would narrow down
the gap between the two concentrations and determine the exact concentration that officially inhibited the
cells.

Introduction:
As the presence of 2, 4- dinitrophenol (2, 4DNP) is constantly increasing in the global
atmosphere due to the use in wood
preservatives, industrial dyes, and as a
pesticide, more and more people have concerns
regarding their current health and long term
affects to their body. 2, 4- DNP is an inhibitor of
the electron transport chain and, in turn, stops

the synthesis of Adenine Triphosphate (ATP),

which is our cells main energy source.
Ethanol is also tested due to its act of inhibiting
cell growth and its effects on the cell membrane,
which is tested as a control factor. Ethanol is
used in many commercial items that are on the
market today such as industrial varnishes;

preservative for biological specimens and used

in many medicines.
Saccharomyces cerevisiae, a common bread
yeast cell, is a single-celled eukaryote. S.
cerevisiae is a model organism for eukaryotic
cells similar to those inside the human body, and
is readily studied due to its rapid growth rates
and its ease and convenience of growing
conditions. By using S. cerevisiae, we are able
to monitor the effects that ethanol and 2, 4-DNP
have on the cells. With the use of multiple
flasks that contain varying concentrations of 2,
4- DNP and Ethanol, we are able to monitor the
effects that the varying concentrations have on
the growth of S. cerevisiae and, in turn, the
amount of 2, 4- DNP and ethanol that it takes to
inhibit the cells growth, and if either or both of
them inhibit the cells growth. Both 2, 4- DNP
and ethanol, at high concentrations, provided
data that shows the effects on S. cerevisiae and
their growth patterns, which we can use as a
model to test other eukaryotic organisms.

to create our yeast cell growth curve. Figure 1

depicts the dilutions and the cells/ml, along with
the stock solution.
The next procedure was to test the yeast cells in
flasks with various concentrations of 2, 4dinitrophenol, diluted with ethanol; YPD broth
control; and finally ethanol control to determine
if it is the 2,4- dinitrophenol inhibiting the cell
growth, or if the ethanol is a contributing factor.
The following table illustrates the flask
designations with the specific treatments.

Figure [3] showing the separate flasks and the

specific treatments assigned to each, along with the
doubling time to show the amount of time for cells to
double, with shorter times showing more rapid
growth[5].

Materials and Methods:

We began our experiment by testing the
absorbance and transmittance of YPD broth,
which is our growth media, which showed at
which absorbance the growth media will have
the least amount of interference with the ability
of the yeast cells suspension to scatter light.
This is shown in Figure [1].
Next we created our broth growth media (YPD
broth), which consists of 2% peptone (w/v), 2%
glucose (w/v) and 1% yeast extract (w/v) and
then autoclaved at 121 degrees Celsius and 15
psi for a minimum of 15 minutes. This is done
to assure all living organisms are killed off to
avoid contamination. After autoclaving, we add
100 g/ ml of ampicillin to kill off any other
contaminants.
Following the sterilization process, we create
dilutions of the YPD broth of 1:3, 1:6, 1:10,
1:25, and 1:50. With each dilution, the cells
were counted using a hemocytometer, giving us
the cell count for the given volume. With each
dilution, we calculated the absorbance at 660 nm

The flasks were then cultured overnight and

incubated at 37 degrees Celsius and 200 rpm in a
rotary shaker. The next morning, 5mL samples
were collected and transferred to a test tube,
adding 100 mL of iodoacetic acid at a
concentration of 250 mM, which terminates the
cells growth to give an accurate reading at the
specific time. The samples are collected every
hour, on the hour for eight hours to be later
tested for the absorbance at 660 nanometers
using a Spec 20 machine.

Results:
This graph shows how the percent transmittance
and absorbance of YPD broth media fluctuates

with the change in wavelength (nm). The

absorbance is at the lowest when it reaches the
range of 625 nm- 700 nm, which gave us a range
to set our wavelength when we tested our
absorbance of later broth growth media.

Figure 2

Figure 1

A larger version of figure 2 is shown following

the results.

A larger version of figure 1 is shown following

the results.
The dilution factors applied to the YPD broth
that was used to count cells at a specific dilution
factor are shown in table 1. With the cell
number and the absorbance, a standard growth
curve was compiled.

Flask designation, with the specific treatments,

is displayed in table 2. The doubling time
determines the amount of time it takes for a cell
to double in size, relatively showing the growth
rate.

Table 2
Flask designation with specific treatments

Table 1

A larger version of table 1 is shown following

the results.
The standard curve for yeast cells of different
dilution factors and the corresponding cell count
per mL yeast stock is shown in figure 2.

Doubling time equation: G= (0.301*t)/ (log (N/N)

T= time elapsed
N= # of cells (Spec 20 reading)
N= initial # of cells (Spec 20 reading)

The yeast growth curve produced from various

concentrations of 2, 4- dinitrophenol, diluted
with ethanol, and an ethanol control is shown in
figure 3.

Figure 3

A larger version of figure 3 is shown following

the results.

Figure 1

Table 1

Figure 2

Table 2

Table 3
A Readings

Figure 3

Discussion:
Initially, we began by creating an absorbance
spectrum of our growth media, which depicted
that the best wavelength range to measure later
in the experiment was between 625- 700 nm as
this is when the absorbance is the least for the
YPD broth media. Within this range, we are
able to determine that 660 nm is the most
favorable wavelength as it gives us little
interference on the ability of a yeast cell
suspension to scatter light.
A direct method in cell counting is to use a
hemocytometer, which counts the individual
number of cells in a defined volume. By taking
dilution factors from our YPD broth media, we
were able to determine the amount of cells in a
defined volume for each dilution, and then take
the absorbance of each to create a standard curve
for yeast cells. By looking at our yeast cell
standard curve, we can conclude that the
standard curve had a correlation coefficient (r2
value) of 0.979, which tells us that it can be used
accurately without having to count cells and use
a hemocytometer each time. Our yeast cell
standard curve is a simple way to see the value
for the number of cells per mL of a solution and
its absorbance.
The yeast growth curve demonstrates that
increasing the concentrations of 2,4dinitrophenol had a negative influence on the
growth of yeast cells, particular between the
concentration of 1.8 mM and 5.4 mM of 2, 4dinitrophenol. As the doubling time, the amount
of time it takes for a cell to double in size,
supports, the cell grows at a rapid rate from 0.2
mM to 1.8 mM, but then is decreased rapidly at
the concentration of 5.4 mM of 2, 4dinitrophenol. This shows that the lethal
concentration of inhibiting S. cerevisiae is
between 1.8 mM and 5.4 mM of 2, 4dinitrophenol.
In contrast to testing the 2, 4- dinitrophenol, we
also tested to see if ethanol, a diluting agent in 2,
4- dinitrophenol, was also an inhibiting factor.
In conclusion, we determined that ethanol did
not inhibit the growth of S. cerevisiae at the

most lethal concentration we tested, 2.16 mL of

95% ethanol as the doubling time stayed
constant. This data shows that S. cerevisiae is
not inhibited by ethanol as their growth rates and
doubling times stay constant.

References:
1.] 2, 4- Dinitrophenol. (1992, April). In United
States Environmental Protection
Agency. Retrieved May 5, 2015,
from
http://www.epa.gov/ttnatw01/hlthef/dini
trop.html
2.] JoVE Science Education Database. Essentials
of Biology 1: yeast, Drosophila and C.
elegans. An Introduction to
Saccharomyces cerevisiae. JoVE,
Cambridge, MA, doi: 10.3791/5081
(2015).
3.] Greenfield, P. F., & Jones, R. P. (1987,
June). Specific and non-specific
inhibitory effects of ethanol on yeast
growth. In Enzyme and Microbial
Technology. Retrieved from
http://www.sciencedirect.com/science/ar
ticle/pii/014102298790055X
4.] Shakhashiri, P. (2009, February 5). Ethanol.
In Chemical of the Week. Retrieved
from
http://www.scifun.org/chemweek/pdf/et
hanol.pdf
5.] Doubling Time. (n.d.) Farlex Partner
Medical Dictionary. (2012). Retrieved
May 7 2015 from http://medicaldictionary.thefreedictionary.com/doubli
ng+time