1. !1
!
!
!
Optimizing the MTT assay to test the Lymphotoxic activities of isolates of
Batrachochytrium dendrobatidis
!
!
!
!
!
!
!
!
!
!
!
!
!
BSCI 283: Independent Laboratory Research (2.0 credit hours) Spring 2015
Mentors: Dr. Louise Rollins-Smith and Laura K. Reinert
20 April 2015
!

2. !2
Abstract
Chytridiomycosis is a fungal disease that has been linked to the decline, or in severe
cases, the extinction of about two hundred species of frogs worldwide (Skerratt et al. 2007). This
emergent disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). In the past,
researchers measured the virulence of B. dendrobatidis by infecting live frogs and observing the
effects. Rather than sacrificing a frog every time researchers wanted to study an isolate of B.d,
the Rollins-Smith lab developed assays to measure one virulence trait (immunotoxicity) using
populations of Jurkat cells (immortal human T cells) as targets. One assay uses 3H-thymidine to
radioactively label the chromosomal DNA of Jurkat cells during cell division. The radioactively
“tagged” cells are then counted using a scintillation beta-counter. Because this assay is more time
consuming and involved handling radioactive material, the MTT assay was developed as a more
efficient alternative for testing the virulence of Bd. The other commonly used assay is an MTT
assay. In this assay, mitochondria in living cells convert the water-soluble MTT reagent to
insoluble purple formazan detectable using a spectrometer to calculate light absorption.
Because numerous isolates of Bd need to be tested for their virulence, it is of great
importance to develop a precise protocol for comparison of these isolates. The studies reported
here examined several variables in the MTT assay in an effort to develop a standard optimized
protocol. The new MTT assay protocol was used to analyze the virulence of an isolate of Bd
from South Korea in comparison with other previously studied isolates. The revised MTT assay
protocol now examines the activity of Bd isolates at a temperature that is within the tolerance
limits of the pathogen (at 27°C) for three days. The MTT protocol also uses a higher
concentration of etoposide for the negative control. After using this improved MTT assay on

3. !3
KR-323, the South Korean isolate, the results indicated that the strain is not effective at
prohibiting Jurkat proliferation. For this assay, the Jurkat cells actually experienced more growth
when combined with the KR-323 compared to the samples of Jurkat cells alone. Due to the high
variability of the results of the KR-323 MTT assay, more tests need to be run on this isolate to
ensure the data trends are accurate.
!
Introduction
Since 1980, hundreds of species of amphibians have experienced rapid declines. Up to
approximately two hundred of these amphibian species declines are thought to be due to the
rapid spread of chytridiomycosis, the lethal disease caused by the fungus, Batachochytrium
Dendrobatidis (Bd) (Skerratt et al. 2007). Bd is highly transmissible due to its ability to survive
in water, on the skin of mature amphibians, and inside the mouths of tadpoles (Longcore et al.,
1999). Bd cells inhibit lymphocyte proliferation and induce apoptosis, severely crippling the
host’s immune system (Fites et al. 2013). Different lineages of Bd have been detected in
geographically diverse locations, and some have been designated global panzootic lineages
(GPL). Recently, in a study performed at Seoul National University, unique and possibly less
virulent strains were discovered in South Korea (Bataille et al. 2013). The found that Korean
isolates of Bd had about a 40% prevalence rate in 11 out of the 17 amphibian species native to
South Korea (Bataille et al. 2013). Along with the high Bd prevalence, this study also found a
low infection intensity, suggesting that some South Korean isolates are less virulent than other
lethal panzootic strains. After reading the Bartaille study on South Korean isolates of Bd,
Rollins-Smith lab decided to study the virulence of newly discovered Korean Isolates in

4. !4
comparison with other isolates at the lab.
To test the virulence of Bd isolates, two types of in vitro growth inhibition assays were
used: an MTT assay and a tritiated thymidine assay. Both assays test the ability of Bd to suppress
the proliferation of Jurkat cells, an immortalized line of human T cells. During my time working
in the Rollins-Smith lab, my focus was on modifying the currently used MTT assay to yield the
most precise results so I could then use the assay to test the virulence of a South Korean isolate
of Bd. Because persistent variation has been observed repeatedly with the use of MTT assays, my
project sought to alter variables in the assay to produce the most optimal results.
!
Materials & Methods
MTT Assay
The MTT assay is an in vitro assay that measures the ability of Bd strains to inhibit
proliferation of Jurkat cells, an immortalized line of human T-lymphocytes. MTT (3-(4.5-
Dimethyltriazol-2-yl)-2.5-Diphenylterazolium Bromide is a yellow solution that is converted to a
colored formazan product by mitochondrial enzymes in viable cells (Sladowski et al. 1992).
These purple-blue MTT formazan crystals can be solubilized with the addition of DMSO
(dimethylsulfoxide). The intensity of the solution’s color correlates with the number of living
cells present; the intensity of the color is measured using a spectrometer set at a constant
wavelength. In samples with low optical density readings, Jurkat proliferation was inhibited
more successfully than samples with high OD readings. One can infer that greater suppression of
Jurkat growth corresponds to greater possible virulence of the Bd isolate. Before performing the

5. !5
co-culture Bd and Jurkat MTT assay, the Jurkat cells and Bd zoospores must be cultured,
counted, and prepped for the assay.
a. Culture and Preparation of the Jurkat Cells
The Jurkat cells were used as targets to quantify the virulence (immunotoxicity) of
various Bd strains as well as serve as the positive control for the MTT assay. Because Jurkat cells
are such an important component of the MTT assay, one must ensure there is adequate Jurkat
growth in fresh medium. Jurkat cells were grown in an RPMI medium supplemented with 100
IU/mL penicillin, 100 ug/ml streptomycin, and 10% fetal calf serum (complete RPMI). This
RPMI medium was also refreshed every 2 weeks with 2mM L-Glutamine, an amino acid mixture
that maximized Jurkat cell growth. The Jurkat cells were subcultured every 3 days at a 1:10
dilution in fresh RPMI medium. They were incubated in a 5% CO2 chamber at 37°C. For the
MTT assay, the viable Jurkat cells were counted using trypan blue and resuspended at a density
of 1x106 cells/mL.
b. Culture and Preparation of the B. dendrobatitis zoospores
Three chytrid fungal isolates of were used in this project: JEL 197 and KR-323. JEL 142
is a closely related but non-pathogenic chytrid fungus, Homolaphlyctis polyrhiza. JEL 197 and
JEL 142 both belong to the Rhizophydiales order and have been studied extensively in the
Rollins-Smith lab. The JEL 197 and JEL 142 were subcultured every 3-4 days to supply the cells
Figure 1: This is a photo of a 96-well plate
after an MTT assay was performed. The
cell concentrations increase as one moves
from left to right across the plate. After the
plate was incubated for 2 hours MTT, the
purple formazan product was extracted
using Dimethyl sulfoxide. As one can see
from the different intensities of the purple
color, higher amounts of cells result in a
darker and stronger purple color due to the
higher formazan production.

6. !6
with fresh, nutrient-rich media. The JEL 197 was subcultured with 1% tryptone broth (T-Broth)
at a 1:10 dilution and the JEL 142 was subcultured with peptonized-milk-tryptone-glucose agar,
at a 1:100 ratio. The KR-323 isolate was subcultured with the same 1% tryptone broth. About 2
mL of one-week-old Bd cultured cells were seeded onto 1% tryptone-broth or PMTG plates,
sealed, and left to grow in the 26°C incubator. In order to test the virulence of Bd using an MTT
assay, the Bd zoospores must mature into thalli and zoosporangia. To collect zoospores for the
assay, the plates were flooded with about 2ml of 1% T-Broth which was pipetted off and
collected. The plates were flooded again and the broth was left to sit on the plates for
approximately 20 minutes. To remove the cells from the plate, the plate was flooded a third time
and the broth was removed promptly. The broth and Bd cell preparation was filtered using 20um
pore filters, that separate the zoospores from the more mature cells in T-Broth (zoospores pass
through and mature cells are retained on the filter). Once the zoospores were filtered from the
zoosporangia, the were counted using the hemocytometer slide. Using the cell count and the total
volume of Bd from the sample, I was able to dilute the culture in T broth or PMTG (for JEL 142)
to a cell density of 10 x 106 cells/mL. With the culture at a new density of 10 x 106 cells/mL, 1ml
of Bd cells would be combined with 9 mL of T-Broth in a sterile flash to bring the density in each
flask to 1 x 106 zoospores/mL. Three flasks were incubated at 19°C for 48 hours. This method
was designed to ensure that all Bd spores were at the same life stage, 48-hour-old germling so
that any variability of virulence would be due to genetic differences rather than differences in
developmental rate in culture during the MTT assay.
After the 48hour incubation, the cells were harvested and resuspended in new medium.
The first step of this process was to scrape the sides of the flasks with cell scrapers to ensure

7. !7
collection of all Bd cells. Then the flasks were rinsed with .05 to 1 mL of RPMI, and the contents
were transferred into separate 50mL conicle centrifuge tubes. These centrifuge tubes were spun
at 1500 RPMI for 20 minutes to collect the cells into a pellet at the bottom of the concicle. The
majority of the supernatant in the tubes was removed, leaving only about 1 ml remaining so as to
not disturb the pellet of cells. The pellet and reminaing superrnatant were vortexed to resuspend
the pellet. I counted the cells and the volumes were then adjusted to achieve concentrations of 5
x 106 cells/mL, 2 x 106 cells/mL, and 1 x 106 cells/mL for co-culture with Jurkat cells.
c. Co-Culture Assay
Co-culture assays of Bd and Jurkat cells were carried out in flat bottom 96-well plates
that each contained six replicates of a blank (medium only), a positive control (Jurkat cells
only), and a negative control (with added etoposide), as well as the experimental co-cultures. The
blank column on each plate was 100ul of RPMI to control for the color of the solution without
any cells in it and any background signal. For the positive control, 50 ul of Jurkat cells at 1 x 106
cells/ml were combined with 50ul of RPMI in 6 replicate wells. The negative control was 50 uL
of Jurkat cells at 1 x 106 cells/mL combined with 50 ul of etoposide, a small molecule that
prohibits DNA synthesis and leads to cell death. My project determined that the most optimal
concentration of etoposide to use as the negative control was 125 ug/mL.
Along with the positive and negative controls, the plates contained six replicates of co-
cultures of Jurkat cells with JEL 197, JEL 142, and KR-323 at cell densities of 5 x 106cells/mL,
2 x 106 cells/mL, and 1 x 106 cells/mL. The isolates I used for each assay, as well as the cell
densities, varied due to different variables of the assay along with the cell count. In order to
control for potential interference of Bd mitochondrial activity with Jurkat cell mitochondrial

8. !8
!
activity, each Bd isolate was also plated in complete RPMI without any Jurkat cells. Figure 1
shows a plating diagram of a typical MTT assays.
MTT Development
The plates were incubated at 26°C for 3 days in a 5% CO2 incubator. Following the
incubation, 100 ul of sterile MTT in PBS (500 ug/ml) was added to each well using a
multichannel pipet. The plates were then returned to the 37°C incubator for 2.5 hours to allow
the samples enough time develop. The plates were then centrifuged at 3000 RPM for 30 minutes
to collect the remaining cells at the bottom of each well. The supernatant remaining after
centrifuging was removed with one inverted shake of the plate into the hazardous waste
container. The contents were resuspended in 100ul of DMSO (dimethyl sulfoxide) carefully to
avoid the introduction of air bubbles which could invalidate the data. The plate was incubated at
37°C for about 10minutes or until the crystals appeared to have been disassociated. The
Figure 1: This is an example of a
typical plate diagram used in an MTT
assay.
!
Column 2: 100 ul RPMI
Column 3: 50 ul Jurkat, 50 ul RPMI
Column 4: 50 ul JEL 142, 50ul RPMI
Column 5: 50 ul JEL 142 50ul Jurkat
Column 6: 50 ul JEL 197, 50 ul
RPMI
Column 7: 50 ul JEL 197, 50 ul Jurkat
Column 8: 50 ul Jurkat, 50 ul
etoposide
!

9. !9
Absorbance was read using the Biotek plate reader at an optical density of 570 nm after a fast 2
min shake.
Results
*Statistical Test: Growth inhibition of Jurkat cells in comparison with the positive control was
compared by an unpaired Student’s t-test. A p value of ≤ 0.05 was considered significant.
The results from this project can be divided into two sections: results from experiments
optimizing the MTT assay and data from testing the virulence of the Korean isolate. The three
main variables I tried to alter in the MTT assay were: incubation temperature after plating,
concentration of the chemical inhibitor, etoposide, used in the negative control, and length of the
incubation period. Using the results from these three experiments, I was able to optimize the
MTT assay and use the improved MTT protocol to test the virulence of KR-323.
I. Optimizing the MTT assay
a. Incubation Temperature
I and other members at the Rollins-Smith lab were curious about the incubation
temperatures of cell cultures during the MTT assay. Previous experiments in the Rollins-Smith
lab have shown that Jurkat cells grow best at 37°C, which is equivalent to 96°F or optimal
human body temperature. Bd zoospores prefer to mature and divide at lower temperatures.They
are killed at temperatures above 30°C. The previous protocol for the MTT assay calls for the
plate with the Jurkat/Bd co-cultures to be incubated at 37°C. Although Bd cells are killed at this
temperature, their inhibitory products would still be released. This part of my project tested the
extent to which Jurkat cells would grow at 26°C compared to 37°C at varying concentrations.
For this experiment, I prepared both plates with three different concentrations of Jurkat cells as

10. !10
well as a blank column of media-only and a negative control. Each plate contained 5 replicates of
each treatment.
Jurkat growth at 50,000cells/well at 26°C compared with the negative control of Jurkat cells in
the presence of 12.5ug/mL of etoposide were significantly different (p ≤ 0.0001). Because there
was a statistically significant difference between the positive and negative controls at 26°C, the
Rollins-Smith lab decided that the incubation temperature for the MTT assay could be adjusted
to 26°C. At 26°C, etoposide is still able to substantially inhibit Jurkat growth. The lower
Figure 3: This figure is a bar graph illustrating the results of two MTT assays performed at
26°C and 37°C. Standard errors are denoted for each treatment. The high optical density (OD)
readings signify more mitochondrial activity from Jurkat cells. Therefore, low optical density
readings correlate with little to no Jurkat mitochondrial activity. The best Jurkat growth was
seen at 37°C at 50,00cells/well or a concentration of 1 x 106 cells/mL. Jurkat growth at 1 x 106
cells/mL at 26°C was still very high and could potentially be used as a positive control in an
MTT assay at 26°C.
!0.05%
0%
0.05%
0.1%
0.15%
0.2%
0.25%
0.3%
0.35%
0.4%
0.45%
Jurkat%at%50,000cells/well% Jurkat%at%20,000cells/well% Jurkat%at%10,000cells/well% 50ul%etoposide%and%10,000%
Jurkat/well%
OD#(570nm)#
Treatment#
OD#Readings#at#37°C#vs#26°C#
37%C% 26°C%

11. !11
temperature would maximize the results by disadvantaging Jurkat cells but providing a more
optimal temperature for the growth of Bd.
b. Length of incubation period for MTT assay
After changing the incubation temperature for the MTT assay to 26°C, I then investigated
the possibility of shortening the time period it takes to culture the Jurkat cells and still obtain
significant inhibition from virulent Bd isolates. The traditional time it takes to culture the Jurkat
cells is three days, but if this time could be shortened MTT assays could be performed in
significantly less time. For this experiment I prepared three 96-well plates with the following
treatment groups:
• 100 ul RPMI (blank)
• 50 ul Jurkat at 1 x 106 cells/mLand 50ul RPMI
• 50ul Jurkat at 1 x 106 cells/mL and 50ul etoposide at 12.5ug/ml
All plates were incubated in the 5% CO2 chamber at the previously determined 26°C for 1, 2, or
3 days.
!
!0.1%
!0.05%
0%
0.05%
0.1%
0.15%
0.2%
0.25%
0.3%
0.35%
1%Day% 2%Day% 3%Day%
Od#Reading#(570nm)#
Incuba4on#Period#
Jurkat#Prolifera4on#over#different#Incuba4on#Periods#
Jurkat+RPMI%
Jurkat+Etoposide%
Figure 4: This figure provides
a graphical representation of
the comparison between
positive and negative controls
for the MTT assay over three
incubation periods. The one
day incubation period samples
showed no growth while the
two and three day plates
exhibited high rates of
mitochondrial activity in
Jurkat cells.

12. !12
One of the first conclusions myself and others at the Rollins-Smith lab drew about this
experiment was that a one-day incubation period was definitely not enough time for the cells to
proliferate. The two-day incubation period samples showed a great deal of mitochondrial Jurkat
activity but very little inhibition by the etoposide. Thirdly, the three day sample of Jurkat cells
displayed fairly high Jurkat growth and much more evident inhibition of growth by the
etoposide. Because the etoposide failed to inhibit Jurkat proliferation during the one and two day
incubation periods, we determined the three day incubation was necessary to obtain a significant
inhibition in comparison with the negative control.
c. Concentration of Etoposide used for negative control
The surprising results regarding the lack of etoposide inhibition on Jurkat cell growth
caused the Rollins-Smith lab to experiment with various concentrations of etoposide to use as the
negative control. The third experiment for this study analyzed the effectiveness of different
concentrations of etoposide at inhibiting Jurkat growth over 1, 2 and 3 day incubation periods.
With this experiment design, two variables could be analyzed together to determine the
incubation time and concentration with the lowest Jurkat growth, or highest inhibition by
etoposide. Once again I prepared three plates to incubate at 26°C in a 5% CO2 chamber over 1, 2
and 3 days, respectively. I compared the growth of Jurkat cells at a concentration of 1 x 106
cells/mL to two fold dilutions of etoposide at concentrations of 0 to 125 ug/mL. Each plate
contained 6 replicates of 5 treatments as well as a blank column to calibrate the spectrometer.

13. !13
By analyzing each variable individually, one can see that the three-day incubation period
is definitely the most effective incubation time for inhibition of Jurkat growth by etoposide.
Although the three-day incubation period positive control did not achieve much growth during
this experiment, I and others at the Rollins-Smith lab have observed significant Jurkat growth
using the three day incubation period. The highest concentration of etoposide, 125 ug/mL was
most effective at inhibiting Jurkat growth. By combining these two variables, I inferred the
negative control would be most effective using etoposide at 125 ug/mL and a three day
incubation period. A two-tailed, unpaired, Students t-test the difference was performed testing the
significance of the difference between the three day positive conttrol and the negative control at
Figure 5: This figure is a
graphical representation of the
effectiveness of various
concentrations of etoposide at
inhibiting Jukat growth over
different incubation periods. It is
clear that the longer incubation
periods and higher concentrations
of etoposide are more effective at
inhibiting Jurkat growth.
!0.1%
0%
0.1%
0.2%
0.3%
0.4%
0.5%
0.6%
%Jurkat%0%ul%etoposide% Jurkat%and%Etoposide%at%
15.625ug/ml%
Jurkat%and%Etoposide%at%
31.35ug/ml%
Jurkat%and%Etoposide%at%
62.5ug/ml%
Jurkat%and%Etoposide%at%
125ug/ml%
OD#reading#
Treatment#
Effects#of#Culture#Period#and#Etoposide#Concentra9on#on#Jurkat#
Growth#
1%day%
incuba@on%
2%day%
incuba@on%
3%day%
incuba@on%
0"
0.02"
0.04"
0.06"
0.08"
0.1"
0.12"
0.14"
0.16"
0.18"
0.2"
Day"1" Day"2" Day"3"
OD#(570nm)##
Treatment#
Effect#of#Culture#Period#on#Etoposide#Effec=veness#at#
Prohibi=ng#Jurkat#Growth##
Jurkat"and"Etoposide"at"125ug/ml"
50ul"Jurkat"50"ul"RPMI"
Figure 6: This bar graph illustrates
the effectiveness of etoposide at
125 ug/mL at inhibiting Jurkat
proliferation. This data rules out
the possibility of performing an
MTT assay with a 1-day
incubation period because the
negative control is unable to
effectively inhibit Jurkat cell
growth.

14. !14
125ug/mL. The calculated P-value is less than 0.0001. By conventional criteria, this difference is
considered to be extremely statistically significant. Although the positive control of Jurkat and
RPMI did not proliferate as well as it has in the past, there was still a statistically significant
difference between the Jurkat and RPMI versus the Jurkat and 125ug/mL of etoposide over the
three-day incubation period. These results, combined with those from the previous experiment
regarding length of incubation period, led us to keep using the three day incubation period for the
MTT assay. However, because the higher concentrations of etoposide were much more effective
at inhibiting Jurkat growth than the previously used 12.5 ug/mL, the MTT assay protocol now
calls for using a negative control of Jurkat combined with etoposide at a concentration of 125 ug/
mL.
II. Virulence of Korean Bd Isolate KR-323
To test the virulence of KR-323, 2 standard MTT assays was performed using the updated
protocol. The assays compared the virulence of KR-323 with two other previously tested
isolates: JEL 197 and JEL 142. Previous studies have shown specific South Korean isolates of Bd
to be less virulent and have a unique and complex haplotype (Batialle et al. 2013). I hypothesized
that this SouthnKorean isolate would also be less virulent due to the genomic differences
between selected Korean isolates and other strains of Bd worldwide. The MTT assays performed
used Jurkat cells at a concentration of 1 x 106 cells/mL co-cultured with JEL 197, JEL 142, and
KR-323 at 2 x 106 cells/mL. Along with these 3 treatments, a positive control of Jurkat cells and
a negative control etoposide at 125 ug/mL and Jurkat were also included in each assay. For both
assays, there were 6 replicates of each treatment.
!

15. !15
!
A two tailed unpaired T-test was run to test if the inhibition of KR-323 was statistically
significant compared to the positive control. The two-tailed P value equals 0.0669 and using a P-
Value of .05 as the significance threshold, the difference between these two means is not
statistically significant; this supports the hypothesis that KR-323 does not significantly inhibit
Jurkat growth. When this MTT test was repeated for a second time, it produced similar results
also supporting the claim that KR-323 does not suppress Jurkat growth.
Discussion
Before examining parameters associated with B. dendrobatidis in MTT assays, I focused
on obtaining maximal Jurkat growth for the positive control and minimal Jurkat growth for the
negative control. The positive control for the currently used MTT assay is Jurkat cells at a
density of 1x106 cells/ml with RPMI growth medium at 37°C and 5% CO2. Although the Jurkat
cells have been known to grow well at 37°C, B. dendrobatidis is more effective at inhibiting
Jurkat cell proliferation at 26°C, as this temperature is more favorable to the survival of the
pathogen. A previous study that investigated the impact of temperature on Bd effectiveness by
Figure 6: This is a graphical
representation of the results from one
of the MTT assays performed testing
the virulence of KR-323 in comparison
with other, previously studied strains.
These preliminary results supported my
hypothesis that KR-323 is less virulent
than JEL 197 and JEL 142.

16. !16
inoculating live frogs. The frogs were infected with Bd and stored in a 22°C chamber. Within 35
days after metamorphosis, 50% of the frogs housed at 22°C died, meaning the Bd proliferated
and were very effective at killing their hosts at this temperature (SE Andre et al. 2008). I
performed an experiment testing the extent at which Jurkat cells supplemented with RPMI would
proliferate at 26°C and 5% CO2 to determine if this was an appropriate temperature for
incubating Jurkat and Bd co-cultures. At 26°C, the Jurkat cells exhibited a fairly high
proliferation which led the Rollins-Smith lab to change the incubation temperature for the MTT
assay from 37°C to 26°C.
Due to the increasing number of Bd isolates being harvested worldwide, it is of great
importance to test the virulence of these isolates. I investigated whether it would be possible to
shorter the incubation period of the MTT assay to one or two days. Although the highest Jurkat
growth was observed at the two day incubation time, the three day incubation period is necessary
to obtain signifiant growth inhibition by the negative control. A study titled “The Course of
Etoposide-induced Apoptosis from Damage to DNA” investigated etoposide effectiveness at
inducing apoptosis in fibroblasts. This found etoposide was significantly more effective at killing
fibroblasts after a 72 hour incubation compared to 24 hour and 48 hour incubation periods.
Although this study was performed on tissue cells rather than lymphocytes, the results coincide
with my data that states etoposide is most effective at inhibiting cell growth at 72 hour
incubation period.
Thirdly, I tested the effectiveness of various concentrations of the negative control on
inhibiting Jurkat cell growth. The negative control is a combination of Jurkat cells and etoposide,
a small molecule that prohibits DNA synthesis and leads to cell death. Previously the MTT assay

17. !17
protocol called for the concentration of etoposide to be 12.5 ug/ml. Using serial dilutions, I was
able to test various concentrations of etoposide against with Jurkat cells and determine the
optimal concentration for inhibiting Jurkat cell growth. I found that the highest concentration of
etoposide, 125 ug/ml, was the most effective at prohibiting Jurkat cell proliferation. Data was
also collected using a tritiated thymidine assay to provide an effective comparison for the
effectiveness of the MTT assay.
After making the necessary adjustments to the MTT assay protocol, I tested the virulence
of a isolate of B. dendrobatidis from South Korea, KR-323. I found that the isolate did not show
any virulence and did not significantly inhibit the growth of Jurkat cells. The extremely high
optical density reading for the Jurkat and KR-323 wells could have been due to: plating error,
synergistic effects between Jurkat cells and KR-323, the Biotek plate reader counting
mitochondrial activity of KR-323 cells, or a combination of these possibilities. Although the
claim KR-323 is not virulent definitely needs further testing, it coincides with the previous
studies that show isolates of Bd from South Korea are not significantly virulent in comparison to
isolates from other parts of the world.
!

18. !18
References
Andre, S., Parker, J., & Briggs, C. (2008). Effect of Temperature on Host Response to
Batrachochytrium dendrobatidis Infection in the Mountain Yellow-legged Frog
(Rana muscosa). Journal of Wildlife Diseases, 44(3), 716-720. Retrieved April 12,
2015, from US National Library of Medicine.
Bataille, A., Fong, J. J., Cha, M., Wogan, G. O. U., Baek, H. J., Lee, H., Min, M.-S. and
Waldman, B. (2013), Genetic evidence for a high diversity and wide distribution
of endemic strains of the pathogenic chytrid fungus Batrachochytrium
dendrobatidis in wild Asian amphibians. Molecular Ecology, 22: 4196–4209. doi:
10.1111/mec.12385
Fites, J. S., Ramsey, J. P., Holden, W. M., Collier, S. P., Sutherland, D. M., Reinert, L. K.,
…Rollins-Smith, L. A. (2013). The invasive chytrid fungus of amphibians
paralyzes lymphocyte responses. Science (New York, N.Y.), 342(6156), 366–369.
doi:10.1126 science.1243316
Karpinoich, N., Tafani, M., Rotham, R., Russo, M., & Farber, J. (2002). The Course of
Etoposide-induced Apoptosis from Damage to DNA and p53 Activation to
Mitochondrial Release of Cytochromec. JBC Papers.
Skerratt, L., Berger, L., Speare, R., Cashins, S., McDonald, K., Phillott, A., . . . Kenyon,
N. (2007). Spread of Chytridiomycosis Has Caused the Rapid Global Decline and
Extinction of Frogs. EcoHealth, 4, 125-134. Retrieved April 9, 2015, from
SpringerLink.

19. !19
Sladowski, D., Steer, S., Clothier, R., & Balls, M. (1993). An improved MTT assay.
Journal of Immunological Methods, (157), 203-207. Retrieved April 10, 2015,
from US National Library of Medicine.
!
!