Hashna Manoharan

Report 17
Monday 6 8:50
Purpose
The purpose of the lab conducted was to test the sensitivity of bacteria to
ultraviolet light, thereby determining the effects of UV light on bacterial growth,
and to relate the effects of the exposure to the repair mechanisms present in
the bacteria.
Theory
Ultraviolet radiation, while a form of non-ionizing radiation, still has the
potential to be extremely harmful to bacteria due to the biological effects that it
has on organisms. UV radiation is commonly split into three types: UV-A, UV-B,
and UV-C radiation.
UV- A radiation, which possesses the longest wavelength of all UV
varieties, is not directly absorbed by nucleic acids. Thus, it does not actively
affect DNA, and often indirectly induces damage due to the activation of
photosensitizer proteins. UV-A radiation is commonly utilized in forensic testing,
such as in fluorescence inspection methods. UV-B radiation is directly absorbed
by nucleic acids. When absorbed by DNA, UV-B radiation initiates the formation
of potentially lethal molecular lesions known as pyrimidine dimers, in which
adjacent thymine or cytosine bases bond to each other on the same strand, thus
distorting DNAs natural right-handed double helix configuration. The distortion

of the shape of the DNA strand halts and disrupts the progress of DNA
polymerase, which aids in the replication of DNA, thus potentially leading to
mistakes such as areas of single stranded DNA. UV-C radiation can be thought
of as an amalgamation of those of UV-A and UV-B, because it is absorbed by all
of the components in a cell, including nucleic acids. Although it has the potential
to be highly dangerous, it is almost completely absorbed by ozone and oxygen
present in the Earths atmosphere. UV-A has the longest wavelength, and is
emitted at approximately 320 to 400 nanometers, while UV-B and UV-C are
emitted at wavelengths of 290 to 230 nm and 200 to 280 nm, respectively.
Due to the constant threat of ultraviolet damage, organisms combat UV
damage with different repair mechanisms. Light repair is a method used by
many organisms which, as the name suggests, requires light energy in order to
function. Also known as photoreactivation, this type of repair uses energy
harnessed from light to repair pyrimidine dimers by splitting dimers with the aid
of photolyase enzymes. Another commonly found UV repair mechanism is that
of dark repair. Unlike light repair (which repairs dimers), dark repair, which is the
repair mechanism used by human cells, destroys the damaged portion of DNA
with the help of activated endonucleases. The gap that is created is then filled
by DNA polymerase and DNA ligase. Unlike light repair, dark repair does not
require light to function, but can also function in the presence of light.
Experiment
In the experiment, a labeled plate of Luria agar was divided in three
sections, and the three sections were each divided in half. Each of the three

sections was plated with a different type of bacteria. The three types of bacteria
used were Escherichia Coli, Staphylococcus Aureus, and XL1 blue II cells.
XL1 blue II cells are mutant E.Coli cells in which the RecA gene has
mutated and is no longer effective. Thus, they are the auxotroph counterparts to
the prototroph or wild type of E.Coli. RecA genes code for RecA proteins, which
regulate DNA recombination and the repair of DNA anomalies caused by UV
radiation. Thus, XL1 blue II cells, which lack efficient repair systems, are more
susceptible to UV radiation than their normal E.Coli counterparts.
After plating, the plate was placed in a cross linker, an instrument whose
purpose is to expose samples to controlled amounts of UV radiation for the
purposes of experimentation. Before turning on the cross linker, half of the plate
was covered with cardboard, to allow for that portion to serve as a control. Each
of the groups performing the experiment placed their samples in the cross liker
for varying amounts of time, so that when the data is combined, it becomes
possible to draw conclusions about the amount of exposure to UV necessary to
cause lethal damage.
After the plates were removed from the cross linker, they were incubated
overnight at 37 C.
Data/Observations
Group
1
2
3
4

Time (second)
6
12
18
30

E. Coli
xxx
xx
x
o

S.Aureus
xxx
xx
x
o

XL1.blue
xxx
o
o
o

My group placed our sample in the cross linker for eighteen seconds, and
when our incubated plate was examined. Our plate exhibited growth on the all
three sections that were covered up by the cardboard, slight growth on the
E.Coli and S.Aureus sections that were exposed to the UV radiation, and no
growth on the XL1 blue section that was exposed.

Discussion
Out of the three strains of bacteria tested, the E.Coli and S.Aureus has
consistently similar results, for all of the recorded times. This can be attributed
to their similar RecA repair mechanisms, which cause them to repair the
damage caused by UV in a similar manner. Up to a certain amount of exposure,
they were able to repair the UV damage, leading to some growth, although it
was diminished when compared to the control. After a certain point, however,
the damage was too severe to repair, causing no growth. According to the data,
this occurred somewhere between 18 and 30 seconds of exposure to UV
radiation via the cross linker device. Although E.Coli and S.Aureus are gram
negative and gram positive, respectively, this did not appear to have an effect
on growth, as this did not affect the repair mechanism.
The XL1 blue cells, which have mutated and ineffective RecA repair
systems, were not able to repair the UV damage as the other two strains of
bacteria could. The cells exhibited growth at 6 seconds, because the length of
time of exposure was not enough to have an effect, but for the remaining times,
no growth was exhibited due to the ineffective repair mechanisms.

The results were similar to those that were theoretically expected,

because the XL1 blue cells exhibited less growth that the other two strains, and
after a certain amount of time, when the damage was too severe to repair, even
the S. Aureus and E. Coli, which had properly function repair mechanism
systems, could not grow.

Extra Credit
The growth under the cardboard control was used to compare the results
from the side that was exposed to UV. A control is needed to endure that a lack
of growth on the exposed side is actually due to the exposure, and not because
the media is ineffective and cannot support the growth of the organisms, for
example.
Works Cited
General Microbiology Laboratory Manual, 3 Edition to accompany MCB 3020L.
Daniela Scheurle. Florida Atlantic University
rd

Microbiology, 8 Edition, Joanne M. Willey, Linda M. Sherwood, Christophe J.

Woolverton.
th