The Effect of Sunglass Lenses on the Growth Inhibition of the Bacterium Serratia
marcescens Against Ultraviolet Radiation
Myka Cabuhat, Justin Euperio and Peter Huang
Department of Biological Sciences
Saddleback College
Mission Viejo, CA, 92692
The sun is the primary source of energy, heat and light, however the sun's energy in
the form of radiation has been shown to be problematic. Too much exposure to UV
radiation raises the risks of diseases including cataracts, macular degeneration, and
cancer, and is also antimicrobial because it damages DNA molecules (Zion, et al., 2006).
Sunglass lenses play a major role in protecting against the effects of UV radiation. In this
study, the growth of Serratia marcescens in nutrient broth tubes was tested with different
sunglass lenses (Gray Polarized, Gray Tint, Transition Gray, Clear UV400) against
exposure to UV radiation. It is predicted that bacterial growth inhibition will not be
significantly different based on lens opacity. Gray polarized (GP) lens culture had 66.26
x 108 ± 6.99 bacterial cells (± se), the gray tint (GT) lens culture had 44.67 x 10 8 ± 4.81
bacterial cells (± se), the transition gray (GTran) lens culture had 65.33 x 10 8 ± 6.96
bacterial cells (± se), the clear UV400 lens had 28.73 x 108 ± 3.25 bacterial cells (± se),
and the control broth culture had 12.93 x 108 ± 0.91 (±se). The means were compared
using an ANOVA with a Post HOC Bonferroni correction (p=1.90×10-11). The results of
this study supported this hypothesis, as not all lenses were capable of producing
significant growth inhibition.
Introduction
UV radiation has been shown to be both
positive and negative. It is a source of
energy, heat and light, and is also known for
being a large source of Vitamin D.
However, too much exposure to UV
radiation increases the chance of developing
a wide range of diseases. According to Kuijk
(1991), photochemical damage is induced by
relatively long-term exposure to lower levels
of light in the UV and blue regions of the
spectrum and are thought to initiate
chemical reactions. This UV radiation has
been attributed to the damage of germinal
epithelial cells such as corneal or lens cells
by their interference with and mutagenesis
of DNA (Behar-Cohen, et al., 2013). The
cumulative effects of UV radiation have
therefore been linked to being a cause of
cataracts, keratitis, age-related macular
degeneration and various eye and skin
cancers. However, the use of sunglasses
could greatly decrease these damaging
effects. The purpose of this study is to
determine if levels of bacterial growth are
significantly different between the different
colorations of lenses. The study’s hypothesis
is that there is no significant difference in
bacterial growth post UV radiation based
upon the opacity of the lens.
Materials and Methods
Nutrient broth for bacteria cultures was
prepared by mixing 8.0 g of Criterion
nutrient broth powder with 1.0 L of
deionized water. Test tubes (n=25) were
filled with 10.0 mL of the prepared nutrient
broth. Test tubes for serial dilutions (n=175)
were filled with 9.0 mL of sterile deionized
water using the Integra Biosciences Dose-it
803 auto pipette machine. Both the nutrient
broth tubes and sterile water tubes were
placed in the autoclave to ensure sterility.
The nutrient broth tubes were inoculated
with prepared cultures of Serratia
marcescens
via
aseptic
technique.
S.marcescens
was
chosen
for
its
pigmentation and ability to colonize on the
cornea, as it is frequently isolated from
lenses of patients with contact lensassociated corneal infiltrates (Zhou, et al.,
2012). The 25 cultured nutrient broth tubes
were separated into 5 groups of 5 tubes
each. Each lens type (Polarized Gray, Gray
Tint, Transition Gray, Clear UV400) was
then placed over their respective group of
culture tubes (n=5 per lens), while one
group of culture tubes were fully exposed
without a lens to serve as a positive control.
All culture tubes were placed into the Lab
Con Co® Ultraviolet light cabinet and were
placed at an optimal distance of 25-30 cm
from the light source. The culture tubes were
then exposed to Ultraviolet light with a
wavelength of 260 nm for a duration of 5
minutes. After exposure, culture tubes were
incubated for 48 hours at 30°C. After the 48
hours incubation period, each culture tube
was then serially diluted in sterile water
(1x107). Pour plates for colony counts
(n=75) were prepared by aseptically placing
0.1 mL of each of the culture tube's 107
dilution along with one full type of liquefied
nutrient agar into Petri dishes marked with
the type of lens used and the dilution factor
(107). The pour plates were then left on the
tabletop to solidify. After the plates had
solidified, they were then inverted and
placed into Petri dish holders and incubated
for 48 hours at 30°C before bacterial counts
were performed. Bacteria counts were
performed using the viable plate count
method, which utilizes the Quebec colony
counter (Clark, et al., 2014). Countable
numbers were defined as being between 0300 colonies. Viable bacteria in the original
broth culture was calculated using the
following formula: Avg. # of Colonies x
Dilution Factor x Plating Dilution. Data was
transferred to Microsoft Excel 2007 for data
analysis. An analysis of variance (ANOVA)
was used to compare bacterial colony counts
of each lens and the control. A Post HOC
(Bonferroni Correction) test was then
indicated by the p-value, and implemented
to determine if there was a significant
statistical difference between groups.
Results
On average, the gray polarized (GP) lens
culture had 66.26 x 108 ± 6.99 bacterial cells
(± se), the gray tint (GT) lens culture had
44.67 x 108 ± 4.81 bacterial cells (± se), the
transition gray (GTran) lens culture had
65.33 x 108 ± 6.96 bacterial cells (± se), the
clear UV400 lens had 28.73 x 108 ± 3.25
bacterial cells (± se), and the control broth
culture had 12.93 x 108 ± 0.91 bacterial
cells (±se). The p-value was reported to be
p=1.90×10-11 indicating a significant
difference in Serratia marcescens colonies
between all lenses against control colonies.
A Post Hoc test however, determined no
statistical difference (p>0.05) between the
GP and GTran, GT and GTran, GT and
Clear, and the Clear and Control lenses.
Conversely, a statistical difference (p<0.05)
was found between the GP and GT, GP and
Clear, GP and Control, GT and Control,
GTran and Clear, GTran and Control lenses.
Figure 1. Mean number of Serratia marcescens
colonies against the type of lens blocking exposure to
the Ultraviolet light.
Discussion
The opacity of the lens should in theory
block the penetration of Ultraviolet light,
more so with increasing opacity. If that UV
exposure is limited, more bacterial growth
should result as less bacterial DNA is
damaged. DNA synthesis inhibition is
attributed by this damage as UV irradiation
results in the formation of thymine dimers in
polynucleotide chains, ultimately halting
protein translation (Setlow, et al., 1963).
Oxygen radicals are also generated which
can cause lipid peroxidation and protein
modification (Kuijk, 1991). With less
mRNA production and more protein
misfoldings, fewer viable colonies would be
present.
In this experiment however, it was
expected that the different opacities and
types of lenses used would not significantly
alter bacterial growth levels after exposure
to Ultraviolet light. The results of this study
supported this hypothesis, as not all lenses
were capable of producing significant
growth inhibition of Serratia marcescens.
We therefore believe this indicates that
although sunglasses do limit UV penetration
to an extent, that penetration is not highly
affected by the lens type or color, suggesting
all sunglasses to be relatively equal in
protection. Similar results could be
translated from the experiments done by
both Rosenthal, et al. (1988), and Dongre et
al. (2007), as they discovered that UV
protective sunglasses, such as those used in
this experiment, decreased penetration of
UV rays to anywhere between 2-14%, in
comparison to the potential 100%
penetration, granted no natural or superficial
uv light protection. Of course the efficacy of
sunglasses in protecting against UV rays
depends on a number of mechanical factors
such as their size, shape, wearing position,
and reflection from the posterior lens
surface, as well as personal factors such as
latitudinal residency, outdoor vs. indoor
occupations, working in open or reflective
environments (such as sand or water), and
extensive outdoor leisure activities, all of
which can greatly increase exposure
(Rosenthal, et al., 1988). Despite these
factors, the range of protection offered by
sunglasses does not prove all sunglass types
to be significantly effective.
Although significance cannot be
noted between the different lenses, it is
important to still note that there was a
significant difference in bacterial growth in
the broth cultures protected by a lens versus
the control broth culture which was fully
exposed to UV radiation without a lens.
These results prudently signify the
importance
of
wearing
sunglasses
altogether. The importance of this will be
greatly increased in the future as global
warming may introduce more and more UV
radiation on Earth, imminently requiring
similar experiments to be done in order to
engineer more enhanced forms of UV
protection that will maximize the level of
UV radiation attenuation.
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Acknowledgements
Special thanks to Professor Teh for his time
and patience in assisting with our
experiment and answering any questions we
had. Special thanks to LensCrafters and the
Biological
Sciences
Department
at
Saddleback College for providing us with
the required materials to carry out this
project.