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Exposure to Low Intensity UV Light Does Not Cause

Significant Resistance to Higher Intensity UV Light
Nathan Zaroban, Joon Young Lee, Christine Solomon
12-6-15

Introduction
Exposure to UV light is known to cause mutations in cells by forming crosslinks between two adjacent thymine base nucleotides in a DNA strand. These
mutations cause severe issues in the transcription of DNA to RNA, thus proving fatal
to the cell (Grinnell College Bio-251 Lab Manual 2015). Thus, UV-C light (UV of
germicidal intensity) has been used a method of bacterial disinfection. It has been
most widely used and studied as an alternative to chemical purification in waste
water treatment (Koivunen, Heinonen-Tanski, 2005)(Oliver, Carey, 1976). Other
studies have shown that UV-C light has also been effective in the deactivation of
airborne Serratia marcescens (Lai, Burge, and First, 2004). Recently it has been
studied as a method of disinfecting surfaces of hospitals and medical clinics.
Hospitals are known to be breeding grounds for bacterial diseases, and many
studies have been conducted to try to find a way to effectively reduce bacterial
counts within hospitals. The use of antimicrobial copper alloy surfaces has been
studied and confirmed to reduce bacterial counts on surfaces such as handrails and
doorknobs (Schmidt, Attaway III, Fairey, et al 2013). However, this method can only
be effective on the areas covered with antimicrobial materials. Some medical
facilities have begun to use automated short-wave UV-C light machines in hospital
rooms in which infected patients stayed. These mobile machines expose the entire
room to UV-C, with light reflecting off surfaces to expose surfaces not in direct line
of sight with the machine. Studies have confirmed their success in reducing

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bacterial counts of many different strains to nearly zero (Nerandzic et. al. 2010)
(Rutala, Gergen, Weber, 2010).
This sounds like a fantastic solution. However, we questioned the long term
effectiveness of UV as a bacterial disinfectant and sought to figure out whether
exposure to multiple rounds of UV light would build a bacterial resistance to UV. This
led us to our experimental question: Would a strain of Serratia marcescens cells
exposed to four rounds of 100 J/m2 UV light have better resistance to higher
intensity UV than wildtype S. marcescens. We hypothesized that the S. marcescens
cells pre-exposed to UV would yield higher cell survivorship (CFU/mL) when exposed
to higher intensity UV than a wildtype strain of S. marcescens. We selected S.
marcescens as our model organism because it is known to be affected by UV
exposure (Grinnell College Bio-251 Lab Manual 2015).

Materials and Methods

Preparation of Pre-UV Exposed Cells
We grew a culture of wildtype S. marcescens in PG broth for 24 hours. One
mL of this culture was micro-centrifuged at 14,000 rpms for one minute. The
supernatant was decanted off the pellet and discarded. We re-suspended the pellet
in one mL of sterile saline and transferred this cell suspension into a 60 mm petri
dish. We exposed the cells to 100 J/m 2 of UV light in a UV cross-linker. We introduced
the exposed cell solution into fresh PG broth. This new broth was incubated for four
hours at 30 degrees Celsius in a roller drum. We repeated this process using the
newly grown broth containing cells exposed to one round of UV as the starting
culture. This was repeated until we had a culture of cells that had been exposed to
four rounds of 100 J/m2 UV light (Henceforth referred to as R4). R4 cells served as

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our experimental group in the experiment. Wildtype S. marcescens cells
(Henceforth referred to as WT) served as the control group in the experiment.
Resistance Testing
We centrifuged both the R4 culture and a WT culture at 8,000 x g for 10
minutes. The supernatants were decanted off both pellets and discarded. We resuspended both pellets in approximately five mL of sterile saline. When necessary,
we diluted the suspension with further saline so that they had roughly the same
visual transparency and thus similar cell densities. For both R4 and WT cell
solutions, we exposed 500 L samples to 0-300 J/m2 UV light. Four biological
replicate samples of each cell type and UV intensity combination were performed.
We created 100-108 serial dilutions for samples exposed to 0 J/m2 UV light and 100105 dilutions for samples exposed to 100-300 J/m 2. Five 10 L spots of each dilution
were plated on PG agar plates. We incubated the plates at 30 degrees Celsius for 24
hours then calculated the CFU/ml of each sample. An ANOVA test was run to
determine the significance of differences in cell survivorship between each sample.

Results
This experiment compared the cell survivorship (CFU/mL) of wildtype S.
marcescens (WT) and mutant S. marcescens that had been previously exposed to
four rounds of 100 J/m2 UV light (R4) when exposed UV light of increasing
intensities. Figure 1 shows that for each UV intensity tested, R4 cells had slightly
better survivorship than WT cells. However, an ANOVA showed that there was no
significance in these differences.

Discussion

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These results did not support our hypothesis that R4 cell would be
significantly more resistant to further UV treatments than WT cells, even at higher
intensities. We can note that in Fig. 1 we do see a common trend that R4 cells have
better percent survival rates than WT cells. A decrease in both R4 and WT
survivability can be seen as UV intensity increases, except 300 J/m 2, which has
higher survivability in both cell types than 200 J/m 2.This is extremely odd as would
expect less survivability in 300 J/m2 UV than 200 J/m2 for both cell types (Chang et
al, 1985). One problem with this is that scope of this experiment was constrained by
time, thus sample sizes were rather small. Larger sample sizes could potentially
correct this issue.
We should also note that the results of the WT survivability conflict with
previous studies conducted by Grinnell Colleges BIO-251 lab, in which they found
WT to have approximately -3.0 (LOGTEN) % survivorship when exposed to 100-300
J/m2 UV compared to 0 J/m2 UV exposure. If we were to compare our R4 survivability
to the WT results of that experiment, the difference in survivability would be much
greater.
Alternatively, one potential flaw to consider in this experiment would be the
differences in cell density between the initial cultures of R4 and WT cells (CFU/ml of
R4 and WT cells exposed to O J/m2). The R4 culture had a slightly higher cell density
than the WT culture. Thus, we could interpret the difference in survivorship two
different ways. One would be that higher cell density better protects cells from UV
exposure. The other is that R4 cells did build slight UV resistance. To eliminate the
cell density variable, we could make sure the initial cell densities were equal by
using optical density analysis.

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Looking forward with this experiment in mind, it would be interesting to test
how many rounds of 100 J/m2 UV exposure it would take to produce a significantly
resistant S. marcescens strain. Another option would be to test the effects of
increasing the UV intensity of pre-exposure cells. This type of experiment would also
need to be conducted on other model organisms as UV resistance may build
differently from one organism to another.
As these current results of this experiment stand, UV treatment appears to
be an excellent method of disinfection of S. marcescens, as it effectively killed at
least 98% of cells, and multiple exposures did not build significantly resistant
strains. However, this study should be refined and continued on a much larger
scale. Further advancements of this experiment should include: further rounds of
pre-exposure to low intensity UV, equal cell densities of initial WT and pre-exposed
cells, much larger sample sizes, and repetition with different model organisms.

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Fig. 1. % of cell survival of R4 and WT cells vs exposure to increasing UV light

intensity. R4 0 n=4. WT 0 n=6. R4 100-200/WT 100-200 n=5. R4 300/WT 300 n=3.
ANOVA test shows that groups sharing the same letter have no significant difference
(p-value>.05), while groups that do not share the same letter are significantly
different (p-value<.05). Error bars= 1 SEM.

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References
Chang, J., Ossoff, S., Lobe, D., Dorfman, M., Dumais, C., Qualls, R., and Johnson J.
1985. UV inactivation of pathogenic and indicator microorganisms. Applied
Environmental. Microbiology. June 1985 Vol. 49 No. 6 pp. 1361-1365.
Grinnell College Biology 251 Laboratory Manual 2015. Pp. 17
Koivunen, J., Heinonen-Tanski, H. 2005. Inactivation of enteric microorganisms with
chemical disinfectants, UV irradiation and combined chemical/UV treatments. Water
Research .Volume 39, Issue 8, pp. 15191526.
Lai, K., Burge, H., and First, M. Size and UV Germicidal Irradiation Susceptibility
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Environmental Microbiology. Volume 70, Issue 4. Pp. 20212027.
Nerandzic, M., Cadnum, J., Pultz, M., Donskey, C. 2010. Evaluation of an Automated
Ultraviolet Radiation Device for Decontamination of Clostridium difficile and Other
Healthcare-Associated Pathogens in Hospital Rooms. BMC Infections Diseases.
Volume 10, Issue 197.
Oliver, B., Carey, J. 1976. Ultraviolet Disinfection: An Alternative to Chlorination.
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Rutala, W., Gergen, M., Weber, D. 2010. Room Decontamination with UV Radiation.
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Acknowledgements
We would like to thank fellow classmates, Haonan Sun, Tristan Aschittino,
Sydney Quartey, Marie Spychala, Jamie Schafroth, and Jason Jennings for allowing
us to use their wildtype S. marcescens strains in our experiments. We would also
like to thank Ms. Bosse and Professor Praitis for their guidance throughout this
project.