Austin Van Niel

TA: Carmen Elenberger

Lab: Friday 8:00am, 710 Behrakis
Submitted: 10/30/15

Ultraviolet Mutagenesis of Serratia Marcescens

Introduction:
 Light exists in wavelengths outside of the visible light spectrum of humans, which is

around 700nm to 400nm in wavelength. The ultraviolet (UV) light spectrum is at a higher energy

than what humans can perceive at around 400nm to 10nm in wavelength (Nave). Due to the

higher energy of UV radiation it has a mutagenic effect on DNA, following the trend of the

electromagnetic light spectrum that as the wavelength and therefore the energy of the particles

increases, the more harmful the radiation to living cell DNA, but the less ability it has to

penetrate obstacles to reach DNA (Begley 25).

UV radiation causes the formation of base pair dimers, particularly between adjacent

thymine bases, adjacent adenine bases, and between adenine and vicinal thymine. Having a

few of these mutations are not necessarily an issue when it comes to the biological viability of

the cell as the error correcting mechanism of exonuclease activity will be able to correct these

errors during replication. However, if there is enough exposure to UV radiation then there will be

more mutations than the exonucleases can handle and it will have a deleterious effect to the cell

(Cadet et al. 2004).

On Earth, the atmosphere will absorb most of the UV radiation from the sun before it

reaches ground level and much of the UV radiation that does reach ground level is readily

absorbed by the abiotic environment. Since there is minimal exposure to harmful levels of UV

radiation on the surface, most organisms do not have substantial defense mechanisms to

protect against UV exposure. Single celled organisms are particularly sensitive to UV radiation

as they have only one set of DNA, and if it is damaged beyond repair the entire organism will

die instead of just a few cells as would happen in a multicellular organism if the exposure was

acute. The scientific industry has capitalized on this deleterious effect on microbes in order to

sterilize or greatly reduce the microbiome of solids and liquids. UV lamps are used in areas like

operating rooms, food preparation kitchens, and other areas where bacteria are highly

undesired (Begley 25).

 This study seeks to explain how the distance of a UV lamp above the surface of a

culture plate containing Serratia marcescens (S. marcescens) affects the growth of the bacteria

or if it causes any non-deleterious mutations. S. marcescens was chosen for its distinctive red

color as this will will allow for easily identifying any contaminants on the culture plates as well as

if there are any color mutations caused by UV light. S. marcescens is also a bacteria that is

often associated with healthcare related infections since its incubation temperature is 37C

(body temperature), as well as resistance to traditional (non-UV) antimicrobial compounds

(Falkiner and Herra).

In order to determine a proper length of exposure that should be used for this

experiment, an initial experiment to find the killing curve of S. marcescens must be performed.

This data will allow the calculation of at which point the distance from the plate overcomes the

effectiveness of UV radiation at killing S. marcescens (Begley 26). It was predicted that as the

distance between the UV lamp and the culture plate increased, the rate of mutation would

decrease and therefore the farther away the UV lamp was located, the more colonies of S.

marcescens would be expected on the plate.

Materials and Methods:

For the first experiment of this study, a killing curve needs to be determined. The dosage

of UV radiation exposure will be varied by exposure time while the height of the lamp will be

held constant. A note card will be held over half of the plate to be the positive control, that is to

say that since UV rays cannot penetrate the paper to damage the DNA of the S. marcescens. If

there are no extraneous factors present in the plate, there will be a normal pattern of growth on

that side and an abnormal pattern of growth on the exposed side. The negative control will be

the plate exposed for 3 minutes, which is plenty of exposure to completely kill the S.

marcescens on the exposed side of the plate.

 The plates will be labeled and exposed to UV rays as seen in Table 1. They will be

inoculated with S. marcescens from a liquid culture in Luria broth onto a tryptic soy agar

substrate. While inoculating the plates it is imperative that aseptic technique be followed,

meaning that the opening of the culture vial should be passed through a flame before and after

the sterile swab is dipped into the culture solution. Every plate should be inoculated with a

separate swab to prevent cross contamination. When exposing the plates to UV radiation the

lamp is to be held 5 inches from the surface of the substrate post inoculation for the requisite

amount of time (Begley 26).

After the killing curve is determined from the data gathered and the length of exposure to

be used in the second experiment is extrapolated, another set of plates should be set up using

the same culture of S. marcescens and following aseptic technique. These plates will all be

exposed for the same amount of time except this experiment will vary the height of the lamp

above the substrate. The positive control will again be the side of the plate covered by the note

card and the negative control will the the plate where the lamp is placed directly on the rim of

the culture plate (demarked with 0 inches) as this will surely kill the exposed bacteria. The

plates will be labeled and exposed as outlined in Table 2.

Table 1: Exposure Setup for Killing Curve

Plate Length of Exposure (Seconds)

1 15

2 30

3 60

4 120

5 180
Plate labels and length of exposure.

Table 2: Setup for Height Varying Exposure

Plate Height of Exposure (inches)

A 0

B 2

C 4

D 6

E 8
Plate labels and height of exposure.

Results:

The results of the killing curve determination experiment are found in Table 3. These

results demonstrate how the bacteria were only able to colonize the exposed half of the culture

plate when the exposure time was less than 30 seconds. Which could be posited to mean that

as the length of exposure increases so do the deleterious effects of UV light. Figure 1

graphically illustrates the killing curve and how it is at 30 seconds of UV exposure.

The colonies grown on plate 1, which were exposed UV light, were phenotypically

similar to the control albeit that they were more isolated. The individual colonies were a dark red

color that showed a gradation in color the further from the center of the colony. The darker

center surrounded by a lighter halo of cells is probably due to the concentration of cells and not

to some mutation brought on by UV radiation. Which makes sense because there had to be a

single cell present to begin the colony and since S. marcescens reproduces through mitosis, the

daughter cells will be concentrated around the central parent cell and expand outwards slowly.

Every single one of the plates in this trial had some growth on the perimeter of the plate

where the substrate met the wall of the dish. This was expected on the control side of the plate

which was not exposed to UV radiation but it was also found on the exposed half of the plate.

The growth was anywhere from 3-30% of the perimeter and not necessarily all one colony as

demonstrated by the growth pattern on plate 2 where there were three established perimeter
colonies and none of them were connected to the control side indicating that they must have

been established separately from the colonies on the control side.

Based on the results of the killing curve, the exposure time used when varying the height

of the UV lamp was be 30 seconds because at that was the minimum point at which the UV

radiation was able to kill the bacteria. This height variance tested whether height will improve or

worsen the effects of UV light on the DNA of S. marcescens. The results of this experiment are

shown in Table 4. Overall, the phenotypes of the control sides of the plates were identical to

both sides of the killing curve bacteria. On the exposed portions of the height varying

experiment there were some differences.

On plates B,C,D, and E there were white colonies on the UV exposed sides of the plate.

They expressed the same color gradients as the red colonies, as in they are more yellow to off-

white in the center and a white color towards the perimeter. They are interspersed with the wild

type cells without any particular pattern and, given the mutagenic effects of UV light, it could be

posited that these are color mutants of S. marcescens and not contaminant bacteria. These

plates also all have the growth around the edge, possibly indicating that it may be an issue

related to the experimental procedure since it was present in both experiments.

Table 3: Results of Killing Curve Experiment

Plate Length of Exposure Percent Coverage of Percent Coverage of

(Seconds) Side Exposed to UV Side Not Exposed to
Radiation UV Radiation

1 15 30% 85%

2 30 3% 80%

3 60 0% 90%
4 120 0% 95%

5 180 0% 97%
Results of the killing curve experiment showing the control and experimental results of the UV
exposure of different times

Figure 1:

Results of Killing Curve experiment, shows that S. marcescens are no longer able to colonize
after 30 seconds of UV exposure.

Table 4: Results of Height Variance on Exposure

Plate Height of Exposure Percent Coverage of Percent Coverage of

(inches) Side Exposed to UV Side Not Exposed to
Radiation UV Radiation

A 0 0% 97%

B 2 1% 95%

C 4 20% 90%
D 6 50% 90%

E 8 85% 97%
Results of varying height of UV lamp above substrate on control and experimental sides of the
culture plates, showing that as height increases so does the level of colonization.

Discussion and Conclusions:

The purpose of these experiments was to determine how the height of a UV lamp above

a culture of S. marcescens would affect the growth and colonization of the bacteria. At the

beginning of this study it was hypothesized that as the distance between the UV lamp and the

culture plate increased, the rate of mutation would decrease and therefore the farther away the

UV lamp was located, the more colonies of S. marcescens would be expected on the plate. On

the surface it appears that this hypothesis was confirmed by the experiments performed in the

study, but there were a few developments that raised a few questions.

The controls for this experiment were effective but they could have been improved. It is

obvious that the negative controls for this study worked as there was no growth on the exposed

half of the control plates. The positive controls were very accurate since they were on the same

plate that the experiment was being performed on, and therefore were a direct representation of

what the plate would have looked like had it not been treated with UV light. There was not a

straight line as would be expected demarcating where the control half of the plate ended and

where the experimental side began. This pattern was expected because the UV light could not

penetrate the note card, which has a straight edge. As it turns out the explanation for this

phenomena could also explain why there was growth on the perimeter of the culture plate

seemingly under beneath the surface of the substrate.

This growth pattern may be explained through the phenomena of bacterial swarming. S.

marcescens are flagellated bacteria with motile abilities which may have allowed multiple cells

to swarm from the control side of the plate to the experimental side. This is not a particularly fast
process, especially across a porous surface like agar, which is why the results are not more

dramatic. It works because individual S. marcescens cells are competing for a finite amount of

nutrients in the agar plate, and when the nutrients get scarce the bacteria try and look for more.

By rotating their flagella they can move to an area less populated by bacteria (Kearns).

This effect is more dramatic around the edge of the culture plates where there is even

less surface area for the bacteria to absorb nutrients. The parent cells originally got there when

the plate was inoculated and turned over. Since it was a liquid culture, when the plate was

turned over some of the broth containing the bacteria must have fallen into the space between

the agar and the dish wall (Kearns). Since UV light does not have a great penetrating ability

these cells were spared the harmful effects from the light and through bacterial swarming

moved along the edge of the plate causing the perimeter colonies (Begley 25). This would also

explain the sectioning of the colonies on the exposed side when a few did end up growing, they

were somehow protected from the radiation more than the neighboring cells which isolated them

and created individual colonies.

The cells that were not spared of the effects of UV light due to the light being blocked

were subject to highly deleterious mutations. Both UVA and UVB radiation cause the formation

of dimers between base pairs which at environmental levels are easily fixed by exonuclease

activity without any interruption to cellular functioning. When DNA is exposed to excessive

amounts of UV radiation like they were in this experiment, exonuclease activity is not enough to

compensate for the damage done and the cell dies.

When the exposure is lessened like they were in the plates exposed from heights above

2 inches in the height varying experiment then there can be mutations that are not completely

deleterious. This may be an explanation for the white colonies, whose DNA was damaged by

the UV light, but not to the point of cell death. The UV light could have theoretically damaged a

portion of the DNA responsible for the surface protein that results in the distinctive red color

(Paruchuri & Harshey 1987). This is more likely than contamination as the same procedure was
followed in the killing curve experiment and no color mutants were found. This coupled with the

phenotypic appearance of the colonies being so similar it becomes even more plausible that

these are color mutants and not contaminant bacteria.

The data from the height varying experiment was counterintuitive to the first experiment,

there were more bacterial colonies than expected. In the killing curve experiment there was a

100% death rate for bacteria on the exposed side at 30 seconds when the lamp was held 5

inches from the substrate. For some reason the bacteria was able to cover 20% of the plate

when the lamp was 4 inches from the plate during the second experiment. The only

explanations would be an error in the timing of exposure that led to the bacteria being exposed

for shorter than 30 seconds, or for an error in the lamp. For example that the light was not as

intense as the killing curve experiment due to a dying UV bulb.

A major improvement for this study would have been a clamp for the UV lamp. This

would have allowed for the precise height of each trial to be maintained for the specified

duration as human hands have the tendency to move when they are required to hold still. This

clamp would also ensure that the light stays at a constant angle parallel to the bench and none

of the light makes it under the note card. An electronic timer in the UV lamp would also help so

that the lag time between registering that the trial is over and turning off the lamp would be held

constant or eliminated. To avoid bacterial swarming it may also be pertinent to either use a

different bacterial culture without flagella or a transgenic model of S. marcescens. without

flagella. This way it can be ensured that all of the bacteria on the experimental side of the plate

were exposed to UV light and not simply control bacteria that migrated looking for more

nutrients. Changing the UV light between experiments may also be pertinent.

A subsequent experiment may look at the effects of bacterial swarming and at which

point the effects of UV sterilization of surfaces are negated by the colonization of a few cells.

These few cells would have the ability to spread out across the surface and recolonize

depending on the length of their exposure. In this experiment the substrate will have to be
particularly nutrient poor to encourage the cells to look for more nutrients. A curve will have to

be determined to see at which point the damage done by UV light causes enough mutations in

the flagella that the S. marcescens are not motile any longer but the light has not caused

enough damage to cause cell death. This experiment will have some healthcare applications as

the goal of a sterilization may not to be to kill all of the bacteria but rather to retard their spread

rather than their growth. As a common and cheap sterilizer UV light would be the perfect

mutagen to explore this with.

Literature Cited:

Begley, Gail S. and Charles H. Ellis Jr. 2013. Genetics and Molecular Biology Laboratory
Manual. Sagamore Beach, MA: Academx.

Cadet, Jean, Evelyne Sage and Thierry Douki. 2004. Ultraviolet Radiation-mediated
Damage to Cellular DNA. Mutation Research/Fundamental and Molecular Mechanisms
of Mutagenesis. Available online 2005. Accessed 28 Oct. 2015.
http://www.sciencedirect.com/science/article/pii/S0027510704004798

Falkiner, Frederick R. and Celine Herra. 2010. Serratia marcescens. Antimicrobe.

Accessed 28 Oct. 2015. http://www.antimicrobe.org/b26.asp

Kearns, Daniel B. 2010. A field guide to bacterial swarming motility. Nature Reviews.
Microbiology. NCBI. 634-644. Available online 2010. Accessed 29 Oct. 2015.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135019/

Nave, R. n.d. Electromagnetic Spectrum. Georgia State University. Accessed 28 Oct.

2015. http://hyperphysics.phy-astr.gsu.edu/hbase/ems3.html
Paruchuri, D and R. Harshey. 1987. Flagellar Variations in Serratia Marcescens is
Associated With Color Variation. Journal of Bacteriology. Available online 2008.
Accessed 29 Oct. 2015. http://jb.asm.org/content/169/1/61.short