Zachary Stayn and John Fu

Testing the Effect of Different UV Light Intensities on Pseudomonas Fluorescens Bacterial Biofilm Formation and Growth

Abstract Video:

Biology DYO.mp4

Background:

Collections of living, reproducing microorganisms called biofilms are everywhere! From dental plaque on your teeth to penicillium roqueforti bacteria in the cheese you eat to the slime on barnacles in the ocean, these communities offer bacteria, fungai, and protists many advantages, such as protection from external threats (i.e., lack of water, high or low pH, and the presence of toxic substances like antibiotics) by forming a protective layer called a matrix, the possibility for quorum sensing (i.e., cooperative behavior to organize necessary tasks), and greater access to life sustaining resources like food and water (1). These communities form by attaching to a surface using a sticky, sugar and protein packed substance called an EPS, then grow and mature, and finally disperse to establish another biofilm (2).

Despite all that is known about biofilms, however, a new, unsolved problem is now at the forefront of biofilm research. The overuse of antibiotics in medicine today has rapidly accelerated harmful bacteria’s ability to resist treatment and form large, intrusive biofilms, throughout the body (3). A new method is needed to combat this type of biofilm formation, especially to solve illnesses like Ultraviolet (UV) light, a form of electromagnetic radiation with shorter wavelengths than the visible spectrum, coupled with antibiotics is a promising way to eradicate biofilm in vivo, though has not been thoroughly investigated (4). Specifically, UV light may target systems in microorganisms that are not designed for that type of radiation, thus possibly damaging bacteria’s ability to properly function and form a biofilm. Additionally, manipulating the UV light intensity (energy of light received per unit of bacterial area) may also affect microorganism biofilm formation.

Introduction:

Multiple studies have already demonstrated that exposing bacteria to different wavelengths of UV light can both produce no effect, or negatively affect biofilm formation and growth (5, 6). In addition, studies have shown that UV light source, whether a mercury lamp, a xenon lamp, or UV LED, can drastically alter the ability to control biofilm (7). However, little is known about how biofilm formation is affected when the intensity of the UV light (an unrelated variable to wavelength or UV source) is altered. And, even in the few studies that touch on wavelength, these studies do not use pseudomonas fluorescens bacteria. So, we were curious about this un-investigated part of biology, and wanted to investigate.

This experiment aimed to discover the relationship between UV light intensity and bacterial biofilm formation in vitro by repeatedly zapping pseudomonas fluorescens bacteria with different intensities of UV light as individual colonies tried to form and grow biofilms on plastic beads.

Methods:

30 Second Methods Overview:

Our procedure was conducted over a 7 day period of time, and we used the Evolving STEM Evolution Protocol and Crystal Violet Assay Protocol to guide our work.

In the first five days of the experiment, we selected for bacteria that would form biofilms on glass beads, and we irradiated these beads with varying intensities of UV light on two days throughout the biofilm formation and growth process.

Then, we plated bacteria on the final glass beads and conducted a crystal violet assay to evaluate biofilm formation on each of the variously treated beads. The results came in the form of different absorbance values, where higher light absorbances indicated more biofilm growth since bacterial biofilms are murky.

Days 1-5: Cultivating the Bacterial Biofilms and Exposing Them to UV Light

Day 1--Inoculation

On a sterilized lab table, four culture tubes (labeled no UV, low UV, medium UV, and high UV) were each filled with 5 ml of QB medium, and a white bead was added to each tube using sterilized forceps. Then, five Pseudomonas fluorescens colonies from a starter plate were placed into each of the five culture tubes, respectively, using 5 plastic inoculation loops, and the tubes were incubated on an orbital shaker for 24 hours to enable the formation of a biofilm on the white bead.

Day 2--Bead Transfer

Four more culture tubes (labeled in the same way as Day 1) were each filled with 4.5 ml of QB and a new white bead. Each of the white beads from the day 1 tubes were then transferred to separate microcentrifuge tubes, each containing 950 µL QB. These tubes were each vortexed for one minute to remove all bacteria from the bead, and then 50 µL of liquid from each microcentrifuge tube was transferred to the corresponding Day 2 culture tube (e.g., day 1-no UV liquid to Day 2-no UV tube). The tubes were then put in the orbital shaker for 24 hours to enable biofilm formation on another bead.

Day 3--Bead Transfer and Low UV Exposure

Four culture tubes (labeled in the same way as day 2) were filled with 5 ml of QB and a black bead was added to each tube. The white beads in each of the day two culture tubes were then placed onto empty, separate sterile petri dishes, respectively. Each bead (on each dish) was then placed into a preheated UV crosslinker, and exposed to either 0 µJ/cm² (no UV bead), 300 µJ/cm² (low UV bead), 1200 µJ/cm² (medium UV bead), or 2400 µJ/cm² (high UV bead). For each run, the bead was zapped 4 times and rolled around using sterile forceps between UV exposures to ensure all sides of the beads were exposed. Each bead was then transferred from the petri dish to the corresponding Day 3 culture tube (e.g., day 2 no UV bead to day 3 no UV culture tube), and the tubes were incubated on the orbital shaker for 24 hours.

Day 4--Bead Transfer and Higher UV Exposure

Four culture tubes (labeled in the same way as day 3) were filled with 5 ml of QB and a black bead was added to each tube. The black beads in each of the day two culture tubes were then placed onto empty, separate sterile petri dishes, respectively. Each bead (on each dish) was then placed into a preheated UV crosslinker, and exposed to either 0 µJ/cm² (no UV bead), 3,000 µJ/cm² (low UV bead), 12,000 µJ/cm² (medium UV bead), or 24,000 µJ/cm² (high UV bead). For each run, the bead was zapped 4 times and rolled around using sterile forceps between UV exposures to ensure all sides of the beads were exposed. Each bead was then transferred from the petri dish to the corresponding Day 4 culture tube (e.g., day 3 no UV bead to day 4 no UV culture tube), and the tubes were incubated on the orbital shaker for 24 hours.

Day 5--Plating of Final Biofilms

Four microcentrifuge tubes (labeled in same way as day 4) were filled with 950 µL of PBS buffer solution, and each of the white beads from the day 4 culture tubes were then transferred to corresponding Day 5 microcentrifuge tube using sterile foreceps. Each of these tubes was vortexted for one minute to remove all bacteria from the beads, leaving an undiluted sample (10⁰). Then, a serial dilution was conducted: 100 µL from each 10⁰ tube was transferred to other, separate microcentrifuge tubes with 900 µL PBS, forming a 10^-¹dilution. Then, 100 µL from each of the 10^-1 tubes was transferred to other tubes, forming 10^-2 dilutions. Finally, 100 µL from each of the 10^-2 tubes was transferred to 10^-3 tubes. 100 µL of solution from the 10^-3 tubes were then transferred to agar plates. The liquid on each plate was spread evenly with a separate, plastic plate spreader, and the plates were put on the orbital shaker for 24 days.

Days 6-7: Evaluating Biofilm Growth After UV Exposures Using Absorbance

Days 6-7--Crystal Violet Assay

2 ml of Queens B medium (QB) was added to each of five culture tubes. One single Pseudomonas fluorescens colony from each of the 4 agar plates were placed into the corresponding culture tubes, respectively, using plastic inoculation loops. Then, these culture tubes were incubated on an orbital shaker. 3 days later, the liquid in each of the culture tubes was carefully pipetted out, and 3 ml of crystal violet solution was added to each of the culture tubes to stain the biofilms. Crystal violet solution was also added to an empty tube to serve as a technical control to indicate a baseline absorbance without bacteria present and account for any effect plastic-crystal violet solution interactions have on absorbance. The tubes were then incubated at room temperature for 15 minutes to allow the dye to stain the tubes. Next, the crystal violet solution was pipetted out of all of the tubes in the same way. 10 ml of deionized water was then added to each culture tube and the control to gently wash away excess crystal violet solution, and the tubes were incubated for 1 minute before pipetting out all of the liquid from the tube. Then, 1 ml of ethanol was added to each of the culture tubes and the technical control, and the tubes were vortexed to wash out the remaining stain. 200 µL from all tubes was then added to 5 separate, corresponding wells in the 96 well plate (25 wells total). Finally, the plate was placed in the spectrophotometer and absorbance was measured at 595 nm. Since a bacterial biofilm is murky, more biofilm formation and growth should yield higher absorbances.

Results:

Figure 1: Impact of UV Light Intensity on Pseudomonas Fluorescens Biofilm Formation and Growth--Measured Through Absorbance. Each of the first four bars represents the average absorbance of bacterial samples exposed twice to one of four sets of UV intensities over a five day period. The fifth bar represents a control with no bacteria. Uncertainty bars represent +/- AAD. Unpaired t-tests to evaluate significance between all UV intensities and the control were calculated a p<0.05 for all but one comparison (ns).

Discussion of Results and Significance:

The results yielded 2 major findings:

Finding 1: Increasing Intensities of UV light generally decrease Pseudomonas fluorescens biofilm formation and growth.

As the intensity of UV light increased from none to low to medium, the amount of biofilm formation and growth decreased significantly from an 595 nm light absorbance of 0.876 to 0.078 to 0.678, respectively (p<0.05, Fig 1). On a molecular level, UV light deactivates the DNA in microorganisms at the beginning stages of attachment to a surface to prevent the formation of a protective biofilm matrix (8) Specifically, in bacteria, UV light causes two of the nucleotides (building blocks) of DNA, cytosine and thymine, to bond covalently together, forming a cyclic ring structure called a dimer. This results in these two pyrimidine bases being unable to bind to the other two complementary nucleotide, purine bases: adenine and guanine, in the double helix (9). Thus, an unstable structure is formed, leading a molecular rearrangement that results in a kink in the fundamental DNA structure, stopping transcription and translation (protein synthesis). Typically, through a process called nucleotide excision repair involving a nuclease enzyme and polymerase, cells can immediately repair damage caused by UV light. But, as UV light is increased, and more of these damaging dimers are formed, the repair mechanism can’t get to all of the damage, or starts to repair the damage incorrectly (10). Thus, cells exposed to high amounts of UV cannot make crucial proteins, and the cells immediately die, preventing biofilm formation.

Finding 2: Above a certain threshold of UV exposure, there is no difference in Pseudomonas fluorescens biofilm formation and growth.

Specifically, there was no statistical difference in biofilm formation between the medium and high intensity UV exposures (p=0.48). The literature, however, does not support out finding. Multiple studies have demonstrated that the matrix that biofilms form out of extracellular polymeric substances (EPS) offers some protection to UV doses, as UV light just can’t penetrate through the increasingly thick layer that forms as bacteria multiply and produce more EPS (11). But, this layer only offers protection against low intensities of UV light that can’t penetrate through the matrix (11). At higher intensities, the UV rays should be able to more thoroughly and completely penetrate through the protective layer, thus further damaging bacterial DNA, killing cells, and preventing further biofilm formation or growth (11). Furthermore, the trends in the literature suggest that, with high enough UV intensities, biofilm formation and growth should be virtually undetectable (12). In our experiment, this result would be evident if the highest UV intensities produced absorbances similar to the crystal violet control (where no bacteria were present). Notably, those findings do not match this experiment’s results, likely because the UV intensities tested in this experiment were not high enough to produce such results.

Conclusions, Uncertainties and Future Experimentation:

In conclusion, the data suggest that [1] increasing intensities of UV radiation lead to decreased Pseudomonas fluorescens biofilm formation and growth, and [2] UV intensities above a certain threshold do not further prevent biofilm formation and growth. This means UV light might be a promising method of eliminating harmful biofilms and could possibly be used to an extent as part of medical treatments.

Some uncertainties that could have affected results were [1] the imprecision in the dozens of volume measurements throughout the experiment that could have led to lost liquid and could have affected final absorbance measurements, [2] the technique of rolling the glass beads around between UV exposures that could have led small amounts of biofilm to come off of the beads, and [3] the overall sterility of the environment since bacteria were exposed to open, contaminant-filled air for long periods of time throughout the procedure

Additional experimentation is needed to determine if this relationship holds true against bacteria that are more readily found in the human body (since Pseudomonas fluorescens is not readily present in humans). Specifically, research into the effect of UV radiation against bacteria in the oral cavity, in the lungs of cystic fibrosis patients, and in the urinary system would be useful.

Here is the link to our full lab report if you are interested:

https://docs.google.com/document/d/1a3iV6ETSwsDyGh5cgxsSxHvwzA9fmv6zJDCzzAFFbdM/edit?usp=sharing

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