Myka Cabuhat, Justin Euperio and Peter Huang

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 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 (±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 lens-associated 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 0-300 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.

Literature Cited

Behar-Cohen, F., Baillet, G., de Ayguavives, T., Ortega, P.G., Krutmann, J., Peña-García, P., Reme, C., Wolffsohn, J.S. (2013). Ultraviolet Damage to the Eye Revisited: Eye-Sun Protection Factor (E-SPF®), a New Ultraviolet Protection Label for Eyewear. Dovepress: Clinical Ophthalmology, 8, 87-104. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3872277/

Clark, J., Friedrich, M., Ininns, E., Moloznik, K., Bandekar, A., Wrightsman, R. (2014). Effects of Ultraviolet Light & The Viable Plate Count Method of Counting Bacteria. Laboratory Manual for Bio 15 - General Microbiology, 74-78 & 92-93.

Dongre, A., Pai, G., Khopkar, U. (2007). Ultraviolet Protective Properties of Branded and Unbranded Sunglasses Available in the Indian Market in UV Phototherapy Chambers. Indian Journal of Dermatology, Venereology and Leprology, 73(1), 26-28. Retrieved from http://www.ijdvl.com/text.asp?2007/73/1/26/30647

Kuijk, F. (1991). Effects of Ultraviolet Light on the Eye: Role of Protective Glasses. Environmental Health Perspectives, 96, 177-184. Retrieved from http://www.jstor.org/stable/3431229.

Rosenthal, F., Bakalian, A., Lou, C., Taylor, H.R. (1988). The Effect of Sunglasses on Ocular Exposure to Ultraviolet Radiation. American Journal of Public Health, 78(1), 72-74. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1349214/

Setlow, R., Swenson, P., Carrier, W. (1963). Thymine Dimers and Inhibition of DNA Synthesis by Ultraviolet Irradiation of Cells. American Association for the Advancement of Science, 142, 1464-1466. Retrieved from http://www.jstor.org/stable/1711999

Zion, M., Guy, D., Yarom, R., & Slesak, M. (2006). UV radiation damage and bacterial DNA repair systems. Journal Of Biological Education (Society Of Biology), 41(1), 30-33.

Zhou, R., Zhang, R., Sun, Y., Platt, S., Szczotka-Flynn, L., Pearlman E. (2012). Innate Immune Regulation of Serratia marcescens-induced Corneal Inflammation and Infection. Investigative Opthalomology & Visual Science, 53(11), 7382-7388. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23033384

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.

Review Form

Department of Biological Sciences

Saddleback College, Mission Viejo, CA 92692

Author (s): Myka Cabuhat, Justin Euperio and Peter Huang

Title: The Effect of Sunglass Lenses on the Growth Inhibition of the Bacterium Serratia marcescens Against Ultraviolet Radiation

Summary

Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.

The investigators did their research and experiment on sunglasses of different colors and tints, and their relative effectiveness of protecting against UV radiation damage. They predicted that they would find no significant difference in growth, post UV exposure, between lenses of different opacities. They used three different tinted lenses, one clear lens and an uncovered control. They placed each lens over a group of five test tubes inoculated with Serratia marcescens. Then they exposed each lens and the control to UV radiation, at a wavelength of 260 nm for five minutes. They then incubated the test tubes for 48 hours at 30˚ C, they then serially diluted each test tube contents in sterile water. After this they created pour plates from the serially diluted test tube contents, and preformed Bacteria counts after a second incubation. They utilized the Quebec colony counter method.

Based upon the data, and analysis run the investigators suggested that their hypothesis had been proven. They also noted that there was a great difference between tinted lenses and the uncovered control, therefore confirming the value of sunglasses. They then proposed further studies in order to maximize UV protection capabilities of sunglasses.

General Comments

Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.

This project is interesting and very relevant to anyone living in southern California. The paper is well organized, clear, and easy to follow. I found it helpful to know the specific advantages of certain lens types.

However the paper has some issues that need to be addressed. This paper had a few formatting errors which have been noted in the comments. The word choice of many of the portions makes it slightly confusing, and could possibly detract from readers’ comprehension. I found the Intro, Material & Methods, and Results sections to be very good, with a few minor mistakes. However, the Discussion section seemed to miss the mark. They mistakenly said that they hypothesis was confirmed. This needs to be fixed. The other qualms I have with the discussion section have been commented on above. The literature cited was in correct order and cited well.

Technical Criticism

Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.

The paper had very few technical errors. Although, changes in word choice could give the readers a better appreciation of the study.

This paper was a final version This paper was a rough draft

Recommendation

o This paper should be published as is

o This paper should be published with revision

o This paper should not be published