The Effect of UVC Radiation on the Prevalence of CAMRSA in high school athletic departments

Specimen Collection. Under surveillance for two years, a population showing the appropriate characteristics for CAMRSA infestation was determined. Characteristics included a high density of participants, lack of cleanliness within a communal area, lack of personal hygiene among participants, and one or more participants showing symptoms of CAMRSA infection. Observations displayed that a high school football athletic department exemplified these characteristics. After a potential population was determined, an exploratory specimen was taken from the helmet of a member of the population. Since Staphylococcus aureus is known to be found in the anterior nares and on the face, the helmet was the prime area of data collection. The polyester applicator was extracted from its package by peeling back the handle end of the package and pulling the applicator by the handle. The helmet was thoroughly swabbed at the chin strap, forehead pad, cranium pads, and ear pads. After collecting the exploratory specimen, the applicator was placed cotton-side down into its respective package. The identification number, time of collection, and date of collection were then written on the applicator package, allowing for efficiency and organization. The applicator was placed in a 2-ply sealed biohazard bag and placed in a cooler for transport to the lab for inoculation to broth. The specimen was placed in a culture tube containing 3ml of BBL Nutrient Broth and incubated for 48 hours at 35oC. After 48 hours, the specimen was shown to have growth; therefore, the population could be tested. The one hundred eleven specimens were collected on two occasions over a period of three weeks, utilizing the same collection procedure as the exploratory specimen. 50 specimens were collected on the first and second days, with eleven collected on the third. For statistical purposes, each sample was chosen using cluster and stratified random sampling. The football department’s locker room was randomly divided into eleven clusters (ten clusters with ten students; one with eleven). A cluster was randomly chosen. From that cluster, five random students were then chosen. This process was repeated until the desired number of specimens was collected. Prevalence. Samples that were collected were placed in culture tubes containing 3 ml of BBL Nutrient Broth. Then, the identification number of the specimen was written on the tube. The tubes were then organized numerically and placed in an incubator. A culture tube that was not inoculated with a specimen was placed in the incubator as a control. The growth in the tubes was then monitored at the 24-hour and 48 hour mark. After 48 hours, the specimens that showed growth were heat smeared and Gram stained using the proper methods. The slides were divided into four sections, each containing a specimen. Stock specimens of Staphylococcus aureus ATCC 25923 and E. coli ATCC 25922 were used as positive and negative controls to ensure proper Gram staining technique. The specimens that were determined to be cocci were then inoculated to Oxacillin Plates to selectively isolate those that were methicillin resistant. These plates were divided into regions, with one region representing one specimen. The cocci specimens were spot inoculated onto their respective region and incubated. An Oxacillin plate that was not inoculated was incubated as a control, to ensure acceptable media. The specimens that were found to be methicillin resistant were then subbed onto mannitol salt agar for selecting Staphylococcus specimens and differentiating between Staphylococcus aureus and coagulase negative Staphylococcus. Each plate grew one specimen, for adequate isolation of colonies. A mannitol salt agar plate that was not inoculated was used as the control. Specimens that presented a yellow halo around the colonies (signaling Staphylococcus aureus) had a coagulase test performed to confirm findings. UV Experimentation. Once a prevalence of CAMRSA has been determined in a population, UV radiation was tested as a potential solution. A culture tube of Staphylococcus aureus ATCC 25923 was made by inoculating the broth with the microbe from a stock blood agar plate. The broth was incubated via a standardized incubator for 48 hours, with a culture tube with no inoculants used as the control. After the S. aureus has grown in the culture tube, three standardized 1L calibrated loops are placed in the broth and then the bacteria is inoculated to three Flinn Scientific Nutrient Agar plates as comparison controls. Using McFarland Standards, these nutrient agar plates should contain 1.5*105 CFU/mL. In order to ensure proper McFarland standards, the calibrated loop was placed into the broth to where only the loop is submerged; all parts of the handle remain dry. A nutrient agar plate with no inoculants was used as a negative control. Once a calibrated loop is used, it is either disposed in a biological hazard trash bin, or is autoclaved at 121oC for 15min and then disposed of in a trash bin. Then, sixty nutrient agar plates are inoculated with respective 1L calibrated loops via the same process. The plates are all placed in an incubator at 35oC for 48 hours. After all 64 plates have been incubated and have shown equal growth, they are taken to a SterilaserTM for testing. The SterilaserTM is a mat cleaner that has UV-C light fixtures attached to a wheeled container. The SterilaserTM was placed on top of two pillars to ensure stability. This makes the UV cleaner three inches from the ground. The comparison controls were placed aside. The SterilaserTM was turned on without any plates underneath and allowed to warm up, to ensure the same light intensity throughout the experiment. Sixty plates were divided into twenty data sets, with one data set comprised of three plates. Each individual dataset was placed under the SterilaserTM for their respective exposure times. Exposure times ranged from 1sec to 20sec in increments of 1sec. Once each dataset has been exposed to its exposure time, it was moved away and the number of visible colonies was counted and multiplied by 1.5*105 CFU/mL to determine the new bacterial concentration. This process was repeated for all twenty datasets.