What You’re Swimming With Matt Fox and Erin Wright Materials and Methods: Two- 20 ml samples were taken from the filter and wall of a hot tub. 2 ml of each sample were mixed with 10 ml of TSB media with 5% glucose and 5% sucrose and placed into a Petri dishes containing coverslips to provide a surface for biofilm growth. The dishes were then incubated for 24 hours at 35  C on a Big Bill Thermolyne® rotary shaker. The two samples were also inoculated onto TSA plates in order to grow colonies for the following day. The colonies grown from the filter sample and from the wall sample were used to inoculate plates of mannitol salt agar, blood agar, starch, pseudomonas isolate agar, eosine methylene blue, and spirit blue lipid hydrolysis. Dilutions of to were made by scraping coverslips of each sample to determine the number of organisms on the 4.4 cm 2 coverslip. Coverslips were then stained with DAPI and fluorescent gram stain was completed (Molecular Probes, Biofilm Preparation for Staining, 2004). Results: Table 1: Agar plate results from Isolate # 1 and 2 from bacterial growth from the filter sample. The biochemical tests indicate that Isolate #1 is likely a Pseudomonas. It grew very well on PIA and Blood agar showing that it could be harmful. Isolate #2 does not appear to be harmful from the biochemical tests run. Introduction: A report released from the Centers for Disease Control proposed that hot tubs are not as harmless as they appear. The article talks about outbreaks of pseudomonas aeruginosa dermatitis associated with hot tub use. In fact, it suggests that the microorganism is present in the biofilm when sanitizer levels are inadequate of the hot tub has not been treated properly with chemical cleaners (pseudomonas dermatitis in hot tubs, 2000). It is believed that hot tubs have a biofilm in them that may contain pseudomonas aeruginosa or other potentially harmful microorganisms. The Center for Biofilm Engineering also describes how formerly healthy patients who used indoor hot tubs had developed a progressive cough, fever and fatigue, and eventually needed lung biopsies. However after removing the hot tubs from the houses, the patients recovered from what researchers termed “hot tub lung”(Germs throw cold water on hot tub hygiene, 2004). This would make a strong argument that the hot tub is the reason the patients are sick. It is hypothesized that a biofilm grown from a sample taken from a hot tub will contain Pseudomonas aeruginosa or another microorganism that could be harmful to humans. Conclusions: Filter sample showed more bacterial growth than that from the wall. Bacteria observed was all gram-negative. Pseudomonas was found to be present in the hot tub biofilm. Potentially harmful biofilms do exist in hot tubs. References: “Germs throw cold water on hot tub hygiene.” (2004, June). Retrieved June 27, 2004 from –sw/recent-media-cov/ “Pseudomonas Dermatitis in hot tubs.” (200, Dec). Weekly. 49(48), pp Ventullo, R. (2004). Biofilm preparation for staining. Handout from June 29, Figure 1, The biofilm viewed under phase shows just how many organisms are present. It indicates that a biofilm can grow in a hot tub and that there are potentially harmful microorganisms in the biofilm. Figure 2, Fluorescent gram stain of the biofilm. All of the cells are green and this means all of the bacteria are gram negative. Figure 3, DAPI stain of the biofilm. The various microorganisms can be seen. One stained blue and the other stained yellow.