Biofilm Growth in Rat Water Bottles Liz Ericson, Stephanie Fisk, Terri Lechtenberg, & Matt Lucas Introduction This past term, it was discovered that the rats used in our senior research had a “slime” growing in their water bottles. This slime was on both the glass and plastic bottles. The “slime” decreased the water quality and caused the researchers to consider outside variables in the water. The researchers began to wonder whether or not there were harmful microorganisms or toxins in the water. A study found in Applied & Environmental Microbiology, indicated that most bacteria found in water bottles were proteobacteria and mostly harmless (Schmeisser, et al, 2003). Another study found in the same journal concluded that after only a single cell attached to the surface, an entire independent microcolony can develop (Martiny, Jargensen, Albrechtsen, & Arvin, 2003). This leads to the production of a biofilm. A biofilm is defined as “an assemblage of microbial cells that is irreversibly associated (not removed by gentle rinsing) with a surface and enclosed in a matrix of primarily polysaccharide material” (Donlan, 2002). The rate of growth in aqueous mediums is dependent on pH, nutrients levels, ionic strength, and temperature (Donlan, 2002). Based on these characteristics, it is hypothesized that there is a biofilm growing in the water bottles. Materials and Methods Three plastic water bottles that had been used in the rat experiment were obtained. These water bottles were washed, rinsed, and bleached. They were filled with approximately 450 ml of tap water. Two plastic coverslips were placed in each water bottle. The water bottles were then inverted and placed in a rat cage (with live rat). One week later, the plastic coverslips were removed. The slides were stained with DAPI according to the procedures found in the lab manual. The coverslips were then viewed under an Olympus fluorescent microscope. Images were captured at 40x and 100x. Results Figure 1 Figure 2 Figure 3 Figures 1, 2, and 3 -- (40x) -- These are fluorescent micrographs of the biofilm growing on the rat water bottles. The green is the bacteria and the blue is the “slime” produced. Conclusion There are microorganisms present in the rat water bottles. They produced a “biofilm” that was visible under fluorescent microscopy. We were not able to determine what type of bacteria these were. The numbers were higher in some water bottles than in others, possibly indicating an unknown variable. References Donlan, R.M. (2002). Biofilms: Microbial Life on Surfaces. Emerging Infectious Diseases. [Electronic Version] Retrieved from the World Wide Web on March 23, 2004 fromhttp://www.cdc.gov/ncidod/eid/ vol8no9/pdf/02-0063.pdf Figure 4 -- (100x) -- A more magnified view of one of the bacteria. Martiny A.C., Jargensen, H., Arvin, E., & Molin, S.A. (2003). Long-Term Succession of Structure and Diversity of a Biofilm Found in a Modified Drinking Water Distribution System. Applied & Environmental Microbiology, 69, 6899-6906. Schmeisser, D., et al. (2003). Metagenome Survey of Biofilms in Drinking-Water Networks. Applied and Environmental Microbiology, 69, 1298 - 1310.