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EFFECTS OF POLYFIBER AND CARBON SUBSTRATES ON THE GROWTH AND DIVERSITY OF BIOFILMS IN A FISH TANK FILTRATION SYSTEM Annie Reiher & Jack Kampf Introduction Materials and Methods Until recently, history tells us that microorganisms have previously been characterized as freely suspended cells whose growth depends on rich culture media. It has since been discovered that microorganisms attach to surfaces and develop into a group of surface-associated microbial cells in an extracellular matrix. This tendency of microorganisms is called a biofilm and this discovery holds great importance for the field of microbiology and general public health. Biofilms form on a variety of surfaces including living tissues, indwelling medical devices, industrial or potable water system piping, and natural aquatic systems. Although commonly unseen, these biofilms can host harmful bacteria on surfaces usually considered uncontaminated such as faucets, swimming pools, and water filters. Biofilms can form on vital surfaces in the human body such as a pacemaker, causing an infection by pathogenic microorganisms Staphylococcus aureus or Enterococcus spp. Carbon filters are used to eliminate water impurities in a fish tank. This carbon filter is composed of an exterior polyfiber pad and an interior carbon component. The polyfiber pad in the fish tank filter will show a greater amount and larger diversity of microorganisms when compared to the inner carbon material. The polyfiber pad on the exterior of the filter traps dirt and large debris thus supplying a more suitable environment for microorganisms to form a biofilm. The carbon located inside the filter removes organic matter and waste from the water. While microorganisms are capable of living on this organic matter, it may not be sufficient to sustain as large of a diversity compared to the polyfiber pad. The significance of this experiment is to determine what types of microorganisms grow on fish tank filters. This information will signify the importance of proper fish filter maintenance. By changing the filter every 2-4 weeks as directed, the growth of pathogenic microorganisms will be reduced. This will prevent dangerous levels of microorganisms and infectious disease from developing inside the filter and tank. Two biofilm reactors were constructed according to standard biofilm reactor construction to keep constant flow over inoculated media on seven slides. Each sample was obtained by blending either the carbon filter or polyfiber pad in PBS. In each biofilm reactor, 7 ml of sample was added to 500 ml of TSB media. After 72 hours of continuous flow, one of the slides from each biofilm reactor was taken and a 3 cm by 2 cm section where flow had occurred was scraped with a scalpel and added to 4.5 ml of 0.9% NaCl water. From this stock, dilutions of 10-1 to 10-8 were plated according to standard TSA plate count procedure. From these plates, isolated colonies will be obtained, extracting the two most reoccurring colonies and performing various biochemical tests to determine their identity. The plates were stored in a 37ºC incubator overnight. Isolated cultures were then taken and streaked onto EMB, PIA, and MSA plates, which were then left to incubate overnight before being read. Another slide was taken from each biofilm reactor and a gram stain was preformed on each. A third slide was then taken from each biofilm reactor and a DAPI stain was completed to find the total number of bacteria. This was accomplished by fixing the cells with 3.7% formaldehyde and staining with DAPI solution according to the DAPI staining procedure. Results The original samples of 1.3 cm2 per ml from both the carbon and polyfiber were taken and serial dilutions were performed to 1.3 x 10-8 cm2 per ml in 10-1 increments. Microorganisms were found to 1.3 x 10-4 cm2 per ml in the carbon sample and to 1.3 x 10-7 cm2 per ml in the polyfiber sample. This indicates that there was a larger amount of microorganisms found on the polyfiber filter. In both the carbon and polyfiber samples, no color difference was observed but differences in size were evident in the isolated colonies. The isolated colonies were found in 1.3 x 10-3 cm2 per ml in both. Two different types of isolated colonies were found in each sample and cultured for further tests. Isolated Culture 1 Isolated Culture 2 Gram Stain (-) (-) MSA (-) (-) PIA (-) (-) EMB (+) (+) Carbon Filter Figure 1: Carbon Filter at 40x Figure 2: Carbon Filter at 40x stained with DAPI. This DAPI test indicates the total count stained and the blue color indicates DAPI stained DNA. It is evident that the amount of microorganisms is smaller compared to the polyfiber filter and shows less diversity. stained with DAPI. This DAPI test indicates the total count stained and the blue color indicates DAPI stained DNA. This slide shows a possible fungal presence indicated by the long fibrous strands similar to hyphae. Table 1: Characteristics of Isolated Colonies (IC). Isolated cultures 1 & 2 appear to be short gram (-) rods. The MSA test indicates IC-1 is E. coli. The PIA test was negative for both IC’s showing no sign of pseudomonas. The EMB test for IC-1 indicates E. coli and IC-2 indicates Salmonella typhimurium. Isolated Culture 1 Isolated Culture 2 Gram Stain (-) (+) MSA (-) (+) PIA (-) (-) EMB (+) (-) Polyfiber Filter Table 2: Characteristics of Isolated Colonies (IC). Isolated culture 1 Figure 3: Polyfiber Filter at 40x Figure 4: Polyfiber Filter at 40x stained with DAPI. This DAPI test indicates the total count stained and the blue color indicates DAPI stained DNA. The yellow areas could be from dying or new microorganisms with less DNA. This slide indicates a larger amount and greater diversity of microorganisms were isolated from the polyfiber filter. stained with DAPI. This DAPI test indicates the total count stained and the blue color indicates DAPI stained DNA. This is another example of greater amount and larger diversity of microorganisms found in the polyfiber filter. is gram (-) while isolated culture 2 is gram (+). The MSA test for IC-2 is positive representing Staphylococcus aureus. The PIA test was negative for both IC’s showing no sign of pseudomonas. The EMB test for IC-1 is positive indicating E. coli. Conclusions The polyfiber pad did have a greater number and diversity of microorganisms than the carbon filter. The organism types that grew on both samples were mostly gram (-), although some gram (+) were found on the polyfiber pad. Filters should be changed as directed to prevent the overgrowth of harmful bacteria such as E. coli and other gram (-) bacteria.