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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.