Variation in Bacterial Load Across Lake Munson

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Variation in Bacterial Load Across Lake Munson

• An investigation by Cain Norris
• and Sam Hyson

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Acknowledgements
We would like to thank Dr. Thomas Miller of the
FSU Ecology department for providing with the
equipment, materials, and expertise needed to
conduct our experiment.
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Question
How does the bacterial load vary across Lake
Munson, from inflow to outflow?
Sub-questions How does bacterial
concentration vary across the lake? How does
species diversity vary throughout the lake?
What species are consistently present throughout
the lake, and are any of these fecal bacteria?
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Hypothesis
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Variables

• Independent variable sample site, qua distance
  through lake.
• Dependent variables bacterial concentration,
  species diversity.
• Controlled variables strength of dilution,
  volume of sample tested, incubation time, all
  environmental factors during sampling.

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Materials

• 25 sterile sample vials (1.5mL microcentrifuge
  vials)
• GPS receiver
• Wading pants, canoe and ephemera
• 50 Petri dishes
• Agar
• Autoclave
• 1L Erlenmeyer flask
• 25 Dilution vials
• 75 sterile tips

• P200 Pipetman micropipette
• Bunsen burner
• Glass spreader
• Alcohol (IPA)
• Vortex mixer
• Incubator
• Refrigerator
• 3 XLD agar plates
• Inoculating loop

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Procedure

• Selected 5 locations on Lake Munson.
• Visited sites via canoe or wading pants and
  recorded exact locations with GPS.
• Filled 5 sample vials at each location.
• Prepared 50 agar plates at lab.
• Prepared one 10-4 dilution for each sample.
• Agitated samples with vortex mixer to ensure even
  bacteria concentration.
• Pipetted 1 mL of each sample/dilution onto agar
  plate and spread evenly with a sterilized glass
  spreader.
• Incubated agar plates for approximately 72 hours.

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• Visually differentiated the species of bacteria,
  fungi, etc. that had grown on the agar plates.
• Discarded uncountable plates and plates without
  sufficient colony growth.
• Counted (or estimated) the number of colonies of
  each species for each sample. (When a species
  appeared on both the original sample and its
  dilution, it was typically only counted on one of
  the two plates.)
• Selected six species of bacteria that were
  prevalent, consistently appearing, or otherwise
  stood out, and transferred samples of these to
  specially prepared XLD agar plates to test for
  fecal bacteria.

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Species classified
shiny white orange matte
tan purple white fungus matte
white pink strawberry yellow red
black fungus pink fungus clear yellow
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Results!
Except for the anomaly of sample site 3, organism
(and hence bacterial) concentration decreases
across the lake.
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Results, cont.
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Results, cont.
Species diversity also decreases across the lake
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We used specially prepared agar plates (agar with
xylose lysine deoxycholate) to determine if
shiny white, clear yellow, pink,,purple,
or red were fecal bacteria (i.e. E. coli).
These species were either consistently present or
otherwise notable. If any were fecal, they would
be identified by a greenish sheen.
None of these bacteria were identified as fecal.
However, the fact that they grew at all on the
XLD plates suggests they are all Gram-negative,
thus probably pathogenic.
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Conclusion
Our analysis of the data supported our
hypothesis. Generally, both the bacterial
concentration and species diversity diminish
across the lake, confirming that the lake acts as
a filter. Lake Munson serves to partially cleanse
the water of likely pathogenic bacteria before it
flows on to Wakulla Springs.
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Limitations and Improvements

• We only distinguished the bacteria colonies
  visually. This may have been a source of
  inaccuracy, which could have been prevented by
  the addition of DNA sequencing to the procedure.
• Colonies were sometimes so numerous that we could
  only estimate their magnitude. Various methods
  exist to cope with this, including taking
  photographs of the plates and magnifying them to
  improve differentiable detail.