Diversity of Culturable Bacteria Isolated from Freshwater Sediment

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Diversity of Culturable Bacteria Isolated from
Freshwater Sediment

• Kimberly Van Demark
• Department of Biological Sciences
• Minnesota State University, Mankato

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• Sampling Regime
• Collected samples Aug, Nov, Dec, 2000 and Jan,
  Feb, 2001 from Hiniker Pond Mankato, Minnesota.
• Methodology
• Used a general 10-fold dilution scheme and
  plated the dilutions on LB, EMB, and SS agar. LB
  is a general complex nutrient medium that allows
  for majority of organisms to grow. EMB is
  selective for gram-negative bacteria and
  differentiates between lactose and non-lactose
  fermenters. SS is selective for gram-negative
  bacteria and differentiates between Salmonella
  and Shigella.
• I chose the selective media because Hiniker Pond
  is a recreational area and the EMB and SS are
  selective for possible human pathogens.
• Isolates that were morphologically different
  over the course of the study were chosen for
  further analysis.

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Hiniker Pond

• Year-round recreational
• area
• Former gravel pit
• Located in Mankato, MN

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?
• Are there different culturable bacteria present
  during the course of the study?

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?
• Are there different culturable bacteria present
  during the course of the study?
• Can the identity of the bacteria be determined by
  the traditional microbiological tests
  (biochemical, physiological, and morphological)?

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Methods
10-fold dilutions (10-1-10-4) With 1 gram sediment
Plate on EMB, SS, and LB Agar
Isolation of morphologically different organisms
Plate counts
Biochemical Tests and Presumptive Identification
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Isolates as they would appear on SS (left) and
EMB (below).
This is a colony-forming unit (CFU). Each colony
represents an individual organism which I am
referring to as an isolate.
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• The next several slides will show the data that
  was collected to answer
• the research questions.
• Question 1
• For the plate counts on LB agar, a statistical
  analysis was performed on all samples except for
  August 2000 (because of fungal contamination on
  the agar plate). It was found that November was
  statistically different from the other months and
  December 2000 was different from February 2001.
• The next slide shows single data points for EMB
  and SS agar plate counts for the study time
  period. The same trend seen on LB for November
  was also seen on EMB for November 2000.

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?
• Are there different culturable bacteria present
  during the course of the study?
• Can the identity of the bacteria be determined by
  the traditional microbiological tests
  (biochemical, physiological, and morphological)?

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Months with the same letters are not
significantly different from each other (p lt 0.5).
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1 EMB is a selective media for gram-negative
bacteria and also differential for lactose
and non-lactose fermenting organisms. 2 SS is a
selective media for gram-negative bacteria and
differential for Salmonella and Shigella.
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• Question 2
• The traditional microbiological test results
  were used to answer this question.
• The next several slides show a flow chart of the
  experimental design that was used for this study.
  
• All selected isolates were characterized for
  variety of tests an example for two isolates is
  shown on slide 20. As shown on this slide these
  two isolates are different based on these test
  results.

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?
• Are there different culturable bacteria present
  during the course of the study?
• Can the identity of the bacteria be determined by
  the traditional microbiological tests
  (biochemical, physiological, and morphological)?

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Traditional Microbiological Tests
Biochemical
Morphological
Physiological
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Traditional Microbiological Tests
Morphological
Colony color, texture, shape Rod, coccus, or
spirillum Cell wall structure
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Traditional Microbiological Tests
Physiological
Respiration enzymes Growth temperatures
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Traditional Microbiological Tests
Biochemical
Glucose fermentation Tryptophan utilization
Extracellular enzymes
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Traditional Microbiological Tests
Biochemical
Morphological
Physiological
Glucose fermentation Tryptophan utilization
Extracellular enzymes
Respiration enzymes Growth temperatures
Colony color, texture, shape Rod, coccus, or
spirillum Cell wall structure
20
December Organism 2
August Organism 1
Microbiological Tests
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• Question 3
• A similar method as seen for question 2 was used
  to presumptively identify the isolates to at
  least a genus level.
• This sequence of slides shows step by step how
  the traditional microbiological tests were used
  for identification purposes. Results were
  compared with those found in Bergeys Manual of
  Determinative Bacteriology.
• Ten of 23 isolates were presumptively identified
  to either the genus level or genus and species as
  seen on slide 28.

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Research Questions

• Does the abundance of culturable bacteria show
  seasonal shifts?
• Are there different culturable bacteria present
  during the course of the study?
• Can the identity of the bacteria be determined by
  the traditional microbiological tests
  (biochemical, physiological, and morphological)?

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Organism A
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Traditional Microbiological Tests
Morphological

• Colony color, texture, shape
• Rod, coccus, or spirillum
• Cell wall structure

• Organism A
• White
• Rough and irregular
• Gram positive rods

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Traditional Microbiological Tests
Physiological
Respiration enzymes Growth temperatures

• Organism A
• Negative for catalase
• Negative for oxidase
• Grew at 50oC

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Traditional Microbiological Tests
Biochemical
Glucose fermentation Tryptophan utilization
Extracellular enzymes

• Organism A
• Positive for glucose
• fermentation
• Negative for tryptophan
• utilization
• Positive for lipase
• Negative for amylase and
• gelatinase

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Organism A
Presumptive Identity Bacillus pumulis
Presumptive identification was based on comparing
laboratory microbiological test results to
Bergeys Manual of Determinative Bacteriology
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(No Transcript)
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Research Questions Summary

• Does the abundance of culturable bacteria show
  seasonal shifts?
• A statistical analysis of the number of
  culturable bacteria cultivated on LB agar
  (complex media) found that samples from November
  were significantly greater than those from
  December, January, and February. It was also
  found that December and February were
  statistically different.

-Seasonal shift Fall (Nov) Winter (Dec, Jan,
Feb) -Monthly shift 1. November compared
to other months 2. December compared to
February
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Research Questions Summary

• Are there different culturable bacteria present
  during the course of the study?
• The traditional microbiological test results
  showed metabolic and morphological diversity
  between isolates on a month to month basis and
  even within the same month.

August 2000 8 November 2000 8 December 2000
4 January 2001 3 Total 23 different
isolates
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Research Questions Summary

• Can the identity of the bacteria be determined by
  the traditional microbiological tests
  (biochemical, physiological, and morphological)?
• Based on a comparison of data obtained from this
  study with that of known bacteria referenced in
  Bergeys Manual of Determinative Bacteriology, 10
  of the 23 isolates were identified.

• Enterobacter agglomerans -Klebsiella
• Micrococcus luteus -Bacillus spp
• Bacillus pumulis -Bacillus spp.
• -Micrococcus roseus -Streptomyces
  -Pseudomonas spp. -Pseudomonas spp.

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Acknowledgements

• College of Graduate Studies
• Faculty Research Grant "Population Dynamics of
  
• Cultured Bacteria Isolated from a Freshwater
  Lake",
• Fall 2000
• Undergraduate Research Conference
• Scholarship
• Department of Biological Sciences
• Laboratory materials and equipment, computer use
• Faculty Advisor
• Dr. Elaine O. Hardwick