Soil Microorganisms and Antibiotics

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Soil Microorganisms and Antibiotics

• December 6, 2004
• Kenice Frank, Allison Johnson, Ruben Krantz,
  Hannah Wilbur

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Soil bacteria
Soil environments are host to a great number of
bacterial species

• Habitats
• Water films
• Need water for metabolic processes
• On surface of organic matter
• Need surface on which to grow
• In rhizosphere

• Competition
• Compete with surrounding bacteria and fungi
• Production of antibiotics by competing bacteria

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Antibiotics

• Can be used as
• Fungicide, etc for farming
• Antibiotics for humans and animals

• From Strepomyces species alone
• 500 antibacterial products identified

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Objectives

• Identify through laboratory testing members of
  the soil communities
• Isolate and cultivate antibacterial producing
  bacteria from soil
• Observe and understand members of different
  communities of soil bacteria

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Soil Experiment

• Methods and Materials

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Methods and Materials

• The first step of our experiment was to chose 3
  different locations/environments to obtain soil
  samples. We chose a river, marsh and forest.

Marsh
Forest
River

• We then used sterile tubes to obtain the sample

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Methods and Materials

• We added sterilized H2O to each soil sample and
  streaked 500 ml of each soil type onto 4
  starch-casein agar for each soil type.
• We incubated these plates at room temperature.

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Methods and Materials

• Week 2
• We took 100 ml of forest sample and diluted it
  with 400 ml more of sterile H2O
• We isolated 4 visually different colonies from
  each soil type and streaked each colony type onto
  individual starch casein agar
• -We did a smear of the entire forest plate and
  the entire river plate because there were no
  identifiable, separate colonies
• Incubate at room temperature

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Methods and Materials

• Week 3
• Got results from previous weeks plates
  examined for any Streptomyces by looking for any
  areas of inhibition (clear areas surrounding
  colonies)
• Using soil plate prepared for the general lab, we
  tried to isolate antibiotic producing organism
  again

Agar plates showing areas of inhibition
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Methods and Materials

• We streaked the lab specimen and a sample from
  each soil plate from last week down the middle of
  a BAP (blood agar plate) and then used method for
  testing sensitivity to observe for any areas of
  inhibition or hemolysis
• We then streaked S.aureus and E.coli on starch
  casin agar plates perpendicularly to a center
  streak of each isolated colony type without
  touching it

Blood Agar Plate (BAP)
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Methods and Materials

• We performed Gram staining on
• -3 forest plates
• -2 river plates
• -4 marsh plates
• Based on our results from the Gram staining, we
  decided that we would need to identify whether we
  had any bacilli or enterics by performing some
  tests
• 1.Bacillus- endospore staining, catalase test
• 2.Enterics- oxidase test, TSI test
• Make decision about what colonies were growing on
  the plates

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Soil Experiment

• RESULTS

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Starch Casein Plates

• 1st plating cultures too thick
• 2nd plating individual colonies observed
• Gram Stain
• Marsh A Gram () Gram (-) rods
• Marsh B Gram () rods
• Marsh C Gram (-) cocci
• River A Gram (-) cocci in clusters
• River B Gram (-) cocci in clusters
• Forest A Gram (-) rods
• Forest BGram () ovals
• Forest C Gram (-) cocci in clusters

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Sensitivity/Inhibition Testing

• No inhibition observed

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Blood Agar Plate

• Marsh B total hemolysis
• Forest A total hemolysis

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Oxidase Test Catalase Test

• Forest A (Gram neg. rods) Oxidase positive

• Forest A Catalase positive
• Marsh B Catalase positive

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Triple Sugar Iron Test

• Forest A Red/Yellow (K/A)
• Glucose and 1 other sugar fermented

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Endospore Staining

• Red bacillus cells
• No endospores observed

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Soil Experiment

• Discussion

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Discussion

• The first goal was to properly identify
  Streptomyacin, or other inhibiting agents
  produced by the bacteria in the soil that would
  supposedly combat against E. coli and S. aureus.
• These attempts failed, as both species of
  bacteria sustained growth.

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Discussion

• The experiment was also intended to isolate
  certain colonies from three different
  environments forest, marsh, and river.
• After single colonies of the first plating were
  isolated physically, a gram stain from the marsh
  indicated that bacillus and enteric species were
  present.
• Tests resulted negative for enteric bacteria and
  positive for bacillus, however the endospore
  stain resulted negatively.

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Tests for Streptomyacin

• What should have happened
• After the second plating the colonies of E. coli
  and S. aureus should have showed suppressed
  growth to any inhibiting factors that the samples
  produced.

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Tests for Streptomyacin

• What went wrong
• The procedure for isolating the bacteria should
  have been done using selective and differential
  media in order to eliminate any other
  contamination in the culture.
• Other possibilities are that streptomyacin
  producing bacteria did not, in fact, reside at
  the chosen locations.

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Bacterial Identification

• Enteric bacteria are gram negative rods. They are
  usually Oxidase negative and Catalase positive.
  They are nitrate reducers, as they are commonly
  found in some soils. They are also known to
  ferment glucose.
• Bacillus species are gram positive rods. They are
  endospore forming and are Catalase positive. They
  are also hemolytic.

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Bacterial Identification

• What went wrong
• The enteric tests showed Oxidase positive which
  is not a characteristic of enteric bacteria.
• The tests for the Bacillus colonies were all
  correct, however the endospore stain did not show
  spore production. This could be because the
  bacteria was not in an environment that spore
  production was needed.
• The second plating also should have been done
  using selective and differential agar to remove
  any contaminations.
• The secong plating should have been inoculated
  from the same spot on the first petri dish.
• Other possibilities are that these could be
  mutants.

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References

• Fenchel T. 2001. Bacterial Ecology. In
  Encyclopedia of Life Sciences. www.els.net
• Madigan, MT, Martinko M, Parker J. 2003.
  Filamentous, High GC, Gram-Positive Bacteria
  Streptomyces and other Actinomycetes. In Brock
  Biology of Microoganisms. pp. 416-420. New
  Jersey Pearson Education, Inc.
• Davelos AL, Kinkel LL, Samac DA. 2004. Spatial
  Variation in Frequency and Intenstiy of
  Antibiotic Interactions among Streptomycetes from
  Prairie Soil. Applied and Environmental
  Microbiology 70(2) 1051-58.