Transcript Organisms in Soil and Antibiotics

Soil Microorganisms and
Antibiotics
December 6, 2004
Kenice Frank, Allison Johnson,
Ruben Krantz, Hannah Wilbur
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
Antibiotics
• Can be used as:
– Fungicide, etc for
farming
– Antibiotics for humans
and animals
• From Strepomyces
species alone:
– 500 antibacterial
products identified
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
Soil Experiment
Methods and Materials
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.
River
Marsh
Forest
•We then used sterile tubes to obtain the sample
Methods and Materials
• We added sterilized
H2O to each soil
sample and streaked
500 ml of each soil
type onto 4 starchcasein agar for each
soil type.
• We incubated these
plates at room
temperature.
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
Methods and Materials
Week 3
• Got results from previous
week’s 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
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)
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
Soil Experiment
RESULTS
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 B:Gram (+) ovals
Forest C: Gram (-) cocci in clusters
Sensitivity/Inhibition Testing
• No inhibition observed
Blood Agar Plate
• Marsh B: total
hemolysis
• Forest A: total
hemolysis
Oxidase Test
Catalase Test
• Forest A (Gram neg.
rods): Oxidase
positive
• Forest A: Catalase
positive
• Marsh B: Catalase
positive
Triple Sugar Iron Test
• Forest A: Red/Yellow
(K/A)
Glucose and 1 other
sugar fermented
Endospore Staining
• Red bacillus cells
• No endospores observed
Soil Experiment
Discussion
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.
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.
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.
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.
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.
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.
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.