Soil Microorganisms and Antibiotics

December 6, 2004

Kenice Frank, Allison Johnson, Ruben Krantz, Hannah Wilbur

Soil bacteria

• 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

Soil environments are host to a great number of bacterial species

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.

•We then used sterile tubes to obtain the sample

River Marsh Forest

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.

Methods and MaterialsWeek 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 MaterialsWeek 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 test2.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

DiscussionThe 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.

DiscussionThe 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.