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

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 MarshForest

Methods and Materials We added sterilized H 2 O 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 Materials Week 2 We took 100 ml of forest sample and diluted it with 400 ml more of sterile H 2 O 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 1 st plating: cultures too thick 2 nd 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

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