Purpose: The purpose of this experiment was to enrich Pseudomonas bacteria, isolating a species of Pseudomonas and identifying it using phenotypic properties and DNA sequencing as an existent or completely new and undiscovered species of Pseudomonas.
Overview: Genus Pseudomonas is a chemoheterotrophic bacteria found in soil and water. They are Gram negative, motile, paired rods that are also oxidase-positive. Pseudomonas species are well known for their ability to metabolize many different organic nutrients, allowing them to be viable in multiple, diverse conditions. The Pseudomonas organisms had to initially be enriched from a soil sample using a basal medium and an additional Carbon and Nitrogen source. These enriched Pseudomonas were then streaked on a plate and viewed with microscopy to ensure a Pseudomonas colony was identified correctly. Further confirmation came from Gram staining and oxidase testing. The confirmed culture was then inoculated in a broth culture and its DNA was purified. Plasmids were checked for and the culture was inoculated with various test media to discover phenotypic properties. Polymerase chain reaction (PCR) was then used to amplify the 16S rRNA gene sequence and that was purified. The narG gene was then amplified and this was run on agarose gel. The sequence was compared to the Ribosomal Database Project (RDP) and the sequence was found to match P. taiwanensis.
Methods and Materials: Enrichment: 25 mL of basal medium (33 mM Na-K-phosphate, pH 6.8; 0.05%MgSO4; 0.005% FeCl3; 0.0005% CaCl2) along with 5 drops sodium benzoate (0.25 mL) and 5 drops ammonium chloride (0.25mL) was added to a large flask. 0.5 g of soil from the Olentangy River was added to the flask. The flask was covered and incubated at 30°C for 2 days. This enrichment was then streaked on a sodium benzoate enrichment plate and inoculated at 30°C for 5 days.... [continues]
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