Pseudomonas taiwanensis – Enrichment, Isolation and Identification Microbiology 521
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.
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.
Isolation: A presumptive identification for Pseudomonas strains was the use of microscopy using wet mounts. One colony was prepared and viewed under a microscope at 1000X. After confirmation, the remainder of the colony was three-phase streaked on Pseudomonas Isolation Agar (PIA), a selective medium for Pseudomonas. A true confirmation of Pseudomonas was obtained using an oxidase test and a Gram stain test. The oxidase test examines an organism’s ability to produce cytochrome oxidase by smearing several colonies of the Pseudomonas onto filter paper and adding a drop of oxidase reagent. Bubbling occurs within 30 seconds if an organism does produce cytochrome oxidase. The Gram stain test was done by smearing a colony across a slide with water (since the colony was from a plate). The smear dried and was heat fixed. The plate was flooded with crystal violet and washed with water after one minute. The mordant Gram’s iodine is added next to retain the purple color in Gram positive bacteria and washed with water after one minute. Then, an acetone-alcohol destain occurred for 3 seconds on the slide before being washed with water. Finally, the dye safranin was added and washed away with water after one minute. The slide was then viewed under bright field microscopy in search of pink bacteria signifying Gram negative bacteria, the type Pseudomonas are. Following confirmation the Pseudomonas isolate was three-phase streaked on LB agar for growth of the isolate. The plates were incubated at 30°C for 2 days. A broth culture with a loopful of isolate was incubated at 30°C for one day so that the experiment could continue with genomic DNA extraction.
Purification of Genomic DNA: 1.5 mL of broth cultured cells were centrifuged for one minute to allow for separation of the unwanted liquid and the important pellet. The liquid was discarded and the pellet was resuspended in 180 μL Buffer ATL. 20 μL protinease K was added and after mixing, was incubated at 56°C...
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