Question

How would one isolate and culture Pseudomonas putida from soil sample contaminated with hydrocarbons

Answer 1

Isolation and culture of pseudomonas putida from samples contaminated with hydrocarbons methodology includes:-

Sample collection- surface sedimented sample collected over a period of 12 months. The collected samples were pooled and transferred to pre-sterilized , labeled, and maintained untill analysis.

THB population- THB population is enumerated by pour plate method . 1 ml of the aliquots from each of the dilution was inoculated by pour plate method into NA in duplicates. The plates were incubated at 37 degree Celsius for 24-48 hours.

HDB population- this is done by spread plate techniques by inoculating aliquot into sterile BHA.

Identification of THB and HDB population- the enumerated bacteria were isolated and and stored in a NA on the basis of colony and morphology and further performing a series of biochemical tests. The isolates which grow on both the agar plates were confirmed as hydrocarbons.

P.putida grows perfectly on LB medium on agar plates in shake flasks and bioreactors. Composition per liter is , 5g yeast extract and 10g Nacl . 2g/L glucose to accelerate growth. Using 230 rpm , 30 degree Celsius and baffled shake flasks. And it has been shown that flageller motion is not crucial for survival under lab conditions as chemotaxis is usually not necessary in rich medium.The study monitored the isolation and culture formation of the hydrocarbon containing P.putida as the efficient among the various strains isolated towards spill treatment.