This article outlines a method for isolating hydrocarbon-degrading bacteria from seawater samples. By utilizing selective media and specific carbon sources, researchers can enrich for microbial communities capable of metabolizing hydrocarbons.
Begin with a seawater sample containing diverse microbial communities, including bacteria capable of degrading hydrocarbons.
Perform serial dilutions of the sample in saline to reduce microbial density.
Add crude oil to sterile distilled water and agitate the mixture to partially solubilize hydrocarbon components of the oil.
Allow the mixture to settle, separating it into water-soluble and water-insoluble oil fractions.
Prepare selective agar plates using each oil fraction as the sole carbon source to enrich for bacteria that degrade either soluble or hydrophobic hydrocarbons.
Plate the diluted seawater samples onto the selective media and incubate at ambient temperature.
Colonies that emerge rely on enzymes such as hydroxylases and dioxygenases to metabolize hydrocarbons.
Pick morphologically distinct colonies and restreak them onto fresh media containing the same oil fraction to obtain pure cultures of hydrocarbon-degrading bacteria.
This method enables the selective isolation of oil-degrading marine bacteria.
To prepare minimum media containing an oil-water soluble fraction and an oil-water insoluble fraction as the only carbon source, add 1-2% crude oil to 500 milliliters of sterile distilled water in a filter flask containing a valve on the bottom. Keep the flask under constant agitation at 24 to 28 degrees Celsius at 150 times g for 48 hours. After that, place the flask on a stable surface and wait 10 to 20 minutes to allow the soluble and insoluble fraction to separate.
Then open the bottom filter flask to transfer the oil-water soluble fraction to a new sterile flask saving the oil-water insoluble fraction. Then prepare two bottles of Bushnell Haas agar minimum medium. Autoclave it.
Combine with the oil-water soluble fraction as the only carbon source to make 500 milliliters of agar medium. Do the same with the oil-water insoluble fraction and transfer approximately 25 milliliters of each medium onto individual Petri dishes. To isolate oil-degrading bacteria, reuse previously diluted samples and pipette 100 microliters of each dilution onto the Petri dishes containing agar medium and plate them.
Remember to keep negative controls for your plates. Incubate the dishes for one to three days at the target temperature and repeat the isolation procedures as previously.
繁體中文
