This study outlines a method for isolating and characterizing bacteria capable of degrading environmental pollutants, specifically EE2. By utilizing serial dilutions and selective media, researchers can identify and cultivate pollutant-degrading bacterial isolates.
Take a seawater sample containing diverse microbes, including environmental pollutant-degrading bacteria.
Serially dilute the sample to reduce microbial density and plate it onto a nutrient medium.
Post-incubation, select morphologically distinct colonies, which may still harbor genetically and metabolically diverse bacterial types.
Streak each selected colony to disperse individual cells and incubate.
Each cell forms a colony, representing a genetically pure bacterial isolate with a distinct metabolic profile.
Inoculate each isolate into a growth medium and incubate to increase biomass.
Centrifuge the culture, discard the supernatant, and wash with a buffer to remove the residual medium.
Resuspend the cells in the buffer and inoculate them into a selection medium containing EE2, an environmental pollutant.
As the sole carbon source in the medium, EE2 selects bacteria that can metabolize it for energy and growth.
Turbidity in the medium indicates bacterial growth, confirming the isolation of the pollutant-degrading bacterial isolate.
To select bacteria, first perform serial dilutions up to 10 to the negative nine in sterile saline solution for coral samples and up to 10 to the negative six for water samples. Pipette dilutions up and down five times before discarding the tip.
Then, vortex each one. Vortex the samples for five seconds every time before performing the next serial dilution. Pipette 100 microliters of each dilution on Petri dishes containing 3% sodium chloride lysogeny broth agar medium and plate them.
Incubate the plates for one to three days at the target temperature, for example 26 degrees Celsius. Check the plates once a day. Select and isolate the colonies presenting distinct growth morphologies on new plates using the streak plate technique.
Repeat this step as many times as needed to have pure colonies growing on the plates. Store the isolates at four degrees Celsius or in glycerol as described in the manuscript. To perform EE2 degradation ability test, pick a single colony from the fresh plates and inoculate in two milliliters of sterile LB medium in a tube.
In case it is a glycerol stock, take 10 microliters of glycerol bacterial stock and streak onto LB agar plates. Incubate at 24 to 28 degrees Celsius overnight to have single colonies growing. Place the tube containing LB medium inclined under constant agitation at 150 times g at 24 to 28 degrees Celsius overnight.
After bacterial growth, pellet the cells by centrifuging them at 8,000 times g for eight minutes at room temperature. Discard the supernatant. To wash the remaining LB broth, add two milliliters of saline water to resuspend the cells.
Repeat the washing twice to guarantee that there are no traces of carbon source. Inoculate the washed and resuspended cells in minimum Bushnell Haas culture medium containing EE2 as the only carbon source. Assess bacterial growth by optical density at 600 nanometers and our colony forming units on LB agar medium after 16 to 72 hours of incubation.
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