1. Water and coral collection and storage for microbial isolation
NOTE: It is essential to take the coordinates and temperature of the sampling sites. If possible, metadata such as salinity, pH, depth, and light intensity can also help in finding fine-tuned cultivation approaches and future interpretation of data. For reliable results, keep the samples stored for the minimum length of time possible. The water/coral microbiomes may change considerably if the samples are not kept at the right temperature and/or are stored for long periods. If the isolation step is not performed instantly after collection, it is crucial to maintain samples at 4 °C until processing. The longer the samples are stored, even at 4 °C, the more the microbial community will change.
- Sample and store seawater.
- Collect 500 mL samples of water in at least triplicate from each targeted sampling site. Preferably use sterile bottles with screw caps.
- If processing the water instantly after collection, keep the bottles at RT for a short interval. If sample processing is happening later, keep the bottles at 4 °C.
- Sample and store the coral.
- Use a sterile pair of pliers to cut coral fragments from the same sampling site of the water samples. To avoid contamination, touch corals only with sterile gloves.
- Rinse the sampled coral fragment using 20 mL sterile saline solution (3% NaCl in distilled water) or artificial seawater to get rid of the loosely attached free-living bacteria of the seawater.
- Using forceps, place each coral fragment into a sterile 250−500 mL container with a screw cap containing sterile saline solution.
- In the laboratory, using sterile forceps and pliers, weigh 5 g of coral fragments using sterile 100 mm x 20 mm Petri dishes on a weighing scale.
- Transfer the 5 g of coral sample to a sterile mortar and macerate it using a sterile pestle.
- Using a sterile spatula, transfer the macerated sample to a sterile culture flask containing 45 mL of 3% NaCl sterile solution and 10−15 glass beads of 5 mm. Use some of the 45 mL sterile saline solution to wash the mortar and recover the maximum amount of the macerate.
- Keep the flasks under constant agitation (150 x g) for 16 h at the water temperature of the sampling site.
NOTE: For shallow-water corals, the optimum temperature will range from 24−28 °C. This step will detach microorganisms from different coral compartments, such as those attached to the host cells, or the ones living inside the tissue and the skeleton. After this step, the coral macerates should not be stored, and the isolation step must be instantly performed.
2. Isolation of synthetic estrogen 17a-ethinylestradiol (EE2)-degrading bacteria from seawater and/or corals
- Select bacteria.
NOTE: After steps 1.1.2 and 1.2.7, the concentration of microorganisms in seawater that have detached from the different coral macerates will be unknown and variable. In order to guarantee the isolation of individual microbial colonies in Petri dishes containing agar media, serial dilutions are needed.- Perform serial dilutions up to 10-9 in sterile saline solution for coral samples and up to 10-6 for water samples. Pipette dilutions up and down 5x before discarding the tip. Vortex samples for 5 s every time before performing the next serial dilution.
- Pipette 100 µL of each dilution on Petri dishes containing 3% NaCl lysogeny broth (LB) agar medium, and plate them.
NOTE: Use marine agar (MA) as an alternative medium. Triplicates of each dilution are required for reliable results. - Incubate the plates for 1−3 days at the target temperature (e.g., 26 °C). 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.
- If the procedure is not instantly continued to step 2.3.1, store isolates at 4 °C or in glycerol as described in section 2.2.
- Prepare glycerol stocks.
NOTE: This step is optional and can be used for long-term bacterial stocks storage.- Pick single colonies from the fresh plate or from the plates stored at 4 °C and independently inoculate them in 2 mL of sterile LB medium.
- Place tubes under constant agitation (150 x g) at 24−28 °C overnight (ON).
- Add 1 mL of the bacterial cultures from step 2.2.2 and sterile glycerol to a final concentration of 20% to the 2 mL cryovials.
- Leave the cryovials ON at 4 °C.
- Place the glycerol bacterial stocks at -80 °C until needed.
- Perform the EE2-degradation ability test.
- Activate the isolates in LB broth or alternative media. For this, pick a single colony from the fresh plates or the plate stored at 4 °C, and inoculate 2 mL of sterile LB medium. In case it is a glycerol stock, first streak it on LB agar plates and incubate at 24−28 °C ON to have single colonies growing. Place the tube containing LB medium inclined under constant agitation (150 x g) at 24−28 °C ON.
- After bacterial growth, pellet the cells by centrifuging them at 8,000 x g for 8 min at room temperature (RT). Discard the supernatant and, gently pipetting up and down, resuspend the cells in an equal volume (2 mL) of saline water to wash the remaining LB broth.
- Repeat step 2.3.2 twice to guarantee that there are no traces of carbon source, resuspending the cells in an equal volume of saline solution. For instance, if it was started with a 2 mL culture, resuspend the cells in a final volume of 2 mL saline solution.
- Inoculate the washed and resuspended cells in minimum Bushnell Haas culture medium (BH Broth) containing EE2 as the only carbon source.
NOTE: EE2 is dissolved in ethanol at a final concentration of 5 mg/L in the culture medium. Make changes in the pollutant type and/or concentration if needed. - Assess bacterial growth by optical density at 600 nm and/or colonies forming units (CFU) on LB agar medium, for 16−72 h of incubation.
NOTE: Alternatively, the microorganisms can be directly isolated on minimum media containing EE2 or other compounds as the only carbon source. This step would direct the selection and avoid undesirable growth.