TITLE: Isolating bacteria from algal monocultures
AUTHORS: Emily Aguirre

[DESCRIPTION]
To isolate bacteria from non-axenic, algal (we did this with Symbiodinium but may work with other algae) monocultures. Our aim is to isolate bacterial members of the phycosphere to use in re-inoculation and genome wide association studies to better understand how microalgal-bacterial relationships influence marine ecosystems.

This protocol was conducted using a three-year, laboratory monoculture of Symbiodinium linucheae, isolated from the anemone Aiptasia pallida, as described previously (https://www.protocols.io/edit/isolating-non-axenic-monoclonal-symbiodinium-cultu-cg9stz6e). 

[BEFORE_START]
NOTE: Assure you have a non-axenic, monoclonal Symbiodinium (or other dinoflagellate/algal) culture that has never been subjected to thermal stress or antibiotic treatments! 

The algal phycosphere may range according to available exudates and organic substances. Therefore, the bacterial community may differ between varying stages of algal growth (lag, log, stationary and death). Please keep in mind the health of your culture, along with the appropriate date of collection for your individualized experiment. 

We collected at ~ stationary phase (1.5 month old culture). 

[STEPS]
SECTION: Initial isolation from Symbiodinium
1. Transfer 1 mL of a non-axenic Symbiodinium culture to a 2mL microcentrifuge tube. Centrifuge the algae and bacteria at 10,000 rpm for 2 minutes. 
SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
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SECTION: Initial isolation from Symbiodinium
2. Discard the liquid and rinse the pellet with 1 mL of 0.2 µm FSW. Vortex. Repeat. 
SECTION: Initial isolation from Symbiodinium
3. Add 2 - 3, 4.5 mm sterile glass plating beads to the microcentrifuge tube and vortex at maximum speed for 1.5 minutes. This breaks down the Symbiodinium cell but leaves most bacteria intact.
SECTION: Initial isolation from Symbiodinium
4. Transfer the slurry (omit the beads) onto a 3mL syringe, attached to a sterile 0.45 µm PES, 30mm filter. 
SECTION: Initial isolation from Symbiodinium
5. Pass the slurry through the filter and into a new, sterile microcentrifuge tube. 
SECTION: Initial isolation from Symbiodinium
6. This is your concentrated slurry (100) . Dilute this to 1:100, 1:1000 and 1:10000 in three different microcentrifuge tubes. 
SECTION: Initial isolation from Symbiodinium
7. Aliquot 50 µL of the concentrated slurry onto R2A marine agar plates (triplicates). This will be the "positive control" to compare growth, but single colonies will likely not be distinguishable (nor should you use this plate to pick single colonies).
SECTION: Initial isolation from Symbiodinium
8. Aliquot 50 µL of the 1:100, 1:1,000 and 1:10,000 dilutions onto R2A marine agar plates (each dilution in triplicates).
SECTION: Initial isolation from Symbiodinium
9. Spread the cells in the plates with 4-6, 4.5 mm sterile glass plating beads, each, by shaking gently until the liquid has been adsorbed on the agar. Seal with parafilm.

Incubate all plates at 27ºC on a 14:10, light: dark cycle (since these are the conditions they are used to from co-existing with the algae) for 5-10 days until visible colonies appear.

  

 *However, if you would like to attempt culturing more diverse members of the phycosphere, I'd suggest various strategies such as variable media and temperatures as high as 34°C

SECTION: Isolating single colonies
10. Using a sterile filter-capped pipet tip (200 µm), pick a defined single colony and streak it on a new R2A plate. 

Do this for each morphologically distinct colony. Keep a record of the colony morphology, color, texture and smell (if any). Label each distinct colony with a unique sample identifier, like a number.
SECTION: Isolating single colonies
11. Incubate at 27°C on a 14:10, light: dark cycle until colonies are visible or up to 5 days. 
SECTION: Isolating single colonies
12. Repeat steps 10-11 until it is visibly clear that the plate contains a bacterial monoculture. Collect as many as you'd like!
 
SECTION: Growing bacterial monocultures in liquid media for downstream processes
13. Aseptically pick a colony from a plate containing the bacterial monoculture and inoculate it into 100 µL of 0.2 µm FSW. Break up the colony gently by pipetting up and down until the liquid is turbid. 
SECTION: Growing bacterial monocultures in liquid media for downstream processes
14. Pipet a 25 µL aliquot of the bacterial solution from step 13 and inoculate in 25 mL of R2A marine broth. 
SECTION: Growing bacterial monocultures in liquid media for downstream processes
15. Incubate at 27°C on a 14:10, light: dark cycle for 2-5 days* or until peak density is approximately 107-109 cells/mL. Allow oxygen to pass through the cultures, and unless the vial contains a cap with a 0.22 µm filter, loosely cap the vials.

*at this point, most bacteria have acclimated to agar or high nutrient conditions like those in media, so dense, visible growth will be seen in many bacteria monocultures by the second day, although other colonies may take longer.
SECTION: Glycerol stocks for cryo-preservation
16. Prepare 40 mL of 40 % glycerol solution in 0.2 µm FSW.

SECTION: Glycerol stocks for cryo-preservation
18. Centrifuge 1.5 mL of the bacterial monoculture obtained from steps 13-15 at 13,000 rpm for 2 minutes. Discard the liquid and add 1.5 mL of fresh R2A marine broth. Mix by pipetting up and down until the media is turbid. 
SECTION: Glycerol stocks for cryo-preservation
19. Transfer 500 µL of the bacterial suspension in a cryovial containing 500 µL of the 40 % glycerol solution. Mix by pipetting up and down, or cap securely and vortex for 3 seconds, max. The final concentration of glycerol in these cryostocks should be 20%.

Repeat in triplicates or more for each bacterial monoculture. 
SECTION: Glycerol stocks for cryo-preservation
20. Allow the cells to acclimate to the glycerol for 10 minutes prior to storing at -80°C. No need to snap-freeze prior to storage. 

Store at -80ºC, indefinitely. 
SECTION: Glycerol stocks for cryo-preservation
17. Transfer 500 µL of the 40 % glycerol solution to a sterile, pre-labeled cryovial tube. For each bacterial monoculture, prepare triplicates (or more). 

Cap and set aside.
SECTION: DNA extractions
21. Centrifuge 5 mL of the bacterial monoculture (in a 15 mL Eppendorf) obtained from steps 13-15 at 13,000 rpm for 5 minutes to obtain a pellet. Do this in triplicates. 
SECTION: DNA extractions
22. Extract DNA with method of choice. 

We used DNeasy Power Biofilm Kit (Qiagen, Germany) or GenElute Bacterial Genomic DNA Kit (Millipore Sigma, USA), both are good choices for this step, but if you're considering doing a genome, we recommend the latter.
SECTION: 16S rDNA PCR
23. To identify the bacteria, we amplified the 16S rDNA region with the DNA obtained (steps 21-22) using the "full-length" primer pair 27F/1492R.  PCR products were Sanger sequenced and the resulting sequences were blasted against the NCBI database.

SECTION: 16S rDNA PCR
23.1. 16s rDNA primers:
27F: 5'-AGAGTTTGATCCTGGCTCAG-3'
1492R: 5'-GGTTACCTTGTTACGACTT-3'