TITLE: Collection and processing of intertidal seagrass tissues for bacterial and fungal culturing
AUTHORS: Cassie Ettinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details collection and processing of intertidal seagrass tissues for bacterial and fungal culturing.</div></div>

[STEPS]
?. [Field collections]
Put on gloves.
?. [Field collections]
Walk to a location that is submerged (~0.5 m).
?. [Field collections]
Using a coring device, take a sample of an individual seagrass plant and its associated sediment.
?. [Field collections]
Place core in dark box  until transport back to the lab.
on ice
?. [Field collections]
Replace gloves and repeat to obtain multiple cores.
?. [Field collections]
Collect nearby seawater in  Nalgene bottles (or similar) as needed for media recipes.
1 L
?. [Field collections]
Using new gloves, carefully pull up additional seagrass plants as needed for media recipes and/or inoculation, place into sterile ziplock bags and then put .
on ice
?. [Field collections]
Once back at the lab, place cores, bulk tissues and seawater into  fridge or cold room and use as needed within - of collection.
4 °C
?. [Field collections]
Insert a clear PVC pipe with a  diameter that has been modified such that one end of the pipe is cut at an angle to make insertion into the sediment easier.
2.375
?. [Field collections]
Once inserted, Cap the PVC pipe, pull the core up and then cap the bottom of the core.
?. [Sample processing and inoculation on culture media]
Rinse the tissue with autoclaved nanopure water to remove loosely associated sediment for ~.
Plant tissues (leaf, root, rhizome, matte).Before starting, prepare plates with your favorite culture media (e.g. see Ettinger & Eisen 2020 for possible media choices previously used for seagrass-associated work)
?. [Sample processing and inoculation on culture media]
Cut ~ pieces of tissue using flame sterilized scissors
1
?. [Sample processing and inoculation on culture media]
Place a subset of these tissue segments directly on plates using flame sterilized tweezers (1–3 segments/plate).
?. [Sample processing and inoculation on culture media]
Take another subset of tissue segments and place these segments into  centrifuge tubes with  of autoclaved nanopure water.
1.5 mL
1 mL
?. [Sample processing and inoculation on culture media]
Vortex the centrifuge tubes for ~.
1.5 mL
?. [Sample processing and inoculation on culture media]
Directly plate intact rinsed tissue segments on media using flame sterilized tweezers (1–3 segments/plate).
?. [Sample processing and inoculation on culture media]
Pipette  of wash liquid from rinsed segments or of homogenized smashed tissue directly on plates.
350 µl
As needed, you can also pipette 1:10 and 1:100 dilutions of wash liquid on plates.
?. [Sample processing and inoculation on culture media]
One protocol is to bleach tissues prior to plating, for example:
If of interest, one can try to remove epiphytes from tissues through several wash steps before plating.
?. [Sample processing and inoculation on culture media]
For a subset of segments, homogenize using a sterile pestel, while for another subset leave the segments intact.
?. [Sample processing and inoculation on culture media]
First, immerse segments for  in  0.5% NaOCl (~10% bleach).
1 mL
?. [Sample processing and inoculation on culture media]
Then immerse in  of 95% EtOH for .
1 mL
?. [Sample processing and inoculation on culture media]
Next immerse in  autoclaved nanopure water for .
1 mL
?. [Sample processing and inoculation on culture media]
Finally directly plate intact bleached tissue segments on media using flame sterilized tweezers (1–3 segments / plate).
?. [Sample processing and inoculation on culture media]
Another, slightly longer protocol to surface clean tissues, involves:
?. [Sample processing and inoculation on culture media]
First, immerse segments in  95% ethanol for ~.
500 µl
?. [Sediment]
Place sediment into  centrifuge tubes.
1.5 mL
?. [Sediment]
Add  of autoclaved nanopure water to tubes.
1 mL
?. [Sediment]
Vortex tubes for ~.
?. [Sediment]
Pipette  of sediment suspension directly onto plates.
350 µl
As needed, you can also pipette 1:10 and 1:100 dilutions of sediment suspension on plates.
?. [Seawater]
Pipette  of seawater directly onto plates.
350 µl
As needed, you can also pipette 1:10 and 1:100 dilutions of seawater on plates.
?. [Sample processing and inoculation on culture media]
Then, immerse in  0.5% NaOCl (∼10% bleach) for .
500 µl
?. [Sample processing and inoculation on culture media]
Next, immerse in  70% ethanol for .
500 µl
?. [Sample processing and inoculation on culture media]
Finally, directly plate intact surface cleaned tissue segments on media using flame sterilized tweezers (1–3 segments /plate).
?. [Sample processing and inoculation on culture media]
Then rinse segments with autoclaved nanopure water for 0w 0d 0h 1m 0s