meth_1.8 Coastal Seawater Sampling for Downstream DNA Analyses for Marine Microbes Coastal UPDATED SEPT 2019 TT

Contributor(s):

Citation:

AM Project ID: Legacy

Method:

  1. Wash three 3-4 L containers at least five times with seawater from the site and label with numbers 1-3. Drop containers separately attached to a rope to ~2 m depth and retrieve. Take note of the time you took the three individual samples. If possible, this is best done from a boat. Alternatively, collect water from a depth of ~2 m using a foldable, acid-washed Again, take note on when you took each sample. These three sample represent replicates and should be handled separately in the following steps.
  2. Suck 50 mL of seawater from the container into a syringe. Wash syringe a few times. Attach a 0.45 µm syringe filter and push a few millilitres into three separate 50mL Falcon tubes. Wash tubes with filtrate three times. Then fill each tube with 40 mL of filtrate, close tube and keep on ice. You should have three 50 mL tubes per sample replicate. Samples are then frozen once returned to the laboratory. (-20 °C). These tubes are for nutrient analysis and please label them according to the instruction given in the spreadsheet “sampling_schedule”.
  3. Use the 50 mL syringe from step 2 to 5 times filter 50 mL (i.e., total of 250 mL) of seawater (from the container of step 1) through a syringe filter holder containing a 25 mm Whatman GF/F filter. If the filter does not show colour, then filter more water through it. Take note of the volume of water filtered. At the end, press air through it to dry filter. Place GF/F filter into pre-labelled cryovial and snap-freeze in liquid N on site. These filters will be used for chlorophyll analysis.
  4. Pipette 1 mL of seawater (from the container of step 1) in a pre-labelled cryovial with paraformaldehyde (1% v/v final concentration). Invert tube a few times and then snap-freeze in liquid N on site. Repeat three times. Use gloves and dispose of pipette tip into a sealed container for safe disposal back in the lab. These samples will be used for cell counts.
  5. For microbe sampling, attach a prefilter to the intake side of a peristaltic pump tubing. The filter can be simply built by cutting a hole in the cap of a 15 Falcon tube and the placing a 100 micron mesh of the threaded part of the lid. Put the lid back on to jam the mesh between the lid and the tube. Cut the bottom of the tube and attach it to the intake side of the peristaltic pump line. Filter ~2 L of seawater within 2 hours using a peristaltic pump onto 0.22 micron sterivex filter. Use speeds of up to ~120 rpm and platinum cured silicone pump tubing (8.0 mm dia. Internal diameter 3.2 mm e.g., Masterflex L/S 16) or similar.
  6. When the 2 L has been filtered continue to run the pump for 1-2min to remove all of the liquid. If the filter gets blocked before, then note the approximate volume filter, disconnect the inlet and try to pump air through the filter for 1-2 min to remove remaining liquid.
  7. Ensure that the sterivex is labelled according to the instruction given in the spreadsheet “sampling_schedule”. Cap both ends of the sterivex filter [alternatively mould some clean blutak around the ends to seal if you have the version with a luer lok on one side only]. Place the filter in a ziplock bag.
  8. Freeze immediately at -80 °C.