Wash a 5 L Niskin with seawater from the site. Collect water sample from surface and bottom at each site. Take three independent samples and note at which time each sample was taken. Assign each replicate to a unique BPA ID. Perform the following steps for each niskin separately.
Flow cytometry samples: Wearing nitrile gloves, fill a 2L acid-washed bottles to the brim with seawater from each replicate niskin collection using acid washed funnel. Pipette 1 mL of this seawater into a pre-labeled cryovial (BPA ID and word “flow”) and add 63 µL 16% paraformaldehyde (1 % v/v final concentration) in fume hood. Invert tube a few times, incubate for 15 min in the fridge and then snap-freeze in liquid N2. Dispose of pipette tip into a sealed container (Formaldehyde waste) for safe disposal back in the lab. Repeat for each replicate water sample.
Microbial samples:
Push a 10 mL serological pipette (filter removed with sterile tweezers) into each inlet tubing end and place in the 2L bottle. Run ~200 mL seawater through tubing (with pump flow controller set at midpoint) then pause the pump.
For each water sample at a site, attach a 0.22 µm sterivex filter to the luer lock outlet of the peristaltic pump tubing. Filter 2 litres of seawater using a peristaltic pump using speeds of up to ~120 rpm (Pharmed pump tubing 8.0 mm dia. Internal diameter 3.2 mm e.g., Masterflex L/S 16 or similar). If the filter gets blocked before 2 litres are filtered, please note the volume filtered.
When all water is filtered, disconnect the inlet and pump air through the filter for 1-2 min to remove any remaining liquid.
Disconnect filter and label with the BPA ID. Cap both ends of the sterivex filter. Wrap filters in foil and place in a liquid nitrogen (LN2) resistant whirlpak bag.
Snap freeze in LN2 by dipping with a canister, and then place in -20 °C freezer until transfer to -80 °C
To clean the pump tubing after use, run 200 mL 5-10 % bleach solution through tubing, followed by 200 mL milliQ or distilled water, and lastly 200 mL 70 % EtOH (to dry the tubing and prevent biofilm growth between uses).
Nutrient Samples as per AIMS WQ Program:A separate niskin cast was done at each site/depth and this single cast was used to do:
1 x salinity (refrigerated) – 250 mL seawater into acid-washed bottle.
1 x CDOM (refrigerated) – 50 mL filtered through 0.2 µm Pall Acropak supor filter into amber glass bottle. Keep additional filtrate (>200 mL) for wet filter blanks – 10 mL for each duplicate GFF sample below (total 8 blanks) and 60 mL for each duplicate TSS sample (2 blanks).
GFFs(using vacuum manifold) plus 2 wet filter blanks for each:
2 x Chl-a (frozen at -20 °C) – 100 mL filtered through 25 mm Whatman GF/F. Filters folded in half (sample side inwards) and wrapped in foil.
2 x Particulate C (frozen at -20 °C) – 500 mL filtered through 25 mm Whatman GF/F. Filters folded in half (sample side inwards) and wrapped in foil.
2 x Particulate N (frozen at -20 °C) – 500 mL filtered through 25 mm Whatman GF/F. Filters folded in half (sample side inwards) and wrapped in foil.
2 x Particulate P (frozen at -20 °C) – 250 mL filtered through 25 mm Whatman GF/F. Filters folded in half (sample side inwards) and wrapped in foil.
2 x TSS (pre weighed filters in 20 mL scint vials) – 1 L filtered through 47 mm polycarbonate 0.4 µm filter. Filters folded in half and half again (sample side inwards) placed in a Scint vial and stored frozen at -20 °C. Also 2 x wet filter blanks as described above with 60 mL 0.22 µm filtered seawater passing through each.
Dissolved nutrients:
2 x DIN (frozen at -20 °C) – 10 mL 0.4 µm filtered seawater into acid-washed sample tubes
2 x TDN (frozen at -20 °C) – 10 mL 0.4 µm filtered seawater into acid-washed sample tubes
2 x Silica (refrigerated) – 10 mL 0.4 µm filtered seawater into acid-washed sample tubes
2 x DOC (refrigerated, HCl added) – 10 mL 0.4 µm filtered seawater into acid-washed sample tubes with 100 µL hydrochloric acid added. Do not freeze.
1 x alkalinity/DIC – 250 mL seawater into acid-washed Schott bottle filled to the brim. 125 µL mercuric chloride added. Store at room temperature (RT) in cool environment.
