meth_1.5.1 Botany Bay NSW and Port Phillip Bay VIC

  1. Wash a ~20 L bucket or water sampler (e.g., Niskin system) at least five times with seawater from the site. Drop bucket/water sampler attached to a rope to ~2 m depth and retrieve. If possible, this is best done from a boat. Alternatively, collect water from a depth of ~2 m using a foldable, acid-washed canister. Take three independent buckets and note at which time each sample was taken. Assign each bucket to one of the unique BPA IDs. Perform the following steps for each bucket separately.
  2. Nutrient samples: Ensure sample is well mixed before collection. Draw entire 50 mL syringe full of seawater from bucket or canister and dispense to waste x 2 (If the syringe was hermetically sealed this rinsing step is not necessary). Draw syringe full a third time and use the water to rinse out the sample tube and lid with 3 x 10 mL rinses. Draw the syringe full again and attach 0.45 µM filter. Discard first 5-10 mL to waste, then fill sample tube. Fill as close to the 30 mL graduation mark as possible ±2 mL, close tube and keep on ice. Repeat process for the duplicate sample. The same filter can be used for duplicate samples. Samples are then frozen once returned to the laboratory. (-20 °C). These two tubes are for nutrient analysis and please label them with the appropriate BPA ID and the word “nutrients”. Only one of the duplicates is to be sent to CSIRO for processing. The duplicate should remain in storage at -20 °C  until successful analysis has been confirmed by CSIRO. 

    Preventing contamination: Wear disposable vinyl or nitrile gloves if possible. Some gloves require rinsing with water and drying prior to use to remove soapy residues. Do not touch the filter or syringe tips, or the insides of the syringe, sample tube, or lid. Do not smoke near where samples will be collected or near the sampling equipment. Cigarette residue on fingers and clothes will show up in the sample. Ensure that the sampling equipment and container to transport the samples are clean and kept clear of fish products.

     

  3. Chlorophyll samples: Use the 50 mL syringe from step 2 to 5 times filter 50 mL (250 mL in total) of seawater (from the container/bucket of step 1) through a syringe filter holder containing a 25 mm Whatman GF/F filter. If the filter does not show color, then filter more water through it. Take note of the amount of water filtered, if it is not 250 mL. At the end, press air with the syringe through the filter to dry it. Place GF/F filter into pre-labelled (BPA ID and word “chlorophyll) cryovial and snap-freeze in liquid N2.
  4. Flow cytometry samples: Pipette 1 mL of seawater (from the container/bucket of step 1) in a pre-labeled cryovial (BPA ID and word “flow”) with paraformaldehyde (1% v/v final concentration). Invert tube a few times, incubate for 15 min on ice and then snap-freeze in liquid N2. Use gloves and dispose of pipette tip into a sealed container for safe disposal back in the lab.
  5. Microbial samples:
    1. 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 (0.5-1 cm) in the cap of a 15 Falcon tube and the placing a 100 micron mesh over 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 litres of seawater using a peristaltic pump onto a 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. If the filter gets blocked before 2 litres are filtered, then note the volume filtered.
    2. When filtration is completed, disconnect the inlet and pump air through the filter for 1-2 min to remove any remaining liquid.
    3. Disconnect filter and label with the BPA ID. 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.
    4. Freeze immediately at -80 °C.