meth_1.18 Coastal Water Sampling and Filtering for DNA Analysis in the Derwent Estuary

Contributor(s): Jodie van de Kamp (jodie.vandekamp@csiro.au)

Citation:

AM Project ID: AM0016

Note that monthly water sample collection occurs as part of the Derwent Estuary Program Ambient Water Quality Monitoring Program (https://www.derwentestuary.org.au/water-quality/). Procedures for biochemical and physical water sampling can be found in the State of the Derwent Update 2020 Appendices A-C (DEP 2020; https://www.derwentestuary.org.au/state-of-the-derwent/).

Equipment:

For DEP sampling trip

  • 14 x seawater containers (plastic; ~2 L capacity)
  • 2 x large eskis to carry 2 L plastic containers
  • Ice (provided by DEP)

In the Laboratory

  • Peristaltic pump, Pump head & tubing with sterivex luer attachment
  • Laboratory single-use gloves (e.g. latex)
  • Millipore Sterivex GP 0.22 µm filters (Cat. # SVGPL10RC) – one per sample
  • Waterproof marker pen
  • Large snap-lock bag big enough to hold 14 x sterivex filters
  • 10% bleach
  • 70% EtOH

Protocol

At site

  1. Conduct Niskin bottle cast(s) to pre-determined depths.
  2. 2 L containers are pre-labelled with site and depth, e.g. B1 surface, B1 bottom
  3. Fill appropriate 2 L container (including the neck space) at each site and depth, surface and bottom, and store in eski.
  4. Surround containers with ice and keep eski out of direct sunshine to minimize sample temperature shifts prior to sample processing.

In the laboratory:

  1. To clean the pump tubing (in case of prior use by others), run a total of 2 L 10% bleach solution through tubing, followed by 2 L milliQ or distilled water. This is done by placing all 8 lines in a container of 2 L capacity (or if neck of container not wide enough, 4 lines in 2 x containers with 1 L of fluid each).
  2. Pump settings – ensure the pump is set to 300, NO HIGHER.
  3. To filter samples, place one end of pump tubing in the 2 L sample bottles. Allow the seawater in container neck to flow through tubing till it reaches the 2 L mark on container.
  4. Attach Sterivex filter (labeled: station e.g. B1; depth e.g. surf or bott; date) to fitting (luer lock), being careful not to touch lock end, and pass the remaining 2 L seawater through filter. If filter blocks before 2 L has passed through, stop pump and record volume on the sterivex and make a note to add to the sample spreadsheet of: volume filtered, if the sample has a lot of particulates.
  5. After 2 L has passed through filter, let the pump continue for ~1 min to assist in drying filter.
  6. Disconnect each filter from tubing, giving each filter several “flicks” to remove as much residual water from inside the filter housing as possible.
  7. Cap both ends of filter with the relevant caps (1 x inlet; 1 x outlet).
  8. Put capped filters into one larger snap-lock bag (labelled DEP; Date) and place at -80 °C as soon as possible after processing.
  9. To clean the pump tubing after use, run a total of 2 L 10% bleach solution through tubing, followed by 2 L milliQ or distilled water. This is done by placing all 8 lines in a container of 2 L capacity (or 4 lines in 2 x containers with 1L fluid each). Lastly run 500 mL 70% EtOH (to dry the tubing and prevent biofilm growth between uses), let pump run till the 70% EtOH has run through fully.