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TidePoolSampling_SOP.md

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Standard protocol for Tide Pool Biogeochemical and Microbial Sampling

Original: 20190120 Last Revised: 20190529

Contents

  1. Materials
  2. Protocol
  3. References
  1. Materials

General

  • 500 mL plastic Erlenmeyer flask with nozzle
  • Size 7 rubber stopper with and without hole
  • ¼ in clear vinyl tubing (for Erlenmeyer to sample water) with weight
  • 7/16 in clear vinyl tubing (for hand pump to Erlenmeyer)
  • Vacuum hand-pump
  • Buckets
  • YSI multi-parameter pro meter (for salinity, conductivity, Dissolved oxygen, and temperature
  • Probe stand holder
  • Various styrofoam holders for tubes
  • Clipboard and data sheets
  • Drying sheets

Nutrients samples

  • 50 mL centrifuge tubes
  • 0.7 μm 25 mm glass fiber filters (GF/F)
  • 4-way stopcock
  • Filter holders
  • Luer-Lock tip 60 mL syringes
  • Cooler
  • Forceps
  • -20°C freezer

Total Alkalinity Samples

  • 250 mL nalgene bottles
  • 50% saturated Mercuric chloride solution
  • Repeater pipette with tips

In situ pH

  • 50 mL centrifuge tubes
  • Orion Star Multiparameter Meter with a ROSS Ultra glass electrode (for pH)
  • Traceable digital thermometer
  • NBS pH buffer solutions
  • Container of DI water and kim wipes

Microbial samples

  • Small cooler
  • 0.2 um Sterivex filter (for eDNA)
  • Pipette with 1000μL pipette tips for field
  • 2.0 mL microcentrifuge tubes (microbial counts)
  • Paraformaldehyde (PFA; 32% stock solution)
  • Pipette and pipette tips for PFA (in lab)
  • Luer-Lock tip 60 mL syringes
  • 20 mL vial (flourescent dissolved organic matter)

  1. Protocol

    1. 24 hours prior: Calibrate probes and place 16μL of the paraformaldehyde (PFA) stock (32%) in 2 mL microcentrifuge tubes (this must be done in fume hood)
    2. Arrive at site several hours before start of sampling
    3. Rinse all biogeochemistry containers (erlenmeyer flasks, tubing, 50 mL centrifuge tubes, 250 mL nalgene bottles, syringes and holders) three times with ocean water. Do not put waste water in tide pools.
    4. Chemistry lab set-up: probe stand with pH and temperature probes in 50 mL centrifuge cap and tube in tupperware on cooler for nutrient samples; pipette set to 1000μL with tips; 250 mL bottles for TA; 50 mL centrifuge tubes in styrofoam holders; 2 mL tubes in holders pretreated with 1mL paraformaldehyde; fDOM containers; filters (0.7 and 0.22), syringe with holders and stopcocks.
    5. Water collection set-up: Buckets of Erlenmeyer flasks with stopper without holes and multi-parameter probe.
    6. Set out tubing with weights and rubber stopper at each pool near the benthos next to the experimental chamber
    7. At sampling time in each pool and the ocean, use multiparameter probe next to tubing to measure DO, conductivity, conductivity, salinity and temperature
    8. Keep person consistent with dissolved oxygen (DO) probe measurements. Wait until the DO starts to stabilize then record data
    9. After recording values, remove cap of Erlenmeyer flask and replace it with the cap connected to the tubing.
    10. Attach tube with hand pump to Erlenmeyer flask and hand pump 550 mL on biogeochemistry/microbial sampling points (purple line) or 400 mL (black line) into flask.
    11. Remove tubing connected to hand pump vacuum first then remove cap with tubing and replace with the non-holed rubber cap.
    12. For ocean sample at each time point, bungee cord erlenmeyer flask to lacrosse stick basket. Rinse flask three times then fill with ocean water and cap it.
    13. Take filled flask to chemistry lab station.
    14. Repeat with other pools.
    15. At next time point, hand pump 10x to flush tubing. Dispose of water on the rocks.
    16. At chemistry lab: Have filters already in filter holder and station organized by order of samples.
    17. Receive capped erlenmeyer flask from field team.
    18. Pore around 20 mL into 50 mL centrifuge tube and twist on to cap with pH and temp probes (cover probes fully)
    19. When pH stabilizes, record temperature first then pH.
    20. Fill 250 mL bottle for total alkalinity measurements, leaving a little head space for mercuric chloride addition.
    21. Pour 50 mL of seawater into syringe connected to filter holder with stopcock in locked position. Place plunger of syringe on then unlock stopcock to side and push water through the filter holder with already pre-loaded GF/F filter. Use same filter for 3 samples.
    22. Place nutrient samples back in cooler after 2 rounds of sampling.
    23. On microbial sample times (1 & 5), pipette 1mL of unfiltered seawater into 2mL microcentrifuge container pretreated with PFA. Use another syringe with sterivex filter to push 2 x 60 mL onto rocks then 20 mL into fDOM flask, then the remainder on to the rocks. Plunge one syringe full of air to flush water out of sterivex. Place sterivex filter and microcentrifuge tubes into small cooler with dry ice after sampling.
    24. Place 100 μl of 50% saturated HgCl2 with repeater pipette in each TA samples to preserve the water. Parafilm container tops
    25. Place nutrient samples with holders in -20 degree freezer within 4 hours of collection.
    26. Place sterivex filters and 2 mL microcentrifuge container in -80 degree freezer as soon as possible.
    27. Place fDOM samples in fridge.
  1. References

  • Silbiger, N. J., and C. J. B. Sorte. 2018. Biophysical feedbacks mediate carbonate chemistry in coastal ecosystems across spatiotemporal gradients. Scientific Reports 8:1–11.

  • Dr. Craig Nelson and Dr. Linda Kelly