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Sample Collection Protocols

1.1 CTD Water Filtration (MBARI) Authors: Walz, K., Sparks, H., Closek, C. and Nye, C.

A. Prior to collection:

• This protocol is for water samples collected from Niskin bottles on a CTD rosette (although it can be applied to other water sample collection methods) and assumes that sterile techniques are practiced for all disposable items
• Prepare the following 50mL falcon tubes (one set per sampling day):
  1. 50mL of RNase Away (x1)
  2. 50mL of MilliQ / DI water (x2)
  3. 50mL of 70% ethanol (x1)
• Remove cotton from the ends of the sterile serological pipettes with clean forceps
• Assemble Swinnex filter cartridges and load them with a 25 mm Millipore filter (0.22uM pore diameter). This assumes we are using a peristaltic pump for water filtration and that we use 25 mm sized filters. A vacuum pump can also be used as well as other filter sizes (e.g. 47 mm). A key element of all of the filtration/handling is to avoid contamination from biological/DNA sources outside of the sample (e.g. bacteria, human, net tow material) or from a previous sample that was filtered on the same rig.

B. Supplies:

• Google spreadsheet of supplies

C. Sampling Notebook and Storage Setup:

• In field notebook, note the station, above labeling scheme, date, max depth, time of CTD cast
• Create a list of anticipated samples by listing Depths(m), Niskin #, Cast #, Notes (to be filled in as collection and filtering occurs)
• Label cryovials (apply horizontally on standing cryovial), labelling scheme is CRUISEc##_BB_eDNA (where ## is cast number and BB is niskin bottle number, i.e. CN18Sc01_12_eDNA is cast 1, bottle 12). This labeling is specific to MBARI cruises and should be adapted for each campaign. It is key to be able to have the correct information (metadata) to be able link the sample to location, date/time, water filtered (and associated protocol) and environmental information from CTD (or other sources).

D. Sampling Area Staging:

• Spray 10% bleach (or RNase Away) onto benchtop where filtering work will be conducted
• Wipe away bleach with clean paper towel and rinse with MilliQ / DI water; dry with paper towel
• Use a 70% solution (or disinfectant wipes) to further clean surface; let dry
• Cover clean area with disposable absorbent under pad (this is changed daily; occasionally more frequently if water is spilled in excess)
• Place peristaltic pump and pumpheads on pad and secure to bench; the pump should be placed next to a sink or deep trough for running sample water through tubing and to prevent potential spills
• Set the prepared 50mL tubes in a falcon tube holder or stand; these should be changed out daily
• Sterilize forceps by first placing into RNAse Away for at least 1 minute, dipping into each tube of water (allow to drip before transferring), and finally set into the tube of ethanol; forceps cleaned in the prior steps can stay in ethanol while doing other preparation activities Cleaned forceps must be dried with a Kimwipe before contacting a filter

E. Peristaltic Array Setup:

• Each peristaltic pump can hold six sets of tubing each (2 per pump head; 3 pump heads per device) for a maximum of 12 water samples at a time
• Set the 63cm / 25 inch MasterFlex tubing in the pump heads, taking care to cover both ends with paper towels or other sterile supplies (i.e. labware plastic bags) to reduce contamination. (As indicated above a vacuum pump can also be used for all of the steps instead of a peristaltic pump)

F. Collection Blank:

• Run 1L of MilliQ / DI water through one pump and loaded Swinnex cartridge; this is to serve as a negative collection blank
• Immediately place Millipore filter into a labeled cryovial and store in a liquid nitrogen dewar or a -80°C freezer
• Run a new collection blank daily

G. Water Sampling Setup:

• Label Whirlpak disposable bags to correspond with Niskin bottle number
• Fold sterile stand-up Whirl-Pak bag lengthwise and insert into 2L wide-mouth square plastic bottle (this provides structural support for bag).
• The Whirlpak bags are used to avoid contamination and the need to wash bottles between samples. Water collection can be made with other bottles if care is taken to avoid contamination. MBARI historical sample collections made before targeted eDNA sampling were done with reusable plastic bottles that were rinsed three times before sample collection and filtered using vacuum pumps.

H. Niskin Sampling:

• At the Niskin sampling area, open the Whirlpak from the top; fill with water from Niskin up to bottle neck line, roll bag down, fold in twist-tie, and twist to re-seal
• Total volume collected should be within 1.3 - 1.5 liters
• Repeat until all of the corresponding bottles have been collected
• Carry the filled Whirlpaks inside of the 2L bottles back to the sampling area inside the wet lab (a crate may be useful for handling multiple)

I. Peristaltic Pump Sampling:

• Stage the water samples near the peristaltic arrays; six samples per array
• Insert the pointed end of the serological pipette into the “wide” end of the peristaltic pump tubing
• Insert the other end of the pipette into the water sample and wrap Whirlpak seal around the pipette to reduce contamination from air particles
• Repeat steps 6 and 7 for every water sample; once staged, start the pump
• Ensure the water flow is moving towards the sink / waste collection and allow ~0.3 liters of sample water to flow through the MasterFlex tubing and into the waste unimpeded; this is to inoculate the tubing with sample
• Once 0.3 liters has passed, stop the pump
• Place the preloaded Swinnex filter cartridges on the tubing outflow end; attach 15cm vinyl outflow tubing to the opposite end of the Swinnex cartridges to direct water flow into 1L collection bottles
• Pump 1L of sample water through the Swinnex cartridge; stop the pump when finished
• Remove the cartridge from the tubing and use a sterile syringe to blow air through the cartridge to remove remaining water droplets on the Millipore filter.
• For vacuum filtration measure the appropriate volume and pour through disposable filter holders.
• Using sterile forceps, remove the filter from the cartridge and carefully insert into a pre-labeled 2mL cryovial
• Immediately place loaded cryovials into a liquid nitrogen dewar (preferred) or a -80°C freezer.
• If a liquid nitrogen dewar or -80°C freezer is not available, add DNAGuard to cryovial so the filter is covered. Store cryovial in the refrigerator (4°C).

J. Cleanup:

Clean used tubing and Swinnex cartridges with a 10% bleach solution at 3 MilliQ water rinses; let air dry in a plastic tray covered in paper towels or Kimwipes; if necessary, clean the outside of the MasterFlex tubing with a disinfectant wipe.

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