eDNA Water Collection & Filtration

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Materials:

• 1L sterile bottles (glass for filtering in the lab and nalgene for water collection in the field)
• ice
• eDNA citizen scientist sampler
• packs/bags with sterile forcepts, filter housing (with filter and filter backing already within), and tubing
  • 1 μm filters
  • drain disc/filter backing
• 2 ml or 5 ml tubes
• gloves
• container for dirty instruments & filter housings
• field materials (listed under “filtering in the field/off boat” in “need:” section)

Prep

Bleaching

(eg. filter holders, other filtering materials, bottles, and stoppers)

• 1. after use (esp if filtering salt water), rinse with water in sink
• 2. store in “dirty contrainer” if not bleaching immediatly
• 3. if ready to bleach, change gloves and place equipment (eg. filter equipment: filters housings, plastic focepts, and housing clear plastic tubes or water collection equipment: nalgene bottles and stoppers) into 10% bleach bath for at least 10 mins (make sure they are submerged)
• 4. after 10 mins, put clean gloves on and transfer everything into MilliQ bath
• 5. remove equipment from water bath and place into clean plastic container in fume hood to dry

Bleach & MilliQ bath set up:

NOTES:

• before starting bleach process, make sure drying contrainers have been recently wiped down with bleach, DI water, & 70% ethanol
• the bleach and DI water baths should be changed out at least monthly (more frequently if cleaning a lot of stuff)
• when making new bleach bath make at least 5 Liters
  • 5 L of 10% bleach solution: 500 ml bleach with 4500 ml milliQ H20

Prepping bottles and filter housings (once dry)

• 1. store bottles sealed in clean cooler (plastic cap if not collecting water soon, change to sterile stoppers when collecting water)

Cooler with bottles capped:

Bottle with stopper:

• 2. prep filters:
  • 2.1 using clean forcepts and wearing clean gloves, place 1 polyester (PETE) Drain disc (mesh spacer) onto metal mesh on black filter housing piece
  • 2.2 then place 1 cellulose nitrate membrane filter on top of drain disc
  • 2.3 take beige housing piece and click onto black piece
  • 2.4 place into clean ziplock bag
  • 2.5 add 1 pair of sterile plastic forcepts and 1 clear hard plastic tubing into same bag
  • 2.6 seal and store on shelf
  • 2.7 change gloves and clean forcepts frequently

filter packs:

supplies cleaned but not prepped

supplies cleaned and prepped with filters

NOTES:

• filters and filter backers are seperated by paper discs (usually blue) and these should be discarded to get to the next filter or backer
• 1 μm filters
• drain disc/filter backing

drain disc and filter boxes:

Water collection in field (eg. off boat)

Need:

• cooler with ice
• sterilized nalgene bottles
• sterilized stoppers
• water collection device (+ both ropes)
• field box:
  • gloves
  • pencils and pens
  • bag of extra sterile stoppers
  • bag for trash/dirty gloves
  • bag for dirty stoppers
  • wet wipes, alcohol wipes, or hand sanitizer
• BSB data sheet printed on water proof paper

water collection device:

Steps:

• 1. place sterile bottles in cooler with sterile stoppers already pressed in
• 2. surround bottles with ice
• 3. once you have reached the collection site, mark down site info onto data sheet and choose a bottle to collect water (each are marked with a number)
• 4. wearing gloves place sterilized bottle between hose clamps and tighten them
• 5. push down stopper to check that it is snug
• 6. clip smaller rope onto the stopper (making sure rope is coming down from top of device, not sides)
• 7. lower device into water and let it go down until bubbles stop/you think it has reached the bottom
  • 7.1 make sure smaller rope has slack so stopper is not prematurely pulled
  • 7.2 count tape markers to estimate depth (every 10 feet)
• 8. once device has reached the bottom, pull on smaller rope to pull out stopper
• 9. wait ~1 min before pulling up device
• 10. when device it at the top of the water slowly pull it up onto the boat keeping the bottle upright
• 11. put on gloves and screw lid of bottle on
• 12. take bottle out of device and add back to the cooler (make sure it is surrounded with ice)
• 13. change gloves then repeat steps 3-12 at next site

Next Step:

• bring bottles on ice back to the lab
• filter water same day then store in -20 C freezer (see: filtering in the lab below)

filtering in the lab

Need:

• eDNA citizen scientist sampler (make sure battery is charged)
• ice
• packs/bags with sterile forcepts, filter housing (with filter and filter backing already within), and tubing
• 2 ml microcentrifuge tubes (label before starting filtering process)
• gloves
• container for dirty instruments & filter housings
• 2 1L sterile glass bottles filled with milliQ water

filtering water in lab:

Steps:

• 1. Wipe down bench area with 10% bleach, DI water, then 75% ethanol (repeat wipedown with bleach within each filter)
• 2. Wearing clean gloves, attach filter housing (black side) to long tube attached to the eDNA citizen scientist sampler
• 3. Attach clear hard plastic tube to beige part of the filter housing
• 4. Turn on vaccume with plastic tube sticking upwards before placing into bottle with water
• 5. Once water gets low in bottle take out plastic tube and slowly pour rest of water into the tube and filter housing until the filtered water reaches the 900 ml mark on the sampler storage container (water in long tube should make it reach 1 L)
• 6. Keep vaccume on and hold hard plastic tube upwards until all of the water in the long tube has reached the storage container
• 7. One vaccume is shut off, pull filter housing off long tube and take off the hard plastic tube (place in dirty container)
• 8. Gently open filter housing and using the plastic forcepts in the bag, fold filter and its backing in half like a taco then fold 2-3 more times until it looks like it will fit into 2ml microcentrifuge tube
• 9. Place filter into 2ml microcentrifuge tube then discard forcepts and both sides of the filter housing into the dirty container
• 10. Place microcentrifuge tube on ice
• 11. Pour storage container water down sink
• 12. Place storage container back citizen scientist sampler (with lid screwed on)
• 13. wipe down bench area with 10% bleach
• 14. Change gloves
• 15. repeat steps for all samples
• 16. place samples in freezer until ready to extract

NOTES:

• start and end filtering process by filtering 1 L bottle of milliQ H20 stored in sterile glass bottle (use for extraction neg control, 2 total)
• Keep bottles of water in ice cooler while working

filtering water in the field (eg. Grabowski tanks at MSC)

Need:

• eDNA citizen scientist sampler (make sure battery is charged)
• ice in small blue ice bucket
• packs/bags with sterile forcepts, filter housing (with filter and filter backing already within), and tubing
• 2 or 5 ml microcentrifuge tubes (label before starting filtering process)
• gloves
• container for dirty instruments & filter housings (and dirty gloves)
• gray bin
• 2 1L sterile glass bottles filled with milliQ water (to use for extraction negative control)

Prep Tanks

• 1. Prep tanks for experiment
  • 1.1 clean tanks with scrub brushs (algae growth) and remove particulates/litter/substrate (eg. dirt, leaves, rocks, shells, bugs…) with nets and water pump
  • 1.2 fill tanks with water
  • 1.3 standardize tanks
    • 1.33 water height: adjust standpipe/outflow tube angle/height (eg. pipe height = 50 cm)
    • 1.32 water flow rate: time how fast a 5 gallon bucket fills up with water (eg. 5 gallons in 1:30 mins +/- 10 seconds)
    • 1.33 ziptie mesh over standpipe/outflow tube opening
    • 1.34 use clamps to cover tanks with mesh (prevent birds from entering & organisms from escaping)
  • 1.4 add organisms to tanks
    • 1.41 decide # of organisms per tank
    • 1.42 figure out how to standardize (eg. size, gender…)
    • 1.43 have empty tank for control (could also have other tanks with different organisms)
  • 1.5 decide how many hours after organism addition or removal you want to filter water
  • 1.6 add temp loggers (can be borrowed from Grabowski lab)
• 2. when ready to filter, place ice bucket, gloves, dirty container, filter pack bags, and tubes on tube rack into gray lab bin

Prepped Bin:

Filtering

• 1. filter 1 L of milliQ water (stored in sterile glass bottle) in the lab then store on ice
• 2. Transport supplies (in gray bin) and eDNA citizen scientist sampler to filter site
• 3. Wearing clean gloves, attach filter housing (black side) to long tube attached to the eDNA citizen scientist sampler
• 4. Attach clear hard plastic tube to beige part of the filter housing
• 5. Turn on vaccume with plastic tube sticking upwards for a few seconds before placing into water
• 6. Remove from water once filtered water reaches the 900 ml mark on the sampler storage container (water in long tube should make it reach 1 L)
• 7. Keep vaccume on and hold hard plastic tube upwards until all of the water in the long tube has reached the storage container
• 8. One vaccume is shut off, pull filter housing off long tube and take off the hard plastic tube (place in dirty container)
• 9. Gently open filter housing and using the plastic forcepts in the bag, fold filter and its backing in half like a taco then fold 2-3 more times until it looks like it will fit into 2ml microcentrifuge tube
• 10. Place filter into 2ml microcentrifuge tube then discard forcepts and both sides of the filter housing into the dirty container
• 11. Place microcentrifuge tube into ice
• 12. Pour storage container water out (either back into same tank you took it from, or onto grass/rocks)
• 13. Place storage container back into citizen scientist sampler and screw lid back on
• 14. Change gloves
• 15. repeat steps for all tanks/samples sites
• 16. transport supplies and samples back to lab
• 17. filter 1 L of milliQ water (stored in sterile glass bottle)
• 18. transfer all samples from ice to -20 freezer until ready to extract

Next Steps:

• lysis and extract DNA from filters
• bleach nalgene bottles, stoppers, and filter supplies (see: Prep section)
• prep filter bags (see: Prep section)