Plate Scrapings

Important consideration:

Make sure that all the containers and tools used have been bleached and rinsed between ARMS processing. Wear gloves at all times; do not touch the water or the plates with bare skin.

Materials:

During this step, all sessile communities will be bulk collected from all the ARMS plates to assess their overall taxonomic diversity using next-generation sequencing. Bulk samples must be treated with care as to not introduce exogenous DNA in these fractions.

  • Laboratory gloves
  • Clean photo tray
  • Clean paint scrapers, new work best - maintained clean edge important to scrape well
  • 1 squeeze bottle containing filtered seawater
  • 1 squeeze bottle containing 90% ethanol
  • 1 food blender capable of blending ice
  • 1 40-microns nytex mesh net
  • 1 waste bucket
  • 3 50 ml falcon tubes (6 tubes if 2 preservatives are used DMSO + ethanol)
  • 2 Spatulas (bleached or disposable)
  • 150 ml Salt saturated DMSO buffer (optional: 150 ml 90% ethanol) - Click here to access the DMSO buffer recipe
  • 1 2-Liters whirlpack or bigger depending on the overall volume of the sessile community
  • 1 pencil
  • 1 marker
  • Laboratory tape
  • Parafilm

Procedures:

  1. Using gloves, place the first plate in the clean photo tray and scrape all sides (including the fine edges - 6 surfaces total - 2 big and 4 edges) into the photo tray
  2. If needed, use the squeeze bottle to quickly rinse off the plate with filtered seawater. Only use a small quantity of water
  3. Repeat step 1 and 2 for all the plates. If several persons are scraping, 2 photo trays can be used
  4. Transfer the content of the tray carefully into the blender and blend the sessile fraction at full strength for 15 seconds or until the fraction is well homogenized
  5. Pour the blended fraction into the mesh net on top of the waste bucket and wash several times with filtered seawater. The sludge may need to be stirred to let the water go through more easily
  6. Squeeze the liquid through the mesh to dry the fraction as much as possible.
  7. Transfer the dried fraction back into the photo tray and mix to homogenize with the spatula
  8. Place ~10 ml of matter into each of the three falcon tubes and fill with DMSO. Shake vigorously to homogenize
  9. Label the tubes and seal closed tubes with parafilm. Place in the freezer
  10. If possible, place another ~10 ml of matter into three additional falcon tubes and fill with 90% ethanol. Shake vigorously to homogenize
  11. Label the tubes and seal closed tubes with parafilm. Place in the freezer
  12. The remaining matter can be dried immediately and weighed for biomass quantification and/or put into a whirpack bag and kept frozen until further analysis

Illustrations:

All sides of the plates are scraped into a clean photo tray

The sessile matter is blended in a strudy blender until well homogenized

The blended fraction is poured into a mesh, rinsed with clean, filtered sea water and dried out by squeezing the water out of the mesh

Once the fraction doesn't contain water anymore, it is placed back into the tray and mixed using a single-use or sterile spatula

Up to 10-mL of sessile scrapings are preserved into 50-mL Falcon tubes. Three subsamples are preserved in DMSO buffer and three subsamples are preserved in 95% Ethanol

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