meth_1.14 Coral Sampling for Marine Microbes Coastal Project

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Citation:

AM Project ID: Legacy

General Notes:

  1. Relative abundance of chosen coral species at each site should be contextualised on each visit by 10 randomly thrown 50 x 50 cm quadrats.
  2. Select several colonies that are of dominant and consistent appearance (colour morph, growth pattern etc.) and document each colony with a photo.
  3. Three different healthy specimens are analysed at each sampling event.
  4. Record the following parameters for each specimen: size (approximate in cm); any contact with other benthic, sessile organisms (yes, no; if yes, specify); morphological condition (discolouration; outgrowth, spots etc.); epiphytic growth (type and coverage (%)), evidence of grazing (presence/ absence and estimated % affected)

Method:

  1. Collect coral mucus samples of 3 colonies with autoclaved cotton swabs placed in 2 mL Eppendorf tubes (see picture). Invert those tubes and open underwater next to the coral surface to minimize contamination with microbes from the surrounding seawater. Swabs should be gently rolled over the coral surface and then placed back in the tube. Labelled tubes according to the instruction given in the spreadsheet “sampling_schedule”.
  2. Once on the surface, remove excess seawater from the tube without touching the swab and snap freeze sample in liquid Nitrogen.
  3. Also remove 3 random samples per coral colony of the same 3 colonies (avoid sampling of the swabbed location). Enclose the 3 random samples separated for each colony inside press-seal bags (or Falcon tubes) with surrounding seawater in situ and bring to the surface. Each sample should comprise of a fragment 5-10 mm in length or a small branch (species dependent). Labelled tubes according to the instruction given in the spreadsheet “sampling_schedule”
  4. Water is emptied before sample is snap frozen in liquid nitrogen to preserve the mucus layer onsite (liquid N is transported in dry shipper).
  5. In the laboratory, remove mucus and tissue with an air gun and 1X PBSE pH 7.4 (137 mM NaCl (8.07 g/L), 2.7 mM KCl (0.201 g/L), 4.3 mM Na2HPO4 (0.611 g/L), 1.4 mM KH2PO4 (0.191 g/L), 10 mM EDTA (20 mL 0.5 M EDTA). Liquefy coral tissue by repeatedly drawing into a 5 mL syringe or using a hand-held homogenizer. Divide samples into 1.5 mL cryo-tubes and centrifuge at 13,000 rpm for 10 minutes. Remove top liquid phase and discard keeping the cell pellet. Freeze samples at -80 °C (or -20 °C).