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Environmental Sample Collection

\n
    \n
  1. Transfer 1 ml of whole seawater to a 2 ml cryovial.
    2. \n
  2. Add 20 ul of 25% glutaraldehyde for a final concentration of 0.5%.
    3. \n
  3. Incubate at 4 degrees celsius for 30 min.
    4. \n
  4. Flash freeze in liquid N2\u00a0and store at -80 degrees celsius.
    5. \n
\n

Fluorescent DNA staining (for bacterial and viral abundances)

\n
    \n
  1. Thaw samples.
    2. \n
  2. To 20 ul of sample, add 980 ul 1X TE buffer with SYBR Gold (see recipe below)\u00a0
    3. \n
  3. Heat to 80 degrees celsius for 10 min in the dark
    4. \n
  4. Cool at RT for 5 min
    5. \n
  5. Analyze via flow cytometry
    6. \n
\n

Analysis (for bacterial and viral abundances)

\n

Samples are analyzed on Influx Model 209S Mariner flow cytometer using BD Software (BD Biosciences).

\n
    \n
  1. An initial Forward Scatter (FSC) vs Side Scatter (SSC) configuration is determined using Molecular Probes Flow Cytometry Sub-micron particles size reference kit (Cat#F13839) consisting of 0.02, 0.1, 0.5, 1.0 and 2.0 um fluorescent beads.
    2. \n
  2. A gating hierarchy is established using both beads and previously determined virus and bacteria populations as\u00a0reference\u00a0(Sybr Gold Fluorescence versus SSC cytogram).
    3. \n
  3. Samples are analyzed using a 488 nm laser for excitation and a minimum trigger threshold is established using 542/15 nm (SYBR Gold) emission.
    4. \n
\n

TE buffer with SYBR Gold recipe

\n

1X TE (for 100\u00a0mls)
\n1 ml of 1M Tris, pH 8.0
\n1 ml of 0.5 mM EDTA
\n98\u00a0mls\u00a0MQ water
\nStore 4 degrees celsius

\n

1X TE + SYBR Gold (for 10\u00a0mls)

\n
    \n
  1. Filter 10\u00a0mls\u00a01 TE buffer, 0.22 um filter
    2. \n
  2. 1:20,0000 dilution of SYBR Gold stock (Molecular Probes) (0.5 ul stock to 10\u00a0mls\u00a0TE buffer)\u00a0
    3. \n
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Bacteria and virus abundance data from the MV1405 cruise. Samples were collected by CTD.

","@type":"rdf:HTML"}],"http://www.w3.org/2000/01/rdf-schema#label":[{"@value":"IrnBru MV1405 - Bacteria and Virus Abundance","@type":"xsd:string"}],"http://ocean-data.org/schema/hasProcessingDescription":[{"@value":"

DMO notes:

\n

- added cruise_id column
\n- changed column names to meet BCO-DMO standards
\n- added ISO_DateTime_UTC column
\n- reformatted lat/lon to appear in decimal degrees
\n- removed prefix \"stn\" and \"sta\" from numbers in station column

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