Dataset: MUSiCC OC1504A - Bacteria Virus and Chlorophyll Containing Cell Abundance
Deployment: OC1504A

Environmental Sample Collection

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

Fluorescent DNA staining (for bacterial and viral abundances)

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

Analysis (for bacterial and viral abundances)

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

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. A gating hierarchy is established using both beads and previously determined virus and bacteria populations as reference (Sybr Gold Fluorescence versus SSC cytogram).
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.

Analysis (for chlorophyll containing cells)

Samples are analyzed on a BD Accuri C6. Fixed, frozen samples are thawed and analyzed immediately.

1. An initial Forward Scatter (FSC) vs Side Scatter (SSC) configuration is determined using various sized fluorescent beads as reference points (1.0, 2.0, 3.0, 6.0 and 10 um).
2. Gating is established using both beads and previously determined phytoplankton populations as reference (Chlorophyll Fluorescence versus FSC cytogram).
3. Samples are analyzed using 488nm laser for excitation and the default BD Accuri threshold (80,000 RFU) on FSC is used.

TE buffer with SYBR Gold recipe

1X TE (for 100 mls)

1 ml of 1M Tris, pH 8.0
1 ml of 0.5 mM EDTA
98 mls MQ water
Store 4 degrees celsius

1X TE + SYBR Gold (for 10 mls)

1. Filter 10 mls 1 TE buffer, 0.22 um filter
2. 1:20,0000 dilution of SYBR Gold (Molecular Probes) stock (0.5 ul stock to 10 mls TE buffer)