Dataset: Bergen MesoHux 2017 Bacteria and Virus Abundance

Environmental Sample Collection

1. Transfer 1 ml of whole seawater to a 2 ml cryovial.
2. Add 20 µl of 25% glutaraldehyde for a final concentration of 0.5%.
3. Incubate at 4°C for 15 min.
4. Flash freeze in liquid N2 and store at -80°C.

Fluorescent DNA staining (for bacterial and viral abundances)

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

Analysis (for bacterial and viral abundances)

Samples are analyzed on Influx Model 209S Mariner flow cytometer using BD Sortware (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 µm 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. Flow rates are determined using the volumetric method.
4. Collected data are analyzed using FlowJo v9.9.6. Virus Like Particles (VLPs) are differentiated from bacteria using the gating hierarchy established at time of collection.

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 4ºC

1X TE + SYBR Gold (for 10 mls)
1. Filter 10 mls 1 TE buffer, 0.22 μm filter
2. 1:20,000 dilution of SYBR Gold stock (Molecular Probes) (0.5 μl stock to 10 mls TE buffer)