Samples were collected in triplicate with a surface water pump (unless depth was specified, then it was collected by CTD), filtered with 0.2um sterivex filters (Millipore Cat #: SVGP 010 50) and fixed with 0.5% final concentration gluteraldehyde. Samples were frozen in liquid nitrogen, and stored at -80.
After thawed, SYBR Gold and stock beads were added to the sample as done in Brussaard 2004. Samples were diluted to get ~200-1000 events per second on a BD FACSCaliber Flow Cytometer looking at fluorescence and side scatter. Contact for Brian Zielinski for journal-quality methods if needed (bzielins@mail.usf.edu).
All the stations represent 2-3 true replicates, each replicate consisted of three separate readings. The reason not all of the stations had 3 replicates was because one day the machine malfunctioned and all the samples to be run that day needed to be discarded. The counts usually are very accurate and precise; however it seems that with these samples the precision was not as good as I would hope to see. This can be caused by two things. 1) small organics/cell debris from the plume water could have been included in the viral counts, or 2) I have only briefly used these methods previously and these counts were taken along my learning curve.
Related files and references:
1. Brussaard CPD (2004) Optimization of procedures for counting viruses by flow cytometry. Applied and Environmental Microbiology 70: 1506.
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