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# CTD Rosette Bottle Sampling on NES-LTER cruises
Samples were collected from the water column at multiple depths using Niskin bottles on a CTD rosette system. A small number of samples were collected from the ship’s underway science seawater near the surface. All samples were collected into 2 ml sterile cryovials and stored in a dark container avoiding exposure to sunlight.
# Sample preservation
Samples were brought into lab and preserved using a 50/50 mix of 25% Glutaraldehyde and 2% Kolliphor P 188, for a final sample concentration of 0.125% Glutaraldehyde and 0.01% Kolliphor. Prior to May of 2019, samples were preserved with only 0.125% Glutaraldehyde. Ten minutes after addition of preservative and gentle mixing, samples were flash frozen in liquid nitrogen where they remained until analysis.
#Sample processing
Samples were thawed immediately prior to analysis. Each sample was pulled by syringe through a 150-micrometer Nitex mesh pre-filter to avoid aspirating overly large particles that might clog the flow cytometer. For heterotrophic bacterial analysis, 500 microliters were placed in a separate tube with 0.5 ul 500x SYBR stain in DMSO (a 1:20 dilution of Invitrogen SYBR Green I Nucleic Acid Gel Stain, 10,000X concentrate in DMSO). The remainder of the sample was placed back in the original tube. Bacterial samples were mixed with a vortex genie and allowed to stain in the dark for at least 10 minutes before analysis.
A ThermoFisher Attune NxT Flow Cytometer equipped with two lasers, green (532 nm, 100mW) and blue (488 nm, 50mW) was used for analysis. The flow cytometer focus fluid (sheath) was milli Q water with 3% Sodium chloride (30 ppt) and 0.1% 2-Phenoxyethanol. For quality control, the Attune flow cytometer Performance test (bead REF# 4449754) was run at least once per week and a beads mix (see recipe), was run daily for normalized size calibration. Instrument cleaning was performed at the end of each analysis day using 25% Hellmanex III detergent.
Each discrete sample was analyzed with 2-3 different configurations shown in the table Settings_configuration_history_discrete_analysis. The specific fcs file used for quantification of each cell type is listed in the data table. Often multiple cell types were quantified with the same fcs file. FCS files are provided in zipped files organized by cruise.
#Instrument configuration and calibration
Instrument configuration settings and calibration are provided in the Settings_configuration_history_discrete_analysis file. The table has a row for each run type used for each research cruise, showing relevant instrument settings or major changes in optical filter configuration, bead runs for calibration, cruise sample collection date, and range of analysis dates.
Recipe for FCB bead mix:
-100ml salt sheath with 0.1% 2 phenoxyethanol
-400 ul diluted stock 0.5 um beads Polysciences Inc #19507
-5 ul 1 um beads Polysciences Inc #23517
-5 drops invitrogen Alignflow plus flow cytometry alignment beads, 6 um #C47397
# Data processing
Cells were identified and enumerated from the flow cytometry (.fcs) data files based on their scattering, SYBR (525 nm), phycoerythrin (575 nm) and chlorophyll (680 nm) fluorescence signals. Gating was completed manually in the Attune NXT software interface, yielding Attune NXT workspace (.aws) files that were exported and interpreted in matlab for enumeration in the provided data table.
The size of each cell was estimated from side-angle light scattering. Side scattering signals were normalized using the side scattering signal of 1-um beads (Flow Check High Intensity Alignment Grade Particles, Polysciences) which were part of the bead mix that was run daily during analysis. Bead-normalized scattering signals were converted to cell volume estimates based on a calibration curve derived from phytoplankton cultures independently sized on a Coulter Multisizer. Finally, cell volume was converted to cell carbon following the relationships described by Menden-Deuer and Lessard (2000). Precision for carbon concentration is provided to two decimal places. Carbon concentration for heterotrophic prokaryotes was estimated from cell concentration and constant carbon per cell ratio of 20fg per cell from Lee and Furham (1987) who report that bacterial biomass is relatively invariant to cell size. The selected term, “Heterotrophic prokaryotes” from the Marine Microbial Flow Cytometry Standardised Group Names, encompasses both bacteria and archaea, and in some cases overlaps with Prochlorococcus. For the purposes of this package, we refer to this group as heterotrophic bacteria.
For all sample runs, the first 20 percent of the volume was omitted from analysis and cell quantification to avoid underestimation. The volume analyzed columns account for this adjustment and reflect the true volume analyzed, rather than the total volume run.
# Quality control
During the manual gating process, if data quality appears poor, gating information (.aws file) is not exported, and data subsequently are not reported. Although this is rare, reasons would be obvious Attune malfunction or lack of preservative. After all manual gates (.aws files) are exported, they are processed with matlab matching them to corresponding .fcs files. In this step, we generated a list of fcs files that did not match to an aws file and ensured the list is as expected. Next, we cross referenced the list of gated samples with our cruise sample log and corrected any mistakes; for example if the log indicates that an alternate niskin was used when that in the filename misfired. The filenames themselves are unchanged during this manual reassignment, so the adjustments are trackable by comparing the filenames to the cast and niskin columns. Finally, we output .pngs of cytograms generated from the matlab processing of fcs and aws files to check for and correct gating errors. At each step, we reprocessed as needed.
#Data Package Assembly
We add CTD bottle metadata (time, latitude, longitude, depth, potential temperature, salinity) from the REST Application Programming Interface (API) of the NES-LTER data system.
Code for data package assembly, including metadata from templates, is available on GitHub (https://github.com/WHOIGit/nes-lter-attune-fcm-discrete-transect).
# References
Lee, S. & Fuhrman, J. A. (1987) Relationships between biovolume and biomass of naturally derived marine bacterioplankton. Appl Env. Microbiol 53: 1298–1303.
Menden-Deuer, S. and Lessard, E.J. (2000) Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnology and Oceanography. 45(3): 569–579.