Analytical procedure:

Microbial cell abundances were determined using an Influx flow cytometer. Briefly, pigmented groups (Prochlorococcus, Synechococcus, and picoalgae) were enumerated in unstained samples by their chlorophyll and forward scatter signatures (Marie et al. 1999). The high phycoerythrin signal in Synechococcus was used to distinguish this group from Prochlorococcus and picoalgae.

To visualize non-pigmented picoplankton (bacteria) and heterotrophic protists, samples were stained with SYBR Green I DNA dye (SG, 1:10000 final concentration, for 10 min at 4C in the dark) (Zubkov et al. 2007, Christaki et al. 2011). Because bacteria and Prochlorococcus groups exhibit overlapping signal characteristics in surface samples after SG staining, the abundances of bacteria in surface samples were calculated by subtracting the Prochlorococcus abundance, determined in the unstained aliquot, from the total SG-stained group abundance. An internal standard of 1-um diameter fluorescent microspheres (Fluoresbrite, Polysciences) was added to each sample.

Sampling and Analytical Methodology:

Seawater samples were acquired from bottle samples collected by a CTD rosette during the OUTPACE cruise. 1.8 ml water samples were collected in duplicate, fixed with 0.25% (w/v) paraformaldehyde, flash frozen, and preserved at -80C. One of the duplicate samples was used to count phytoplankton (Prochlorococcus, Synechococcus, picoalgae), and the other was used to count bacteria and protists.

For pigmented groups (Prochlorococcus, Synechococcus, and picoalgae), cells were excited using a combination of two lasers (488+456 nm) focused into one pinhole; while for non-pigmented groups (bacteria and heterotrophic protists), cells were excited using the 488 nm laser only. For calibration, a solution of 1-um diameter fluorescent microspheres (Fluoresbrite, Polysciences) was used.

References:

Christaki U, Courties C, Massana R, Catala P, Lebaron P, Gasol JM, Zubkov MV (2011) Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I. Limnology and Oceanography-Methods 9:329-339. https://doi.org/10.4319/lom.2011.9.329

Marie D, Partensky F, Vaulot D, Brussaard C (1999) Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples. Current Protocols in Cytometry:11.11.11-11.11.15. https://doi.org/10.1002/0471142956.cy1111s10

Zubkov M, Burkill PH, Topping JN (2007) Flow cytometric enumeration of DNA-stained oceanic planktonic protists. Journal of Plankton Research 29:79-86. https://doi.org/10.1093/plankt/fbl059