Samples were taken using a rosette of Niskin bottles, fixed with freshly titrated paraformaldehyde (pH 7.4–8.1, 0.1% final concentration), held in the dark for 10 min, frozen in liquid nitrogen, and stored in a -80 deg C freezer (CH0409 samples) or in liquid nitrogen (CH0510 samples) until analysis. Preserved samples were analyzed by dual beam flow cytometry on a modified Coulter-EPICS 753 flow cytometer (Binder et al. 1996). Samples were chosen in random order, defrosted in a 30°C water bath (just long enough to melt, ~5 min), and stained with the DNA-specific stain Hoechst 33342 (0.5 ug mL-1 final concentration) (Invitrogen, Carlsbad, California) for a minimum of 20 min in the dark. Prior to analysis, polystyrene fluorescent beads (Flow Check® 1.0 um (YG) and 0.494 um (BB); Polysicences Inc., Washington, PA, USA), were added to each sample, and used to normalize cellular light scatter, red (chlorophyll-derived) fluorescence, and Hoechst fluorescence.
Samples were run at an infusion rate of 10 uL min-1 for 10 to 50 min, depending on cell abundance within the sample. A minimum of 10,000 Prochlorococcus cells were analyzed, except for samples in which low Prochlorococcus concentrations made this impractical.
DNA frequency distributions for Prochlorococcus cells were obtained from Hoechst-derived blue fluorescence. These frequency distributions were deconvoluted into their component cell cycle stages (G1, S, G2) using Modfit software (Verity Software House, Topsham, ME, USA), and assuming a simple model comprised of two Gaussian populations (G1 and G2) and a broadened rectangle (S).
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