Syringe pumped high speed flow cytometry of oceanic phytoplankton

Background: Nanophytoplankton (2-20 mu m) are less numerous than picophytoplankton (< 2 mu m) in the oceans but their biomass and production are comparable and sometimes higher. The accuracy of cytometry-based enumeration of phytoplankton ultimately depends on cell abundance and sample flow rate. Commercial flow cytometers in which sheath and core streams are driven by air pressure cannot produce sufficiently high, stable sample flow rate. The present study demonstrates the applicability of a syringe pump for flow cytometric enumeration of oceanic nanophytoplankton on two meridional transects across the Atlantic Ocean. Methods: Commercially available syringe pumps were used to deliver live phytoplankton samples into a flow cell of standard flow cytometers (FACSort, FACSCalibur, 1313) with increased flow rate of > 1.0 ml min(-1) compared to the normal air pressure sample delivery of < 0.1 ml min(-1). An auxiliary application of syringe pump flow cytometry for calibrating 0.5 mu m bead concentration standards is also discussed. Results: The results demonstrated that flow cytometry of samples injected at rates above 0.1 ml min(-1) is achievable and worthwhile. Counts of phytoplankton in air and syringe pumped samples agreed closely. Syringe pumping of samples offered a broader range of flow rates up to 0.8-1.0 ml min(-1) without detrimental effect on flow cytometric enumeration of cells. The increased number of coincidences at high flow rates led to an approximate 10% decrease of Cyanobacteria counts when the acquisition rate approached 1,000 particles s(-1), but seemed to have a lesser effect on counting rarer phytoplankton. The syringe pump flow cytometry allowed enumeration of phytoplankton groups at concentrations of 5-100 cells ml(-1), cell concentrations equivalent to those of Cyanobacteria in the twilight deep ocean. Conclusion: The proposed syringe pump modification of a FACS instrument represents a significant improvement for accurate enumeration of the less abundant phytoplankton and so gives better estimations of phytoplankton distribution and standing stocks.