Zubkov Mikhail V, Burkill Peter H
National Oceanography Centre, Southampton, UK.
Cytometry A. 2006 Sep 1;69(9):1010-9. doi: 10.1002/cyto.a.20332.
Nanophytoplankton (2-20 microm) are less numerous than picophytoplankton (<2 microm) 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.
Commercially available syringe pumps were used to deliver live phytoplankton samples into a flow cell of standard flow cytometers (FACSort, FACSCalibur, BD) 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 microm bead concentration standards is also discussed.
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.
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.
在海洋中，微型浮游植物（2 - 20微米）的数量比微微型浮游植物（<2微米）少，但其生物量和生产力相当，有时甚至更高。基于流式细胞术的浮游植物计数准确性最终取决于细胞丰度和样品流速。由气压驱动鞘流和芯流的商用流式细胞仪无法产生足够高且稳定的样品流速。本研究证明了注射泵在横跨大西洋的两条子午线上对海洋微型浮游植物进行流式细胞术计数的适用性。
使用商用注射泵将活的浮游植物样品输送到标准流式细胞仪（FACSort、FACSCalibur、BD）的流动池中，与正常气压下小于0.1毫升/分钟的样品输送相比，流速提高到>1.0毫升/分钟。还讨论了注射泵流式细胞术在校准0.5微米珠浓度标准方面的辅助应用。
结果表明，以高于0.1毫升/分钟的流速注入样品的流式细胞术是可行且有价值的。气压注入和注射泵注入样品中的浮游植物计数非常接近。注射泵注入样品可提供高达0.8 - 1.0毫升/分钟的更宽流速范围，且对细胞的流式细胞术计数没有不利影响。当采集速率接近1000个颗粒/秒时，高流速下重合事件数量增加导致蓝细菌计数大约减少10%，但对较罕见浮游植物的计数影响似乎较小。注射泵流式细胞术能够对浓度为5 - 100个细胞/毫升的浮游植物类群进行计数，该细胞浓度与深海弱光层中的蓝细菌浓度相当。
所提出的对FACS仪器的注射泵改进对于准确计数数量较少的浮游植物是一项重大改进，从而能更好地估计浮游植物分布和现存生物量。
