Pernice Massimo C, Gasol Josep M
Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar-CSIC, Barcelona, Spain.
Front Microbiol. 2023 Jan 6;13:1064112. doi: 10.3389/fmicb.2022.1064112. eCollection 2022.
On a standard oceanographic cruise, flow cytometry data are usually collected sparsely through a bottle-based sampling and with stations separated by kilometers leading to a fragmented view of the ecosystem; to improve the resolution of the datasets produced by this technique here it is proposed the application of an automatic method of sampling and staining. The system used consists of a flow-cytometer (Accuri-C6) connected to an automated continuous sampler (OC-300) that collects samples of marine surface waters every 15 min. We tested this system for five days during a brief Mediterranean cruise with the aim of estimating the abundance, relative size and phenotypic diversity of prokaryotes. Seawater was taken by a faucet linked to an inlet pump ( 5 m depth). Once the sample was taken, the Oncyt-300 stained it and sent it to the flow cytometer. A total of 366 samples were collected, effectively achieving a fine-grained scale view of microbial community composition both through space and time. A significative positive relationship was found comparing data obtained with the automatic method and 10 samples collected from the faucet but processed with the standard protocol. Abundance values retrieved varied from 3.56·10 cell mL in the coastal area till 6.87 10 cell mL in open waters, exceptional values were reached in the harbor area where abundances peaked to 1.28 10 cell mL. The measured features (abundance and size) were associated with metadata (temperature, salinity, conductivity) also taken in continuous, of which conductivity was the one that better explained the variability of abundance. A full 24 h measurement cycle was performed resulting in slightly higher median bacterial abundances values during daylight hours compared to night. Alpha diversity, calculated using computational cytometry techniques, showed a higher value in the coastal area above 41° of latitude and had a strong inverse relationship with both salinity and conductivity. This is the first time to our knowledge that the OC-300 is directly applied to the marine environment during an oceanographic cruise; due to its high-resolution, this set-up shows great potential both to cover large sampling areas, and to monitor day-night cycles .
在一次标准的海洋学巡航中，流式细胞术数据通常通过基于瓶子的采样稀疏收集，且采样站相隔数公里，这导致对生态系统的认识支离破碎；为提高该技术产生的数据集的分辨率，本文提出应用一种自动采样和染色方法。所使用的系统由一台流式细胞仪（Accuri - C6）连接到一个自动连续采样器（OC - 300）组成，该采样器每15分钟采集一次海洋表层水样本。我们在地中海的一次短暂巡航中对该系统进行了为期五天的测试，目的是估计原核生物的丰度、相对大小和表型多样性。海水通过连接进水泵的水龙头采集（深度5米）。样本采集后，Oncyt - 300对其进行染色并送至流式细胞仪。总共采集了366个样本，有效地实现了对微生物群落组成在空间和时间上的细粒度尺度观察。将自动方法获得的数据与从水龙头采集但按标准方案处理的10个样本进行比较，发现了显著的正相关关系。检索到的丰度值在沿海地区从3.56·10⁶个细胞/毫升到开阔水域的6.87·10⁶个细胞/毫升不等，在港口区域达到了异常值，丰度峰值达到1.28·10⁷个细胞/毫升。所测量的特征（丰度和大小）也与连续采集的元数据（温度、盐度、电导率）相关，其中电导率是最能解释丰度变异性的因素。进行了完整的24小时测量周期，结果显示白天的细菌丰度中值略高于夜间。使用计算细胞术技术计算得到的α多样性在纬度高于41°的沿海地区值更高，并且与盐度和电导率都呈强烈的负相关关系。据我们所知，这是OC - 300首次在海洋学巡航期间直接应用于海洋环境；由于其高分辨率，该设置在覆盖大面积采样区域以及监测昼夜循环方面都显示出巨大潜力。
