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Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses

机译：用于船载分析的高通量微生物自动计数系统的改进

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In the age of ever-increasing “-omics” studies, the accurate and statistically robust determination of microbial cell numbers within often-complex samples remains a key task in microbial ecology. Microscopic quantification is still the only method to enumerate specific subgroups of microbial clades within complex communities by, for example, fluorescence in situ hybridization (FISH). In this study, we improved an existing automatic image acquisition and cell enumeration system and adapted it for usage at high seas on board an oceanographic research ship. The system was evaluated by testing settings such as minimal pixel area and image exposure times ashore under stable laboratory conditions before being brought on board and tested under various wind and wave conditions. The system was robust enough to produce high-quality images even with ship heaves of up to 3 m and pitch and roll angles of up to 6.3°. On board the research ship, on average, 25% of the images acquired from plankton samples on filter membranes could be used for cell enumeration. Automated enumeration was highly correlated with manual counts ( r ~(2) > 0.9). Even the smallest of microbial cells in the open ocean, members of the alphaproteobacterial SAR11 clade, could be confidently detected and enumerated. The automated image acquisition and cell enumeration system developed here enables an accurate and reproducible determination of microbial cell counts in planktonic samples and allows insight into the abundance and distribution of specific microorganisms already on board within a few hours.IMPORTANCE In this research article, we report on a new system and software pipeline, which allows for an easy and quick image acquisition and the subsequent enumeration of cells in the acquired images. We put this pipeline through vigorous testing and compared it to manual microscopy counts of microbial cells on membrane filters. Furthermore, we tested this system at sea on board a marine research vessel and counted bacteria on board within a few hours after the retrieval of water samples. The imaging and counting system described here has been successfully applied to a number of laboratory-based studies and allowed the quantification of thousands of samples and FISH preparations (see, e.g., H. Teeling, B. M. Fuchs, D. Becher, C. Klockow, A. Gardebrecht, C. M. Bennke, M. Kassabgy, S. Huang, A. J. Mann, J. Waldmann, M. Weber, A. Klindworth, A. Otto, J. Lange, J. Bernhardt, C. Reinsch, M. Hecker, J. Peplies, F. D. Bockelmann, U. Callies, G. Gerdts, A. Wichels, K. H. Wiltshire, F. O. Gl?ckner, T. Schweder, and R. Amann, Science 336:608–611, 2012, http://dx.doi.org/10.1126/science.1218344). We adjusted the standard image acquisition software to withstand ship movements. This system will allow for more targeted sampling of the microbial community, leading to a better understanding of the role of microorganisms in the global oceans.
机译：在不断增长的“-组学”研究时代，经常复杂的样品中微生物细胞数量的准确和统计学上可靠的确定仍然是微生物生态学中的关键任务。显微镜定量仍然是通过例如荧光原位杂交（FISH）枚举复杂群落中微生物进化枝的特定亚组的唯一方法。在这项研究中，我们改进了现有的自动图像采集和像元枚举系统，并使其适合在海洋研究船上的公海使用。通过在稳定的实验室条件下测试设置（例如最小像素面积和岸上图像曝光时间）对系统进行评估，然后将其带上船并在各种风浪条件下进行测试。该系统具有足够的鲁棒性，即使船舶起伏不超过3 m，俯仰角和横滚角不超过6.3°，也可以产生高质量的图像。在研究船上，平均而言，从滤膜上的浮游生物样本获取的图像中有25％可用于细胞计数。自动枚举与人工计数高度相关（r〜（2）> 0.9）。即使是公海中最小的微生物细胞，也就是α变形细菌SAR11进化枝的成员，都可以放心地检测和列举。此处开发的自动图像采集和细胞计数系统可以准确，可重复地确定浮游生物样品中的微生物细胞数量，并可以在数小时内洞悉船上已经存在的特定微生物的数量和分布。在新的系统和软件管道上运行，可以轻松，快速地获取图像并随后枚举所获取图像中的单元格。我们对该管道进行了严格的测试，并将其与膜过滤器上的微生物细胞的手动显微镜计数进行了比较。此外，我们在海上研究船上在海上测试了该系统，并在取回水样后数小时内对船上的细菌进行了计数。此处描述的成像和计数系统已成功应用于许多基于实验室的研究，并允许对数千种样品和FISH制剂进行定量（例如，参见H. Teeling，BM Fuchs，D。Becher，C。Klockow， A. Gardebrecht，CM Bennke，M。Kassabgy，S。Huang，AJ Mann，J。Waldmann，M。Weber，A。Klindworth，A。Otto，J。Lange，J。Bernhardt，C。Reinsch，M。Hecker， J. Peplies，FD Bockelmann，U.Callies，G.Gerdts，A.Wichels，KH Wiltshire，FO Gl？ckner，T.Schweder和R.Amann，Science 336：608–611，2012，http：// dx .doi.org / 10.1126 / science.1218344）。我们调整了标准的图像采集软件以承受船的移动。该系统将允许对微生物群落进行更有针对性的采样，从而更好地了解微生物在全球海洋中的作用。

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