Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses
Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses
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DOI：
10.1128/aem.03931-15
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发表时间：
2016-06-01
影响因子：
4.4
通讯作者：
Fuchs, Bernhard M.
中科院分区：
文献类型：
--
作者：
Bennke, Christin M.;Reintjes, Greta;Fuchs, Bernhard M.
在“组学”研究不断增长的时代，对通常复杂的样品中的微生物细胞数量进行准确且统计稳健的测定仍然是微生物生态学的一项关键任务。显微定量仍然是通过荧光原位杂交 (FISH) 等方法枚举复杂群落内微生物进化枝特定亚群的唯一方法。在这项研究中，我们改进了现有的自动图像采集和细胞计数系统，并将其改造为在海洋学研究船上在公海上使用。该系统通过在稳定的实验室条件下测试最小像素面积和岸上图像曝光时间等设置进行评估，然后再带上船上并在各种风浪条件下进行测试。该系统足够强大，即使在船舶起伏高达 3m、俯仰角和横滚角高达 6.3 度的情况下也能生成高质量图像。在研究船上，从滤膜上的浮游生物样本获取的图像平均有 25% 可用于细胞计数。自动计数与手动计数高度相关（r(2)>0.9）。即使是公海中最小的微生物细胞（α变形菌 SAR11 分支的成员）也可以被自信地检测和计数。这里开发的自动图像采集和细胞计数系统能够准确且可重复地测定浮游样品中的微生物细胞计数，并允许在几个小时内深入了解船上已存在的特定微生物的丰度和分布。 重要性在这篇研究文章中，我们报告了一个新的系统和软件管道，它允许轻松快速地采集图像并随后对所采集图像中的细胞进行计数。我们对该管道进行了严格的测试，并将其与膜过滤器上微生物细胞的手动显微镜计数进行了比较。此外，我们在一艘海洋研究船上测试了该系统，并在取回水样后的几个小时内对船上的细菌进行了计数。这里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，并允许对数千个样品和 FISH 制剂进行定量（参见，例如，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.克林德沃斯，A. 奥托，J. Lange、J. Bernhardt、C. Reinsch、M. Hecker、J. Peplies、F. D. Bockelmann、U. Callies、G. Gerdts、A. Wichels、K. H. Wiltshire、F. O. Glockner、T. Schweder 和 R. Amann，《科学》336：608-611，2012 年， http://dx.doi.org/10.1126/science.1218344）。我们调整了标准图像采集软件以承受船舶运动。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。
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 3m and pitch and roll angles of up to 6.3 degrees. 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.IMPORTANCEIn 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. Glockner, 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.