Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures
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DOI：
10.3389/fmicb.2019.00720
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发表时间：
2019-04-09
影响因子：
5.2
通讯作者：
Lever, Mark Alexander
中科院分区：
文献类型：
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作者：
Deng, Longhui;Fiskal, Annika;Lever, Mark Alexander
沉积物中微生物种群的流式细胞仪（FCM）定量的准确性随FCM设置、细胞提取和染色方案以及样品类型而变化。在本研究中，我们提高了准确性的FCM计数微生物栖息在不同的湖泊和海洋沉积物类型的基础上，广泛的测试与FCM设置，提取缓冲液的化学成分，细胞分离方法，染色程序。对FCM设置的测试（例如，采集时间、事件速率）和提取溶液的盐度分别对FCM计数和细胞提取的产率显示出较小的影响。现有的方法，包括氢氟酸（HF）处理释放沉积物附着的细胞到溶液中证明是有效的海洋和淡水样品。然而，不同的染色技术（细胞提取物的直接染色，膜过滤细胞提取物的染色）在细胞数量估计中产生明显的差异。我们证明，虽然劳动密集型膜染色产生高细胞染色效率和准确的细胞计数，是一致的FCM和基于荧光显微镜（EFM）的定量方法，准确的细胞计数确定更省时省力的直接染色需要考虑染料浓度，样品稀释度和岩性。然而，两种染色方法之间的良好一致性可以通过在直接染色期间对染料浓度和样品稀释度进行样品特异性调整来实现。因此，我们提出了一个完整的方案，FCM为基础的细胞定量，包括从最初的样品固定到最终计数的所有步骤，与缓冲液组合物的建议，直接和基于膜的染色程序，和最终的FCM测定。该协议是通用的，准确的，可靠的，是显而易见的，从良好的协议与细胞定量EFM和定量聚合酶链反应（qPCR）的1 6S rRNA基因在广泛的沉积样品类型。
The accuracy of flow cytometric (FCM) quantifications of microbial populations in sediments varies with FCM settings, cell extraction and staining protocols, as well as sample types. In the present study, we improve the accuracy of FCM for enumerating microorganisms inhabiting diverse lake and marine sediment types based on extensive tests with FCM settings, extraction buffer chemical compositions, cell separation methods, and staining procedures. Tests on the FCM settings, (e.g., acquisition time, rates of events) and salinity of extraction solutions show minor impacts on FCM enumerations and yields of cell extraction, respectively. Existing methods involving hydrofluoric acid (HF) treatment to release sediment-attached cells into solution prove effective on both marine and freshwater samples. Yet, different staining techniques (direct staining of cell extracts, staining of membrane-filtered cell extracts) produce clear differences in cell number estimates. We demonstrate that, while labor-intensive membrane-staining generates high cell staining efficiency and accurate cell counts that are consistent across FCM and epifluorescence microscopy-based (EFM) quantification methods, accurate cell counts determined by more time- and labor-efficient direct staining require consideration of dye concentration, sample dilution, and lithology. Yet, good agreement between the two staining methods can be achieved through samplespecific adjustments of dye concentrations and sample dilutions during direct staining. We thus present a complete protocol for FCM-based cell quantification, that includes all steps from the initial sample fixation to the final enumeration, with recommendations for buffer compositions, direct and membrane-based staining procedures, and the final FCM assay. This protocol is versatile, accurate, and reliable, as is evident from good agreement with cell quantifications by EFM and quantitative polymerase chain reaction (qPCR) of 1 6S rRNA genes across a wide range of sedimentary sample types.