Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM). We initially examined the efficiency of microbial cell staining at various dye/sediment ratios (volume ratio of SYBR-I/sediment [vSYBR/vSed]). Non-cell particles in sediment strongly and preferentially adsorbed SYBR-I dye, resulting in the unsuccessful staining of microbial cells when an insufficient ratio (<1.63 vSYBR/vSed) of SYBR-I dye was present per volume of sediment. SYBR-I dye at an abundance of 10 vSYBR/vSed successfully and stably stained microbial cells in green fluorescence, while the fluorescent color of non-cell particles red-shifted to yellow-orange with the overaccumulation of SYBR-I dye. A low vSYBR/vSed ratio was quickly recognized by a colorless supernatant after centrifugation. At the appropriate vSYBR/vSed ratio, FCM-measured cell concentrations in subseafloor sediments were consistently similar to microscopy counts (>106 cells cm-3). Samples with low cell abundance (<105 cells cm-3) still require cell separation. This modified staining allows us to efficiently process and perform the microbial cell counting of sediment samples to a depth of a few hundred meters below the seafloor with a higher throughput and capability to scale up than procedures employing microscopy-based observations.

中文翻译：

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微生物细胞的简单液体染色，用于流式细胞术定量海洋海底沉积物中的微生物种群。


微生物细胞计数为研究细胞丰度分布和与周围环境的生物地球化学相互作用提供了重要信息。然而，它通常需要劳动密集型且耗时的过程，特别是对于海底沉积物样品，其中含有丰富的非细胞颗粒。我们开发了一种快速、简单的方法，通过 SYBR Green I (SYBR-I) 对微生物细胞内 DNA 进行染色，从而通过流式细胞术 (FCM) 对细胞进行计数。我们最初检查了不同染料/沉积物比率（SYBR-I/沉积物的体积比[vSYBR/vSed]）下微生物细胞染色的效率。沉积物中的非细胞颗粒强烈并优先吸附SYBR-I染料，导致比例不足（<1 id=9>106个细胞cm-3）时微生物细胞染色不成功。细胞丰度较低（<105 个细胞 cm-3）的样品仍然需要细胞分离。这种改进的染色使我们能够有效地处理和执行海底以下几百米深度的沉积物样本的微生物细胞计数，与采用基于显微镜观察的程序相比，具有更高的通量和扩大能力。