Simple In-liquid Staining of Microbial Cells for Flow Cytometry Quantification of the Microbial Population in Marine Subseafloor Sediments

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.