Abstract
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 [(v)SYBR/(v)Sed]). 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 (v)SYBR/(v)Sed) of SYBR-I dye was present per volume of sediment. SYBR-I dye at an abundance of 10 (v)SYBR/(v)Sed 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 (v)SYBR/(v)Sed ratio was quickly recognized by a colorless supernatant after centrifugation. At the appropriate (v)SYBR/(v)Sed ratio, FCM-measured cell concentrations in subseafloor sediments were consistently similar to microscopy counts (>10(6) cells cm(-3)). Samples with low cell abundance (<10(5) 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.