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

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 [^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.

SUBMITTER: Mori F 

PROVIDER: S-EPMC8446754 | biostudies-literature |

REPOSITORIES: biostudies-literature