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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

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Project description: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 sample-specific 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 16S rRNA genes across a wide range of sedimentary sample types.

Dataset Information

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

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