- 著者
- Yuki Morono Kengo Kubota Daisuke Tsukagoshi Takeshi Terada
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.35, no.3, pp.ME20062, 2020 (Released:2020-06-27)
- 参考文献数
- 20
- 被引用文献数
- 5
Fluorescence in situ hybridization (FISH) is a widely used molecular technique in microbial ecology. However, the non-specific adsorption of fluorescent probes and resulting high intensity of background signals from mineral particles hampers the specific detection of microbial cells in grain-rich environmental samples, such as subseafloor sediments. We herein demonstrated that a new buffer composition containing EDTA efficiently reduced the adsorption of probes without compromising the properties of the FISH-based probing of microbes. The inclusion of a high concentration of EDTA in the buffer in our protocol provides a simple and effective approach for reducing the background in FISH for environmental samples.
- 著者
- Fumiaki Mori Tomoya Nishimura Taisuke Wakamatsu Takeshi Terada Yuki Morono
- 出版者
- Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles
- 雑誌
- Microbes and Environments (ISSN:13426311)
- 巻号頁・発行日
- vol.36, no.3, pp.ME21031, 2021 (Released:2021-08-24)
- 参考文献数
- 22
- 被引用文献数
- 3
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.