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Figure F2. Positive and negative control experiments on flow cytometry using SYBR Green I fluorescent dye. A. Surface seawater sample collected from Site U1368. Microbial cells in the seawater sample were stained with SYBR Green I. The high cell density (~105 cells/mL) does not show significant interference from background fluorescent signals. B, C. Cell-free SYBR Green I solution diluted with (B) Tris-EDTA (TE) buffer (1:400) and (C) phosphate-buffered saline (PBS) buffer (1:400). Both negative control samples showed unstable fluorescent background signals derived from unknown chemical reaction of SYBR Green I. These background signals interfere with accurate cell enumeration, especially for low-biomass samples, such as South Pacific Gyre subseafloor sediment. D. Image of SYBR Green I–derived precipitates in TE buffer, PBS buffer, and 2.5% NaCl + 2% formalin solution. Even at 1:1000 dilution, red to orange precipitates are visually observed. These precipitates are barely removable by washing steps and hence significantly hamper detection of cell-derived fluorescence using flow cytometry.

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