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Standardization of flow cytometry cell count protocol for South Pacific Gyre sediment

Subsamples were taken from the innermost part of whole-round core sediment samples by a tip-cut sterilized syringe on the catwalk or in the core refrigerator of the R/V JOIDES Resolution as soon as possible after core recovery (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Two cubic centimeters of sediment was transferred to a sterile-filtered 15 mL centrifuge tube containing 8 mL of 0.2 µm filtered, 2.5% (w/v) NaCl solution with 2% (v/v) formalin as a fixative and thoroughly shaken to form a homogeneous suspension. The slurry sample was tested with the cell detachment and cleaning steps for FCM cell count according to the following experimental protocols.

Protocol FCM-A (basic protocol prior to modifications)

  1. Depending on the expected cell abundance, 50–500 µL of the (1:5) slurry is used for the extraction. When sample volumes are <500 µL, 2.5% NaCl solution is added to adjust the sample volume to 500 µL. Then, 50 µL each of detergent mix (100 mM disodium EDTA dihydrate, 100 mM sodium pyrophosphate decahydrate, 1% [v/v] Tween 80) and methanol is added, followed by vigorous vortex mixing for 30–60 min. In cases of extremely low cell abundances, the volume of slurry and all reagents can be increased proportionately.

  2. The sample is underlain with a cushion of Nycodenz (50%) and centrifuged (10 min at 3000 × g) to separate microbial cells and sediment particles by density.

  3. The supernatant is carefully removed with a syringe and stored in separate vial. The pellet is suspended in 400 µL of 2.5% NaCl solution, followed by addition of 50 µL each of detergent mix and methanol.

  4. The slurry is subjected to an ultrasonic treatment at 20 W for 30 s.

  5. The sample is placed on a vortex mixer for 10–20 min followed by density separation as before (Step 2).

  6. The second supernatant is recovered as described before and pooled with the first one.

  7. To the pooled supernatants, 100 µL of 1% HF is added and the solution incubated for 20 min at room temperature.

  8. To remove HF from the suspension, 120 µL of a 0.5 M each CaCl2/Na acetate solution is added. Precipitation of CaF2 occurs immediately.

  9. To remove CaF2 precipitates, the solution is underlain by a cushion of Nycodenz (50%) and centrifuged (10 min at 3000 × g). The clear supernatant is removed as described before.

  10. The recovered supernatant is diluted (1:5) with 2.5% NaCl solution to decrease density and centrifuged again (10 min at 3000 × g).

  11. Most of the supernatant is discarded and only ~100 µL of the solution at the bottom, containing the cell pellet is kept (in most cases, the pellet is invisible).

  12. SYBR Green I solution (2.5 µL; Lonza Rockland Inc., Maine [USA]), is added and let to sit for 10 min in darkness at room temperature.

  13. Tris-EDTA (TE) buffer (pH 8.0; 400 µL) is added. The cell suspension is ready for FCM cell count analysis.

Protocol FCM-B (protocol without CaF2 precipitation)

For the Steps 1–7, follow Protocol FCM-A.

  1. Tris-base solution (100 µL; 1.5 M) is added to neutralize the solution pH without CaF2 precipitation.

Continue with Step 10 in Protocol FCM-A.

Protocol FCM-C (protocol without HF treatment)

Follow Steps 1–6 in Protocol FCM-A. Skip Steps 7–9 and continue with Steps 10–13.

Protocol FCM-D (final improved protocol with HF treatment)

Follow Steps 1–11 in Protocol FCM-A.

  1. Add 400 µL of 1:40 diluted SYBR Green I in TE buffer and let sit for 10 min at room temperature.

  2. Centrifuge for 20 min at 3000 × g and discard supernatant.

  3. Wash the precipitates with 1 mL TE buffer.

  4. Centrifuge for 20 min at 3000 × g and discard supernatant.

  5. Repeat the washing step (Steps 14 and 15) two more times.

  6. Add 500 µL TE buffer to the solution.

During Expedition 329, a C6 flow cytometer (Accuri Cytometers, Inc.) equipped with a 96-well autosampler for FCM cell count experiments was used. All cell extraction experiments and FCM operations were performed in the Paleontology Preparation Laboratory on the JOIDES Resolution. On the flow cytometer, the stained cells were excited with a 488 nm laser and the forward-scatter, side-scatter, and fluorescent signals at 530 and 610 nm were detected and counted.

Quality assurance and control and flow cytometry cell count data processing

For quality assurance and quality control, all reagents were filtered through 0.2 µm filters. Twice-filtered (0.2 µm) 18.2 MΩ water was used for the blank and FCM-sheath solution. For negative control samples, “cell/DNA-free” kill-control samples were prepared by (1) treating some core sediment with an equal volume of bleach-based commercial cleaning product and (2) treating other sediment by heating at 450°C for 5 h. Both kill-control samples were washed and neutralized twice with TE buffer and subjected to FCM cell counting and direct microscopic cell counting (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). The flow cytometer was calibrated prior to data collection for accurate volume measurement using fluorescent microsphere standards at known concentrations (Accuri Volume Validation Beads, Accuri, QA120). Because ship heave prevented automatic volume calibration procedure during FCM, volume-validation bead counts were used to calibrate the flow volume.