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Results and discussion

Prior to Expedition 329, the FCM cell counting technique was tested with several organic-rich continental margin sediments (e.g., Nankai Trough and Aarhus Bay) onshore. Using Protocol FCM-A as the basic method, the successful separation of microbial cells from SYBR-SPAM signals on the same flow cytometer was confirmed; these FCM cell count data were stable and highly consistent with direct microscopic cell counts (data not shown). However, at the beginning of Expedition 329, significant differences between shore-based FCM counts and shipboard results on the JOIDES Resolution were observed. For example, unexpectedly high background signals with the same or similar fluorescence wavelength of SYBR-stained cells were observed in early shipboard experiments using the South Pacific Gyre sediment. Several factors may contribute to this difference in performance:

  1. Cell concentrations in South Pacific Gyre sediment are significantly lower than those that were observed onshore in organic-rich continental margin sediment;

  2. Sedimentological characteristics are very different between smectite-rich young continental margin sediment and geologically old zeolite-rich metalliferous sediment of the South Pacific Gyre; and

  3. Experimental conditions such as cleanliness, reagent qualities, and machine stability may differ significantly between shore-based and shipboard laboratories.

Nevertheless, Expedition 329 provided an excellent opportunity to establish a more robust fieldwork protocol for FCM cell counting, using various types of cored sediment from the extensive area of the South Pacific Gyre. Here a site-by-site effort to develop the best protocol for FCM cell counting with low background and high sensitivity (i.e., low detection limit) is reported.

Flow cytometry quality control

To verify absolute mechanical background signals of FCM under onboard laboratory conditions, null-control (water) during protocol development and sample measurements were run repeatedly. A “hat-trick cleaning step” for FCM was conducted according to the manufactures’ recommendation when relatively unstable and/or high background signals were observed during the experiment. Overall, these null-control experiments showed negligible numbers (10 signals per milliliter of water) within the range of fluorescent wavelength of SYBR-stained cells. Surface seawater was used as a positive control sample for FCM cell counting; these surface water counts consistently matched direct microscopic cell count (~105 cells/mL; data not shown). Based on these seawater sample tests and the standard bead counts on FCM, the effect of ship movement (i.e., pitch and roll) was negligible for onboard FCM cell counting during Expedition 329.

Site U1365

At Site U1365, FCM cell counting Protocol FCM-A was first tested for sediment samples from just below the seafloor to just above the sediment/basement contact. Unexpectedly high background signals were observed, which resulted in a minimum detection limit (MDL) of ~104 cells/cm3. Using Protocol FCM-A, estimates of cell concentrations in sediment <10 meters below seafloor (mbsf) were in good agreement with direct counts using epifluorescence microscopy (Fig. F1; see also Fig. F61 in the “Site U1365” chapter [Expedition 329 Scientists, 2011b]). However, at greater depths no reliable data could be obtained using Protocol FCM-A because all FCM counts were below MDL. These results indicate that (1) cell abundance in organic-poor sediment of the South Pacific Gyre is overall significantly lower than cell abundances in previously studied organic-rich continental margin sediments (D’Hondt et al., 2009), and (2) Protocol FCM-A needs to be improved by lowering the MDL in order to assess more precisely the microbial biomass in this extreme subseafloor habitat.

The reason for high background signals was unclear at this point. One possible factor is interference by CaF2 precipitates that remained in the final suspension after density centrifugation. The fluorescent particles were examined with epifluorescence microscopy, but neither visible particles nor SYBR-stained cells were observed, suggesting that the fluorescence interference might instead be caused by unknown inorganic precipitates that could only be detected with the relatively wider signal detection capacity of FCM with a very short excitation time. To solve this problem, it was necessary to improve Protocol FCM-A by addressing possible causes of the interference one-by-one during Expedition 329.

Site U1366

Because FCM measurements from Site U1365 had high background signals caused by potential interference of CaF2 precipitates, Protocol FCM-A was modified by eliminating the addition of 0.5 M each of calcium chloride and sodium acetate and replacing them with 1.5 M Tris-buffer (see Protocol FCM-B). However, the results from Site U1366 contained remarkably high background signals that interfered with cell-derived SYBR fluorescence. Consequently, no or very little improvement of the results was achieved with Protocol FCM-B. These high background signals cannot result from CaF2 in Protocol FCM-B. Instead, they may be due to fine chemical particles (e.g., silicates and mineral salts) that are sensitive to pH change during the Tris-buffer neutralization step.

Given the high background signals, FCM counts were ~1.5–2 orders of magnitude higher than microscopic direct counts. Because these abiotic background signals concealed cell-derived SYBR-fluorescent signals, no reliable onboard cell counts using FCM for Site U1366 sediment samples were obtained. Clean-up steps on the flow cytometer were repeatedly performed according to the manufacturer’s instruction, and the null controls (i.e., 18.2 MΩ water and TE buffer) showed negligible signals of SYBR-specific fluorescence wavelength.

Site U1367

At Site U1367, the sample preparation protocol was refined for FCM-based cell counts. Given the results from previous sites, cell-extract solutions without HF treatment steps were prepared (see “Protocol FCM-C (protocol without HF treatment)”). This acid-wash treatment is standardized for better recovery of cells from smectite-rich sediment on continental margins and significantly reduces fluorescent backgrounds caused by amorphous silica when using an epifluorescent microscope (Morono et al., 2009). For FCM counts in South Pacific Gyre sediment, the protocol without HF treatment was found to reduce background signals, and consequently the MDL was lowered to 2.2 × 103 cells/cm3. Using Protocol FCM-C for Site U1367 sediment samples, a decreasing trend in FCM counts with depth were observed. However, these counts were still ~1.5–2 orders of magnitude higher than direct microscopic counts, suggesting that FCM-cell count numbers using Protocol FCM-C may still overestimate the “true” cell numbers with faint nonbiological (i.e., mineral) fluorescence signals from cell extracts. Despite the low MDL, SYBR-SPAM interfered significantly with identification of SYBR-stained cells for unknown reasons. Nevertheless, the preliminary FCM count indicated that cell abundance in South Pacific Gyre sediment (Sites U1365 and U1366) is overall significantly lower than any other previously studied subseafloor sediment.

Site U1368

At Site U1368, the cell-staining steps with the SYBR Green I fluorescent were examined. To examine the extent of background fluorescent signals caused by the SYBR Green I dye, a dilution series of SYBR Green I dye solution (1:200, 1:400, 1:1000, 1:10000, and 1:100000) in a phosphate-buffered saline (PBS) buffer, TE buffer, and 2.5% NaCl solution containing 2% formalin was prepared. These solutions were analyzed by FCM without any addition of cells. The analyses indicated that background signals were unexpectedly high in ~1:1000 dilution in PBS and TE buffers. The pattern of background signals using FCM varied in each solution and appeared to overlap with the area of SYBR-stained cell fluorescence (Figs. F2A, F2B, F2C). The formation of precipitates in PBS buffer was especially critical because PBS buffer is commonly used for many fluorescent dye–staining experiments in medical and molecular ecological studies. This has not been observed in previous experiments using SYBR Green I (Invitrogen) on shore. The result clearly indicates that the quality of SYBR Green I dye solution is most likely different between the dye from Lonza Rockland, Inc. (used during Expedition 329), and the dye from Invitrogen (used in previous experiments). Although the background signals in 1:1000-diluted solution with 0.5% NaCl plus 2% formalin were less obvious than those in other buffers (Fig. F2D), the slightly acidic pH (~5.0) weakened the fluorescence of, or even degraded, SYBR Green I. During Expedition 329, only the SYBR Green I fluorescence dye from Lonza Rockland, Inc., was available.

SYBR Green I–derived precipitates were observed on a 0.22 µm filter under epifluorescence microscopy. The precipitates were obviously not cells; instead they appeared as thin squared or dendritic crystals that produce weak green fluorescence. Because FCM is highly sensitive to even weak fluorescence in extremely short exposure time, these chemical precipitates are automatically detected by FCM as “fluorescent particles,” like SYBR-stained cells. However, these backgrounds are clearly distinguishable from cell-derived SYBR fluorescence by morphology and fluorescent intensity; hence, the effect of these background signals on FCM are not critical for microscopic direct counts.

Given these results, the nonbiological SYBR precipitates were washed three times after staining with TE buffer (i.e., over 1:100000 dilution) to remove the chemical precipitates as completely as possible. Using modified Protocol FCM-D, samples from Site U1368 were processed for FCM cell counting. The results are in relatively good agreement with the trend of microscopic direct counts (Fig. F1). However, FCM background signals were still inconstant, hampering statistically reliable data production under these experimental conditions.

Site U1369

At Site U1369, the background signals produced by SYBR Green I were examined using surface seawater. It was found that SYBR Green I produced by Lonza Rockland, Inc., was highly sensitive to solvent salinity. Visible precipitants of SYBR Green I in both PBS and 3% NaCl were detected in the solutions. In addition, the color and DNA-staining capacity of SYBR Green I disappeared in TE buffer (1:10000) when the solution was stored at 4°C for a few days. This indicates that even though no visible precipitates in TE were observed, SYBR Green I might form crystal precipitates in the solution and interfere with analysis on FCM.

Another finding is that it is extremely difficult to eliminate the SYBR-derived precipitates from the suspension. Although attempts to remove these precipitates by quadruplicate washing with TE buffer were performed, only a slight decrease in the background signals was observed (data not shown). Even worse, often a significant decrease in cell-derived signals (e.g., SYBR-stained Escheria coli or seawater cells) during the washing step was observed, likely because of cell destruction caused by osmotic and centrifugation stresses.

Subsequently, Protocol FCM-D with no modification for samples from Site U1369 and kill-control samples from Sites U1365–U1369 was applied. Although reasonable FCM-based cell counts in near-surface sediment (~2 mbsf) were obtained, because of the relatively high number of cells (>104 cells/cm3), background signals were still high enough to conceal the low abundance of cells in deeper South Pacific Gyre subseafloor sediment.

Sites U1370 and U1371

Given the data described above, a new SYBR Green I solution for further methodological improvements of FCM-based cell counting was required, which is impossible to achieve in the middle of the South Pacific Gyre, the place furthest away from continents (and, consequently, the place furthest away from replacement supplies). Because of this, the onboard FCM experiment was terminated. Further analysis will be carried out at a shore-based laboratory.