Microbiological sampling of Site 1253 covered the carbonate sediment intervals (370.0-400.5 and ~430-442.0 mbsf) and the more altered lower igneous section, Subunit 4B (544.4-598.7 mbsf). For sediments, 5-cm-long whole rounds were removed from Cores 205-1253A-2R, 3R, 4R, 10R, and 11R adjacent to IW whole rounds and processed in the microbiology laboratory. Samples from the lower Subunit 4B in the vicinity of filled veins or fractures were obtained from Cores 205-1253A-32R through 34R, 36R through 39R, and 41R through 43R. One piece of igneous rock was taken from each core and transferred to the anaerobic chamber in the microbiology laboratory to break off a ~40-cm3 whole round, using sterile techniques at all times. Digital photographs were taken prior to and after the initial breaking of the core (Figs. F76, F77). The unused portion was returned to the core liner, and the sample was subdivided for multiple analyses (see "Microbiology" in the "Explanatory Notes" chapter).
The perfluorocarbon tracer (PFT) was run continuously during coring, with a target concentration of 1 ppm in the drilling fluid, as developed by Smith et al. (2000). The Hewlett-Packard 8059 gas chromatograph designated for PFT quantification was calibrated for the range of 4.4 x 10-15 to 4.4 x 10-7 g PFT. The 10-9 dilution calibration standard sets the detection limit for PFT at 0.045 無. Catwalk sampling for PFT levels allowed rapid encapsulation of sediment plugs, and PFT measurement proceeded as detailed in "Microbiology" in the "Explanatory Notes" chapter. Analyses showed arrival of PFT along the length of the cores, with variable amounts of intrusion, as anticipated with RCB coring methods. Fluid intrusion was not consistent down the cored section. Drilling fluid intrusion of 0.05-0.62 無 PFT/g sediment was observed at the innermost part of the core for more lithified intervals (Cores 205-1253A-2R through 4R), whereas less indurated samples, Cores 10R and 11R, showed intrusion at the innermost core of 2.6 and 105 無 PFT/g of sediment, respectively (Table T12).
The PFT contamination results for the sediment sections highlight three major issues necessary for microbiological investigations of such deep-sea sediments. First, cell counting of background drilling fluid samples is necessary to constrain the impact, in terms of estimated cell numbers, because of fluid intrusion in the range of 0.05 to 105 無 PFT/g of sediment calculated from PFT data. Second, for tracer sampling in all future assays, intact whole-round samples must be used and sampled in the interior. Third, some contamination penetrates even the interior samples with RCB coring of nonindurated sediments.
PFT delivery was confirmed in all of the igneous Subunit 4B cores. Swabs of the inner core liner during catwalk sampling showed PFT concentrations significantly above the detection limit of 0.045 無. The swabs are used to determine the concentration of PFT on the outside of the gabbro core.
Given these detection limits, the results of PFT samples taken from the inside of the gabbro core indicate a fair amount of drilling fluid intrusion, ranging from 0.61 to 510.37 無 PFT/g of rock (Table T13). These calculations are corrected for background PFT contamination occurring on the catwalk where initial cutting of the core liner and sampling took place, as well as the background in the anaerobic chamber where the core was cut and subsampled. In most cases, the background on the catwalk was either below or slightly above the detection limit of 0.045 無. The background in the anaerobic chamber ranged from low to fairly high PFT concentrations and did not increase in any systematic way. In Core 205-1253A-38R, the background PFT concentration in the chamber and on the catwalk was higher than the inner core concentration, indicating significant volatilization of the PFT during sample distribution.
The PFT results indicate a fair amount of contamination in all samples. However, there are several limitations in using these results quantitatively for the igneous samples. The time between cutting the core liner for sampling, crushing the sample, and distributing the subsamples (including the sample for PFT analysis) was ~1 hr. The PFT volatilizes at room temperature, so it is difficult to determine if the tracer penetrated the sample with the drilling fluid or during the 1-hr sampling procedure. Also the exteriors of the samples were rinsed with a sterile salts solution (see "Microbiology" in the "Explanatory Notes" chapter) before crushing, but they were not flamed or sterilized to more effectively rid the sample of excess tracer. In addition, the outer portion of the core was not removed, as the gabbro was fresh and difficult to break. Thus, the samples used to measure drilling fluid intrusion into the center of the core may contain a large portion of PFT transferred from the outside of the core during handling. Nevertheless, the samples (including the outer portions of the core) used for measuring the tracer concentration were from the same mixture used for inoculating media and for genetic analyses, therefore, providing a qualitative assessment of drilling contamination. Additionally, sampling of veins and fractures is required to determine the impact that microorganisms have in the weathering of the rock; these veins may act as weak boundaries that easily break and as a conduit for the flow of drilling fluids containing PFT or volatilized PFT in the ambient air. Because of the numerous unknowns involved prior to analyzing the tracer, the calculated contamination in the gabbro will be used in a qualitative sense rather than a quantitative one.
Heat-sealed Whirl-Pak bags of fluorescent microspheres were deployed in every other core catcher during sediment coring and in every core catcher for igneous cores. Adjacent to microbiological sampling sites, toothpick samples of sediments at the core interior and exterior were taken and suspended in 0.5 mL of synthetic seawater solution. The small sample volume and prolonged exposure of the microspheres to natural and artificial light precluded rigorous microsphere counting at this site. To minimize this problem at future sites and to preserve microsphere fluorescence, particulate tracer Whirl-Paks will be kept refrigerated for as long as possible prior to deployment and core samples for microsphere counts will be kept refrigerated at all times except during microscope observation. The original protocol for microsphere sampling (Smith et al., 2000) was modified during recent legs to increase the sensitivity of counting assays after analyses at the site. The updated method will be employed at future sites. Sediment plugs of 3 cm3 will be placed in 25 mL of saturated salt solution in a 50-mL Falcon centrifuge tube, mixed, and centrifuged to settle the large grain sizes only. The supernatant will be filtered onto a black polycarbonate filter, and the microspheres will be counted.
Fluorescent beads were successfully deployed in all igneous cores except for Core 205-1253A-33R. A sterile minimal salts solution used for rinsing the gabbro core was collected and used as a proxy for outer core contamination. The crushed sample, primarily the interior of the core with small amounts of the outer core, was also mixed with the minimal salts solution to make a slurry for microsphere counts, representing intrusion of the drilling fluids into the core. For bead counting, the filters were viewed under a 100x objective with the area for one field of view = ~3.14 x 104 痠2. Beads were detected on the outside of all of the cores where samples were taken except for Core 205-1253A-43R (Table T14). For the interior of most cores, significantly fewer microspheres were observed or there was a complete absence of microspheres.
Because the gabbro was so fresh and difficult to break, the outer edge of the core could not be excluded from the samples. Thus, some amount of contamination was expected and the results are to be used qualitatively rather than quantitatively. However, of the techniques used here, microsphere tracers may more accurately indicate contamination by microorganisms from the drilling fluid. This is because PFT is soluble at room temperature, can travel through very small pore spaces (smaller than bacteria), and is found in the laboratory air and on the hands of anyone who has handled a core liner. The presence of beads is a strong indication that contamination by microbe-sized particles from the drilling fluid has occurred. Nevertheless, the absence of microspheres alone cannot confirm that a sample is uncontaminated.
Five sediment cores (Cores 205-1253A-2R, 3R, 4R, 10R, and 11R) were sampled for microbial community assessment. For each core, a ~5-cm3 whole round was obtained adjacent to the IW whole round to relate microbiological and interstitial water chemistry results. From the centermost part of the cleaned whole round, two ~5-cm3 sediment plugs were taken with sterile, cut-off syringes, one for postcruise DNA evaluation and one for postcruise ATP quantification. Both sets of plugs were frozen at -80蚓. Formalin-fixed 0.5-cm3 sediment samples from the same sites were refrigerated for postcruise cell counting.
After sampling a ~40-cm3 whole round from the core in an aerobic glove box, the sample was crushed using sterile techniques into small fragments. Some fragments were set aside for postcruise analyses involving fluorescent in situ hybridization, microscopy, or chemical mapping using the microprobe. The rest of the fragments were subsequently crushed with a sterile mortar and pestle. Half of the fine-grained sample was stored for genetic analyses or for PFT analyses. The sterile, anoxic minimal salts solution was added to the other half of the crushed sample and used to inoculate enrichment media or for microsphere tracer analyses.
Cultivations were conducted with slurries prepared from the crushed gabbro and sterile, anoxic marine salts solution (see "Microbiology" in the "Explanatory Notes" chapter). The enrichment media were inoculated with ~0.5 mL of slurry or with the drilling fluids for a control and were incubated at 8蚓. The media used were selective for primarily anaerobic chemolithotrophic and heterotrophic microorganisms such as methanogens, sulfate reducers, sulfur oxidizers, iron(II) oxidizers, and iron(III) reducers. Further subculturing and molecular analyses will be performed postcruise.
DNA was extracted using the freeze-thaw protocol outlined in "Microbiology" in the "Explanatory Notes" chapter. The DNA obtained is a mixture from all of the microorganisms present in the sample, including that from any contamination introduced during the drilling process. Agarose gel electrophoresis of total community DNA, before performing polymerase chain reaction (PCR), did not show any banding in the gels. This result may mean that there was no DNA in the sample or, more likely, that the amount of DNA was so small that it could not be observed.
DNA was amplified with PCR using Taq polymerase and universal 16S ribosomal ribonucleic acid primers. PCR was performed several times using differing concentrations of template DNA and primers. Agarose gel electrophoresis was also used to quantify the PCR products. All PCR results came back negative (no DNA present). One possible explanation for this is an absence of total community DNA. Another explanation is that the primers, polymerase, and/or nucleotides degraded during shipping because a positive control did not work on several occasions. One more possibility is that there is some substance (for example, a high metal concentration) in the sample that is inhibiting the PCR. Initial DNA extractions and samples will be shipped to shore for purification and further genetic analyses.