The primary microbiology objective for Leg 185 was to determine the types and abundance of microbes in unconsolidated sediments, sedimentary rocks, and igneous rocks of the oceanic crust. To do this, water, sediment, and rock samples were collected to determine cell abundance and total biomass, to extract and characterize DNA contained within the samples, and to establish cultures of microorganisms inhabiting these environments. Interpretation of results is complicated by the possibility of contamination of samples with microbes from the seawater, the drilling fluid, the ship and drilling equipment, and from postcollection processing of samples. A sample handling protocol was established to minimize postcollection contamination and tracer tests were conducted to determine the extent of microbial contamination during drilling and sample preparation. Methods for tracer tests are detailed in "Materials and Methods" in "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" (Smith et al., 2000).
Surface-water samples were collected at each site using a sterile flask lowered from the bow of the ship to avoid encountering waste and cooling water from the ship. In addition, a sample of surface water was collected at Site 801 in a flask lowered from the small launch at ~1 km upwind of the ship. Drill water was collected in a bucket from the drill string when it was opened to retrieve cores.
Water samples from near the bottom of Hole 801C were collected using the WSTP tool (Barnes, 1988). This water had not been disturbed since the hole was logged in June 1992 during Leg 144. The microbial content of the WSTP water sample was compared to that of surface water, sediments, and basement rocks. The chemistry of the WSTP sample was used to determine the origin of the water sampled.
Before deployment, the WSTP tool was flushed with a chlorine dioxide solution (FreeBact, Xenex Europe AB, Stockholm, Sweden; 1 ppm final concentration) and subsequently flushed with filtered (0.2-µm pore size) distilled water. The total abundance of microbial cells in the water used to flush the tool before and after it passed through the WSTP was determined by epifluorescence microscopy. The coil of the WSTP contained filtered distilled water at the time of deployment. Before it was sealed, the WSTP reservoir was flushed with nitrogen gas to reduce the concentration of oxygen. The Hole 801C water sample was taken with the WSTP sampler extended ~50 cm ahead of the bottom of the drill string. Special care was taken not to flush the hole while lowering the drill string. Water from the overflow reservoir was extracted with syringes from the pressure relief valve at the top of the reservoir. Samples from the coil reservoir were extracted in the anaerobic glove box after the coil was removed from the sampler. Temperature measurements were also obtained during the period the WSTP tools were in the borehole.
WSTP, drill water, and seawater samples were fixed in 0.2-µm-filtered formalin (2% final concentration) and stained with DNA fluorochromes 4´,6-diamidino-2-phenylindole (DAPI; 0.1 mg/mL final concentration) or Acridine orange (AO; 0.1 mg/mL final concentration) for 10 min. Stained cells were filtered onto black polycarbonate filters (Poretics or Nuclepore; 0.2-µm pore size) and examined with a Zeiss Axiophot microscope at 1000× final magnification (100× Plan-NEOFLUAR objective) using epifluorescence illumination (100-W Hg bulb) with UV and blue filter sets for DAPI and AO, respectively. Cells were enumerated and normalized to the volume of water filtered.
WSTP, drill water, and seawater samples were inoculated into 12 types of anaerobic liquid culture media. The media were based on the composition of seawater, reduced with sulfide and cysteine, and buffered to pH 8.0. Media also contained vitamins and trace elements. Electron acceptors were Mn(IV), Fe(III), sulfate, and bicarbonate. Electron donors were organic carbon (acetate, lactate, and pyruvate), hydrogen, and methane. Eleven different combinations of electron donors and acceptors were used to enrich manganese reducers, iron reducers, sulfate reducers, acetogens, and methanogens. Fermenters were enriched in the same medium base with the organic nutrients of R2A medium (Reasoner and Geldreich, 1985). One bottle of each medium type was inoculated with 0.25 mL of water from the WSTP coil, or 1 mL of drill water or seawater. Cultures were incubated at 25°C for the WSTP sample and 30°C for the drill-water and seawater samples and monitored for growth. Uninoculated negative controls were incubated at the same temperature. Cultures were shipped to Göteborg University, where growth will be detected by analysis of metabolic products and staining of microbial cells with DAPI and AO.
WSTP and seawater samples
were stored in polycarbonate tubes capped with tight-fitting polyethylene lids
and sealed with at least two layers of stretched Parafilm. Vials were placed
into stainless steel high-pressure vessels with an ID of 2 cm and a tube length
of 10 cm (Fig. F11).
Vials were immersed in water (distilled for the WSTP sample and NaCl solution
(3.1%) for the seawater sample), eliminating air space inside the high-pressure
vessel. High-pressure vessels were capped with a stainless steel plug that was
screwed into the vessel, sealing it while ejecting air and excess water. The
high-pressure seal was accomplished with a Vyton plug-to-wall gasket. Pressure
vessels were connected to 
-in
stainless steel tubing and sealed with a pin valve. Pressurization was
accomplished with a piston-stroke pump, using distilled water as a pressure
medium. Pressures were restored to ~100-200 psi above the in situ conditions.
High-pressure samples were transported to Scripps Institution of Oceanography
for shore-based studies.
Aliquots of water extracted from the WSTP coil and reservoir, as well as the drill-water and seawater samples, were immediately frozen in liquid nitrogen and stored at -70°C. Drill-water and seawater samples were also filtered through capsule filters (Sterivex-GS 0.22 µm; Millipore), and the filters were immediately frozen in liquid nitrogen and stored at -70°C. Frozen samples were shipped to the Graduate School of Oceanography, University of Rhode Island, for DNA extraction and characterization.
Methods for chemical analyses of the WSTP samples are described in "Interstitial Water Chemistry and Headspace Gas".
Whole rounds cut from the cores were first sampled for microbiology and then for interstitial water analyses. The cores were cut and capped on the catwalk and immediately transported to the microbiology lab. Subsamples of the whole rounds were taken using cutoff sterile 1-mL syringes for the analyses listed below. Immediately after microbiological sampling, whole-round cores were taken to the chemistry lab where interstitial water was extracted. When the sediments were too hard to be sampled with a syringe, samples from the interior of the core were taken using sterile spatulas and spoons.
Plugs (0.5 cm3) taken from the interior of the whole rounds were fixed in formalin (2% final concentration) and stored at 5°C. Duplicate samples were stained with AO or DAPI, filtered onto polycarbonate filters, and examined with epifluorescence microscopy. Additionally, separate samples were preserved in formalin to be analyzed by shore-based participants.
Adenosine triphosphate (ATP) was quantified in subsamples from the whole rounds to estimate total biomass. The assays were performed in a luminometer (Turner Designs 20/20) using the luciferin-luciferase analysis method. Sediment slurries were made by diluting 0.5 cm3 of sediment with 1.0 cm3 of autoclaved distilled water. A subsample (50 µL) of the slurry was placed in a 12 mm × 50 mm polypropylene vial. An ATP releasing agent (50 µL; Turner Designs) was added to the vial and mixed, after which 50 µL of HEPES buffer was added to the vial and mixed again. Luciferin-luciferase solution (100 µL) was then added to the vial and mixed, and light production was immediately quantified in the luminometer. Blanks were determined using 50 µL of the sterile, distilled water, and standards were analyzed using purified ATP (Turner Designs).
Subsamples of the plugs taken from the interior of the whole rounds were distributed into anaerobic growth media designed to select for sulfate reducers and methanogens or fermentative microbes. The samples were serially diluted in the growth medium to estimate total abundance using the most probable number method. The tubes were incubated at 10°C.
Selected samples were returned to their ambient pressure using the high-pressure vessels described above. Sediment samples were placed in polycarbonate vials and sealed using Parafilm. The space inside the pressure vessels was filled with anaerobic 0.2-µm filtered seawater. Pressure vessels were stored at in situ temperature and transported at high pressure to Scripps Institution of Oceanography for further study.
Plugs (~1-4 cm3) taken from the interior of the core were placed in prebaked (450°C for 2 hr) glass vials, immediately frozen in liquid nitrogen, and then stored at -70°C. These frozen samples were transported to the University of Rhode Island for DNA extraction and characterization.
Whole-round cores were collected on the catwalk through the ends of unsplit core liners or in the core-splitting room immediately after the core liner was split. Cores were handled with latex gloves, placed in nitrogen-flushed plastic bags (Ziploc), and taken immediately to the anaerobic chamber (Coy) in the microbiology laboratory. Samples were in anaerobic conditions within 40 min of the core arriving on deck.
In the anaerobic chamber, a hydraulic rock trimmer (Ward) was used to split off the outside of the cores. Inside core pieces were handled using sterile techniques. Interior pieces of core, kept moist with 1 mL of culture medium without growth substrates, were crushed in a sterile percussion mortar (Rock Labs). Additional interior pieces of core were used whole as described below.
Crushed rock (1-2 cm3) was preserved in phosphate-buffered saline (PBS; NaH2PO4, 2 mM; Na2HPO4, 8 mM; NaCl, 130 mM; pH 7.2) containing 2% formalin. Cells were stained with AO or DAPI, filtered onto polycarbonate filters, and examined with epifluorescence microscopy. Unstained samples were examined for the presence of fluorescent microspheres, as described in "Quantification of Microspheres" in "Materials and Methods" in "Methods for Quantifying Potential Microbial Contamination During Deep Ocean Coring" (Smith et al., 2000).
The 12 types of anaerobic culture media described above were used to enrich viable microbial populations from the rock samples. Approximately 50 mg of crushed rock was added to each of four bottles of each type of culture media. Formalin (2% final concentration) was added to one of the four bottles of each medium type to inhibit all biological reactions in that bottle. These controls allow differentiation of abiotic and biotic reactions in the growth media. For Site 801, the sample in situ temperatures were calculated according to Larson et al. (1993). Samples were incubated at 25° or 30°C, whichever was nearest to the in situ temperature. For Site 1149, the temperature gradient was estimated using Adara temperature tool (Adara) measurements, and cultures were incubated within 5°C of in situ temperature. Uninoculated negative controls were incubated at each temperature to ensure sterility of growth media. Some culture media were inoculated with crushed rock and incubated at ~9000 psi and 25°C in the high-pressure vessels described above. Growth is confirmed by the accumulation of metabolic products and of microbial cells; these analyses will be performed at Göteborg University.
Rock samples were crushed in the anaerobic chamber and sample splits and individual rock specimens were maintained under high pressure. The samples were placed in polycarbonate tubes and overlaid with filtered (0.2 µm) 3.1% NaCl. The same solution was used to fill the high-pressure vessels, which were pressurized to ~100-200 psi above the in situ conditions. Pressurization was typically achieved within ~1 hr of arrival of the cores at the rig floor. The decompression time from departure from ambient pressures at 6500 meters below sea level to atmospheric pressure at the rig floor is ~30-45 min. Pressurized samples were transported to Scripps Institution of Oceanography for shore-based studies.
Whole-rock pieces split from the interior of cores were preserved for shore-based DNA extraction and in situ hybridization. For DNA extraction, rocks were covered with Cell Resuspension Buffer (20 mM Tris, 20 mM EDTA, 0.35 M sucrose), immediately frozen in liquid nitrogen, and stored in a -70°C freezer. Samples for in situ hybridization were fixed in 4% paraformaldehyde for 5-18 hr at 4°C. The rocks were washed twice with PBS and covered with a 50:50 mixture of PBS and 98% ethanol. Tubes were frozen in liquid nitrogen and stored at -70°C. Samples were transported frozen to Göteborg University for shore-based analysis.
Samples were collected from areas of volcanic ash, glass, crystalline basalt, hydrothermally deposited minerals, fractures, and veins for examination by scanning electron microscopy (SEM) and in thin section for evidence of extant and fossil microbial activity. Some of the samples were subsamples of those collected for other microbial studies. Thin sections were prepared on board ship, and chips were preserved in alcohol. Additional crushed rock was preserved in 0.2-µm sterile filtered 3.1% NaCl containing 2% formalin for future shore-based SEM examination.
Two types of tracer tests were conducted to determine the level of contamination of samples recovered by drilling: perfluorocarbon tracer and 0.5-µm fluorescent microspheres. Methods for these tests are described in "Materials and Methods" in "Methods for Quantifying Potential Microbial Contamination During Deep Ocean Coring" (Smith et al., 2000).
