The primary microbiology objective for Leg 206 was to determine the types and abundance of microbes in igneous rocks formed in fast-spreading oceanic crust. To do this, water and rock samples were collected to prospect for evidence of extant and fossil microbial activity, to extract and characterize deoxyribonucleic acid (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 ODP Technical Note, 28 (Smith et al., 2000).
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 sterile aluminum foil, taken immediately to the anaerobic chamber (Coy) in the microbiology laboratory, and put on blue ice to maintain core temperature at or below in situ temperature. Samples were in anaerobic conditions within ~30 min of the core arriving on deck, and work was performed quickly to avoid warming.
In the anaerobic chamber, samples were rinsed of unwanted debris with a nitrogen-flushed anaerobic marine salts solution (23.5 g NaCl + 10.8 mg MgCl2 · 6H2O per liter). A hydraulic rock trimmer (Ward) was used to split off the outside of the cores. Using sterile techniques, interior pieces of core were crushed by wrapping the rock in sterile aluminum foil and breaking it into several pieces with a sterile chisel and hammer or in a sterile percussion mortar (Rock Labs). Additional interior pieces of core were used as described below, and any unused portions were returned to the core.
A nitrogen atmosphere with 5% CO2 and >2% H2 was maintained in the anaerobic chamber, and several hours before each use the chamber was flushed with this gas mixture to ensure anaerobic handling of the core. Hydrogen is present to combine with residual oxygen in a reaction catalyzed by palladium pellets maintained within the chamber. As an additional precaution to minimize oxygen contamination, tools and glassware to be used for manipulation and storage of samples for strict anaerobic work were stored within the chamber.
Samples were collected for shore-based 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. Additional samples for thin sections were collected during regular shipboard sampling parties, particularly in intervals of interest to others. The samples for SEM analysis were placed in 10- or 50-mL sterile Falcon tubes, depending on sample size. They were then fixed in 2% glutaraldehyde for 4-24 hr before being washed twice and stored in 0.25% glutaraldehyde at 4°C. Samples for shore-based chemical analysis were placed in sterile sample bags, immediately frozen, and stored at -80°C.
Whole-rock pieces and crushed rock split from the interior of cores were preserved for shore-based DNA extraction and in situ hybridization. For DNA extraction, samples were immediately stored in sterile 2-mL Eppendorf tubes and frozen at -80°C for future genetic analyses. Samples for in situ hybridization were fixed in 4% paraformaldehyde in sterile Falcon tubes for 4-24 hr. The samples were then washed twice and stored in a carefully blended 1:1 mixture of phosphate buffered saline/100% ethanol at -20°C.
To enrich microorganisms, culturing media, which were prepared prior to the cruise, were inoculated with 0.25 mL of a slurry from a subsample set aside for culturing work. The slurry was prepared by first crushing the rock fragment into very small pieces (<2 mm) with a hammer or with a percussion mortar. Next, the finely crushed pieces were placed into sterile beakers with anoxic minimal salts solution used for initial rinsing of the core. The beakers were shaken and vortexed to remove microorganisms attached to the rock fragments. Sterile syringes flushed with nitrogen were used to transfer the slurry into capped 5-mL serum vials or into gradient tubes. A sterile needle was used each time to inoculate the various media. Innoculated media and gradient tubes were stored at 4°C
Two types of tracer tests were conducted to check for the potential intrusion of drill water and confirm the suitability of the core material for microbiological research: perfluorocarbon tracer (PFT) and 0.5-µm fluorescent microspheres. The presence or absence of these two tracers also acts as a quality assurance check on core handling methods. Detailed methods for these tests are described in ODP Technical Note, 28 (Smith et al., 2000). Samples of the drilling fluids were also collected upon core retrieval to determine the "background contamination."
PFT was continuously fed to the drill water at a concentration close to the limit of solubility (1 µg/g) and well above the detection limit for gas chromatographic analysis (1 pg/g). Samples for PFT analysis were taken from three cores in Hole 1256C as a test for drill water intrusion and possible contamination. To verify delivery of the PFT, a sterile cotton ball was used to wipe the interior of the liner and then placed in a headspace vial and sealed with a Teflon septum. After processing in the anaerobic chamber, a small rock fragment was taken from the interior of the core (to monitor tracer intrusion) and stored in a similar fashion as the cotton ball. Air samples were occasionally taken in sealed headspace vials on the catwalk and in the core laboratory to monitor the background (blank) levels of PFT. Samples were analyzed by gas chromatography.
Latex fluorescent microspheres (Polysciences, Inc., Warrington, Pennsylvania; 0.5-µm diameter; YG) were used as a particulate tracer complementary to the volatile PFT. Two milliliters of microsphere stock (2.69% solids) was diluted with 40 mL distilled water, sonicated for 2 min, and heat-sealed into a 4-oz Whirl-Pak bag. The bag was then attached with thread to the core catcher, a slight modification from procedures as described in Smith et al. (2000). The bag was positioned to rupture upon impact of the core barrel with the core, where the microspheres mixed with the drill fluid and coated the outside of the core as it was pushed into the liner.
To monitor the successful deployment of the microspheres, samples of the core exterior were taken by washing the core sample with sterile nitrogen-flushed marine salts solution and collecting the fluid in Falcon tubes. Interior core samples were crushed with a steel percussion mortar, mixed with the marine salts solution, and collected in Falcon tubes. The fluid samples were vortexed and allowed ~5 min each for settling of the larger particulates. A 5-mL aliquot was taken from each sample and filtered onto black polycarbonate filters (0.2-µm pore size; Millipore), and the filter was mounted on a clean slide with nonfluorescent immersion oil for microscopic examination.
The usable filter area was ~1.98 x 108 µm2 as determined by the inner diameter of the filtration tower (15.64 mm). Microspheres in slide preparations were counted using a Zeiss Axioplan fluorescence microscope equipped with the Zeiss number 9 filter set (BP 450-490; LP 520), and the number of spheres observed was used to quantify contamination in spheres per milliliter of sample. The 100x objective was used for detecting microspheres, where the area for one entire field of view was ~3.14 x 104 µm2 (diameter for one field of view = 200 µm). A total of 20 fields of view were analyzed for each sample. Comparison of microsphere numbers between paired samples from inner and outer core layers provides a relative measure of fluid intrusion and contamination. A sample with many spheres in outer layers and few or none within may be considered "higher quality" than one with very few spheres in the outer layers and few or none within.