Microbiological samples were successfully collected from APC Holes 1200D and 1200E. Microbiological whole-round core samples were collected adjacent to pore water geochemical whole-round samples in all cases. A total of 18 samples was collected from Hole 1200D and 21 samples from Hole 1200E, with a higher sampling frequency near the surface. The maximum sampled depth was 28.9 and 55.3 mbsf, respectively, in Holes 1200D and 1200E. All microbiological samples were subjected to a series of tracer tests to determine the presence and extent of possible contamination to the exterior and interior of whole-round cores. For both holes, adjacent in situ pore water chemistry and fluorescent microspheres (Cores 195-1200D-5H through 9H and throughout Hole 1200E) were used to detect contamination of samples. In Hole 1200D, PFT was also used as an additional chemical tracer (Smith et al., 2000a, 2000b). All three methods of detection positively demonstrated the contamination of a single whole-round core sample from Hole 1200D, showing relatively low alkalinity, interior fluorescent beads, and interior elevated levels of PFT, respectively (Sample 195-1200D-5H-1, 20-30 cm). All other microbiological samples showed either no direct contamination in terms of pore water chemistry and/or fluorescent microspheres or no detectable levels of PFT. These results demonstrate the potential for the collection of uncontaminated core samples to be used in microbiological research by drilling of deep ocean sediments.
Subsamples from all microbiological samples were collected and processed using a laminar flow hood. These included (1) the preservation of subsamples for molecular biological analyses by quick freezing with liquid nitrogen and then maintenance at -70°C; (2) toothpick scrapings stored at 4°C in sterile phosphate buffered saline for determination of the abundance and distribution of microorganisms by direct counting fluorescence microscopy, along with morphological characterizations by light microscopy; (3) the preservation of subsamples in 2.5% glutaraldehyde for scanning electron microscopy and, potentially, transmission electron microscopy; (4) the preservation of subsamples for flourescent in situ hybridization probing; (5) the aseptic cryopreservation of subsamples placed in 30% glycerol solution and quick freezing in liquid nitrogen for use with multiple enrichment culture procedures; and (6) the preservation of subsamples in serum vials stored at 4°C under nitrogen-flushed headspace, also for use in enrichment culture under anaerobic conditions, which was done for subsamples from Hole 1200D only.