The Juan de Fuca Ridge is a seafloor spreading center off the coast of North America (Fig. 1). Sites 1026 and 1027 were situated ~100 km east of the crest of the Juan de Fuca Ridge and roughly 80 km east of the nearest area of extensive basement outcrop. Site 1026 was located over a basement ridge with a sediment thickness of ~250 m. Site 1027 was located about 2.2 km to the east in the adjacent valley with a sediment thickness of ~600 m. Near-surface sediment temperatures were ~2°C, and the extrapolated basement temperatures were very similar at both sites, 61.4°C at Site 1026 and 62.8°C at Site 1027. Thermal gradients were 0.242° and 0.103°C m-1 at Sites 1026 and 1027, respectively (Davis, Fisher, Firth, et al., 1997).
A total of 45 1-cm3 sediment samples were taken from the two sites (Table 1). Samples were removed from the ends of 1.5-m core sections as they were cut on the catwalk. A thin layer of sediment was removed from the cut surface with a sterile scalpel to expose an uncontaminated surface and a sterile (autoclaved) 5-mL plastic syringe, from which the luer end had been removed, was used to take a 1-cm3 minicore. The sample was sealed with a sterile Suba Seal before being injected directly into a tared serum vial containing 9 mL of filter-sterilized (0.2 µm) 4% formaldehyde in artificial seawater, crimp sealed, and shaken vigorously to disperse the sediment. These preserved samples were returned to the laboratory for analysis.
Acridine orange staining and microscopic quantification were based on the general recommendations of Fry (1988), as outlined in Cragg and Parkes (1994). Preserved samples were vortex mixed, and a 2.5- to 15-µL subsample was added to 10 mL of 2% filter-sterilized (0.1 µm) formaldehyde in artificial seawater. Acridine orange (50 µL) was added to give a final concentration of 5 mg dm-3. After 3 min, the solution was filtered through a 25-mm, 0.2-µm pore size, Nucleopore black polycarbonate membrane (Costar, High Wycombe, U.K.). The filter was rinsed further with 10 mL of 2% filter-sterilized formaldehyde in artificial seawater and mounted in a small amount of paraffin oil under a coverslip. Three replicate filters were prepared from each sample to minimize the variance of the counts (Kirchman et al., 1982). Where 95% confidence limits of the mean count exceeded 0.5 log10 units, further replicate filters were prepared. A minimum of 200 fields of view were counted.
The mounted membrane filters were viewed under incident illumination with a Zeiss Axioscope microscope fitted with a 50-W mercury vapor lamp, a wide-band interference filter set for blue excitation, a 100× (numerical aperture = 1.3) Plan Neofluar objective lens, and 10× eyepieces. Green and red fluorescing objects, which resembled bacteria, were counted. Cells on or off particles were counted separately, and the numbers of those on particles doubled in the final calculations to account for cells hidden from view by particles (Goulder, 1977). Dividing cells (those with a clear invagination) and divided cells (pairs of cells of identical morphology) were also counted. The detection limit for bacterial cells was calculated to be 2.23 × 105 cells cm-3.