Cell Density Measurement

Following 6.5 years of storage at -20°C, cell densities in the non-core catcher sediment samples was determined on the same interior sediment samples used for fluorescent microsphere analysis described above, following published cell counting procedures (Kallmeyer et al., 2008). The formaldehyde-fixed sample slurry (1:6 sediment:buffer) was mixed with acid dissolution and detergent solutions to chemically release cells from the sediment matrix, and also gently sonicated to physically release cells from the matrix. Suspended cells were separated from sediment debris by density gradient centrifugation with Nycodenz. The cleaned cell pellet was concentrated on a 0.2-μm-mesh polycarbonate filter and stained with either acridine orange or propidium iodide DNA stains following methods described previously (Orcutt et al., 2011a), then visualized at 100x objective magnification on an Olympus BX60 epifluorescence microscope. Samples from the core catchers (i.e., U1363F-4HCC and U1363F-6HCC) were not preserved appropriately for direct cell counting.