New cell extraction procedure applied to deep subsurface sediments

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A new method is presented for the separation of cells from a marine sediment matrix. Different methods and reagents were tested for detaching microbial cells from sediment particles; the highest yields were achieved in a solution of EDTA, Tween 80, sodium-pyrophosphate, and methanol plus gentle ultrasonic treatment, followed by density centrifugation through a cushion of Nycodenz. If present, carbonates were dissolved before extraction. Comparison with untreated sediments and pure cultures verified that this technique minimizes cell lysis. The new procedure was tested on seafloor sediment from several locations and water depths (<1 to >4000 m) and subseafloor sediment from the Arctic Ocean (IODP Expedition 302). Cell extraction efficiency was relatively high (65% to 100%) and consistent for each sediment type, with significantly (P < 0.01) lower variability of counts of separated cells compared with conventional counts on slurried sediments. Concentrating cells before enumeration allows for a much lower minimum detection limit and lower uncertainty than the conventional approach of simply slurrying sediment. Resolving relatively small differences in the distribution of microorganisms will allow for comparisons to other parameters (porewater chemistry, lithology, etc). Additionally, this method has potential for the use of molecular techniques that were previously difficult owing to coelution of interfering compounds from the sediment matrix.

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Limnology and Oceanography: Methods