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doi:10.2204/iodp.proc.325.102.2011

Microbiology

During the offshore phase of Expedition 325, microbiology samples were collected at all sites with a suitable soft-sediment lithology. Fluorescent microspheres were used during the coring process to identify possible contamination from drilling fluids, which might adversely affect microbiological studies. A majority of samples were collected for DNA and RNA characterization of microbial populations. Additional samples were collected and preserved for cell enumeration. Further sample collections were made during the onshore phase to determine changes in the microbial community during core storage.

Microbiological sediment sampling from soft sediments

Offshore science party sampling

Cell enumeration—fresh samples

For cell enumeration of fresh samples, 2 g of wet sediment was collected by penetrating a sterile syringe (the forepart of the syringe was cut off) into the fresh sediment core. Collected samples were transferred to a 10 mL plastic vial (prefilled with 2 mL of 4% formaldehyde). The vial was capped and mixed with gentle shaking. The vial was stored at 4°C and transported back to the Bremen Core Repository in the refrigerated container.

Molecular-based community characterization and biomarker analysis

A 5 cm long whole-round sample was sawed from the bottom of a core section, collected (with liner) in Whirl-Pak bags (double bagged), and stored in a –80°C freezer as soon as possible after the core was recovered onto the drill floor. Section lengths were specifically cut so that microbiology samples were not located midsection, which would have required the liner to be split. A list of samples is provided in Table T15. These samples were shipped directly to laboratories in China and the USA on dry ice at the end of the offshore phase of Expedition 325.

Onshore Science Party sampling

Cell enumeration

Core sections directly adjacent to the whole-round samples taken offshore were targeted for cell enumeration. Immediately following core splitting, the sediment was subsectioned using a 5 mL syringe with the end removed. From the syringe, 0.5 g of wet sediment was transferred to a glass vial containing 5 mL of preservation buffer (phosphate buffered saline containing 10% methanol and 10% formalin). Sediment was mixed thoroughly to maximize cell exposure to the preservation buffer. Vials were then frozen at –20°C and shipped on dry ice to the receiving laboratory.

Molecular-based community characterization

Sediment remaining from the cell enumeration sampling sections was collected for molecular-based community characterization. These approximate half-round sections were located adjacent to the molecular analysis whole-round samples removed offshore (see “Molecular-based community characterization and biomarker analysis”). The sediment was transferred to sterilized Whirl-Pak bags, frozen immediately at –80°C, and shipped on dry ice to the receiving laboratory.

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