SAMPLING STRATEGY

To ensure as little damage as possible to the microbiological community present in these cores, a unique sampling strategy is required. Since microorganisms existing at deep-water seafloor temperatures (2°-4°C) can be particularly sensitive to elevated temperature (>10°C), there is a critically acute need to prevent thermal equilibration of the cores after recovery and prior to processing. The detailed example of a shipboard sampling plan and core flow outlined below may be possible to complete before thermal equilibration where core recovery is slow; however, rapid core recovery will likely require refrigeration of cores immediately after recovery until they can be processed.

Sampling guidelines and policy are available at the following World Wide Web site: http://www-odp.tamu.edu/publications/policy.html. The Sample Allocation Committee (SAC), which consists of the two co-chiefs, staff scientist, and ODP curator onshore or curatorial representative aboard ship, will work with the entire science party to formulate a formal Leg 201 specific sampling plan for shipboard and postcruise sampling.

Archives
Because all sites have been previously occupied, a permanent archive for these sites already exists in the ODP repository. With the exception of the whole-round sampling required for microbiological experiments, however, the permanent archive for this cruise will be the ODP-defined "minimum permanent archive" and will be reconstructed postcruise. We will limit all routine and personal shipboard sampling, outside of contamination-free microbiological sampling, to the working halves of the cores.

Example Core Flow and Shipboard Sampling Strategy
Because the outer part of the core will have been bathed in surface seawater (our drilling fluid) and exposed to oxygen during handling, only the inner part of the core is appropriate for many microbiological samples. This necessitates removing whole-round cores (WRC), so that the exterior contaminated part of the core can be pared away in a sterile, anoxic environment to preserve the more pristine interior. We expect that on the order of 1.5 m of WRC will be required to accommodate all the experiments outlined for each sample in the "Scientific Approaches" section above. Ideally, these will all come from the same 1.5-m interval to accumulate as many measurements as possible on the same interval, but poor or disturbed recovery may require sampling over a longer interval to ensure or at least optimize the chance of sampling uncontaminated core.

As soon as the core arrives on deck and is carried to the catwalk, it will be marked by the ODP technical staff into 1.5-m sections. To minimize equilibration of the cores, it may be necessary to modify the standard ODP practice of shelving a recovered core barrel on the rig floor while a new core barrel is deployed and a joint of pipe is added. At deep water sites, we will probably ask the rig floor to deliver the core liner to the catwalk as soon as it can be removed from the core barrel, recognizing this may slow the coring operation. One of the shipboard microbiology contingent will be charged to identify one 1.5-m section for microbiology (MBIO) for rapid microbiological processing (Fig. 5). Once the MBIO section is selected, it and an adjacent 1.5-m section (either above or below, according to placement of the MBIO section) will be wiped with ethanol, labeled with a red permanent marker with orientation and section number, and removed from the core (total 3.0-m section), before any other cut is made. The ends will be covered with plastic caps but not sealed with acetone, and the 3.0-m section will be carried (using clean, ethanol-swabbed gloves) into the core lab. This section will be taken immediately to a sterile, oxygen-free subsampler, and the remainder of the core will be processed according to standard ODP protocol. Routine samples for measurements of ephemeral properties will be collected from the non-MBIO core sections. These measurements include those for organic geochemistry for safety monitoring (free gas and 20-cm³ sediment samples), biostratigraphy (core catcher samples), and physical properties.

The MBIO section will be subsampled in a cutting rig continuously bathed with sterile oxygen-free nitrogen. First, the section adjacent to the MBIO section will be removed and the non-MBIO section returned to the core lab for standard processing. The remaining nominally 1.5-m section will be cut into a series of WRCs and distributed to various parts of the shipboard laboratory. Although we may not envision all the possible types of samples that could be accommodated, what follows is an example of how the 1.5-m section might be distributed (see Fig. 5).

First, two 10-cm WRCs would be removed from the end of the section cut on the catwalk for PFT contamination and interstitial water (IW) analysis. These WRCs would be removed from the end in case the exterior cut was not sterile (not a requirement for the PFT or IW analysis). The next 60 cm would be removed and transported immediately to the radioisotope van (see "Radioisotope Protocols" section below). Ten-centimeter WRCs would be cut for MPN, enrichment cultures, and fluorescent in situ hybridization (FISH) analysis. These, along with 5-cm whole rounds for cell counts, ATP analysis, and additional pore water analysis, would be transported to the anaerobic chamber in the microbiology laboratory for further processing. Two 10-cm WRCs would be removed and placed in the ultra low temperature freezer (-86°C) for postcruise DNA and biomarker analyses and a third would be deep frozen as a residual sample. The outline above is provided to give interested scientists an idea of our core flow strategy and may require modification at sea based on recovery rate, completeness, or disturbance.

Radioisotope Protocols
Since radioisotope studies have never been carried out on board the JOIDES Resolution, we include the following section as our current plan of action for these experiments. The low-energy beta emitters planned for the cruise do not constitute a health hazard, but radioactive contamination could be a potential problem for other research, in particular for the sensitive analyses of natural radioisotopes. A van produced specifically for and dedicated to radioisotope experiments should be on the vessel prior to departure from port call. Dr. Bo Jørgenson, Co-Chief Scientist, is responsible for overseeing radioisotope work at the Max Planck Institute for Marine Microbiology and has agreed to accept senior responsibility for radioisotope work during Leg 201. In addition, any scientists working in the radioisotope facility will be experienced in performing these experiments, and a scientist on each shift will be assigned active responsibility for radioisotope handling and safety.

Once a section has been subsampled for radioisotope studies, the subsection will be hand carried to the van and passed to the scientist in the van. We intend to keep traffic in and out of the van to a minimum, and only personnel trained in radioisotope work should be in the van. Sample processing will be done within a plastic basin with the surrounding bench covered with plastic-backed absorbent paper. All solutions will be stored in tightly capped containers and routine contamination wipe tests will be performed. Any personnel in the van will need to wear gloves, a lab coat, and safety glasses at all times. Radioisotope stock solutions, radiolabeled samples, and contaminated laboratory products do not leave the isotope van at any time during the cruise.

Three types of experiments are planned at this time: (1) determining the rates of sulfate reduction using the radiotracer ³⁵SO₄^2-, (2) rates of methanogenesis with ¹⁴C-labeled HCO₃^- or acetate and anaerobic oxidation of methane with ¹⁴CH₄, and (3) a tritiated (3H) amino acid mixture will be used to determine the number of active cells via microautoradiography. The general procedure will be to put an aliquot of uncontaminated sediment into 12-mL serum vials and store them for a few hours in an incubator at in situ temperature (or potentially some other temperature for experimental purposes). Ten microliters of carrier-free radioisotope will be injected by microsyringe into each sample. At the conclusion of the incubation period, the bacterial activity will be fixed and the sample will be frozen. Samples will then be transferred into 50-mL screw-capped plastic centrifuge vials for safe handling and transport. These samples should be sent by air freight, packed in sturdy containers, and maintained in a frozen condition.

Radiotracers expected to be on board at the beginning of the expedition are stock solutions of 500 MBq of ³⁵SO₄^2- in distilled water, 500 MBq of ¹⁴C-labeled HCO₃^-, 250 MBq of ¹⁴C-labeled acetate, 100 MBq of ¹⁴C-labeled methane, and a total of 10 MBq of a mixture of 15 tritiated amino acids. The total amount of contaminated laboratory products we expect to generate will be <2 L of liquid and as much as 10 kg of solid material. Solid and liquid contaminated laboratory products will be returned to Texas A&M University.

Special Requirements
Special requirements include (1) radioisotope training/advisor for ODP/TAMU, (2) shipping supplies for large frozen shipment, (3) modification of PFT delivery system, and (4) three times the current -86°C freezer space (minimum). Radioisotope van requirements include (1) meets University-National Oceanographic Laboratory System (UNOLS) specifications, (2) additional refrigerator, freezer, and incubators, (3) supplies and safety gear.