Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring

Abstract

INTRODUCTIONThe Ocean Drilling Program (ODP) is committed to deep-biosphere research and has constructed a new microbiological laboratory on board the JOIDES Resolution. The use of the JOIDES Resolution as a platform for deep-biosphere research requires that the recovered cores are suitable for microbiological study. The major concern is whether microbes from the drilling fluid are introduced into the recovered core material during coring. Therefore, it is critical to verify whether recovered cores are contaminated. Here we present details of two tracer methods used to quantify the amount of contamination. These methods were modified from land-based drilling operations for use on the JOIDES Resolution (see review by Griffin et al., 1997). Tracer experiments were first conducted during ODP Leg 185 (Plank, Ludden, Escutia, et al., in press) and involve the delivery of both chemical and particulate tracers during drilling and their quantification in the ODP cores. These tracers were introduced while drilling unconsolidated sediments using the advanced hydraulic piston corer (APC), sedimentary rock using the extended core barrel and rotary core barrel (RCB), and igneous rock using the RCB and diamond core barrel. This technical note presents details on the characteristics, preparation, and delivery of the tracers and their quantification in cores. Suggestions are made regarding sample handling with the goal of minimizing sample contamination. It is strongly recommended that these contamination tests be routinely conducted when coring for microbiological studies. D.C. SMITH ET AL. TRACER TESTS FOR MICROBIOLOGICAL STUDIES 2 MATERIALS AND METHODS Chemical Tracer: Perfluorocarbon CharacteristicsPerfluorocarbon tracers (PFT) have been used extensively in landbased drilling applications (Senum and Dietz, 1991;Russell et al. 1992;McKinley and Colwell, 1996) because they are inert and can be detected with high sensitivity. Perfluoro(methylcyclohexane) is the tracer that has been tested on the JOIDES Resolution. This perfluorocarbon (Aldrich 30293-7) has a molecular weight of 350.05, a boiling point of 76°C, and a density of 1.76 g/mL. Its solubility is ~1 mg/L in water and is 10 g/L in methanol (Colwell et al., 1992). The low solubility in water facilitates gas phase partitioning and quantitative headspace analysis. Preparation and DeliveryThe stock PFT is shipped in sealed ampoules, and it is not necessary to dilute it prior to use. Because the PFT is volatile and can be detected at extremely low concentrations, it is necessary to open the ampoules and transfer the PFT to the carboy used for delivery in a ventilated area well away from the core handling and PFT detection areas. Gloves should be worn during this process and discarded afterward. It is recommended that this transfer be performed on the helicopter deck while the JOIDES Resolution is under way and that all materials that may have been in contact with the PFT be disposed of immediately. These precautions will minimize the probability of obtaining false positi...

Methods

“…Similar methodologies have been applied during previous ocean drilling expeditions (Smith et al, 2000a(Smith et al, , 2000bLever, 2006). The PFC tracer used was perfluoromethylcyclohexane (C 7 F 14 ) (Smith et al, 2000b).…”

“…Similar methodologies have been applied during previous ocean drilling expeditions (Smith et al, 2000a(Smith et al, , 2000bLever, 2006). The PFC tracer used was perfluoromethylcyclohexane (C 7 F 14 ) (Smith et al, 2000b). The PFC was pumped undiluted directly into the main stream of drilling fluid (seawater or drilling mud) at a rate of 0.1 mL/min (0.18 g/min) using a high-performance liquid chromatography (HPLC) pump (Alltech Model 301).…”

“…The PFC was pumped undiluted directly into the main stream of drilling fluid (seawater or drilling mud) at a rate of 0.1 mL/min (0.18 g/min) using a high-performance liquid chromatography (HPLC) pump (Alltech Model 301). The target concentration of PFC in the drilling fluid was 1 mg PFC/L, which is the maximum solubility of perfluoromethylcyclohexane in water (Smith et al, 2000b).…”

“…All measurements were carried out using a gas chromatograph with an electron capture detector (Agilent 7820A GC), following the technical specifications described previously (Smith et al, 2000b) and subsequently used during other ocean drilling expeditions (e.g., Smith et al, 2000a;Lever et al, 2006). However, to ensure full PFC recovery from sediments, all headspace vials containing samples were preincubated for 2 h at 80°C.…”

Methods

“…Contamination will be further assessed by postcruise genomic comparison of the basalt or sediment sample with drilling fluid from the time of coring. The two shipboard techniques have been successfully used on multiple ODP legs and during Expedition 301 (Smith et al, 2000a(Smith et al, , 2000bHouse et al, 2003;Lever et al, 2006). The third technique is common for studies of seafloor samples, in which contamination is ubiquitous and genomic signatures of the contaminating material are subtracted from those of the seafloor sample.…”