T. autotrophicus EF1 cells were grown at in situ temperature (10 °C) in acid-washed (10% HCl) and autoclaved bottles containing copiotrophic seawater media from Puget Sound or oligotrophic seawater media from the Sargasso Sea (Supplementary Table 2 ). Aerobic cultures were maintained in acid washed polycarbonate bottles. Anaerobic cultures were maintained in acid washed and autoclaved 125 ml serum bottles, sealed with 20 mm butyl rubber stoppers (Wheaton, Millville, NJ, USA), then bubbled with an N2:CO2 gas mix (1000 ppm CO2, Praxair, Danbury, CT, USA, Specialty Gas Mix) for 10 min, and headspace sparged for an additional 5 min. Complete removal of oxygen inside serum bottles was confirmed using BD (Franklin Lakes, NJ, USA) GasPak Anaerobic strips added to an un-inoculated control serum bottle. Experiments (aerobic and anaerobic) were started by transferring 1000 cells in early exponential growth phase to new media. Culture purity was checked before and after each physiology study by sequence analysis and TRFLP.
Gaspak anaerobic strips
Manufactured by BD
Sourced in United States
The GasPak Anaerobic Strips are a laboratory equipment used to create an anaerobic environment. They generate an anaerobic atmosphere through a chemical reaction.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using gaspak anaerobic strips
1
Cultivation of T. autotrophicus EF1
2
Cultivation of marine microorganisms
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Cells were grown as previously described (15 (link), 19 (link), 40 (link)), with minor modifications. Media was prepared by filter sterilizing seawater collected from the Puget Sound using a tangential flow filtration system equipped with a 30-kDa filter (Millipore, NJ, USA). Media was stored, checked for purity, and subsequently distributed into acid-washed and autoclaved 125-ml glass serum bottles. Oxic media bottles were covered with sterilized aluminum foil. Anoxic media bottles were sealed with 20-mm butyl rubber stoppers (Wheaton, Millville, NJ, USA) and bubbled with an N2-CO2 gas mixture that contained 1,000 ppm CO2 (Praxair, Danbury, CT, USA) for 10 min, and then the headspace was sparged for an additional 5 min. Oxygen removal was verified by adding GasPak anaerobic strips (BD, Franklin Lakes, NJ, USA) to uninoculated serum bottles. Batch cultures used for cryo-electron tomography and CHN analysis were amended with additional 5 μM NH4+ and 30 μM NO3− to ensure the highest cell yields.
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