Dehalococcoides sp.

1. Prepare media in Balch tubes, with 20 mL of media allowing for 20% headspace in the tube. Add 5 uL cis-1,2-dichloroethene (cis-DCE) to each Balch tube.
2. Pre-reduce all media and supplies 24 hours in advance of opening vial and/or culturing using 3% cysteine and 80% N₂-10%CO₂-10% H₂ gas.
3. Open thawed vial according to enclosed instructions or visit www.atcc.org for instructions. It is highly recommended that all work be performed in an anaerobic chamber.
4. Under anaerobic conditions, aseptically transfer the entire contents of the vial to a tube of #2760 broth prepared as described above. Establish growth in this primary tube before subculturing. Several drops from the primary broth tube can be sued to inoculate a #260 plate to check purity.
5. Incubate in an anaerobic atmosphere at 30°C for 2-4 weeks. Incubate one agar plate aerobically at 37°C to check for contamination.
6. Once growth has been established, additional test tubes can be inoculated by transferring 1 mL from the primary broth tube to secondary broth tubes. Best practice dictates the use of pre-reduced media.

Anaerobic conditions for incubation may be obtained by any of the following: 
Loose screw caps on test tubes in anaerobic chamber, 
Loose screw caps on test tubes in an activated anaerobic gas pack jar, or 
Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.

This strain should be provided with the chloronated electron acceptor cis-DCE every 2-4 weeks.

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