Thermoanaerobacter ethanolicus Wiegel and Ljungdahl

1. Open vial according to enclosed instructions. 
2. Perform all steps under anaerobic conditions (see below).
3. Aseptically transfer 0.5 ml of ATCC Medium #1190 to the vial and rehydrate the freeze-dried pellet, immediately place the rehydrated pellet under a stream of oxygen-free sterile gas.  Then transfer the suspension back into the tube of broth.  Inoculate a plate of non-selective medium with 0.1 of the culture.  Inoculate a non-selective tube of broth.
4. Seal the test tube with a rubber stopper and incubate anaerobically at 55°C.  The rubber stoppers should be tapped down to insure that the anaerobic conditions are maintained during incubation.  Incubate the plate(s) and aerobic broth at 37°C as a purity check.
5. After one or three days, growth should be evident by turbidity through out the broth.  Once growth has been established, the culture should be transferred to fresh broth every 24 to 48 hours.
6. This culture is very sensitive to oxygen; therefore steps should be taken to avoid exposure to oxygen.  When the culture exhibits good growth it will remain viable for up to 1 week if stored at 4°C under anaerobic conditions.

ANAEROBIC CONDITIONS:

• Tubes of media are placed under a gassing cannula system hooked to a source of oxygen free gas.
• All transfers are performed while the test tubes are on the cannula system with a gentle stream of oxygen-free gas flowing through the system.
• As the test tubes are removed from the cannula system each is sealed with butyl rubber stopper thus maintaining the anaerobic headspace.

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