Fibrobacter succinogenes subsp. succinogenes (Hungate) Montgomery et al.

1. Perform all steps under anaerobic conditions.
2. Open vial according to enclosed instructions.
3. Using a single tube of #1943 broth (5 to 6 mL), withdraw approximately 0.5 to 1.0 mL with a Pasteur or 1.0 mL pipette.  Rehydrate the entire pellet.
4. Aseptically transfer the entire rehydrated contents of the vial into 5 to 6 mL of #1943 broth and immediately dilute 1:5 to a second broth tube.  Medium must be absolutely reduced.  Strain grows best in a stabbed slant or in broth that contains very little cryoprotective agent.  Inoculate a plate of a non-selective medium such as Tryptic Soy, Nutrient, or blood agar with 0.1 mL of the cell suspension.
5. Seal the tube with a rubber stopper or use Hungate tubes and incubate anaerobically at 37°C.  Incubate the plate aerobically as a purity check.
6. After 3-4 days, growth should be evident as turbidity throughout the broth. Once growth has been established, the culture should be transferred to fresh broth every 48 hours.  No growth should appear on the plate incubated aerobically.

ANAEROBIC CONDITIONS

• Tubes of media are placed under a gassing cannula system connected 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.
• This strain typically is grown in the presence of 97% carbon dioxide-3% hydrogen as a gas mixture.

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