Clostridium carboxidivorans Liou et al.

1. Open vial according to enclosed instructions.
2. Under anaerobic conditions, withdraw 0.5 mL of the appropriate medium from a single test tube (5 to 6 mL) and rehydrate the entire vial contents.
3. Aseptically transfer this aliquot back into the broth tube.  Additional tubes may be inoculated with 0.5 mL each from the suspension.  Streak onto a blood plate and incubate aerobically to check for purity.
4. Incubate tubes under an anaerobic atmosphere at 37-40°C. 

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.  A 100% nitrogen or 80% nitrogen-20% carbon dioxide gas mixture is typically employed as the oxygen-free gas source.
• Resazurin is a commonly used redox indicator that is pink when the redox potential is above -50 mv., and colorless when the redox potential is below -110 mv. i.e. highly reducing.  Most strict anaerobes require this low redox potential for optimum growth.
• To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added.  Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.  Sodium sulfide solutions are normally made up at a stock concentration of 1.5% while cysteine solutions are normally made at a stock concentration of 3%.  When reducing medium add one drop of reducing agent for each one or two mL of medium. 

To obtain growth on agar, transfer the initial 24-48 hour growth in broth to solid media.  Colonies on Brucella agars are flat, irregular, lobate, and spreading.

In 24-48 hours, growth is evident by turbidity. Culture should be stored in the dark at room temperature due to sensitivity to cold (refrigeration).  No growth should occur on agar plates incubated aerobically. 

Additional information on this culture is available on the ATCC^® website at www.atcc.org.

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