Oxalobacter formigenes Allison et al.

1. To reduce media before inoculation, use 3% Cysteine HCl solution (0.2 mL per 10 mL). Growth is best achieved with the use of Hungate tubes.
2. Exchange the gas in the Hungate tube for an anaerobic mixture containing CO₂. CO₂ is required for growth.
3. Under anaerobic conditions, preferably in an anaerobe chamber, rehydrate the vial and then quickly transfer entire contents into a single Hungate tube of #1352 broth. A second tube of #1352 broth can also be inoculated with 0.5 mL from the original broth.
4. Incubate at 37°C. Initial growth may be observed at 48 hours. Suitable growth for transfer may take up to 2 to 3 days.  Once growth is established, the culture should be transferred to fresh broth every 3-5 days.
5. Agar cultures may require inoculation from an established broth culture and a longer incubation period. 
6. Growth may be evident by light turbidity in the broth; however, a wet mount is usually necessary to observe growth.

ANAEROBIC CONDITIONS:

1. Hungate (or Balch) tube refers to a special type of test tube that is designed to be pressurized and is suited for anaerobic work. The Hungate test tubes can be purchased form Bellco glass (www.bellcoglass.com; stock no. 204800150).
2. 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.
3. To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added. There are several common reducing agents, however, 3% Cysteine HCl solution is recommended for this strain. Add 0.2 mL of reducing agent for each 10 mL of medium. 
4. Syringes can be made anaerobic by one of two methods.

1. Displace the dead space in the syringe with a sterile oxygen-free gas.
2. Displace the dead space in the syringe with a reducing agent.

For further information, reference Dawson KA, et al. Isolation and some characteristics of anaerobic oxalate-degrading bacteria from the rumen. Appl. Environ. Microbiol. 40: 833-839, 1980. PubMed: 7425628 

Always use freshly prepared pre-reduced media or pre-reduced media that has been previously prepared but stored under anaerobic conditions. Resazurin in the media is a color indicator for anaerobic conditions. Observance of pink color in medium before use or during incubation shows anaerobic conditions have not been met and oxidation has occurred. Medium should be discarded.

Growth on plates requires inoculation with a growing broth culture.

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

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