Ruminococcus callidus Holdeman and Moore

1. Keep vial frozen until ready to use. 
2. For optimal growth, do not filter out the meat particles when preparing the #1016 broth.
3. To reduce media before inoculation, use 3.0% Cysteine then gas out vessel. 
4. Add 10% by volume of sterile rumen fluid (0.5 mL per 5 mL broth) before inoculation. 
5. Under anaerobic conditions, preferably in an anaerobe chamber, thaw vial and then quickly transfer entire contents into a single tube of #1016 broth. A second tube of #1016 broth can also be inoculated with 0.2 mL from the original broth.
6. Incubate at 37°C. Initial growth may be observed at 48 hours. Subsequent transfers in broth, may grow faster. 
7. Add 0.1 to 0.2 mL sterile rumen fluid to each Brucella blood plate prior to inoculation. Growth on agar may take up to 72 hours. It may be necessary to inoculate plates from a growing broth culture.
   
   

ANAEROBIC CONDITIONS:

1. 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, ie, highly reducing. Most strict anaerobes require this low redox potential for optimum growth.
2. 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, 3.0% cysteine is recommended for this strain. Add 0.1 to 0.2 mL of reducing agent for each tube of medium. 
3. 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.

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