| | Casitone | 30 | g |
| | Yeast extract | 5 | g |
| | K2HPO4 | 5 | g |
| | Resazurin | 1 | mg |
| | Glucose | 4 | g |
| | Cellobiose | 1 | g |
| | Maltose | 1 | g |
| | Soluble starch | 1 | g |
| | Water containg chopped meat* | 1 | L |
| | | Firstly prepare the water containg chopped meat* and then add other ingredients. To make medium anoxic boil it, cool under nitrogen atmosphere, add 0.5 g/l cysteine hydrochloride and adjust pH to 7.0. Dispense anoxically 7 ml medium into Hungate tubes (for strains demanding meat particles put those first into the tube (use 1 part meat particles to 4 or 5 parts fluid)). Autoclave at 121C for 30 min. As necessary, add 10 ml/l haemin solution** and 0.2 ml/l vitamin K1 solution*** or 0.2 ml/l vitamin K3 solution**** to medium after autoclaving. For agar slants use 15 g agar per 1000 ml medium. |
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| | | *Water containg chopped meat: |
| | | Use lean beef or horse meat. Remove fat and connective tissue before grinding. Mix meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, skim fat off surface, and filter, retaining both meat particles and filtrate. To the filtrate add water to a final volume of 1000 ml. |
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| | | **Haemin solution |
| | | Dissolve 50 mg haemin in 1 ml 1 N NaOH, make up to 100 ml with distilled water and filter sterilize. Store refrigerated. |
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| | | ***Vitamin K1 solution |
| | | Dissolve 0.1 ml of vitamin K1/K3 in 20 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle. |
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| | | ****Vitamin K3 solution |
| | | Dissolve 5 mg/ml of vitamin K3 in 10 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle. |