| Solution A* | 440 | ml | | Solution B** | 200 | ml | | Solution C*** | 300 | ml | | Solution D**** | 50 | ml | | Solution E***** | 10 | ml | | pH 7.0-7.2 | | | | | Mix solutions A, B and C, adjust pH to 7.0-7.2. Dispense the medium into suitable culture vessels under a stream of the N2/CO2 (80/20) mixed gas, seal with butyl rubber stoppers, and autoclave. After autoclaving, aseptically and anaerobically add filter-sterile solutions D and E to the medium using sterile syringes. The sterile syringe, filter and needle are flushed with nitrogen to remove the air before using. During preparation of the medium, precipitation may occur. But it not has much influence on the growth of the bacteria. The color of resazurin becomes colorless within several days after making the medium. | | | | | | | | | | *Solution A | | | | (NH4)2SO4 | 1.3 | g | | | | KH2PO4 | 1.43 | g | | | | K2HPO4·3H2O | 7.2 | g | | | | FeSO4·7H2O | 1.1 | mg | | | | Yeast extract | 4.5 | g | | | | Resazurin | 1 | mg | | | | Distilled water | 440 | ml | | | | | | | | | | **Solution B | | | | MgCl2·6H2O | 2.6 | g | | | | CaCl2·2H2O | 0.13 | g | | | | Distilled water | 200 | ml | | | | Completely dissolve ingredients in distilled water. | | | | | | | | | | ***Solution C | | | | Sodium glycerophosphate | 6 | g | | | | Distilled water | 300 | ml | | | | | | | | | | ****Solution D | | | | Cellobiose | 5 | g | | | | Distilled water | 50 | ml | | | | Completely dissolve ingredient in hot distilled water, and remove air by bubbling with nitrogen gas. | | | | | | | | | | *****Solution E | | | | Glutathione | 0.25 | g | | | | Distilled water | 10 | ml | | | | Completely dissolve ingredient in distilled water, and remove air by bubbling with nitrogen gas. |
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