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Main sol. 497

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Internal Solution-ID: S1014

Equipment needed: Gassing station Hungate tubes Autoclave Filter
Compound Amount Unit Conc. [g/L] Conc. [mM]
Solution A 952 ml - -
Solution B 30 ml - -
Solution C 1 ml - -
Solution D 10 ml - -
Solution E 10 ml - -
1 Solution A is sparged with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then distributed under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclaved. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C and D are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved under 100% N2 gas atmosphere. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.1 - 7.4.
2 Note: Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days. After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium.
497 CLOSTRIDIUM KN MEDIUM (HYDROXYBENZOATE)