To solution A add first solution C and then solution D. Adjust the complete medium to pH 6.9 at 50°C by adding 4.0 to 4.5 ml of sterile 1.0 N KOH. The pH of the medium is critical. Finally add solution B. Swirl medium in flask during dispensing to Petri dishes or tubes to keep charcoal suspended.
Solution A
Distilled water490.0ml
Activated charcoal (Sigma C 5510)2.0g
Yeast extract (Difco 0127)10.0g
*N-2-acetamido-2-aminoethane-sulfonic acid. Dissolve all components by boiling and autoclave at 121°C for 15 min. Cool to 50 - 55°C in a waterbath.
Solution B
Distilled water490.0ml
Autoclave at 121°C for 15 min and cool to 50 - 55°C in a waterbath.
Solution C
Distilled water10.0ml
Cysteine-HCl x H2O0.4g
Prepare a fresh solution and filter-sterilize it.
Solution D
Distilled water10.0ml
Prepare a fresh solution by heating to 50 - 55°C in a waterbath. Filter-sterilize solution separately. Note: Soluble ferric pyrophosphate must be kept dry and in the dark. Do not use if chemical looses its green colour and becomes brown or yellow.

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