Final pH: 7.6 - 8.0 Final volume: 1000 ml FWM stock solution 50.00 ml Distilled water (MilliQ) 1000.00 ml CaCO3 4.00 g 1. Dilute stock solution with MilliQ water (1+19 v/v), add 4 g CaCO3 per litre. 2. Autoclave at 120 °C for 21 min. 3. Cool down to room temperature. 4. After autoclaving add sterile solutions: Trace element solution SL-10 1.00 ml Selenite-tungstate solution 1.00 ml Borate-copper solution 1.00 ml Ammonium chloride solution 10.00 ml 5. Check pH 7.6-8.0 6. Complete medium can be stored at 4°C. 7. Incubate at 42°C without shaking. Activity and growth can be followed by measuring the depletion of ammonium or the production of nitrite and/or nitrate. Higher ammonium concentrations are inhibitive. For that reason our recommendation is follow nitrite content. As soon as nitrite has been produced and is depleting again (to form nitrate) another dose of ammonium can be added. Incubation time may be up to several weeks. We checked the nitrite content (absence/presence) every 5-7 days and added ammonium chloride about every 2-3 weeks. Don't expose cultures to intense light. Borate-copper solution H3BO3 54.00 mg CuCl2 x 2 H2O 18.00 mg Distilled water 1000.00 ml Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. Selenite-tungstate solution (from medium 385) NaOH 0.50 g Na2SeO3 x 5 H2O 3.00 mg Na2WO4 x 2 H2O 4.00 mg Distilled water 1000.00 ml Ammonium chloride solution Ammonium chloride 0.80 g Distilled water 100.00 ml FWM stock solution KH2PO4 1.00 g/l KCl 1.50 g/l MgSO4 x 7 H2O 1.00 g/l NaCl 11.68 g/l 1. Make up to 1000 ml with MQ water. 2. Autoclave at 120 °C for 21 min.
