Final pH: 7.0 - 7.2 Final volume: 1003 ml Solution A 962.00 ml Solution B 20.00 ml Solution C 20.00 ml Solution D 1.00 ml Solution A is sterilized by autoclaving at 121°C for 15 min under a 100% N2 gas atmosphere. Solutions B and D are sterilized by filtration under a 100% N2 gas atmosphere. Solution C is sterilized by autoclaving under an atmosphere of 80% N2 and 20% CO2 gas mixture. Appropriate amounts of solutions B to D are added to the sterile solution A in the sequence as indicated. Check the pH of the complete medium and adjust it to 7.0 - 7.2, if necessary. Solution A KH2PO4 2.00 g KNO3 2.00 g NH4Cl 1.00 g MgSO4 x 7 H2O 0.80 g Trace element solution SL-4 2.00 ml Distilled water 960.00 ml Solution B Na2S2O3 x 5 H2O 5.00 g Distilled water 20.00 ml Solution C Na2CO3 1.00 g Distilled water 20.00 ml Solution D FeSO4 x 7 H2O 20.00 mg H2SO4 (0.1 N) 10.00 ml Trace element solution SL-4 (from medium 14) Na2-EDTA 0.50 g FeSO4 x 7 H2O 0.20 g ZnSO4 x 7 H2O 0.10 g MnCl2 x 4 H2O 0.03 g H3BO3 0.30 g CoCl2 x 6 H2O 0.20 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 x 2 H2O 0.03 g Distilled water 1000.00 ml First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.
