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Medium No.	1319
Medium	Medium for Strain HC45 (Anaerobic cultivation)
Composition
NaCl 20 g   MgCl2·6H2O 3 g   CaCl2·2H2O 0.15 g   Na2SO4 4 g   NH4Cl 0.25 g   KH2PO4 0.2 g   KCl 0.5 g   Glucose 1.8 g   Yeast extract 0.5 g   Resazurin 1 mg   Trace element mixture* 1 ml   Selenite-tungstate solution** 1 ml   Bicarbonate solution (1.0 M)*** 30 ml   Vitamin solution**** 1 ml   Thiamine solution***** 1 ml   Vitamin B12 solution****** 1 ml   Sulfide solution (1.0 M)******* 1.5 ml   Distilled water 963.5 ml   pH 7.0-7.3       Mix ingredients except for yeast extract, bicarbonate, vitamin, thiamine, cyanocobalamin and sulfide solutions. 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 at 121°C for 15 min. Aseptically and anaerobically add other ingredients using sterile syringes. Adjust pH to 7.0-7.3 using compact pH meter.                   *Trace element mixture       HCl (25%; 7.7 M) 12.5 ml       FeSO4·7H2O 2100 mg       H3BO3 30 mg       MnCl2·4H2O 100 mg       CoCl2·6H2O 190 mg       NiCl2·6H2O 24 mg       CuCl2·2H2O 2 mg       ZnSO4·7H2O 144 mg       Na2MoO4·2H2O 36 mg       Distilled water 987 ml     Sterilized by autoclaving.                   **Selenite-tungstate solution       NaOH 0.4 g       Na2SeO3·5H2O 6 mg       Na2WO4·2H2O 8 mg       Distilled water 1 L     Sterilized by autoclaving.                   ***Bicarbonate solution (1.0 M)       NaHCO3 8.4 g       Distilled water 100 ml     Saturated with CO2 gas. Tightly sealed in bottles filled with CO2 gas and then sterilized by autoclaving.                   ****Vitamine solution       p-Aminobenzoic acid 4 mg       Biotin 1 mg       Nicotinic acid 10 mg       Ca-pantothenate 5 mg       Pyridoxine-HCl 15 mg       Sodium phosphate buffer (10 mM; pH7.1) 100 ml     Sterilized by filtration.                   *****Thiamine solution       Thiamine-HCl 10 mg       Sodium phosphate buffer (25 mM; pH 3.4) 100 ml     Sterilized by filtration.                   ******Vitamin B12 solution       Cyanocobalamin 5 mg       Distilled water 100 ml     Sterilized by filtration.                   *******Sulfide solution (1.0 M)       Na2S·9H2O 24 g       Distilled water 100 ml     Sterilized by filtration and stored under N2 gas.
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