| KCl | 0.355 | g | | KH2PO4 | 0.14 | g | | MgCl2·6H2O | 4 | g | | MgSO4·7H2O | 3.45 | g | | NH4Cl | 0.25 | g | | CaCl2·2H2O | 0.14 | g | | NaCl | 6 | g | | Fe(NH4)2(SO4)2·6H2O | 2 | mg | | (NH4)2Ni(SO4)2·6H2O | 20 | mg | | Trace mineral solution* | 10 | ml | | Trace vitamins** | 10 | ml | | NaHCO3 | 5 | g | | Bacto Yeast Extract (Difco) | 2 | g | | Trypticase Peptone (BBL) | 2 | g | | Glucose | 1.8 | g | | Resazurin | 1 | mg | | Na2S·9H2O | 0.5 | g | | Cysteine-HCl | 0.5 | g | | Distilled water | 1 | L | | pH 7.0 | | | | | Mix ingredients except for trace vitamins and Na2S, cysteine and autoclave under N2/CO2 (80/20) condition. Concentrated Na2S and cysteine solutions are sterilized separately by autoclaving under a N2 atmosphere in tightly closed vessels. Aseptically and anaerobically add filter-sterile trace vitamins, Na2S and cysteine solutions. | | | | | | | | | | *Trace mineral solution | | | | Nitrilotriacetic acid | 1.5 | g | | | | MgSO4·7H2O | 3 | g | | | | MnSO4·2H2O | 0.5 | g | | | | NaCl | 1 | g | | | | FeSO4·7H2O | 0.1 | g | | | | CoSO4·7H2O | 0.1 | g | | | | CaCl2·2H2O | 0.1 | g | | | | ZnSO4·7H2O | 0.1 | g | | | | CuSO4·5H2O | 0.01 | g | | | | AlK(SO4)2 | 0.01 | g | | | | H3BO3 | 0.01 | g | | | | Na2MoO4·2H2O | 0.01 | g | | | | NiCl2·6H2O | 0.025 | g | | | | Na2SeO3·5H2O | 0.3 | g | | | | Na2WO4·2H2O | 0.01 | g | | | | Distilled water | 1 | L | | | First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add other minerals. Final pH 7.0 (with KOH). Sterilize by filtration. | | | | | | | | | | **Trace vitamins | | | | Biotin | 2 | mg | | | | Folic acid | 2 | mg | | | | Pyridoxine-HCl | 10 | mg | | | | Thiamine-HCl | 5 | mg | | | | Riboflavin | 5 | mg | | | | Nicotinic acid | 5 | mg | | | | Ca-pantothenate | 5 | mg | | | | Vitamin B12 | 0.1 | mg | | | | p-Aminobenzoic acid | 5 | mg | | | | Lipoic acid | 5 | mg | | | | Distilled water | 1 | L |
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