# 730: FERROGLOBUS PLACIDUS MEDIUM ## Main sol. 730 950 ml **Solution A** 30 ml **Solution B** 1 ml **Solution C** 10 ml **Solution D** 10 ml **Solution E** 1. Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials to 10% volume (e.g., 10 ml medium in 100 ml bottle) and autoclave. Solution B is autoclaved separately after sparging with 80% N2 and 20% CO2 gas mixture. Solution C and D are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved under 100% N2 gas atmosphere. To complete the medium add appropriate amounts of solutions B to E to the sterile solution A in the sequence as indicated. 2. Adjust pH of complete medium to 7.2, if necessary. 3. Note: Cultures are very sensitive to the nitrite produced during growth. Thus, incubate this strain ony a few hours at one time (8-12 h) and thereafter check growth by microscopy. Immediately stop the incubation at 85°C, when the medium begins to show some turbidity. ## Solution A 18 g NaCl 4.3 g MgCl2 x 6 H2O 0.14 g CaCl2 x 2 H2O 0.34 g KCl 0.14 g K2HPO4 0.24 g NH4Cl 1 g KNO3 10 ml **Modified Wolin's mineral solution** 940 ml Distilled water ## Solution B 1.5 g Na2CO3 30 ml Distilled water ## Solution C 1 ml **Wolin's vitamin solution (10x)** ## Solution D 1 g Na-pyruvate 10 ml Distilled water ## Solution E 0.3 g Na2S x 9 H2O 10 ml Distilled water ## Modified Wolin's mineral solution 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.18 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.18 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.02 g AlK(SO4)2 x 12 H2O 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 H2O 0.4 mg Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water