# 503d: SELENIIVIBRIO MEDIUM (ARSENATE) ## Main sol. 503d 942 ml **Solution A** 30 ml **Solution B** 10 ml **Solution C** 1 ml **Solution D** 10 ml **Solution E** 10 ml **Solution F** 1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic, distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C and F are autoclaved under 100% N2 gas atmosphere. Solutions D and E are prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. Adjust pH of complete medium to 7.2 7.4, if necessary. ## Solution A 0.2 g KH2PO4 0.25 g NH4Cl 1 g NaCl 0.4 g MgCl2 x 6 H2O 0.5 g KCl 0.15 g CaCl2 x 2 H2O 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 0.5 ml Sodium resazurin (0.1% w/v) 940 ml Distilled water ## Solution B 1.5 g Na2CO3 30 ml Distilled water ## Solution C 0.8 g Na-acetate 10 ml Distilled water ## Solution D 1 ml **Seven vitamins solution** ## Solution E 3.1 g Na2HAsO4 x 7 H2O 10 ml Distilled water ## Solution F 0.3 g Na2S x 9 H2O 10 ml Distilled water ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 990 ml Distilled water 1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. ## Seven vitamins solution 100 mg Vitamin B12 80 mg p-Aminobenzoic acid 20 mg D-(+)-biotin 200 mg Nicotinic acid 100 mg Calcium pantothenate 300 mg Pyridoxine hydrochloride 200 mg Thiamine-HCl x 2 H2O 1000 ml Distilled water ## Selenite-tungstate solution 0.5 g NaOH 3 mg Na2SeO3 x 5 H2O 4 mg Na2WO4 x 2 H2O 1000 ml Distilled water