# 739: DESULFONEMA ISHIMOTOI MEDIUM ## Main sol. 739 952 ml **Solution A** 10 ml **Solution B** 1 ml **Solution C** 10 ml **Solution D** 20 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 and reach a pH of around 6, then distribute under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Solutions B and C are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solutions D and F are autoclaved separately under 100% N2 gas. Solution E is prepared under 80% N2 and 20% CO2 gas gas atmosphere. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be at 7.0. After combining solutions the medium should equilibrate overnight and a white precipitate should be apparent. 2. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution freshly prepared under N2 and filter-sterilized) just before inoculation may stimulate growth at the beginning. For transfers use 5 - 10% (v/v) inoculum. ## Solution A 25 g NaCl 5.6 g MgCl2 x 6 H2O 6.8 g MgSO4 x 7 H2O 1.4 g CaCl2 x 2 H2O 0.72 g KCl 0.14 g KH2PO4 0.25 g NH4Cl 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 0.5 ml Sodium resazurin (0.1% w/v) 950 ml Distilled water ## Solution B 2.5 g Na-acetate x 3 H2O 0.1 g Disodium succinate 0.19 ml Isobutyric acid 10 ml Distilled water 1. Adjust to pH 7.0 with 1 N NaOH. ## Solution C 1 ml **Wolin's vitamin solution (10x)** ## Solution D 0.48 g AlK(SO4)2 x 12 H2O 10 ml Distilled water ## Solution E 1 g Na2CO3 20 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. ## Selenite-tungstate solution 0.5 g NaOH 3 mg Na2SeO3 x 5 H2O 4 mg Na2WO4 x 2 H2O 1000 ml Distilled water ## 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