# 169b: LEADBETTERA MEDIUM ## Main sol. 169b 952 ml **Solution A** 20 ml **Solution B** 20 ml **Solution C** 1 ml **Solution D** 10 ml **Solution E** 1. Dissolve ingredients of solution A (except cysteine) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add cysteine and adjust pH 7.2 with KOH, then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere, solution C is autoclaved under 100% N2 gas atmosphere and solutions D and E are sterilized by filtration under 100% N2 gas atmosphere. To complete the medium appropriate amounts of solutions B, C, D and E are added to the sterile solution A in the sequence as indicated. Final pH of the complete medium should be 7.5. ## Solution A 0.04 g CaCl2 x 2 H2O 0.5 g MgSO4 x 7 H2O 4 g Yeast extract (OXOID) 4 g D-Glucose 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 0.5 ml Sodium resazurin (0.1% w/v) 0.5 g L-Cysteine HCl x H2O 950 ml Distilled water ## Solution B 1 g NaHCO3 20 ml Distilled water ## Solution C 16.04 ml K2HPO4 (0.5 M) 3.96 ml KH2PO4 (0.5 M) 1. Combine 16.04 ml of 0.5 M K2HPO4 with 3.96 ml of 0.5 M KH2PO4 stock solution to reach a pH of around 7.4. ## Solution D 1 ml **Seven vitamins solution** ## Solution E 0.15 g DL-Dithiothreitol 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 ## 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