# 1526d: LIMISALSIVIBRIO (L21 LS) MEDIUM ## Main sol. 1526d 60 g NaCl 6 g MgCl2 x 6 H2O 1.5 g KCl 1 g Na2SO4 1 g NH4Cl 0.4 g CaCl2 x 2 H2O 0.4 g K2HPO4 10 ml **Modified Wolin's mineral solution** 0.5 ml Sodium resazurin (0.1% w/v) 1 g Na2CO3 1.6 g Na-acetate 6.4 g Na2-fumarate 1 ml **Seven vitamins solution** 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients except carbonate, acetate, fumarate, vitamins, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add acetate, fumarate, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas (fumarate and vitamins are sterilized by filtration) and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 6.8 - 7.0, if necessary. ## 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. ## 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