# 701: HALOANAEROBACTER CHITINOVORANS MEDIUM ## Main sol. 701 1 g NH4Cl 7 g MgCl2 x 6 H2O 9.6 g MgSO4 x 7 H2O 0.5 g CaCl2 x 2 H2O 3.8 g KCl 0.4 g K2HPO4 x 3 H2O 10 g NaCl 3 g NaHCO3 1 g Na2CO3 0.5 g Na2S x 9 H2O 1 g Yeast extract 0.5 g L-Cysteine 1 mg Resazurin 5 g Substrate (glucose, chitin etc.) 1 ml **Modified Wolin's mineral solution II** 1000 ml Distilled water 1. pH 7.2 2. Prepare the medium without the Na2S, Na2CO3, NaHCO3, L-cysteine and substrate. Boil the medium and cool under a stream of nitrogen. Add the Na2CO3 and NaHCO3 . Adjust the pH to 7.2. If chitin is used as the substrate it must be added before autoclaving. Autoclave the medium and add the Na2S (from a sterile, neutralized stock solution prepared under nitrogen), L-cysteine (from a stock solution prepared under nitrogen), and the glucose or N-acetylglucosamine (if used as substrate) from sterile stock solution prepared under nitrogen. Check the final pH. ## Modified Wolin's mineral solution II 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 0.025 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 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.