# 441: DIAZOTROPHIC MEDIUM (RBA) ## Main sol. 441 953 ml **Solution A** 50 ml **Solution B** 5 ml **Standard vitamin solution** 1. Sterilize solution A separately at 121°C for 15 min., cool to 50°C and then mix aseptically with filter-sterilized solution B and 5.0 ml of filter-sterilized standard vitamin solution (see medium 428). 2. RBA is an ammonium-free medium which has successfully been used for the isolation, growth and purity check of a broad spectrum of nitrogen fixing bacteria (Ref. 3363). For microaerophilic nitrogen-fixing bacteria use semisolid medium with 0.3% end concentration of agar and incubate the liquid cultures under 10% (v/v) air and 90% (v/v) N2. ## Solution A 0.1 g KH2PO4 0.9 g K2HPO4 0.1 g NaCl 0.1 g CaCl2 x 2 H2O 0.1 g MgSO4 x 7 H2O 5 mg Na2MoO4 x 2 H2O 5 mg NaVO3 x H2O 5 mg MnSO4 x H2O 0.01 g FeSO4 x 7 H2O 0.05 g Yeast extract 3 ml **Trace element solution SL-6** 950 ml Distilled water 15 g Agar, if required 1. Adjust pH to 7.3. ## Solution B 1 g Disodium succinate 2 g DL-Malate 1 g Na-pyruvate 2 g D-Mannitol 2 g D-Glucose 50 ml Distilled water 1. Adjust pH to 7.3. ## Trace element solution SL-6 0.1 g ZnSO4 x 7 H2O 0.03 g MnCl2 x 4 H2O 0.3 g H3BO3 0.2 g CoCl2 x 6 H2O 0.01 g CuCl2 x 2 H2O 0.02 g NiCl2 x 6 H2O 0.03 g Na2MoO4 x 2 H2O 1000 ml Distilled water ## Standard vitamin solution 10 mg Riboflavin 50 mg Thiamine-HCl x 2 H2O 50 mg Nicotinic acid 50 mg Pyridoxine hydrochloride 50 mg Calcium pantothenate 0.1 mg Biotin 0.2 mg Folic acid 1 mg Vitamin B12 100 ml Distilled water