# 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	

1. Filter sterilize (0.2 µm pore-size) and store at 4°C.