# 979: PETROBACTER SUCCINIMANDENS MEDIUM


## Main sol. 979

0.3	g	K2HPO4	
0.3	g	KH2PO4	
0.2	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	KCl	
1	g	NaCl	
1	g	NH4Cl	
1	g	Yeast extract	
5	ml	**Wolin's vitamin solution**	
10	ml	**Modified Wolin's mineral solution II**	
1000	ml	Distilled water	

1. When grown aerobically prepare the medium without the vitamins, adjust the pH to 7.0 with 10M KOH, and sterilise for 45 min at 110°C. On cooling add the vitamins from a filter sterilised stock solution. Both Geococcus nitratireducens (DSM 15495) and Petrobacter succinimandens (DSM 15512) are supplied from aerobically grown cultures.
2. Anaerobic growth may be obtained by gassing the medium with N2:CO2 (80:20), to which 10mM KNO3 has been added, and dispensing the medium under N2:CO2 (80:20). Sterilise by autoclaving at 110°C for 45 min , and to the cold medium add the filter sterilised vitamins. A reducing agent is not required.


## Wolin's vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## 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.