# J1307: PPLL MEDIUM


## Main sol. J1307

1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
4	g	Na2SO4	
0.25	g	NH4Cl	
0.2	g	KH2PO4	
0.5	g	KCl	
0.5	g	Yeast extract	
0.89	g	Sodium malate	
1000	ml	Distilled water	

1. Mix components and autoclave. Cool down under a N2 gas stream. Aseptically and anaerobically add per liter the following solutions from sterile anaerobic stocks : 

1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
30	ml	NaHCO3 (8%)	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	

2. Adjust pH to 7.0-7.3. Aseptically and anaerobically distribute the medium into culture vessels (e.g., 10 ml medium in Balch tubes), fill the head space with a gas mixture of N2-CO2 (4:1, v/v), and seal with butyl rubber stoppers. Prior to inoculation, add per liter the following solution. 

5	ml	Na2S x 9 H2O (5%)	
5	ml	L-Cysteine HCl x H2O (5%)	


## Trace element solution

12.5	ml	HCl (25%)	
2.1	g	FeSO4 x 7 H2O	
30	mg	H3BO3	
100	mg	MnCl2 x 4 H2O	
190	mg	CoCl2 x 6 H2O	
24	mg	NiCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
144	mg	ZnSO4 x 7 H2O	
36	mg	Na2MoO4 x 2 H2O	
987	ml	Distilled water	


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Vitamin solution

4	mg	p-Aminobenzoic acid	
1	mg	Biotin	
10	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
10	mg	Pyridoxine hydrochloride	
100	ml	Sodium phosphate buffer (10 mM, pH 7.1)	


## Thiamine solution

10	mg	Thiamine HCl	
100	ml	Sodium phosphate buffer (25 mM, pH 3.4)	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water