# J1223: WIDDEL FRESHWATER MEDIUM WITH LACTATE


## Main sol. J1223

1. Mix components, adjust pH to 7.5 and autoclave under a N2 gas atmosphere. After cooling, add the following solutions from sterile anaerobic stocks (autoclaved or *filter-sterilized): 

1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.25	g	NH4Cl	
0.2	g	KH2PO4	
0.5	g	KCl	
4	g	Na2SO4	
1	mg	Resazurin	
988	ml	Distilled water	
1	ml	**Trace element solution**	
1	ml	**Vitamin solution** (*)	
1	ml	**Thiamine solution** (*)	
1	ml	**Vitamin B12 solution 0.05 g/L** (*)	
1	ml	**Selenite-tungstate solution**	
7.3	ml	L-Sodium lactate (1.0 M)	

2. Readjust pH to 7.5, if necessary. Separately autoclave and dry iron metal (grain). Aseptically distribute the medium and iron metal into culture vessels (e.g., 10 ml medium and 100 mg iron in Hungate tubes) under a N2 gas stream and seal with butyl rubber stoppers. Prior to use, add per liter the following solution: 

2	ml	Na2S x 9 H2O (5%)	


## Trace element solution

12.8	g	Nitrilotriacetic acid	
0.42	g	FeSO4 x 7 H2O	
0.01	g	H3BO3	
0.1	g	MnCl2 x 4 H2O	
0.17	g	CoCl2 x 6 H2O	
0.02	g	CuCl2 x 2 H2O	
0.21	g	ZnSO4 x 7 H2O	
0.01	g	Na2MoO4 x 2 H2O	

1. Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add the minerals and bring volume to 1.0 L.


## 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	


## Selenite-tungstate solution

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