# J462: SALTWATER MEDIUM WITH LACTATE


## Main sol. J462

20	g	NaCl	
4	g	Na2SO4	
3	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.75	g	NH4Cl	
0.6	g	KH2PO4	
0.09	g	KBr	
0.5	g	KCl	
2.5	g	NaHCO3	
1	ml	**Trace element solution**	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	
1	ml	**Riboflavin solution 0.025 g/L**	
1	ml	**Selenite-tungstate solution**	
2.25	g	Sodium lactate	
0.3	g	Na2S x 9 H2O	
1	mg	Resazurin	

1. Dissolve components except NaHCO3, Vitamin solution, Thiamine solution, Vitamin B12 solution, sodium lactate and Na2S x 9H2O in 900 ml distilled water and adjust pH to 7.0. Bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas stream. Distribute the medium into culture vessels (e.g., 9 ml of the medium in Hungate tubes) under a N2-CO2 (4:1, v/v) gas mixture, seal with butyl rubber stoppers and autoclave. Separately autoclave 0.2 M sodium lactate under a N2 atmosphere. After cooling, add 1/9 volume of the sodium lactate solution, and filter-sterilized 8% NaHCO3 solution, Vitamin solution, Thiamine solution and Vitamin B12 solution to the medium. Readjust pH to 7.0, if necessary. Prior to use, reduce the medium with Na2S x 9H2O (3% solution, autoclaved and stocked under N2).


## Riboflavin solution 0.025 g/L

2.5	mg	Riboflavin	
100	ml	Distilled water	


## 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	


## 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