# J1097: SEAWATER PYRUVATE THIOSULFATE MEDIUM


## Main sol. J1097

1. Mix components and autoclave under a N2-CO2 (4:1, v/v) atmosphere. After cooling, add the following solutions (autoclaved or *filter-sterilized): 

20	g	NaCl	
3	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.25	g	NH4Cl	
0.2	g	KH2PO4	
0.5	g	KCl	
10	ml	**RST trace elements**	
1	mg	Resazurin	
922.5	ml	Distilled water	
37.5	ml	NaHCO3 (*, 8%)	
10	ml	**RST vitamin solution** (*)	
20	ml	Sodium pyruvate (1 M)	
10	ml	Yeast extract (1%)	

2. Anaerobically and aseptically distribute the medium into appropriate culture vessels (e.g., 20 ml medium in 60 ml serum bottles) under a N2-CO2 (4:1, v/v) gas stream and seal with butyl rubber stoppers. Finally, add per liter the following solution: 

10	ml	**Reducing agent solution**	


## RST vitamin solution

5	mg	Coenzyme M	
2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water	


## RST trace elements

2	g	Nitrilotriacetic acid	
1	g	MnSO4 x n H2O	
0.8	g	Fe(NH4)2(SO4)2 x 6 H2O	
0.2	g	CoCl2 x 6 H2O	
0.2	g	ZnSO4 x 7 H2O	
0.02	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.02	g	Na2MoO4 x 2 H2O	
0.02	g	Na2SeO4	
0.02	g	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.0 with KOH. Then proceed to add minerals.


## Reducing agent solution

12.5	g	Na2S x 9 H2O	
12.5	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. Autoclave and store under a N2 atmosphere.
2. Comment: For strain JCM 13588, inoculate with a 20% inoculum, and incubate the vessels in inverted position.