# J881: KNH MEDIUM


## Main sol. J881

0.136	g	KH2PO4	
0.535	g	NH4Cl	
3.05	g	MgCl2 x 6 H2O	
0.147	g	CaCl2 x 2 H2O	
2.52	g	NaHCO3	
20.45	g	NaCl	
2.84	g	Na2SO4	
0.656	g	Sodium acetate	
1	ml	**Trace element solution**	
1	ml	**Se/W solution**	
1	ml	**Trace vitamins**	
1	mg	Resazurin	
0.1	g	Sodium thioglycolate	
0.1	g	Ascorbic acid	
1000	ml	Distilled water	

1. Mix all ingredients except sodium thioglycolate and ascorbic acid, and expel dissolving O2 from the solution with deoxygenated N2-CO2 (80:20, v/v). Dispense the solution into vials where deoxygenated H2-CO2 (80:20, v/v) is sparged. Cap with butyl rubber stoppers. Autoclave, then add reducing agent solution (sodium thioglycolate and ascorbic acid, 1% solution each) sterilized with 0.2 �m-filter (0.1 ml in 10 ml medium) and inoculate fully grown culture at 5-10% of final volume. Fill with H2-CO2 (80:20, v/v) gas at 1.5 atm.


## Trace element solution

12.8	g	Nitrilotriacetic acid	
1.35	g	FeCl3 x 6 H2O	
0.1	g	MnCl2 x 4 H2O	
0.024	g	CoCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnCl2	
0.025	g	CuCl2 x 2 H2O	
0.01	g	H3BO3	
1	g	NaCl	
0.12	g	NiCl2 x 6 H2O	
0.026	g	Na2SeO3 x 5 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid first in 200 ml of distilled water, and adjust pH to 6.5 with KOH. Then add the remaining salts and adjust volume to 1.0 L. Readjust pH to 7.0 with KOH.
2. Comment: For strain JCM 10134, incubate with a 10-20% inoculum.


## Se/W solution

4	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Trace vitamins

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 pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water