# J976: THERMOFLEXUS HUNGENHOLTZII MEDIUM


## Main sol. J976

1. Mix components thoroughly and adjust pH to 6.75 with NaOH. Distribute the medium into culture vessels (e.g., 5.0 ml in 25 ml serum bottles/Balch tubes) under a stream of N2, seal with butyl rubber stoppers and autoclave. After cooling, aseptically add per liter the following solutions (filter-sterilized and stored anaerobically): 

3	g	NaCl	
0.15	g	KCl	
0.3	g	Na2SO4	
0.123	g	MgSO4 x 7 H2O	
0.015	g	CaCl2 x 2 H2O	
0.107	g	NH4Cl	
1.38	g	NaH2PO4	
5	ml	**Mineral solution **	
1000	ml	Distilled water	
10	ml	**Trace vitamins**	
10	ml	Peptone (w/v, 10%)	

2. Before inoculation, add filter-sterilized O2 gas or air, at a final concentration of 1% O2 in the gas phase.


## Mineral solution 

3.7	g	EDTA	
1.1	g	FeSO4 x 7 H2O	
98.4	mg	MnCl2 x 4 H2O	
28.8	mg	ZnSO4 x 7 H2O	
23.8	mg	CoCl2 x 6 H2O	
17.2	mg	CuCl2 x 2 H2O	
24.2	mg	Na2MoO4 x 2 H2O	
31	mg	H3BO3	
1000	ml	Distilled water	

1. Adjust pH to 6.0 with KOH.
2. Comment: Consistent growth in serum tubes requires pretreatment of butyl rubber stoppers by boiling in 1% sodium sulfide solution (1 hr) followed by boiling in distilled H2O (1 hr, twice).


## 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