# 1053a: THIOPROFUNDUM LITHOTROPHICUM MEDIUM


## Main sol. 1053a

20	g	NaCl	
0.09	g	K2HPO4	
0.07	g	KH2PO4	
0.8	g	CaCl2	
0.25	g	NH4Cl	
1	g	NaNO3	
4	g	MgSO4 x 7 H2O	
3	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.01	g	Fe2(SO4)3 x H2O	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
3	g	Na2S2O3 x 5 H2O	
1	g	NaHCO3	
1	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except thiosulfate, bicarbonate and vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 6.8 with NaOH, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials up to a volume of 20% and autoclave. Add thiosulfate, vitamins and bicarbonate to the autoclaved medium from sterile anoxic stock solutions. The stock solutions of thiosulfate and vitamins are sterilized by filtration and stored under 100% N2 gas, whereas the stock solution of bicarbonate is prepared under 80% N2 and 20% CO2 gas mixture and autoclaved. Adjust pH of the complete medium to 6.5.
2. After inoculation add 1% (v/v) sterile O2 gas (e.g., by adding 1.50 ml sterile air to 5 ml medium in a Balch-type tube) and pressurize vials to 2 bar overpressure with sterile   80% N2 and 20% CO2 gas mixture.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water