# 171: THERMOANAEROBACTER KIVUI MEDIUM


## Main sol. 171

0.22	g	K2HPO4	
0.22	g	KH2PO4	
4.5	g	NaH2PO4 x H2O	
6.1	g	Na2HPO4 x 12 H2O	
0.31	g	NH4Cl	
0.22	g	(NH4)2SO4	
0.45	g	NaCl	
0.09	g	MgSO4 x 7 H2O	
6	ml	CaCl2 x 2 H2O (0.1% w/v)	
2	ml	**FeSO4 x 7 H2O solution (0.1% w/v)**	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except cysteine and sulfide, sparge medium with 80% H2 and  20%  CO2 gas mixture for 30 - 45 min to make it anoxic, then distribute under same gas atmosphere into Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 6.5.
2. After inoculation pressurize cultivation vessels to one bar overpressure with sterile 80% H2 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.


## FeSO4 x 7 H2O solution (0.1% w/v)

1	g	FeSO4 x 7 H2O	
1000	ml	H2SO4 (0.1 N)	

1. The ferrous sulfate solution is not stable and should be freshly prepared.