# 611: PYROBACULUM (BS) MEDIUM


## Main sol. 611

125	ml	**Synthetic seawater mix**	
10	ml	**Modified Wolin's mineral solution**	
2	ml	Fe(NH4)2(SO4)2 x 6 H2O (0.01% w/v)	
0.25	g	NH4Cl	
0.07	g	KH2PO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	KNO3	
0.5	g	Yeast extract	
2	g	NaHCO3	
875	ml	Distilled water	

1. Dissolve ingredients (except nitrate, yeast extract and bicarbonate) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Adjust pH to 7.0 and dispense under 100% N2 gas atmosphere into serum vials (e.g., 20 ml medium per 100 ml bottle). Replace gas phase with 80% H2 and 20% CO2 gas mixture and autoclave. Prior to inoculation add nitrate and yeast extract from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 7.0 - 7.2.


## Synthetic seawater mix

47.15	g	NaCl	
18.1	g	MgCl2 x 6 H2O	
7	g	MgSO4 x 7 H2O	
3.13	g	CaCl2 x 2 H2O	
3.24	g	Na2SO4	
1.2	g	KCl	
0.1	g	Na2CO3	
0.1	g	NaBr	
80	mg	KBr	
72	mg	SrCl2 x 6 H2O	
52	mg	H3BO3	
8.1	mg	Na2HPO4	
2.4	mg	NaF	
0.4	mg	Na-silicate	
50	µg	KI	
1000	ml	Distilled water	


## 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.