# 700: ALKALIPHILIC SPIROCHAETE MEDIUM


## Main sol. 700

10	g	Na2CO3	
15	g	NaHCO3	
10	g	NaCl	
0.2	g	K2HPO4	
1	g	NH4Cl	
0.2	g	KCl	
1	g	Na2S x 9 H2O	
0.5	g	Yeast extract	
5	g	Sucrose	
10	ml	**Wolin's vitamin solution**	
1	ml	**Modified Wolin's mineral solution II**	
1000	ml	Distilled water	

1. Prepare the medium under N2, omitting Na2CO3, Na HCO3, Na2S, sucrose and vitamin solution.
2. Boil the medium and cool under nitrogen, add the Na2CO3 and NaHCO3 and adjust the pH to 9.7 with 6N NaOH (about 15 ml). Sterilize by autoclaving and add the Na2S (neutralized), vitamins and sucrose from sterile stock solutions.


## Wolin's vitamin solution

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 D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Modified Wolin's mineral solution II

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	
0.025	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 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.