# 1263: SPIROCHAETA DISSIPATITROPHA MEDIUM


## Main sol. 1263

30	g	NaCl	
0.2	g	KCl	
0.2	g	K2HPO4	
0.1	g	MgCl2 x 6 H2O	
1	g	NH4Cl	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
2.76	g	Na2CO3	
24	g	NaHCO3	
0.5	g	Yeast extract	
5	g	Cellobiose	
2	ml	**Wolin's vitamin solution**	
0.4	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, bicarbonate, yeast extract, cellobiose, vitamins and sulfide), then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add solid carbonate and bicarbonate to the medium, dissolve and adjust to pH 9.4. Dispense medium under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum bottles and autoclave. To the autoclaved medium add yeast extract, cellobiose, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Stock solutions of vitamins and cellobiose should be sterilized by filtration.


## 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

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