# 946: NAUTILIA MEDIUM


## Main sol. 946

500	ml	**Synthetic seawater (2 x conc.)**	
0.33	g	NH4Cl	
0.33	g	KH2PO4	
10	ml	**Modified Wolin's mineral solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur (powdered)	
1.5	g	Na2CO3	
3	g	Na-formate	
1	ml	**Wolin's vitamin solution (10x)**	
0.6	g	Na2S x 9 H2O	
500	ml	Distilled water	

1. Dissolve ingredients (except sulfur, carbonate, formate, vitamins and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials that contain already the appropriate amount of sulfur. For sterilization heat cultivation vessels for 1 - 2 hours to 90 - 100°C in a water bath on each of three successive days. Prior to inoculation add carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere and formate, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins are sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary.


## 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.


## Synthetic seawater (2 x conc.)

55.4	g	NaCl	
0.03	g	SrCl2 x 6 H2O	
14	g	MgSO4 x 7 H2O	
0.1	mg	KI	
11	g	MgCl2 x 6 H2O	
20	mg	Na3-citrate	
1.3	g	KCl	
1.5	g	CaCl2 x 2 H2O	
0.2	g	NaBr	
0.06	g	H3BO3	
1000	ml	Distilled water	


## 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