# 766: THERMOCOCCUS CHITINOPHAGUS MEDIUM


## Main sol. 766

0.5	g	(NH4)2SO4	
0.2	g	NaHCO3	
0.5	g	KH2PO4	
0.15	mg	Na2WO4 x 2 H2O	
0.15	mg	Na2SeO4	
0.3	mg	(NH4)2Ni(SO4)2	
4	g	**Chitin stock solution**	
0.5	g	Na2S x 9 H2O	
15	ml	**Modified Wolin's mineral solution II**	
1	mg	Resazurin	
485	ml	**Artifical sea water** (synthetic)	
500	ml	Distilled water	

1. Prepare the medium without Na2S x 9 H2O and NaHCO3, boil and cool under N2. Add the NaHCO3 to the cooled medium and adjust the pH to 6.7. Dispense into Hungate tubes or serum bottles under N2 and autoclave. Reduce the medium before use from a sterile stock solution of neutralised Na2S x 9 H2O.


## Chitin stock solution

20	g	Chitin	
200	ml	HCl (37 %)	
1000	ml	Distilled water	

1. The chitin solution is prepared as follows: mix 20 g chitin (practical grade from crab shells) with 200 ml of 37% HCl (pre-cooled to 4°C) and stir for 1 hr at 4°C. Pour the suspension into 1 litre of distilled water (pre-cooled to 4°C) and filter through filter paper (Schleicher and Schüll Nr. 311853). Wash the residue five times with 500 ml distilled water and resuspend in 1 litre of distilled water. Neutralise the suspension with 10 ml of 5M KOH (final pH 6.5). Filter and wash with 3 litres of distilled water to remove KCl.


## Artifical sea water

23.47	g	NaCl	
3.92	g	Na2SO4	
10.64	g	MgCl2 x 6 H2O	
1.1	g	CaCl2	
192	mg	NaHCO3	
664	mg	KCl	
96	mg	KBr	
26	mg	H3BO3	
24	mg	SrCl2	
3	mg	NaF	
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.