# 1267a: MARIPROFUNDUS (ES) MEDIUM


## Main sol. 1267a

901	ml	**Solution A**	
1	ml	**Solution B**	
100	ml	**Solution C**	

1. Preparation of the bottom layer: Mix 1 volume of solution A (except NaHCO3 and agarose) with 1 volume of solution C and add 1% (w/v) agarose type 1, low EEO. After autoclaving, aseptically fill 1.2 ml of the suspension in sterile screw capped tubes (10 ml total volume). The bottom layer solidifies in approx. 30 min.
2. Preparation of the top layer: Add bicarbonate and low melt agarose to solution A and autoclave. Let the sterile solution cool to 40°C and add 10.00 ml/l of solution B. Sparge solution with sterile CO2 gas until a pH of 6.1 - 6.4 is reached. Then, aseptically pipette aliquots of 6.0 ml over the bottom layer of each tube and let medium equilibrate for at least three hours, but not longer than 12 hours.
3. Inoculation: Inoculate the semisolid top layer with a pipette that is inserted just above the bottom layer; the pipette tip is drawn upward as the inoculum is dispensed.


## Solution A

27.5	g	NaCl	
5.38	g	MgCl2 x 6 H2O	
6.78	g	MgSO4 x 7 H2O	
0.72	g	KCl	
1.4	g	CaCl2 x 2 H2O	
1	g	NH4Cl	
0.05	g	K2HPO4	
1	ml	**Wolfe's mineral elixir**	
0.5	g	NaHCO3 (for top layer)	
1.5	g	Agarose (for top layer, low melt)	
900	ml	Distilled water	


## Solution B

1	ml	**Wolin's vitamin solution (10x)**	


## Solution C

100	ml	**Ferrous sulfide sludge**	


## Wolfe's mineral elixir

30	g	MgSO4 x 7 H2O	
5	g	MnSO4 x H2O	
10	g	NaCl	
1	g	FeSO4 x 7 H2O	
1.8	g	CoCl2 x 6 H2O	
1	g	CaCl2 x 2 H2O	
1.8	g	ZnSO4 x 7 H2O	
0.1	g	CuSO4 x 5 H2O	
0.18	g	AlK(SO4)2 x 12 H2O	
0.1	g	H3BO3	
0.1	g	Na2MoO4 x 2 H2O	
2.8	g	(NH4)2Ni(SO4)2 x 6 H2O	
0.1	g	Na2WO4 x 2 H2O	
0.1	g	Na2SeO4	
1000	ml	Distilled water	

1. First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts.


## 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	


## Ferrous sulfide sludge

15.4	g	FeSO4 x 7 H2O	
12.3	g	Na2S x 9 H2O	
100	ml	Distilled water	

1. Heat distilled water to 50°C in a 250 ml beaker with a stir bar present. While rapidly stirring the water, add the ferrous sulfate followed immediately by the sodium sulfide. The formed black FeS sludge is decanted into a glass bottle that can be stoppered. The FeS is allowed to settle for several hours and then the overlying water is decanted and replaced. This procedure is repeated at least five times to wash the FeS. After washing, the pH of the FeS solution should be close to neutrality. The FeS suspension can be kept in closed bottles or tubes under a nitrogen atmosphere for at least three months.