# 892: METHANOCALCULUS PUMILUS MEDIUM


## Main sol. 892

0.4	g	KH2PO4	
0.9	g	NH4Cl	
0.36	g	MgCl2 x 6 H2O	
10	g	NaCl	
0.5	g	Na-acetate	
2	g	Yeast extract (OXOID)	
2	g	Trypticase peptone (BD BBL)	
10	ml	**Trace element solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
5	g	Na2CO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	Na2S x 9 H2O	
0.5	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, vitamins, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve carbonate, adjust pH to 7.2 then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 7.3 - 7.5, if necessary.
2. After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1.5 - 2 bar overpressure.


## Trace element solution

12.8	g	Nitrilotriacetic acid (NTA)	
1	g	FeCl2 x 4 H2O	
0.1	g	MnCl2 x 4 H2O	
0.03	g	CoCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnCl2	
0.02	g	CuCl2	
0.01	g	H3BO3	
0.03	g	Na2MoO4 x 2 H2O	
0.1	g	NiCl2 x 6 H2O	
1	g	NaCl	
0.03	g	Na2SeO3 x 5 H2O	
0.04	g	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml.


## 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