# 863: METHANOTHERMOBACTER/METHANOBACTERIUM TRANSDUCTION MEDIUM


## Main sol. 863

100	ml	**Basal salts solution**	
10	ml	**Trace elements solution**	
24	ml	Sodium carbonate solution	
5	ml	**Cysteine solutions**	
5	ml	**Na2S Solution**	
856	ml	Distilled water (degassed)	

1. Anaerobically dispense liquid minimal medium in 10-ml portions into 100-ml serum vials under an atmosphere of N2/CO2 (80:20). Seal vials with butyl rubber stoppers secured with aluminium crimp seals. Sterilize by autoclaving for 15 minutes at 121°C. Prior to inoculation, pressurize bottles to 2 x 102 kPa with H2/CO2. For solidified plating medium, add the following components per liter of liquid minimal medium: 

15	g	Agar	
16	ml	**Titanium (III) citrate solution (Zehnder and Wuhrmann 1976)**	

2. Add titanium(III) citrate solution to warm (60°C) agar medium after autoclaving. For preparation of solidified agar medium.


## Basal salts solution

68	g	KH2PO4	
21.2	g	NH4Cl	
10	mg	Resazurin	
1000	ml	Water (Distilled or deionized)	


## Trace elements solution

9.55	g	Nitrilotriacetic acid (Aldrich N8407)	
4.06	g	MgCl2 x 6 H2O	
0.98	g	FeCl2 x 4 H2O	
0.264	g	Na2WO4 x 6 H2O	
0.118	g	NiCl2 x 6 H2O	
0.1	g	Na2Se2O3	
0.024	g	Na2MoO4 x 2 H2O	
0.023	g	CoCl2 x 6 H2O	
1000	ml	Water (Distilled or deionized)	

1. Adjust pH of nitrilotriacetic acid in solution to 7.0 with KOH. Add minerals and adjust the final pH to 7.0.


##  Na2CO3 solution

106	g	Na2CO3	
1000	ml	Water (degassed)	

1. Dissolve 106 g Na2CO3 in 1 liter of degassed H2O. Autoclave in sealed serum bottles under CO2.


## Cysteine solutions

50	ml	Water	
5	g	L-Cysteine HCl	

1. To 50 ml of boiled and cooled distilled or deionized H2O add 5 g of L-cysteine hydrochloride
. Adjust pH to 10 with 3N NaOH and store under N2 in a serum bottle sealed with a butyl rubber stopper.


## Titanium (III) citrate solution (Zehnder and Wuhrmann 1976)

5	ml	Titanium(III) chloride (Merck 808307, 15% solution)	
50	ml	Sodium citrate (0.2M)	

1. Neutralize pH with a saturated sodium carbonate solution and store in a serum bottle under N2.


## Na2S Solution

50	ml	Water	
5	g	Na2S x 9 H2O	

1. To 50 ml of boiled and cooled distilled or deionized H2O add 5 g of Na2S x 9 H2O. Adjust pH to 10 with 3N NaOH and store under N2 in a serum bottle sealed with a butyl rubber stopper.