ATCC medium: 2260 Freshwater medium for Geobacter
NH Cl ....................................0.25 g
4 NaH2PO4 ..................................0.06 g
KCl......................................0.1 g
Wolfe's Vitamin Solution (see below)....10.0 ml
Wolfe's Mineral Solution (see below)....10.0 ml
NaHCO3 ...................................2.5 g
Distilled water........................980.0 mL
Degas under 80% N2, 20% CO2. Autoclave at 121C for 15 minutes.
To the autoclaved medium, add the following compounds to achieve the final concentration noted:
FeNTA (Electron Acceptor) (see below), 10 mM Sodium acetate (Electron Donor) (see below), 10 mM
Wolfe's Vitamin Solution:
Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).
Biotin.......................2.0 mg
Folic acid...................2.0 mg
Pyridoxine hydrochloride....10.0 mg
Thiamine . HCl...............5.0 mg
Riboflavin...................5.0 mg
Nicotinic acid...............5.0 mg
Calcium D-(+)-pantothenate...5.0 mg
Vitamin B12..................0.1 mg
p-Aminobenzoic acid..........5.0 mg
Thioctic acid................5.0 mg
Distilled water..............1.0 L
Wolfe's Mineral Solution:
Available from ATCC as a sterile ready-to-use liquid (Trace Mineral Supplement, catalog no. MD-TMS.)
Nitrilotriacetic acid........1.5 g
MgSO4 . 7H2O .................3.0 g
MnSO . H O ..................0.5 g
4 2 NaCl.........................1.0 g
FeSO4 . 7H2O .................0.1 g
CoCl2 . 6H O .................0.1 g
2 CaCl2 ........................0.1 g
ZnSO4 . 7H2O .................0.1 g
CuSO . 5H O .................0.01 g
4 2 AlK(SO4)2 . 12H2O.............0.01 g
H3BO3 ........................0.01 g
Na MoO . 2H O................0.01 g
2 4 2 Distilled water..............1.0 L
Add nitrilotriacetic acid to approximately 500 ml of water and adjust to pH 6.5 with KOH to dissolve the compound.
Bring volume to 1.0 L with remaining water and add remaining compounds one at a time.
1 FeNTA Solution:
NaHCO3 .............................1.64 g
Trisodium nitrilotriacetic acid....2.56 g
FeCl3 . 6H2O .......................2.7 g
Distilled water to...............100.0 ml
Dissolve NaHCO3 in 80 ml of distilled water in a 100-ml flask.
Add trisodium nitriacetic acid; add FeCl3; bring volume to 100 ml; filtersterilize using a prefilter. Do not autoclave.
This is a 100 mM solution. Add approximately 1 ml of solution to 9 ml of medium to achieve 10 mM.
Sodium Acetate Solution:
Use a 100-200 mM solution of sodium acetate as electron donor. Add enough to achieve a final concentration of 10 mM.