ATCC medium: 2557 Fresh Water Medium for Geosinus fermentans
NaHCO3, 2.50 g
NH4Cl, 0.25 g
NaH2PO4, 0.06 g
KCl, 0.10 g
Wolfe’s Mineral solution (see below), 10.0 ml
Wolfe’s Vitamin solution (see below), 10.0 ml
Distilled water, 800 ml
After all the ingredients are added bring the final volume up to 1 liter with distilled water. The pH should
be 6.8 to 7.0.
Dispense the medium (10 ml) anaerobically using 80:20 N2:CO2 into pressure resistant test tubes.
Continue gassing out the tubes for several minutes after dispensing. Autoclave the medium at 121?C for 20 minutes.
After autoclaving allow the medium to cool. Add 0.10 ml of Cysteine-HCL (3% stock solution anoxic) and exchange the headspace with 80:20 N2:CO2 or 80:20 H2:CO2.
This strain seems to grow better with H2:CO2 gas mixture. Just before inoculation, add 0.10 ml of glucose (stock solution 2.0
M) and 0.10 ml of acetate (stock solution 2.0 M).
This will give a final concentration of 20mM glucose and 20mM acetate.
Wolfe's Mineral Solution:
Available from ATCC as a sterile ready-to-use liquid (Trace Mineral Supplement, catalog no. MDTMS.)
EDTA, 0.5 g
MgSO4 . 7H2O, 3.0 g
MnSO4 . H2O, 0.5 g
NaCl, 1.0 g
FeSO4 . 7H2O, 0.1 g
CoCl2 . 6H2O, 0.1 g
CaCl2, 0.1 g
ZnSO4 . 7H2O, 0.1 g
CuSO4 . 5H2O, 0.01 g
AlK(SO4)2 . 12H2O, 0.01 g
H3BO3, 0.01 g
Na2MoO4 . 2H2O, 0.01 g
Na2SeO3 (anhydrous), 0.001 g
Na2WO4 2H2O, 0.010 g
NiCl2 6H2O, 0.020 g
Distilled water, 1.0 L
Add EDTA 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.
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