Sodium citrate.................................2.94 g
NH4Cl ..........................................0.53 g
KH2PO4 .........................................1.36 g
MgCl2 . 6H2O ...................................2.03 g
CaCl2 . 2H2O ...................................0.15 g
Modified Wolfe's Minerals (see below).........10.0 ml
Wolfe's Vitamin Solution (see below)..........10.0 ml
NaHCO3 .........................................2.5 g
Na2S . 9H2O ....................................0.24 g
Distilled water................................1.0 L
Add all components except sodium bicarbonate and sodium sulfide. Boil and cool medium under 90% N2, 10% CO2.
Add bicarbonate; add sodium sulfide. Adjust medium for final pH 7.5-7.7. Tube under 90% N2, 10% CO2 gas phase using anaerobic techniques.
Autoclave at 121C for 15 minutes.
Modified Wolfe's Minerals:
Na2SeO3 .........................................10.0 mg
NiCl2 . 6H2O ....................................10.0 mg
Na2WO4 . 2H2O....................................10.0 mg
Wolfe's Mineral Solution (see below)............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
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
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.
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