NH4Cl ....................................1.0 g
KH2PO4 ...................................0.33 g
K2HPO4 ...................................0.45 g
MgSO4 . 7H2O .............................0.1 g
Wolfe's Mineral Solution (see below)....20.0 ml
Wolfe's Vitamin Solution (see below)....20.0 ml
Yeast extract............................2.0 g
Fructose (see below)....................10.0 g
Resazurin................................1.0 mg
NaHCO3 ..................................10.0 g
L-Cysteine . HCl.........................0.5 g
Na2S . 9H2O ..............................0.5 g
Distilled water..........................1.0 L
Mix all of the ingredients except of fructose. Equilibrate medium with 80% N2, 20% CO2 gas mixture until a pH of 7.4 is reached. Tube and autoclave under the same environment. Before inoculation adjust the pH to 8.2 by adding sterile anaerobic Na2CO3 (0.25 ml of 5% Na2CO3 per 10 ml medium) and add sterile solution of fructose to a final concentration of 1%. For autotrophic growth omit fructose and use a gas atmosphere of 80% H2, 20% CO2.
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
2 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.