NH4Cl ..........................................0.4 g
NaCl...........................................0.4 g
MgCl2 . 6H2O ...................................0.33 g
CaCl2 . 2H2O ...................................0.05 g
KH2PO4 .........................................1.0 mg
Trace Elements Solution SL-10 (see below)......1.0 ml
Na2SeO3 . 5H2O..................................3.0mcg
Wolfe's Vitamin Solution (see below)..........10.0 ml
Yeast extract..................................2.0 g
Resazurin......................................1.0 mg
Pittsburgh Seam Coal..........................10.0 g
NaHCO3 .........................................3.5 g
Na2S . 9H2O ....................................0.3 g
Distilled water................................1.0 L
Prepare medium anaerobically using Hungate techniques under 80% N2, 20% CO2. Autoclave at 121C for 15 minutes. Final pH 7.3 +/- 0.1.
Trace Elements Solution SL-10:
HCl (25%).........................10.0 ml
FeCl2 . 4H2O .......................1.5 g
ZnCl2 .............................70.0 mg
MnCl2 . 4H2O .....................100.0 mg
H3BO3 ..............................6.0 mg
CoCl2 . 6H2O .....................190.0 mg
CuCl2 . 2H2O .......................2.0 mg
NiCl2 . 6H2O ......................24.0 mg
Na2MoO4 . 2H2O.....................36.0 mg
Distilled water..................990.0 ml
Dissolve FeCl2 in the HCl, dilute with water, add and dissolve the other salts, adjust pH to 6.0 with NaOH, and fill up to 1.0 L.
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