ATCC medium: 1644 Clostridium pfennigii medium
Solution A:
Mineral Solution (See below)..................50.0 ml
SL-10 Trace Elements Solution (See below)......1.0 ml
Yeast extract..................................2.0 g
Sodium vanillate...............................2.0 g
Ferulic acid...................................0.58 g
Wolfe's Vitamin Solution (See below)...........5.0 ml
Resazurin......................................1.0 mg
Rumen fluid..................................300.0 ml
Distilled water..............................540.0 ml
Adjust for final pH of 7.0-7.2. Prepare Solution A using anaerobic techniques under 80% N2, 20% CO2. Sterilize by autoclaving.
Mineral Solution:
KH2PO4 ......................10.0 g
MgCl2 . 6H2O .................6.6 g
NaCl.........................8.0 g
NH4Cl ........................8.0 g
CaCl2 . 2H2O .................1.0 g
Distilled water..............1.0 L
SL-10 Trace Elements Solution: 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 to 1.0 L with distilled water.
2 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
Filter-sterilize and outgas for 20 minutes with the N2, CO2 gas mixture.
Solution C:
L-Cysteine...................0.24 g
Distilled water.............10.0 ml
Autoclave under N2 gas.
Solution D:
Na2S . 9H2O .................78.0 mg
Distilled water..............1.0 ml
Autoclave under N2 gas. Aseptically combine Solutions A through D under 80% N2, 20% CO2. Final pH of complete medium should be 7.0-7.2.