ATCC Medium: 1494 Modified NOS Medium
Heart
Infusion Broth (BD 238400).............................................1.25 g
Trypticase Peptone (BD 211921)..............................................1.0 g
Yeast Extract (BD 211928)........................................................0.25 g
Sodium thioglycollate.................................................................0.05 g
L-Cysteine x HCl........................................................................0.1 g
L-Asparagine..............................................................................0.025 g
D-Glucose..................................................................................0.2 g
Resazurin (0.1% w/v in water)...................................................0.1 ml
DI Water to................................................................................94.0 ml
Combine the above ingredients and bring to a boil in a round-bottom flask while flushing with O2-free gas (10% H2, 10% CO2, 80% N2). Stopper anaerobically with butyl rubber, wire or seal closed, and autoclave at 121?C. After autoclaving, allow to cool, open without admitting oxygen and while flushing with gas, add the following filter-sterilized ingredients:
NaHCO3 (7.5% w/v in water)……………………….....................2.67 ml
TPP/VFA Solution (see below)...................................................2.0 ml
Rabbit serum..............................................................................2.0 ml
TPP-VFA Solution Thiamine pyrophosphate (0.2% w/v in water)............................1.5 ml
VFA Solution (see below)..........................................................1.0 ml
DI Water.....................................................................................8.4 ml
Mix together and filter-sterilize. Store at -20C.
VFA Solution Isobutyric acid.............................................................................0.5 ml
2-Methylbutyric acid....................................................................0.5 ml
Isovaleric acid.............................................................................0.5 ml
Valeric acid.................................................................................0.5 ml
0.1 N NaOH to............................................................................100.0 ml
Add volatile fatty acids to about 70-80 ml of NaOH and mix. Add NaOH to bring final volume to 100 ml. If not used the same day, filter-sterilize and store at 4C.