ATCC Medium: 1869 PRAS-PYG w/Tween 80
Peptone……………………………………..5.0 g
Trypticase…………………………………...5.0 g
Yeast extract………………………………..10.0 g
0.025% Resazurin…………………………4.0 ml
Salt solution (see below)………………….40.0 ml
Hemin solution (see below)……………....10.0 ml
Vitamin K1 solution (see below)………….0.2 ml
Glucose……………………………………..10.0 g
Tween 80……………………………………0.25 ml
L-Cysteine HCl x H2O………..……………0.5 g
DI Water……………………………………..945.55 ml
Boil medium without glucose, tween 80, and cysteine. Cool under 97% N2, 3%H2 and add glucose, tween 80, and cysteine. Adjust pH to 7.0. Dispense anaerobically under same gas phase. Autoclave at 121?C.
Salts Solution CaCl2………………………………………….0.2 g
MgSO4………………………………………...0.2 g
K2HPO4……………………………………….1.0 g
KH2PO4……………………………………….1.0 g
NaHCO3………………………………………10.0 g
NaCl…………………………………………...2.0 g
DI Water……………………………………….1000 ml
Mix calcium chloride and magnesium sulfate in 300 ml of distilled water until dissolved. Add 500 ml of distilled water and add the remaining salts while swirling slowly. Add 200 ml of distilled water, mix, and store at 4?C.
Hemin Solution Hemin…………………………………………..50.0 mg
1N NaOH………………………………………1.0 ml
DI Water………………………………………..99 ml
Dissolve hemin in NaOH and raise volume to 100ml with distilled water.
Vitamin K1 Solution
Vitamin K1………………………………………0.15 ml
95% Ethanol…………………………………….30.0 ml
Dissolve Vitamin K1 in ethanol. Store in brown bottle and keep refrigerated. Discard after one month.