ATCC Medium: 5608 PY Medium (M2-VPI) With D-Glucose w/ Durham Tube
D-Glucose…………………………………………….10.0g
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
Cysteine HCl x H2O..................................................0.5 g
DI Water………………………………………………..950.0 ml
Adjust medium for final pH 7.0 +/- 0.2. Prepare medium anaerobically using a gas phase of 80% N2, 10% CO2 and 10% H2. Add durham tubes and purge air by autoclaving at 110?C for 1-3 minutes.
***** For stabs, do not use durham tubes.
Hemin Solution
Hemin…………………………………………………..50.0 mg
1N NaOH……………………………………………….1.0 ml
DI Water………………………………………………...100.0 ml
Dissolve hemin in NaOH. Add DI Water. Sterilize by autoclaving at 121?C.
Vitamin K1 Solution
Vitamin K1 (2-Methyl-3-phytl-1,4-naphthoquinine)…0.15 ml
95% Ethanol……………………………………………30.0 ml
Refrigerate Vitamin K1 in a brown bottle. Discard after one month.
Salt 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, magnesium sulfate and sodium chloride in 300 ml of distilled water. Mix di- and monopotassium phosphates in 200 ml of distilled water. Dissolve sodium bicarbonate in 500 ml of distilled water. Combine the 3 solutions. Store at 4?C.