To the sterile completed PY medium [ATCC Medium 1524 (see below)], aseptically add the following to the final concentrations indicated: 5% sterile heat-inactivated horse serum and 15 mcg/ml filter-sterilized Nacetyl muramic acid.
ATCC Medium 1524:
Peptone, Bacto (BD 211677).........5.0 g
Trypticase Peptone (BD 211921).....5.0 g
Yeast extract.....................10.0 g
0.025% Resazurin, aqueous..........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
L-Cysteine . HCl...................0.5 g
Distilled water..................950.0 ml
Adjust medium for a final pH of 7.0.
Mix all ingredients except cysteine and bring to boil under 80% N2, 10% H2, 10% CO2.
Add cysteine and readjust pH, if necessary.
Tube medium anaerobically using Hungate methods and the same gas phase.
Autoclave at 121C for 15 minutes.
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
Distilled water....................1.0 L
Mix CaCl2 and MgSO4 in 300.0 ml distilled water until dissolved.
Add 500.0 ml water and the remaining salts while swirling slowly.
Add 200.0 ml distilled water and mix. Store at 4C.
Hemin Solution:
Hemin.............................50.0 mg
N NaOH.............................1.0 ml
Distilled water to...............100.0 ml
Suspend hemin in NaOH; then add water.
Autoclave at 121C for 15 minutes.
Vitamin K1 Solution:
Vitamin K1.........................0.15 ml
95% Ethanol.......................30.0 ml
Store in brown bottle at 4C. Discard after 1 month.