ATCC medium: 1257 ETSA medium Trypticase
Soy Agar (BD 211043)...40.0 g
Yeast extract......................1.0 g
Agar...............................4.0 g
KNO3 ...............................0.5 g
Sodium lactate, 60% syrup..........1.3 ml
Sodium succinate...................0.5 g
Sodium formate.....................0.5 g
Hemin Solution (see below).........1.0 ml
Distilled water..................924.0 ml
Autoclave the above solution at 121C for 15 minutes. Cool to 55C. Aseptically add the following, freshly prepared, filter-sterilized solutions in the order listed:
Menadione Solution (see below).....2.0 ml
4% L-Cysteine . HCl...............10.0 ml
0.5% Dithiothreitol (DTT).........10.0 ml
10% Glucose.......................10.0 ml
1% Sodium fumarate.................2.0 ml
4% Na2CO3 .........................10.0 ml
Defibrinated sheep blood..........30.0 ml
This medium solidifies very quickly and should be maintained at 50-55C while dispensing. Aseptically tube the sterile completed medium under an anaerobic atmosphere of 80% N2, 10% CO2, 10% H2. Plug the tubes with butyl rubber stoppers. A note of caution: Hydrogen gas can be explosive in the concentration used in preparing this medium. Gas tanks should be equipped with spark arrestors.
Hemin Solution: KOH................................1.12 g
95% Ethanol......................100.0 ml
Hemin............................200.0 mg
Distilled water..................100.0 ml
Dissolve KOH in water. Add ethanol and hemin.
Menadione Solution:
Menadione (Vitamin K3)............50.0 mg
95% Ethanol.......................50.0 ml
Distilled water...................50.0 ml
Dissolve menadione in ethanol; then add water. Filter-sterilize solution.