ATCC medium: 2255 Hydantoin minimal medium
Glucose, 40% Solution...................25.0 ml
10X M9 Salts Solution (see below)......100.0 ml
Wolfe's Minerals Solution (see below)...10.0 ml
MgCl2, 1M solution .....................200.0 mcl
CaCl2, 1M solution .....................200.0 mcl
Hydantoin, 4% solution.................250.0 ml
Agar, if required.......................20.0 g
Distilled deionized water..............624.0 ml
Prepare the 6 solutions, autoclave separately, and combine aseptically.
Add sterile water. If agar medium is required, add the agar to the water, boil, autoclave separately, and add aseptically.
10X M9 Salts Solution:
Na2HPO4 .....................60.0 g
KH2PO4 ......................30.0 g
NH4Cl .......................10.0 g
NaCl.........................5.0 g
Distilled deionized water....1.0 L
Adjust to pH 7.4 Wolfe's Mineral Solution:
Available from ATCC as a sterile ready-to-use liquid (Trace Mineral Supplement, catalog no. MD-TMS).
Nitrilotriacetic acid........1.5 g
MgSO4 . 7H2O .................3.0 g
MnSO4 . H2O ..................0.5 g
NaCl.........................1.0 g
FeSO4 . 7H2O .................0.1 g
CoCl2 . 6H2O .................0.1 g
CaCl2 ........................0.1 g
ZnSO4 . 7H2O .................0.1 g
CuSO4 . 5H2O .................0.01 g
AlK(SO4)2 . 12H2O.............0.01 g
H3BO3 ........................0.01 g
Na2MoO4 . 2H2O................0.01 g
Distilled water..............1.0 L
Add nitrilotriacetic acid to approximately 500 ml of water and adjust to pH 6.5 with KOH to dissolve the compound. Bring volume to 1.0 L with remaining water and add remaining compounds one at a time.