Na2HPO4 ................................284.0 mg
KCl.....................................75.0 mg
Wolfe's Mineral Solution (see below).....5.0 ml
Wolfe's Vitamin Solution (see below).....1.0 ml
Resazurin................................1.0 mg
Yeast extract............................5.0 g
Tryptone (BD 211705)....................10.0 g
Distilled water........................850.0 ml
Autoclave basal medium for 15 minutes at 121C. Cool under an atmosphere of 100% N2. Aseptically add Solutions A - D and enough sterile 2N NaOH to give a final pH 10.2 +/- 0.1.
Solution A:
Na2CO3 .......................6.2 g
Distilled water............100.0 ml
Autoclave Solution A under 100% N2 for 15 minutes at 121C.
Solution B:
Glucose......................3.0 g
Distilled water.............20.0 ml
Autoclave Solution B under 100% N2 for 15 minutes at 121C.
Solution C:
L-Cysteine . HCl...........125.0 mg
Distilled water.............10.0 ml
Autoclave Solution C under 100% N2 for 15 minutes at 121C.
Solution D:
Na2S . 9H2O ................125.0 mg
Distilled water.............10.0 ml
Autoclave Solution D under 100% N2 for 15 minutes at 121C. 2 Wolfe's Vitamins: Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).
Folic acid...................2.0 mg
Pyridoxine hydrochloride....10.0 mg
Riboflavin...................5.0 mg
Biotin.......................2.0 mg
Thiamine.....................5.0 mg
Nicotinic acid...............5.0 mg
Pantothenic acid.............5.0 mg
Vitamin B12..................0.1 mg
p-Aminobenzoic acid..........5.0 mg
Thioctic acid................5.0 mg
Distilled water..............1.0 L
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.