ATCC medium: 1871 Acetohalobium medium
NaCl................................... 150.0 g
MgCl2 . 6H2O ............................. 4.0 g
KCl...................................... 0.33 g
NH4Cl .................................... 0.33 g
KH2PO4 ................................... 0.33 g
CaCl2 . 2H2O ............................. 0.33 g
Wolfe's Mineral Solution (see below).... 10.0 ml
Wolfe's Vitamin Solution (see below).... 10.0 ml
Yeast extract............................ 0.05 g
Trimethylamine . HCl *................... 2.4 g
Resazurin................................ 1.0 mg
NaHCO3 ................................... 4.0 g
Na2S . 9H2O .............................. 0.5 g
Distilled water.......................... 1.0 L
Add all components except sodium bicarbonate and sodium sulfide to distilled water. Bring to boiling and cool in an atmosphere of 80% N 2, 20% CO2. Add sodium bicarbonate and equilibrate under same gas phase for 5 minutes. Add sodium sulfide and allow medium to reduce. Adjust medium, if necessary, for final pH 7.8 +/- 0.2 with sodium carbonate. Tube anaerobically under same gas phase. Autoclave at 121C for 15 minutes. *Glycinebetaine may be substituted for trimethylamine at a concentration of 4.0 g/L. Wolfe's Vitamins: Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no.
MD-VS). mg
Folic acid................... 2.0
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.) g
Nitrilotriacetic acid........ 1.5
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.