ATCC medium: 1770 Clostridium halophilum medium
Solution 1: NaCl..........................................60.0 g
MgSO4 . 7H2O ...................................5.0 g
NH4Cl ..........................................1.0 g
CaCl2 . 2H2O ..................................25.0 mg
Trace Elements Solution SL-10 (see below)......1.0 ml
Wolfe's Vitamin Solution (see below)..........10.0 ml
Na2SeO3 . 5H2O.................................15.0 mcg
Resazurin......................................1.0 mg
Betaine........................................5.86 g
Yeast extract..................................1.0 g
L-Alanine......................................2.2 g
Distilled water..............................900.0 ml
Solution 2:
NaHCO3 .............................5.0 g
Distilled water...................80.0 ml
Bubble under N2 for 20 minutes; add to Solution 1.
Solution 3:
K2HPO4 ...........................358.0 mg
KH2PO4 ...........................223.0 mg
Distilled water...................10.0 ml
Autoclave under N2.
Solution 4:
Na2S . 9H2O ......................300.0 mg
Distilled water...................10.0 ml
Autoclave under N2. Boil and cool Solution 1 under 80% N2, 20% CO2. Add Solution 2. Adjust to pH 8.3 +/- 0.1, tube and autoclave under 80% N2, 20% CO2 gas phase.
Add Solutions 3 and 4 aseptically to cooled, sterile medium. Trace Elements
Solution SL-10:
HCl (25%).........................10.0 ml
FeCl2 . 4H2O .......................1.5 g
ZnCl2 .............................70.0 mg
MnCl2 . 4H2O .....................100.0 mg
H3BO3 ..............................6.0 mg
CoCl2 . 6H2O .....................190.0 mg
CuCl2 . 2H2O .......................2.0 mg
NiCl2 . 6H2O ......................24.0 mg
Na2MoO4 . 2H2O.....................36.0 mg
Distilled water..................990.0 ml
Dissolve FeCl2 in the HCl, dilute with water, add and dissolve the other salts; adjust pH to 6.0 with NaOH, and fill to 1.0 L with distilled water. Wolfe's Vitamin Solution:
Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).
Biotin.............................2.0 mg
Folic acid.........................2.0 mg
Pyridoxine hydrochloride..........10.0 mg
Thiamine . HCl.....................5.0 mg
Riboflavin.........................5.0 mg
Nicotinic acid.....................5.0 mg
Calcium D-(+)-pantothenate.........5.0 mg
Vitamin B12........................0.1 mg
p-Aminobenzoic acid................5.0 mg
Thioctic acid......................5.0 mg
Distilled water....................1.0 L