ATCC medium: 1302 Halophilic Clostridium medium
Solution 1:
NaCl...........................................................105.0 g
KCl..............................................................7.5 g
L-glutamic acid (dissolves upon heating of the final mixture)....4.0 g
Yeast extract....................................................2.0 g
Nutrient broth...................................................2.0 g
Casamino acids...................................................2.0 g
FeSO4 . 7H2O .....................................................2.0 mg
Resazurin........................................................1.0 mg
Wolfe's Vitamin Solution (see below)............................10.0 ml
Wolfe's Mineral Solution (see below)............................10.0 ml
2.5 N NaOH......................................................12.5 ml
Distilled water..................................................1.0 L
Solution 2:
MgCl2 . 6H2O ......................20.3 g
CaCl2 . 2H2O .......................7.35 g
Distilled water..................100.0 ml
Dissolve the ingredients in water. Boil Solution 1 under nitrogen until the reasurin turns red, then add 0.5 g L-cysteine . HCl, and continue boiling under nitrogen until the medium is yellow.
(The final volume should be around 900 ml.) Using anaerobic techniques, dispense the medium in 9 ml portions into stoppered serum tubes, cap and autoclave.
Bubble Solution 2 with nitrogen and autoclave. Add 1 ml to each tube of the autoclaved Solution 1.Adjust pH if necessary to 6.2-7.0 using sterile gassed NaOH or HCl.
Note: For solid medium add to Solution 1: 2.0 g soluble starch, 20.0 g Agar, Bacto (BD 214050), and 5.0 g CaCO3, the last compound being added after complete dissolution of the glutamic acid in the presence of the NaOH.
Autoclave and add Solution 2 as instructed above. In the presence of CaCO3, the final pH may be higher than 7.0, but there is no need to adjust it.
2 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
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.