TSD-268 ™
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Grow in recommended media (preparation information listed below), at 35°C, in oxygen free nitrogen, and incubate for 5 to 7 days.
The basal medium used consisted of (1 Litter of distilled water): 0.5 g K2HPO4, 0.5 g KH2PO4, 0.5 g NH4Cl, 0.4 g KCl, 0.05 g CaCl2 . 2H2O, 0.3 g MgCl2 . 6H2O, 0.4 g NaCl, 0.25 g HCl-Cysteine, 1 ml trace element mineral solution (composition below) and 1 ml resazurin 0.1% (w/v). 30g of Fastidious Anaerobic Broth and 2g of Tryptone were added to the medium and the pH is adjusted to 6.5.
The medium then was heated to boiling and until the medium turn colorless. The medium is t then cooled at room temperature under a stream of O2-free nitrogen. The medium is finally sterilized by autoclaving for 20 min at 121 ℃.
After autoclaving, solutions of NaHCO3 (10%) and Na2S.9H2O (2%; pH adjusted) were autoclaved separately and added to the medium (20 mL each) at room temperature. The medium was also supplemented with 1 ml of a filter sterilized (0.22 μm) vitamin solution (composition below). The final pH after adding solutions should be to pH 7.0±0.1.
Element mineral solution composition (Quantity for 1000 ml)
HCl (37%): 6.7 ml
FeCl2. 4H2O*: 1500 mg
H3BO3 3: 6 mg
MnCl2. 4H2O: 100 mg
CoCl2. 6H2O: 190 mg
ZnCl2: 70 mg
NiCl2. 6H2O: 24 mg
CuCl2. 2H2O: 2 mg
Na2MoO4: 36 mg
Vitamin solution composition (Quantity for 100 ml)
Para-amino-benzoic acid: 25 mg
D-biotin: 10 mg
Cyanocobalamin: 0.5 mg
Thiamine-HCl: 25 mg
Riboflavin: 25 mg
Pyridoxal-HCl: 50 mg
DL-pantothenate de calcium: 25 mg
Nicotinic acid (niacine): 25 mg
Folic acid: 10 mg
Lipoïc acid: 25 mg
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