Haloanaerobium praevalens Medium

[所属分类:培养基配方] [发布时间:2021-3-24] [发布人: ] [阅读次数:] [返回]

Haloanaerobium praevalens Medium

培养基配方

Composition per liter:
NaCl..........................................................................................130.0g
Agar ............................................................................................20.0g
Yeast extract..................................................................................2.0g
Pancreatic digest of casein............................................................2.0g
NH 4 Cl ...........................................................................................0.5g
MgSO 4 ·7H 2 O................................................................................0.5g
K 2 HPO 4 .......................................................................................0.35g
CaCl 2 ·2H 2 O.................................................................................0.25g
KH 2 PO 4 .......................................................................................0.23g
FeSO 4 ·7H 2 O...............................................................................2.0mg
NaHCO 3 solution.....................................................................20.0mL
L -Cysteine-sulfide reducing agent...........................................20.0mL
Wolfe’s vitamin solution..........................................................10.0mL
Methanol..................................................................................10.0mL
Resazurin (0.025% solution)......................................................4.0mL
Trace elements solution SL-6 ....................................................3.0mL
pH 6.8 ± 0.2 at 25°C
NaHCO 3 Solution:
Composition per 20.0mL:

NaHCO 3 .................................................................................850.0mg

Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Gas with 100% CO 2 for 20 min.
L -Cysteine-Sulfide Reducing Agent:
Composition per 20.0mL:
L- Cysteine·HCl·H 2 O .....................................................................0.3g
Na 2 S·9H 2 O....................................................................................0.3g
Preparation of  L -Cysteine-Sulfide Reducing Agent: Add  L- cysteine·HCl·H 2 O to 10.0mL of distilled/deionized water. Mix thoroughly. In a separate tube, add Na 2 S·9H 2 O to 10.0mL of distilled/deionized water. Mix thoroughly. Gas both solutions with 100% N 2 and cap tubes. Autoclave both solutions for 15 min at 15 psi pressure–121°C using fast exhaust. Cool to 50°C. Aseptically combine the two solutions under 100% N 2 .
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
Thiamine·HCl............................................................................5.0mg
Riboflavin..................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Calcium pantothenate ................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Thioctic acid..............................................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Cyanocobalamin.....................................................................100.0μg
Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.
Trace Elements Solution SL-6:
Composition per liter:
H 3 BO 3 ...........................................................................................0.3g
CoCl 2 ·6H 2 O..................................................................................0.2g
ZnSO 4 ·7H 2 O.................................................................................0.1g
MnCl 2 ·4H 2 O ...............................................................................0.03g
Na 2 MoO 4 ·H 2 O............................................................................0.03g
NiCl 2 ·6H 2 O.................................................................................0.02g
CuCl 2· 2H 2 O.................................................................................0.01g
Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.
Preparation of Medium: Add components, except NaHCO 3 solution,  L -cysteine-sulfide reducing agent, Wolfe’s vitamin solution, and methanol, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N 2 + 20% CO 2 . Aseptically and anaerobically add the sterile NaHCO 3 solution, the sterile  L -cysteine-sulfide reducing agent, the sterile Wolfe’s vitamin solution, and filter-sterilized methanol. Mix thoroughly. Adjust pH to 6.8. Aseptically and anaerobically distribute into sterile tubes or flasks.
Use:  For the cultivation and maintenance of Haloanaerobium praevalens.