Acetogen Medium

[所属分类:培养基配方] [发布时间:2019-9-18] [发布人:] [阅读次数:] [返回]

山东拓普生物工程有限公司

Shandong Tuopu Biol-Engineering Co.,Ltd

Acetogen Medium
Composition per 421.8mL:
NaHCO 3 ........................................................................................2.4g
NH 4 Cl ...........................................................................................0.2g
Yeast extract..................................................................................0.2g
Stock salts solution #1.............................................................40.0mL
Potassium phosphate buffer.....................................................20.0mL
Clarified rumen fluid ...............................................................20.0mL
Stock salts solution #2...............................................................4.0mL
Trace minerals solution..............................................................4.0mL
Vitamin solution.........................................................................4.0mL
Reducing agent solution ............................................................4.0mL
Tungstate solution......................................................................0.4mL
Resazurin (0.1% solution) .........................................................0.4mL
Potassium Phosphate Buffer:
Composition per 830.0mL:
K 2 HPO 4 .....................................................................................15.68g
KH 2 PO 4 .......................................................................................4.72g
Preparation of Potassium Phosphate Buffer:  Dissolve
K 2 HPO 4 in 600.0mL of distilled/deionized water and KH 2 PO 4 in
230.0mL of distilled/deionized water. Mix the two solutions together
and use.
Stock Salts Solution #1:
Composition per liter:
KCl................................................................................................1.6g
NaCl..............................................................................................1.4g
MgSO 4 ·7H 2 O................................................................................0.2g
Preparation of Stock Salts Solution #1:  Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Stock Salts Solution #2:
Composition per liter:
CaCl 2 ·2H 2 O ..................................................................................0.1g
Preparation of Stock Salts Solution #2: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Trace Minerals Solution:
Composition per liter:
Nitrilotriacetic acid.......................................................................1.5g
MgSO 4 ·7H 2 O................................................................................3.0g
MnSO 4 ·H 2 O..................................................................................0.5g
NaCl..............................................................................................1.0g
NiCl 2 ·6H 2 O...................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
CoCl 2 ·6H 2 O ..................................................................................0.1g
CaCl 2 .............................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
Na 2 SeO 3 ·5H 2 O............................................................................0.01g
CuSO 4 ·5H 2 O...............................................................................0.01g
AlK(SO 4 ) 2 ·12H 2 O.......................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O ..........................................................................0.01g
Preparation of Trace Minerals Solution:  Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Bring volume to 1.0L with distilled/deionized water.
Add remaining components. Mix thoroughly.
Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
Ascorbic acid .............................................................................5.0mg
Calcium pantothenate ................................................................5.0mg
Choline chloride.........................................................................5.0mg
Lipoic acid .................................................................................5.0mg
i-Inositol.....................................................................................5.0mg
Niacinamide...............................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Pyridoxal·HCl............................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Thiamine·HCl ............................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Store fro-
zen.
Tungstate Solution:
Composition per liter:
Na 2 WO 4 ·2H 2 O.........................................................................99.0mg
Preparation of Tungstate Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Reducing Agent Solution:
Composition per 110.0mL:
L- Cysteine·HCl·H 2 O......................................................................2.5g
Na 2 S·9H 2 O....................................................................................2.5g
Preparation of Reducing Agent Solution : Add 110.0mL of dis-
tilled/deionized water to a 250.0mL round-bottomed flask. Boil under
N 2 gas for 1 min. Cool to room temperature. Add  L- cysteine·HCl and
dissolve. Adjust to pH 9 with 5N NaOH. Add washed Na 2 S·9H 2 O and
dissolve. Distribute in amounts needed into tubes or flasks. Autoclave
for 10 min at 15 psi pressure–121°C.
Preparation of Medium: Add components, except NaHCO 3 and
reducing agent, to distilled/deionized water and bring volume to
417.8mL. Mix thoroughly. Gently heat and bring to boiling under 80%
N 2 + 20% CO 2 . Cool to 45°–50°C. Add NaHCO 3 and reducing agent.
Distribute into tubes or flasks under 80% N 2 + 20% CO 2 . Autoclave for
15 min at 15 psi pressure–121°C. After inoculation, exchange head-
space with 80% H 2  + 20% CO 2 .
Use: For the cultivation and maintenance of acetogenic anaerobes
such as some Clostridium species.