Acetivibrio cellulolyticus 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

Acetivibrio cellulolyticus Medium

Composition per 1170.0mL:
Cellobiose or cellulose (MN 300, Whatman
CF II, Kleenex tissue paper,
or HCl-treated cotton).............................................................3.0g
NaHCO 3 ........................................................................................2.0g
L -Cysteine·HCl............................................................................0.25g
Na 2 S·9H 2 O..................................................................................0.25g
FeSO 4 ·7H 2 O................................................................................0.02g
Resazurin ..................................................................................0.001g
Mineral solution 1....................................................................75.0mL
Mineral solution 2....................................................................75.0mL
Cellobiose solution ..................................................................50.0mL
Trace elements solution ...........................................................10.0mL
Vitamin solution.......................................................................10.0mL
Reducing agent solution...........................................................10.0mL
pH 7.2 ± 0.2 at 25°C
Mineral Solution 1:
Composition per liter:
K 2 HPO 4 .........................................................................................3.9g
Preparation of Mineral Solution 1: Add K 2 HPO 4 to distilled/de-
ionized water and bring volume to 1.0L. Mix thoroughly.
Mineral Solution 2:
Composition per liter:
(NH 4 ) 2 SO 4 .....................................................................................6.0g
K 2 HPO 4 .........................................................................................2.4g
MgSO 4 ·7H 2 O................................................................................1.2g
CaCl 2 ·2H 2 O.................................................................................0.72g
NaCl............................................................................................0.59g
Preparation of Mineral Solution 2: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Cellobiose Solution:
Composition per 50.0mL:
D- Cellobiose..................................................................................5.0g
Preparation of Cellobiose Solution: Add cellobiose to distilled/
deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge
under 100% N 2 gas for 3 min. Filter sterilize. Store under N 2 gas.
Trace Elements Solution:
Composition per liter:
MgSO 4 ·7H 2 O................................................................................3.0g
Nitrilotriacetic acid.......................................................................1.5g
CaCl 2 ·2H 2 O..................................................................................1.0g
NaCl..............................................................................................1.0g
MnSO 4 ·2H 2 O................................................................................0.5g
CoSO 4 ·7H 2 O...............................................................................0.18g
ZnSO 4 ·7H 2 O...............................................................................0.18g
FeSO 4 ·7H 2 O .................................................................................0.1g
NiCl 2 ·6H 2 O...............................................................................0.025g
KAI(SO 4 ) 2 ·12H 2 O......................................................................0.02g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O..........................................................................0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Trace Elements Solution : Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add distilled/deionized water to 1.0L. Add remain-
ing components. Mix thoroughly. Adjust pH to 7.0 with 1N KOH.
Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
Calcium  DL -pantothenate...........................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
p-Aminobenzoic acid.................................................................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.
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 flask. Boil under N 2 gas for 1 min. Cool
to room temperature. Add  L -cysteine·HCl·H 2 O and dissolve. Adjust to pH 9
with 5N NaOH. Add washed Na 2 S·9H 2 O and dissolve. Distribute under N 2
gas in 10.0mL volumes into tubes. Autoclave for 10 min at 15 psi pressure–
121°C.
Preparation of Medium: Add components, except cellobiose solu-
tion and reducing agent solution, to distilled/deionized water and bring
volume to 940.0mL. Gently heat and bring to boiling. Continue boiling
for 3 min. Cool to room temperature under 80% N 2 + 20% CO 2 . Adjust
pH to 7.6 by gassing. Distribute anaerobically under 80% N 2 + 20%
CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. After autoclav-
ing, the pH of the medium will be 7.2. Prior to inoculation of cultures,
aseptically and anaerobically add 0.1mL of sterile reducing agent solu-
tion and 0.5mL of sterile cellobiose solution to each tube containing
9.4mL of sterile basal medium.
Use: For the cultivation and maintenance of Acetivibrio cellulolyticus.