KoKo Medium

[所属分类:培养基配方] [发布时间:2021-4-25] [发布人:李佳颐] [阅读次数:] [返回]

KoKo Medium

山东拓普生物工程有限公司 培养基配方 http://www.topbiol.com

Composition per 1020.0mL:

K 2 HPO 4 .........................................................................................1.6g
NaH 2 PO 4 ·2H 2 O.............................................................................1.0g
Peptone, meat................................................................................1.0g
Pancreatic digest of casein............................................................1.0g
Yeast extract..................................................................................1.0g
NH 4 Cl ...........................................................................................0.5g
MgSO 4 ·6H 2 O..............................................................................0.16g
Resazurin ...................................................................................0.5mg
Glucose solution ....................................................................100.0mL
NaHCO 3  solution .....................................................................10.0mL
CaCl 2 ·2H 2 O solution................................................................10.0mL
L -Cysteine·HCl·H 2 O solution ..................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Trace elements solution SL-4 ..................................................10.0mL
Wolfe’s vitamin solution..........................................................10.0mL
pH 7.0 ± 0.2 at 25°C
Glucose Solution:
Composition per 100.0mL:
D -Glucose......................................................................................5.0g
Preparation of Glucose Solution:  Add glucose to distilled/deion-
ized water and bring volume to 100.0mL. Mix thoroughly. Sparge with
100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
NaHCO 3 Solution:
Composition per 10.0mL:
NaHCO 3 ........................................................................................1.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
CaCl 2 Solution:
Composition per 10.0mL:
CaCl 2 ·2H 2 O.................................................................................0.06g
Preparation of CaCl 2 Solution:  Add CaCl 2 ·2H 2 O to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
L -Cysteine·HCl·H 2 O Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O.....................................................................0.3g
Preparation of  L -Cysteine·HCl·H 2 O Solution:  Add  L -
cysteine·HCl·H 2 O to distilled/deionized water and bring volume to
10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min
at 15 psi pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.3g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Before use, neutralize to pH 7.0 with sterile HCl.
Trace Elements Solution SL-4:
Composition per liter:
EDTA............................................................................................0.5g
FeSO 4 ·7H 2 O................................................................................. 0.2g
Trace elements solution SL-6................................................100.0mL
Trace Elements Solution SL-6:
Composition per liter:
MnCl 2 ·4H 2 O .................................................................................0.5g
H 3 BO 3 ...........................................................................................0.3g
CoCl 2 ·6H 2 O..................................................................................0.2g
ZnSO 4 ·7H 2 O................................................................................ 0.1g
Na 2 MoO 4 ·2H 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.
Preparation of Trace Elements Solution SL-4:  Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Filter sterilize.
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Riboflavin..................................................................................5.0mg
Thiamine·HCl ............................................................................5.0mg
Calcium  DL -pantothenate...........................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Wolfe’s Vitamin Solution:  Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N 2 . Filter sterilize.
Preparation of Medium: Prepare and dispense medium under
100% N 2 . Add components, except glucose solution, NaHCO 3  solu-
tion, CaCl 2 ·2H 2 O solution,  L -cysteine·HCl·H 2 O solution, Na 2 S·9H 2 O
solution, and Wolfe’s vitamin solution, to distilled/deionized water and
bring volume to 850.0mL. Mix thoroughly. Adjust pH to 7.0. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Asep-
tically and anaerobically add 100.0mL of sterile glucose solution,
10.0mL of sterile NaHCO 3  solution, 10.0mL of sterile CaCl 2 ·2H 2 O so-
lution, 10.0mL of sterile  L -cysteine·HCl·H 2 O solution, 10.0mL of ster-
ile Na 2 S·9H 2 O solution, and 10.0mL of sterile Wolfe’s vitamin
solution. Mix thoroughly. The pH should be 7.0. A buffer solution of
1% MOPS from a 10% anaerobic solution at pH 6.9–7.0 may be added
aseptically and anaerobically to enhance the buffer capacity of the me-
dium. Aseptically and anaerobically distribute into sterile tubes or bot-
tles.
Use: For the cultivation of Thermoanaerobacter italicus.