Alkaliphilic Spirochete Medium

[所属分类:培养基配方] [发布时间:2019-9-27] [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

Alkaliphilic Spirochete Medium
Composition per 1011.0mL:
Na 2 CO 3 .......................................................................................10.0g
NaCl............................................................................................10.0g
NH 4 Cl ...........................................................................................1.0g
K 2 HPO 4 .........................................................................................0.2g
KCl................................................................................................0.2g
Yeast extract..................................................................................0.5g
NaHCO 3  solution .....................................................................50.0mL
Sucrose solution.......................................................................20.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Wolfe’s vitamin solution..........................................................10.0mL
Trace elements solution SL-6 ....................................................1.0mL
pH 9.7 ± 0.2 at 25°C
NaHCO 3 Solution:
Composition per 50.0mL:
NaHCO 3 ......................................................................................15.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 50.0mL. Mix thoroughly. Filter ster-
ilize. Sparge with 100% N 2 .
Sucrose Solution:
Composition per 20.0mL:
Sucrose..........................................................................................5.0g
Preparation of Sucrose Solution:  Add sucrose to distilled/deion-
ized water and bring volume to 20.0mL. 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....................................................................................1.0g
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.
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.
Filter sterilize. Sparge with 100% N 2 .
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 Medium: Prepare and dispense medium under
100% N 2 . Add components, except NaHCO 3  solution, sucrose solu-
tion, Na 2 S·9H 2 O solution, and Wolfe’s vitamin solution, to distilled/de-
ionized water and bring volume to 910.0mL. Mix thoroughly. Adjust
pH to 9.7 with 6N NaOH (about 15.0mL). Gently heat and bring to
boiling. Cool to room temperature while sparging with 100% N 2 . Au-
toclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobi-
cally add 50.0mL of sterile NaHCO 3  solution, 20.0mL of sterile
sucrose solution, 10.0mL of sterile Na 2 S·9H 2 O solution, and 10.0mL
of sterile Wolfe’s vitamin solution. Mix thoroughly. Aseptically dis-
tribute into sterile tubes or flasks.
Use: For the cultivation of Spirochaeta africana, Spirochaeta alkal-
ica, and Spirochaeta asiatica.