Carbonate-Buffered Medium CMB4 with Glucose

[所属分类:培养基配方] [发布时间:2020-4-7] [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

Carbonate-Buffered Medium CMB4 with Glucose

培养基配方
Composition per 1002.0mL:
NaCl............................................................4.0g
NaHCO3 .........................................................2.5g
Glucose ........................................................1.0g
MgCl2 ·6H2O....................................................0.8g
KCl.............................................................0.5g
NH4Cl ..........................................................0.3g
KH2PO4 .........................................................0.2g
Resazurin .....................................................1.0mg
Modified Wolfe’s mineral solution............................10.0mL
Wolfe’s vitamin solution.....................................10.0mL
Sulfide-calcium solution.......................................2.0mL
pH 6.9 ± 0.2 at 25°C
Modified Wolfe’s Mineral Solution:
Composition per liter:
MgSO4 ·7H2O....................................................3.0g
Nitrilotriacetic acid...........................................1.5g
NaCl............................................................1.0g
MnSO4 ·H2O.....................................................0.5g
CaCl2 ..........................................................0.1g
CoCl2 ·6H2O....................................................0.1g
FeSO4 ·7H2O....................................................0.1g
ZnSO4 ·7H2O....................................................0.1g
AlK(SO4)2 ·12H2O..............................................0.01g
CuSO4 ·5H2O...................................................0.01g
H3BO3 .........................................................0.01g
Na2MoO4 ·2H2O.................................................0.01g
Na2SeO3 .......................................................0.01g
NaWO4 ·2H2O ..................................................0.01g
NiC12 ·6H2O...................................................0.01g
Preparation of Modified Wolfe’s Mineral Solution: Add
nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH
to 6.5 with KOH. Add remaining components one at a time. Add dis-
tilled/deionized water to 1.0L. Adjust pH to 6.8.
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.
Sulfide-Calcium Solution:
Composition per liter:
Na2S·9H2O.....................................................36.0g
CaCl2 ·2H2O ..................................................15.0g
Preparation of Sulfide-Calcium Solution: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Adjust pH to 7.2. Sparge with 100% N2 . Anaerobically distribute into
tubes. Autoclave at 121°C for 15 min.
Preparation of Medium: Prepare and dispense medium under 80%
N2 + 20% CO2 . Add components, except NaHCO3 and sulfide-calcium
solution, to distilled/deionized water and bring volume to 1.0L. Mix
thoroughly. Gently heat and bring to boiling. Continue boiling for 3
min. Cool to room temperature while sparging with 80% N2 + 20%
CO2 . Add NaHCO3 . Mix thoroughly. Anaerobically distribute 10.0mL
volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–
121°C. Aseptically and anaerobically add 0.02mL of sterile sulfide-
calcium solution to each tube. Mix thoroughly. Adjust pH to 6.9.
Use: For the cultivation of Spirochaeta thermophila.