Clostridium thermocellum Medium

[所属分类:培养基配方] [发布时间:2020-5-15] [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

Clostridium thermocellum Medium

培养基配方
Composition per liter:
Filter paper..................................................................18.75g
Na2HPO4 ·12H2O...............................................................4.2g
Yeast extract.................................................................2.0g
KH2PO4 .......................................................................1.5g
NH4Cl ........................................................................0.5g
MgCl2 ·6H2O ................................................................0.18g
Reducing solution...........................................................40.0mL
Wolfe’s modified mineral elixir.............................................5.0mL
Resazurin (0.1% solution) ...................................................1.0mL
Vitamin solution.............................................................0.5mL
Caution: This medium contains Na2S, and H2S production will occur,
especially upon prolonged boiling. H2S is hazardous and preparation of
this medium should be done in a chemical fume hood.
Reducing Solution:
Composition per 200.0mL:
L -Cysteine·HCl·H2O.....................................................2.5g
Na2S·9H2O................................................................2.5g
NaOH (0.2N solution)...................................................200.0mL
Preparation of Reducing Solution: Gently heat the NaOH solu-
tion and bring to boiling. Gas with 95% N2 + 5% H2 . Cool to room tem-
perature. Add the  L -cysteine·HCl·H2O and Na2S·9H2O. Anaerobically
distribute into tubes. Cap with rubber stoppers. Autoclave for 15 min
at 15 psi pressure–121°C.
Vitamin Solution:
Composition per 500.0mL:
Pyridoxine HCl...........................................................0.1g
p-Aminobenzoic acid.....................................................0.05g
Calcium pantothenate ...................................................0.05g
Nicotinic acid..........................................................0.05g
Thioctic acid...........................................................0.05g
Biotin..................................................................0.02g
Folic acid .............................................................0.02g
Riboflavin..............................................................5.0mg
Thiamine·HCl...........................................................5.0mg
Vitamin B 12 ...........................................................1.0mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 500.0mL. Mix thoroughly. Store
solution in the dark at −10°C.
Wolfe’s Modified Mineral Elixir:
Composition per liter:
MgSO4 ·7H2O..............................................................3.0g
Nitrilotriacetic acid.....................................................1.5g
NaCl......................................................................1.0g
MnSO4 ·H2O...............................................................0.5g
CaCl2 , anhydrous.........................................................0.1g
Co(NO3)2 ·6H2O...........................................................0.1g
FeSO4 ·7H2O..............................................................0.1g
ZnSO4 ·7H2O..............................................................0.1g
AlK(SO4)2 , anhydrous....................................................0.01g
CuSO4 ·5H2O.............................................................0.01g
H3BO3 ...................................................................0.01g
Na2MoO4 ·2H2O...........................................................0.01g
Na2SeO3 , anhydrous......................................................1.0mg
Preparation of Wolfe’s Modified Mineral Elixir: Add nitrilo-
triacetic acid to 500.0mL of distilled/deionized water. Dissolve by ad-
justing pH to 6.5 with KOH. Add remaining components. Add
distilled/deionized water to 1.0L.
Preparation of Medium: Add components, except reducing solu-
tion, to distilled/deionized water and bring volume to 1.0L. If medium
is to be distributed into tubes, omit bulk filter paper and substitute one
Whatman #1 filter paper strip (8mm × 70mm) per tube of broth. Gently
heat and bring to boiling under 95% N2 + 5% H2 . Continue boiling until
color changes from blue to pink. Add the reducing solution. The pink
color will disappear, indicating that the solution has been reduced. Dis-
tribute into tubes or flasks under 95% N2 + 5% H2 using anaerobic
techniques. If tubes are used, remember to add Whatman #1 filter paper
strips prior to the addition of broth. Cap tubes with rubber stoppers.
Autoclave for 15 min at 15 psi pressure–121°C.
Use:  For the cultivation and maintenance of Clostridium thermocel-
lum.