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Acetogen Medium

[所属分类:培养基配方] [发布时间:2021-10-27] [发布人:网站管理员2] [阅读次数:] [返回]

Acetogen Medium

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

Composition per 421.8mL:

NaHCO3 ..............................................................2.4g

NH4Cl .................................................................0.2g

Yeast extract........................................................0.2g

Stock salts solution #1 .................................. 40.0mL

Potassium phosphate buffer.......................... 20.0mL

Clarified rumen fluid .................................... 20.0mL

Stock salts solution #2 .................................... 4.0mL

Trace minerals................................................. 4.0mL

Vitamin solution.............................................. 4.0mL

Reducing agent ............................................... 4.0mL

Tungstate solution........................................... 0.4mL

Resazurin (0.1% solution) .............................. 0.4mL

Potassium Phosphate Buffer:

Composition per 830.0mL:

K2HPO4...........................................................15.68g

KH2PO4.............................................................4.72g

Preparation of Potassium Phosphate Buffer:

Dissolve K2HPO4 in 600.0mL of distilled/deionized

water and KH2PO4 in 230.0mL of distilled/deionized

water. Mix the two solutions together and use.

Stock Salts Solution #1:

Composition per liter:

KCl......................................................................1.6g

NaCl....................................................................1.4g

MgSO4·7H2O......................................................0.2g

Preparation of Stock Salts Solution #1: Add

components to distilled/deionized water and bring

volume to 1.0L. Mix thoroughly.

Stock Salts Solution #2:

Composition per liter:

CaCl2·2H2O ........................................................0.1g

Preparation of Stock Salts Solution #2: Add

components to distilled/deionized water and bring

volume to 1.0L. Mix thoroughly.

Trace Minerals:

Composition per liter:

Nitrilotriacetic acid .............................................1.5g

MgSO4·7H2O......................................................3.0g

MnSO4·H2O........................................................0.5g

NaCl....................................................................1.0g

NiCl2·6H2O.........................................................0.1g

FeSO4·7H2O........................................................0.1g

CoCl2·6H2O ........................................................0.1g

CaCl2...................................................................0.1g

ZnSO4·7H2O .......................................................0.1g

Na2SeO3·5H2O..................................................0.01g

CuSO4·5H2O.....................................................0.01g

AlK(SO4)2·12H2O.............................................0.01g

H3BO3 ...............................................................0.01g

Na2MoO4·2H2O ................................................0.01g

Preparation of Trace Minerals: Add nitrilotri

acetic acid to 500.0mL of distilled/deionized water.

Dissolve by adjusting pH to 6.5 with KOH. Bring

volume to 1.0L with distilled/deionized water. Add

remaining components. Mix thoroughly.

Vitamin Solution:

Composition per liter:

Pyridoxine·HCl ..............................................10.0mg

Ascorbic acid ...................................................5.0mg

Calcium pantothenate ......................................5.0mg

Choline chloride...............................................5.0mg

Lipoic acid .......................................................5.0mg

i-Inositol...........................................................5.0mg

Niacinamide.....................................................5.0mg

Nicotinic acid...................................................5.0mg

p-Aminobenzoic acid.......................................5.0mg

Pyridoxal·HCl ..................................................5.0mg

Riboflavin ........................................................5.0mg

Thiamine·HCl ..................................................5.0mg

Biotin ...............................................................2.0mg

Folic acid..........................................................2.0mg

Vitamin B12 ......................................................0.1mg

Preparation of Vitamin Solution: Add compo

nents to distilled/deionized water and bring volume

to 1.0L. Mix thoroughly. Store frozen.

Tungstate Solution:

Composition per liter:

Na2WO4·2H2O...............................................99.0mg

Preparation of Tungstate Solution: Add com

ponents to distilled/deionized water and bring vol

ume to 1.0L. Mix thoroughly.

Reducing Agent:

Composition per 110.0mL:

L-Cysteine·HCl·H2O............................................2.5g

Na2S·9H2O..........................................................2.5g

Preparation of Reducing Agent: Add 110.0mL

of distilled/deionized water to a 250.0mL round-bot

tomed flask. Boil under N2 gas for 1 min. Cool to

room temperature. Add L-cysteine·HCl and dissolve.

Adjust to pH 9 with 5N NaOH. Add washed

Na2S·9H2O and dissolve. Distribute in amounts

needed into tubes or flasks. Autoclave for 10 min at

15 psi pressure–121°C.

Preparation of Medium: Add components, ex

cept NaHCO3 and reducing agent, to distilled/deion

ized water and bring volume to 417.8mL. Mix

thoroughly. Gently heat and bring to boiling under

80% N2 + 20% CO2. Cool to 45°–50°C. Add

NaHCO3 and reducing agent. Distribute into tubes or

flasks under 80% N2 + 20% CO2. Autoclave for 15

min at 15 psi pressure–121°C. After inoculation, ex

change headspace with 80% H2 + 20% CO2.

Use: For the cultivation and maintenance of acetoge

nic anaerobes such as some Clostridium species.

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Acetogen Medium