Methanosarcina barkeri Medium

[所属分类:培养基配方] [发布时间:2021-5-26] [发布人:网站管理员2] [阅读次数:] [返回]

Methanosarcina barkeri Medium

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

Composition per liter:

NaHCO 3 ........................................................................................2.5g
NaCl............................................................................................0.46g
Yeast extract................................................................................0.24g
KH 2 PO 4 .......................................................................................0.23g
K 2 HPO 4 .......................................................................................0.23g
(NH 4 ) 2 SO 4 ...................................................................................0.23g
MgCl 2 ·6H 2 O................................................................................0.09g
CaCl 2 ·2H 2 O.................................................................................0.06g
NiCl 2 ·6H 2 O................................................................................2.0mg
Methanol..................................................................................10.0mL
Trace elements solution ...........................................................10.0mL
Vitamin solution.......................................................................10.0mL
L -Cysteine·HCl·H 2 O solution ..................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Resazurin (0.01% solution)........................................................1.0mL
Preparation of Methanol:  Filter sterilize 10.0mL of methanol.
Trace Elements Solution:
Composition per liter:
MgSO 4 ·5H 2 O................................................................................3.0g
Nitrilotriacetic acid.......................................................................1.5g
NaCl..............................................................................................1.0g
MnSO 4 ·2H 2 O................................................................................0.5g
NaS 4 ·SeO 3 ·5H 2 O...........................................................................0.3g
NiCl 2 ·6H 2 O.................................................................................0.25g
CoSO 4 ·7H 2 O...............................................................................0.18g
ZnSO 4 ·7H 2 O...............................................................................0.18g
CaCl 2 ·7H 2 O...................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
KAI(SO 4 ) 2 ·12H 2 O.......................................................................0.02g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O ..........................................................................0.01g
Preparation of Trace Elements Solution:  Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Mix thoroughly. Add distilled/de-
ionized water to 1.0L. Adjust pH to 6.8.
Vitamin Solution:
Composition per liter:
Calcium  DL -pantothenate..............................................................5.0g
Vitamin B 12 ...................................................................................0.1g
Pyridoxine·HCl........................................................................10.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid .................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Thiamine·HCl ............................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Preparation of Vitamin Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
L -Cysteine·HCl·H 2 O Solution:
Composition per 100.0mL:
L -Cysteine·HCl·H 2 O .....................................................................2.5g
Preparation of  L -Cysteine·HCl·H 2 O Solution:  Bring 100.0mL
of distilled/deionized water to boiling. Cool to room temperature while
sparging with 100% N 2 . Add  L -cysteine·HCl·H 2 O to the 100.0mL of
anaerobic water. Distribute into serum bottles fitted with butyl rubber
stoppers and aluminum seals. Do not grease stoppers. Autoclave for 20
min at 15 psi pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 100.0mL:
NaOH....................................................................................... 1 pellet
Na 2 S·9H 2 O....................................................................................2.5g
Preparation of Na 2 S·9H 2 O Solution:  Bring 100.0mL of dis-
tilled/deionized water to boiling. Cool to room temperature while
sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic wa-
ter. Weigh out a little more than 2.5g of Na 2 S·9H 2 O. Briefly rinse the
crystals in distilled/deionized water. Dry the crystals by blotting on pa-
per towels or filter paper. Weigh out 2.5g of washed Na 2 S·9H 2 O crys-
tals. Add to the 100.0mL of anaerobic NaOH solution. Distribute into
serum bottles fitted with butyl rubber stoppers and aluminum seals. Do
not grease stoppers. Pressurize to 60kPa with 100% N 2 . Autoclave for
15 min at 15 psi pressure–121°C. Store at room temperature in an an-
aerobic chamber.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 . Add components, except methanol,  L -cyste-
ine·HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to distilled/deionized
water and bring volume to 970.0mL. Mix thoroughly. Gently heat and
bring to boiling. Continue boiling for 3 min. Cool to room temperature
while sparging with 80% H 2 + 20% CO 2 . Anaerobically distribute
9.7mL volumes into anaerobic tubes. Autoclave for 20 min at 15 psi
pressure–121°C. Aseptically and anaerobically add 0.1mL of sterile
methanol, 0.1mL of sterile  L -cysteine·HCl·H 2 O solution, and 0.1mL of
sterile Na 2 S·9H 2 O solution to each tube. Mix thoroughly.
Use:  For the cultivation of Methanosarcina barkeri.