Methanosarcina Mass-Culturing 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 Mass-Culturing 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:

NaCl............................................................................................0.58g
NH 4 Cl .........................................................................................0.53g
K 2 HPO 4 .......................................................................................0.44g
KH 2 PO 4 .......................................................................................0.35g
MgCl 2 ·6H 2 O ...............................................................................0.04g
CaCl 2 ·2H 2 O ................................................................................0.03g
Resazurin ..................................................................................0.001g
Trace elements solution...........................................................10.0mL
Vitamin solution.......................................................................10.0mL
Methanol....................................................................................8.0mL
Na 2 S·9H 2 O solution...................................................................8.0mL
Trace Elements Solution:
Composition per liter:
Sodium nitrilotriacetate ..............................................................1.67g
CoCl 2 ·6H 2 O................................................................................0.18g
FeCl 2 ·4H 2 O.................................................................................0.14g
MnCl 2 ·4H 2 O ...............................................................................0.09g
NiCl 2 ·6H 2 O.................................................................................0.09g
ZnCl 2 ...........................................................................................0.09g
CaCl 2 ...........................................................................................0.06g
Na 2 MoO 4 ·2H 2 O........................................................................0.046g
CuSO 4 .......................................................................................0.045g
Preparation of Trace Elements Solution:  Add sodium nitrilotri-
acetate to 500.0mL of distilled/deionized water. Adjust pH to 6.5. Add
remaining components. Add distilled/deionized water to 1.0L. Adjust
pH to 7.0.
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. Filter ster-
ilize.
Preparation of Methanol:  Filter sterilize 10.0mL of methanol.
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. Add 2.5g of washed Na 2 S·9H 2 O crystals to
100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit-
ted with butyl rubber stoppers and aluminum seals. Do not grease stop-
pers. Pressurize to 60kPa with 100% N 2 . Autoclave for 15 min at 15 psi
pressure–121°C. Store at room temperature in an anaerobic chamber.
Preparation of Medium: Prepare and dispense medium under 80%
H 2 + 20% CO 2 . Add components, except methanol, vitamin solution,
and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume
to 974.0mL. Mix thoroughly. Gently heat and bring to boiling. Contin-
ue boiling for 3 min. Cool to room temperature while sparging with
80% H 2 + 20% CO 2 . Anaerobically distribute 9.7mL volumes into an-
aerobic tubes. Autoclave for 15 min at 15 psi pressure–121°C. Asepti-
cally and anaerobically add 0.1mL of sterile vitamin solution, 0.08mL
of sterile methanol, and 0.08mL of sterile Na 2 S·9H 2 O solution to each
tube. Mix thoroughly.
Use:  For the cultivation of Methanosarcina species.