Methanococcus McC Medium

[所属分类:培养基配方] [发布时间:2021-5-24] [发布人:网站管理员2] [阅读次数:] [返回]

Methanococcus McC Medium

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

Composition per 1100.0mL:

NaHCO 3 ........................................................................................5.0g
Yeast extract..................................................................................2.0g
L -Cysteine·HCl·H 2 O.....................................................................0.5g
General salts solution.............................................................500.0mL
NaCl solution...........................................................................75.0mL
Na 2 S·9H 2 O solution.................................................................20.0mL
K 2 HPO 4 solution......................................................................10.0mL
Trace minerals solution............................................................10.0mL
Sodium acetate solution...........................................................10.0mL
Iron stock solution .....................................................................5.0mL
Resazurin solution......................................................................1.0mL
General Salts Solution:
Composition per liter:
MgSO 4 ·7H 2 O................................................................................6.9g
MgCl 2 ·6H 2 O..................................................................................5.5g
NH 4 Cl ...........................................................................................1.0g
KCl..............................................................................................0.67g
CaCl 2 ·2H 2 O.................................................................................0.28g
Preparation of General Salts Solution:  Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
NaCl Solution:
Composition per 100.0mL:
NaCl............................................................................................29.3g
Preparation of NaCl Solution:  Add NaCl to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
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.
K 2 HPO 4 Solution:
Composition per 100.0mL:
K 2 HPO 4 .........................................................................................1.4g
Preparation of K 2 HPO 4 Solution:  Add K 2 HPO 4 to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Trace Minerals Solution:
Composition per liter:
Nitrilotriacetic acid.......................................................................1.5g
Na 2 WO 4 ·2H 2 O..............................................................................1.0g
Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O..................................................................0.2g
Na 2 SeO 3 ........................................................................................0.2g
Na 2 MoO 4 ·2H 2 O ............................................................................0.1g
Mn 4 ·2H 2 O.....................................................................................0.1g
Zn 4 ·7H 2 O ......................................................................................0.1g
NiCl 2 ·7H 2 O...............................................................................0.025g
CuSO 4 ·5H 2 O...............................................................................0.01g
Preparation of Trace Minerals Solution:  Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L. Adjust pH to 7.0.
Sodium Acetate Solution:
Composition per 100.0mL:
Sodium acetate·3H 2 O..................................................................13.6g
Preparation of Sodium Acetate Solution:  Add sodium ace-
tate·3H 2 O to distilled/deionized water and bring volume to 100.0mL.
Mix thoroughly.
Iron Stock Solution:
Composition per 100.0mL:
Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O..................................................................0.2g
Preparation of Iron Stock Solution:  Add Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O
to 5.0mL of distilled H 2 O containing 2 drops of concentrated HCl. Mix
thoroughly. When the Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O has dissolved, bring the
volume to 100.0mL with distilled/deionized water.
Resazurin Solution:
Composition per 10.0mL:
Resazurin.................................................................................10.0mg
Preparation of Resazurin Solution:  Add resazurin to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under 80%
H 2 + 20% CO 2 . Add components, except NaHCO 3 and Na 2 S·9H 2 O so-
lution, to distilled/deionized water and bring volume to 1080.0mL.
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 . Add NaHCO 3 . Mix thoroughly. Anaerobically distribute 9.8mL
volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–
121°C. Aseptically and anaerobically add 0.2mL of sterile Na 2 S·9H 2 O
solution to each tube. Mix thoroughly.
Use:  For the cultivation of Methanococcus species.