Methanogenium Alcohol Medium

[所属分类:培养基配方] [发布时间:2021-5-25] [发布人:网站管理员2] [阅读次数:] [返回]

Methanogenium Alcohol Medium

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

Composition per 1003.0mL:

NaCl..............................................................................................1.0g
KCl................................................................................................0.5g
MgCl 2 ·6H 2 O .................................................................................0.5g
NH 4 Cl ...........................................................................................0.4g
Sodium acetate·3H 2 O....................................................................0.4g
KH 2 PO 4 .........................................................................................0.2g
CaCl 2 ·2H 2 O ..................................................................................0.1g
NaHCO 3 solution.....................................................................60.0mL
2-Propanol..................................................................................5.0mL
Na 2 S·9H 2 O solution...................................................................3.0mL
Cyanocobalamin solution ..........................................................1.0mL
Selenite-molybdate-tungstate solution.......................................1.0mL
Thiamine solution......................................................................1.0mL
Trace elements solution.............................................................1.0mL
Vitamin solution.........................................................................1.0mL
Trace Elements Solution:
Composition per 100.0mL:
FeSO 4 ·7H 2 O.........................................................................1400.0mg
ZnSO 4 ·7H 2 O..........................................................................145.0mg
CoCl 2 ·6H 2 O...........................................................................120.0mg
MnCl 2 ·4H 2 O ..........................................................................100.0mg
NiCl 2 ·6H 2 O..............................................................................50.0mg
H 3 BO 3 ........................................................................................6.0mg
CuSO 4 ·5H 2 O..............................................................................3.0mg
HCl (25%,w/v)...........................................................................8.0mL
Preparation of Trace Elements Solution:  Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Selenite-Molybdate-Tungstate Solution:
Composition per liter:
NaOH............................................................................................0.2g
Na 2 MoO 4 ·2H 2 O.......................................................................40.0mg
Na 2 WO 4 ·2H 2 O.........................................................................33.0mg
Na 2 SeO 3 ·2H 2 O...........................................................................5.0mg
Preparation of Selenite-Molybdate-Tungstate Solution:  Add
components to distilled/deionized water and bring volume to 1.0L. Mix
thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pres-
sure–121°C.
NaHCO 3 Solution:
Composition per liter:
NaHCO 3 ......................................................................................84.0g
Preparation of NaHCO 3 Solution:  Add NaHCO 3 to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Na 2 S·9H 2 O Solution:
Composition per 100.0mL:
Na 2 S·9H 2 O....................................................................................2.5g
NaOH.......................................................................................1 pellet
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 2-Propanol:  Filter sterilize 10.0mL of 2-propanol.
Sparge with 100% N 2 .
Vitamin Solution:
Composition per liter:
Sodium 2-mercaptoethanesulfonate............................................0.25g
Pyridoxine·HCl...........................................................................0.15g
Calcium pantothenate ...................................................................0.1g
Nicotinic acid................................................................................0.1g
p-Aminobenzoic acid...............................................................40.0mg
Biotin .......................................................................................10.0mg
Potassium phosphate buffer (25mM solution, pH 7.0).................1.0L
Preparation of Vitamin Solution:  Combine components. Mix
thoroughly. Filter sterilize. Sparge with 100% N 2 .
Thiamine Solution:
Composition per liter:
Thiamine·HCl ...............................................................................0.1g
Sodium phosphate buffer (0.1M solution, pH 3.6) .......................1.0L
Preparation of Thiamine Solution:  Combine components. Mix
thoroughly. Filter sterilize. Sparge with 100% N 2 .
Cyanocobalamin Solution:
Composition per liter:
Cyanocobalamin ......................................................................50.0mg
Preparation of Cyanocobalamin Solution:  Add cyanocobala-
min to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Filter sterilize. Sparge with 100% N 2 .
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 . Add components, except NaHCO 3 solution, 2-propa-
nol, Na 2 S·9H 2 O solution, cyanocobalamin solution, selenite-molyb-
date-tungstate solution, thiamine solution, trace elements solution, and
vitamin solution, to distilled/deionized water and bring volume to
930.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave
for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add
60.0mL of sterile NaHCO 3 solution, 5.0mL of sterile 2-propanol,
3.0mL of sterile Na 2 S·9H 2 O solution, 1.0mL of sterile cyanocobalamin
solution, 1.0mL of sterile selenite-molybdate-tungstate solution,
1.0mL of sterile thiamine solution, 1.0mL of sterile trace elements so-
lution, and 1.0mL of sterile vitamin solution. Mix thoroughly. Asepti-
cally and anaerobically distribute into sterile tubes or bottles.
Use:  For the cultivation of Methanogenium species.