MTP4 Medium

[所属分类:培养基配方] [发布时间:2021-6-10] [发布人:网站管理员2] [阅读次数:] [返回]

MTP4 Medium

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

Composition per 1001.0mL:

Solution A..............................................................................870.0mL
Solution C..............................................................................100.0mL
Solution D (Vitamin solution)..................................................10.0mL
Solution E ................................................................................10.0mL
Solution B (Trace elements soluiton SL-10) .............................1.0mL
Methanol....................................................................................1.0mL
Methanethiol gas....................................................................1–2.0mL
pH 7.1–7.4 at 25°C
Solution A:
Composition per 870.0mL:
NaCl............................................................................................21.0g
MgCl 2 ·6H 2 O..................................................................................3.1g
KCl................................................................................................0.5g
NH 4 Cl ...........................................................................................0.3g
KH 2 PO 4 .........................................................................................0.2g
CaCl 2 ·2H 2 O.................................................................................0.15g
Resazurin ...................................................................................1.0mg
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 870.0mL. Mix thoroughly. Gently heat and
bring to boiling. Continue boiling for 3-4 min. Allow to cool to room
temperature while gassing under 80% N 2 + 20% CO 2 . Continue gas-
sing until pH reaches below 6.0. Seal the flask under 80% N 2 + 20%
CO 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution B (Trace Elements Solution SL-10 ):
Composition per liter:
FeCl 2 ·4H 2 O...................................................................................1.5g
CoCl 2 ·6H 2 O...........................................................................190.0mg
MnCl 2 ·4H 2 O ..........................................................................100.0mg
ZnCl 2 ........................................................................................70.0mg
Na 2 MoO 4 ·2H 2 O.......................................................................36.0mg
NiCl 2 ·6H 2 O..............................................................................24.0mg
H 3 BO 3 ........................................................................................6.0mg
CuCl 2 ·2H 2 O...............................................................................2.0mg
HCl (25% solution)..................................................................10.0mL
Preparation of Solution B (Trace Elements Solution SL-10):
Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Gas under 100% N 2 . Autoclave for 15 min at
15 psi pressure–121°C.
Solution C:
Composition per 100.0mL:
NaHCO 3 ........................................................................................5.0g
Preparation of Solution C:  Add NaHCO 3 to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.
Gas under 80% N 2 + 20% CO 2 .
Solution D (Vitamin Solution):
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
Calcium  DL -pantothenate...........................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Riboflavin..................................................................................5.0mg
Thiamine·HCl............................................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Solution D (Vitamin Solution): Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution E:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.4g
Preparation of Solution E:  Add Na 2 S·9H 2 O to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Gas under 100%
N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Aseptically and anaerobically combine so-
lution A with solution B, solution C, solution D, and solution E, in that or-
der. Mix thoroughly. Anaerobically distribute into sterile tubes or flasks
under 80% N 2 + 20% CO 2 . Prior to inoculation, aseptically and anaerobi-
cally add 1.0mL of filter-sterilized methanol and 1.0-2.0mL of methane-
thiol gas to each liter of medium. Addition of 10-20mg of sodium
dithionite per liter (e.g., from a 5% solution, freshly prepared under N 2 and
filter-sterilized) may stimulate growth at the beginning.
Use: For the cultivation and maintenance of Methanosarcina species.