Halomethanococcus Medium (Methanohalophilus Medium)

[所属分类:培养基配方] [发布时间:2021-3-26] [发布人: ] [阅读次数:] [返回]

Halomethanococcus Medium

(Methanohalophilus Medium)

培养基配方

Composition per 1030.0mL:
Trimethylamine·HCl.....................................................................2.5g
Na 2 CO 3 .........................................................................................2.0g
NaHCO 3 ........................................................................................2.0g
Casamino acids.............................................................................0.5g
L- Cysteine·HCl·H 2 O......................................................................0.5g
Na 2 S·9H 2 O solution......................................................................0.5g
NH 4 Cl ...........................................................................................0.5g
Pancreatic digest of casein............................................................0.5g
Yeast extract..................................................................................0.5g
K 2 HPO 4 .........................................................................................0.2g
Ammonium-2-mercaptoethanesulfonate ...................................1.0mg
Artificial brine ..............................................................................1.0L
Wolfe’s vitamin solution..........................................................10.0mL
Wolfe’s mineral solution..........................................................10.0mL
Volatile acids solution..............................................................10.0mL
pH 7.1 ± 0.2 at 25°C
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................2.0g
Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.
Artificial Brine:
Composition per liter:
NaCl............................................................................................80.7g
MgCl 2 ·6H 2 O ...............................................................................35.1g
Na 2 SO 4 ........................................................................................12.9g
KCl................................................................................................5.7g
CaCl 2 ...........................................................................................0.55g
LiCl 2 ............................................................................................0.13g
H 3 BO 3 .........................................................................................0.12g
Preparation of Artificial Brine: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl...........................................................................0.01g
Thiamine·HCl ............................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Calcium pantothenate ................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Thioctic acid ..............................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Cyanocobalamin ........................................................................0.1mg
Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Store at 4°C.
Wolfe’s Mineral Solution:
Composition per liter:
MgSO 4 ·7H 2 O................................................................................3.0g
Nitrilotriacetic acid.......................................................................1.5g
NaCl..............................................................................................1.0g
MnSO 4 ·H 2 O..................................................................................0.5g
FeSO 4 ·7H 2 O..................................................................................0.1g
CoCl 2 ·6H 2 O ..................................................................................0.1g
CaCl 2 .............................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
CuSO 4 ·5H 2 O...............................................................................0.01g
AlK(SO 4 ) 2 ·12H 2 O.......................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O ..........................................................................0.01g
Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L.
Volatile Acids Solution:
Composition per liter:
α-Methylbutyric acid.................................................................0.5mL
Isobutyric acid............................................................................0.5mL
Isovaleric acid............................................................................0.5mL
Valeric acid ................................................................................0.5mL
Preparation of Volatile Acids Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Preparation of Medium:  Combine components, except the  L - cysteine·HCl·H 2 O, trimethylamine·HCl, and Na 2 S·9H 2 O solution. Mix thoroughly. Gently heat and bring to boiling. Add  L -cysteine·HCl·H 2 O. Mix thoroughly. Cool in an ice-water bath under 80% N 2 + 20% CO 2 . Add trimethylamine·HCl. Mix thoroughly. Adjust pH to 7.1. Aseptically and anaerobically distribute into tubes in 10.0mL volumes under 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Immediately prior to inoculation, aseptically add 0.25mL of sterile Na 2 S·9H 2 O solution to each tube. Mix thoroughly.
Use:  For the cultivation and maintenance of Methanohalophilus mahii.