Acetitomaculum ruminus Medium (LMG Medium 224)

[所属分类:培养基配方] [发布时间:2019-9-18] [发布人:] [阅读次数:] [返回]

山东拓普生物工程有限公司

Shandong Tuopu Biol-Engineering Co.,Ltd

Acetitomaculum ruminus Medium
(LMG Medium 224)
Composition per liter:
NaCl..............................................................................................7.0g
NaHCO 3 ........................................................................................4.0g
Yeast extract..................................................................................1.5g
MgCl 2 ·6H 2 O..................................................................................1.2g
KCl................................................................................................0.5g
NH 4 Cl ...........................................................................................0.3g
KH 2 PO 4 .........................................................................................0.2g
CaCl 2 ·2H 2 O.................................................................................0.15g
Na 2 SO 4 ..........................................................................................0.1g
Resazurin ...................................................................................0.5mg
Ascorbic acid ............................................................................50.0μg
Myo-inositol..............................................................................50.0μg
Niacinamide..............................................................................50.0μg
Choline chloride........................................................................50.0μg
Glucose solution ......................................................................10.0mL
L -Cysteine·HCl·H 2 O solution ..................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Wolfe’s mineral solution..........................................................10.0mL
Wolfe's vitamin solution ..........................................................10.0mL
pH 6.9 ± 0.2 at 25°C
Glucose Solution:
Composition per 10.0mL:
Glucose .........................................................................................5.0g
Preparation of Glucose Solution:  Add glucose to distilled/deion-
ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with
100% N 2 . Filter sterilize.
L -Cysteine·HCl·H 2 O Solution:
Composition per 100.0mL:
L -Cysteine·HCl·H 2 O .....................................................................3.0g
Preparation of  L -Cysteine·HCl·H 2 O Solution:  Bring 100.0mL
of distilled/deionized water to boiling. Cool to room temperature while
sparging with 100% N 2 . Add  L -cysteine·HCl·H 2 O to the 100.0mL of
anaerobic water. Distribute into serum bottles fitted with butyl rubber
stoppers and aluminum seals. Do not grease stoppers. Autoclave for 20
min at 15 psi pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 100.0mL:
NaOH....................................................................................... 1 pellet
Na 2 S·9H 2 O....................................................................................3.0g
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
water. Weigh out a little more than 3.0g of Na 2 S·9H 2 O. Briefly rinse
the crystals in distilled/deionized water. Dry the crystals by blotting on
paper towels or filter paper. Weigh out 2.5g of washed Na 2 S·9H 2 O
crystals. Add to the 100.0mL of anaerobic NaOH solution. Distribute
into serum bottles fitted with butyl rubber stoppers and aluminum
seals. Do not grease stoppers. Pressurize to 60kPa with 100% N 2 . Au-
toclave for 15 min at 15 psi pressure–121°C. Store at room temperature
in an anaerobic chamber.
Wolfe’s Mineral Solution:
Composition per liter:
MgSO 4 ·7H 2 O................................................................................3.0g
Nitrilotriacetic acid.......................................................................1.5g
NaCl..............................................................................................1.0g
MnSO 4 ·2H 2 O................................................................................0.5g
CoCl 2 ·6H 2 O..................................................................................0.1g
ZnSO 4 ·7H 2 O.................................................................................0.1g
CaCl 2 ·2H 2 O ..................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
NiCl 2 ·6H 2 O...............................................................................0.025g
KAl(SO 4 ) 2 ·12H 2 O.......................................................................0.02g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O..........................................................................0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Wolfe’s Mineral 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 6.8.
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl........................................................................10.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid.................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
Riboflavin..................................................................................5.0mg
Thiamine·HCl ............................................................................5.0mg
Calcium  DL -pantothenate...........................................................5.0mg
Biotin.........................................................................................2.0mg
Folic acid ...................................................................................2.0mg
Vitamin B 12 ................................................................................0.1mg
Preparation of Wolfe’s Vitamin Solution:  Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 . Add components, except bicarbonate, glucose solution,
L -cysteine·HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to distilled/de-
ionized water and bring volume to 970.0mL. Mix thoroughly. Add sol-
id bicarbonate and equilibrate pH to 6.8–7 by gassing. Gently heat and
bring to boiling. Continue boiling for 3 min. Cool to room temperature
while sparging with 80% N 2 + 20% CO 2 . Anaerobically distribute
9.7mL volumes into anaerobic tubes. Autoclave for 20 min at 15 psi
pressure–121°C. Aseptically and anaerobically add 0.1mL of sterile
glucose, 0.1mL of sterile  L -cysteine·HCl·H 2 O solution, and 0.1mL of
sterile Na 2 S·9H 2 O solution to each tube. Mix thoroughly.
Use:  For the cultivation of Acetitomaculum ruminus.