Mycobactin Medium

[所属分类:培养基配方] [发布时间:2021-6-15] [发布人:网站管理员2] [阅读次数:] [返回]

Mycobactin Medium

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

Serum Agar Medium:

Composition per liter:
Noble agar...................................................................................15.0g
Casamino acids.............................................................................2.5g
Na 2 HPO 4 , anhydrous....................................................................2.5g
Sodium citrate...............................................................................1.5g
KH 2 PO 4 .........................................................................................1.0g
MgSO 4 ·7H 2 O................................................................................0.6g
Asparagine....................................................................................0.3g
Crude mycobactin.......................................................................0.16g
Chloramphenicol.........................................................................0.05g
Primaricine (myprozine).............................................................0.05g
Penicillin..............................................................................100,000U
Bovine serum, 56°C-inactivated............................................200.0mL
Tween™ 80 (1% solution).......................................................50.0mL
Glycerol ...................................................................................25.0mL
pH 7.2 ± 0.2 at 25°C
Preparation of Crude Mycobactin: Grow Mycobacterium phlei
in 600.0mL of mycobactin production broth for 2 weeks at 37°C. Auto-
clave the culture for 15 min at 15 psi pressure–121°C. Filter the cells and
wash with distilled/deionized water. Dry cells under CaCl 2 . Treat 100.0g
of dried culture with three successive acetone extractions—500.0mL of
acetone for 30 min in a 1.0L flask fitted with a reflux condenser. Evapo-
rate the acetone to dryness. Extract the residue in a Soxhlet apparatus
with petroleum ether for 18–20 hr at 40°–60°C. A hard red residue will
remain. Dissolve the residue in warm absolute ethanol. Centrifuge for 30
min at 2250 rpm to remove debris. Evaporate the supernatant to dryness.
Grind the residue to a powder of crude mycobactin.
Source:  Purified mycobactin is available from Allied Labs, Inc., 2520
Hunt St., Ames, IA 50010.
Mycobactin Production Broth:
Composition per 600.0mL:
Solution B..............................................................................500.0mL
Solution A..............................................................................100.0mL
Preparation of Mycobactin Production Broth:  Aseptically mix
cooled sterile solution A and solution B.
Solution A:
Composition per 100.0mL:
L- Asparagine .................................................................................5.0g
Na 2 HPO 4 .......................................................................................2.0g
KH 2 PO 4 .........................................................................................1.0g
Glycerol ...................................................................................30.0mL
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C. Cool to 45°–50°C.
Solution B:
Composition per 500.0mL:
Glucose.......................................................................................10.0g
MgSO 4· ·7H 2 O...............................................................................0.2g
Preparation of Solution B:  Add components to distilled/deionized
water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C. Cool to 45°–50°C.
Preparation of Medium: Add components, except penicillin,
chloramphenicol, primaricine, and bovine serum, to distilled/deionized
water and bring volume to 800.0mL. Mix thoroughly. Gently heat with
a minimum of heat. Autoclave for 15 min at 10 psi pressure–116°C.
Cool to 50°C. Aseptically add penicillin, chloramphenicol, primari-
cine, and sterile bovine serum. Mix thoroughly. Adjust pH to 7.2. Dis-
tribute into sterile tubes or flasks.
Use:  For the cultivation and maintenance of Mycobacterium avium and
Mycobacterium paratuberculosis.