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PHB/Pyruvate Medium
(DSMZ Medium 193a)

山東拓普生物工程有限公司 培養(yǎng)基配方 http://qp8008.cn

Composition per 991.0mL:

Solution A..............................................................................870.0mL
Solution C..............................................................................100.0mL
Solution D................................................................................10.0mL
Solution E (Vitamin solution)..................................................10.0mL
Solution B (Trace elements solution SL-10) .............................1.0mL
pH 7.1–7.4 at 25°C
Solution A:
Composition per 870.0mL:
Na-pyruvate ..................................................................................5.0g
NaCl..............................................................................................7.0g
Na 2 SO 4 ..........................................................................................3.0g
MgCl 2 ·6H 2 O .................................................................................1.3g
Poly-hydroxybutyrate (PHB)........................................................1.0g
KH 2 PO 4 .........................................................................................0.2g
NH 4 Cl ...........................................................................................0.3g
KCl................................................................................................0.5g
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.
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. Sparge with 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.
Flush with 80% N 2  + 20% CO 2 to remove dissolved oxygen.
Solution D:
Composition per 10.0mL:
Na-acetate·3H 2 O...........................................................................2.5g
Preparation of Solution D: Add Na-acetate·3H 2 O to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution E (Vitamin Solution):
Composition per liter:
Pyridoxine-HCl........................................................................10.0mg
Thiamine-HCl·2H 2 O..................................................................5.0mg
Riboflavin ..................................................................................5.0mg
Nicotinic acid.............................................................................5.0mg
D -Ca-pantothenate......................................................................5.0mg
p-Aminobenzoic acid.................................................................5.0mg
Lipoic acid .................................................................................5.0mg
Biotin .........................................................................................2.0mg
Folic acid....................................................................................2.0mg
Vitamin B 12 ..............................................................................0.10mg
Solution E (Vitamin Solution): Add components to distilled/de-
ionized water and bring volume to 1.0L. Mix thoroughly. Sparge with
100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Solution F:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.4g
Preparation of Solution F:  Add Na 2 S·9H 2 O to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100%
N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Gently heat solution A and bring to boil-
ing. Boil solution A for a few minutes. Cool to room temperature. Gas
with 80% N 2  + 20% CO 2 gas mixture to reach a pH below 6. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to room temperature. Se-
quentially add 1.0mL solution B, 100.0mL solution C, 10.0mL solu-
tion D, 10.0mL solution E, and 10.0mL solution F. Distribute
anaerobically under 80% N 2  + 20% CO 2 into appropriate vessels. Ad-
dition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution,
freshly prepared under N 2  and filter sterilized, may stimulate growth.
After inoculation, pressurize vessels up to 1 bar H 2 + CO 2 (80% + 20%)
overpressure.
Use: For the cultivation of unclassified bacterium DSM 6754.