Acetobacterium Heterotrophic Medium (DSMZ Medium 135)

[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

Acetobacterium Heterotrophic Medium
(DSMZ Medium 135)
Composition per liter:
NaHCO 3 ......................................................................................10.0g
Yeast extract..................................................................................2.0g
NH 4 Cl ...........................................................................................1.0g
K 2 HPO 4 .......................................................................................0.45g
KH 2 PO 4 .......................................................................................0.33g
MgSO 4 ·7H 2 O................................................................................0.1g
Resazurin ...................................................................................1.0mg
Na 2 CO 3  solution.......................................................................25.0mL
Trace elements solution...........................................................20.0mL
Vitamin solution.......................................................................20.0mL
Fructose solution......................................................................10.0mL
Cysteine solution .....................................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
pH 8.2 ± 0.2 at 25°C
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O....................................................................................0.5g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O to dis-
tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 .
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an-
aerobically.
Cysteine Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O .....................................................................0.5g
Preparation of Cysteine Solution:  Add  L -cysteine·HCl·H 2 O to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Fructose Solution:
Composition per 10.0mL:
Fructose.........................................................................................5.0g
Preparation of Fructose Solution: Add fructose 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. Cool to
room temperature.
Trace Elements 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
CoSO 4 ·7H 2 O...............................................................................0.18g
ZnSO 4 ·7H 2 O...............................................................................0.18g
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
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·4H 2 O ..........................................................................0.01g
CuSO 4 ·5H 2 O...............................................................................0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Trace Elements 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. Mix thoroughly.
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 ................................................................................0.1mg
Preparation of Vitamin Solution:  Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H 2 + 20% CO 2 . Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
N 2 + 20% CO 2 gas atmosphere. Add components, except NaHCO 3 ,
Na 2 CO 3  solution, Na 2 S·9H 2 O solution, vitamin solution, fructose sol-
tuion, and cysteine solution, to distilled/deionized water and bring vol-
ume to 925.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil
for 5 min. Cool while sparging with 80% N 2 + 20% CO 2 . Add 10.0g solid
NaHCO 3 . Equilibrate with 80% N 2 + 20% CO 2 until pH is approximate-
ly 7.4. Distribute into bottles. Autoclave under 80% N 2 + 20% CO 2  for
15 min at 15 psi pressure–121°C. Aseptically and anaerobically add ap-
proximately 0.25mL sterile Na 2 CO 3  solution to each 10.0mL of medi-
um so that pH is adjusted to 8.2. For every 10.0mL of medium inject
0.1mL Na 2 S·9H 2 O solution, 0.1mL fructose solution, 0.2mL vitamin
solution, and 0.1mL cysteine solution.
Use:  For the cultivation and heterotrophic growth of Acetobacterium
spp.