Cryptoanaerobacter Medium (DSMZ Medium 1022)

[所属分类:培养基配方] [发布时间:2020-5-27] [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

Cryptoanaerobacter Medium
(DSMZ Medium 1022)

培养基配方
Composition per liter:
Solution A.............................................................650.0mL
Clostridium sporogenes supernatant.....................................350.0mL
pH 7.5–8.0 at 25°C
Solution A:
Composition per 650.0mL:
Yeast extract...........................................................5.0g
NaHCO3 .................................................................4.0g
Casamino acids..........................................................1.0g
4-Hydroxybenzoic acid..................................................0.45g
KH2PO4 .................................................................0.4g
NH4Cl ..................................................................0.4g
Resazurin..............................................................0.5mg
Vitamin solution......................................................10.0mL
Magnesium chloride solution ..........................................10.0mL
Calcium chloride solution.............................................10.0mL
Trace element solution SL-10...........................................1.0mL
Selenite/tungstate solution ...........................................1.0mL
Selenite/Tungstate Solution:
Composition per liter:
NaOH....................................................................0.5g
Na2WO4 ·2H2O..........................................................4.0mg
Na2SeO3 ·5H2O.........................................................3.0mg
Preparation of Selenite/Tungstate Solution:  Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–121°C.
Trace Elements Solution SL-10:
Composition per liter:
FeCl2 ·4H2O............................................................1.5g
CoCl2 ·6H2O.........................................................190.0mg
MnCl2 ·4H2O ........................................................100.0mg
ZnCl2 ................................................................70.0mg
Na2MoO4 ·2H2O........................................................36.0mg
NiCl2 ·6H2O..........................................................24.0mg
H3BO3 ................................................................6.0mg
CuCl2 ·2H2O..........................................................2.0mg
HCl (25% solution)...................................................10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl2 ·4H2O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–
121°C.
Vitamin Solution:
Composition per liter:
Pyridoxine-HCl......................................................10.0mg
Thiamine-HCl·2H2O..................................................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% H2 + 20% CO2 . Filter sterilize.
Magnesium Chloride Solution:
Composition per 10.0mL:
MgCl2 ·6H2O..............................................................0.08g
Preparation of Magnesium Chloride Solution:  Add
MgCl2 ·6H2O to distilled/deionized water and bring volume to 1.0L.
Mix thoroughly. Sparge with 100% N2 . Autoclave for 15 min at 15 psi
pressure–121°C.
Calcium Chloride Solution:
Composition per 10.0mL:
CaCl2 ·2H2O..............................................................0.06g
Preparation of Calcium Chloride Solution:  Add CaCl2 ·2H2O
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Solution A: Add components, except bicarbonate,
magnesium chloride and calcium chloride solution, to distilled/deion-
ized water and bring volume to 630.0mL. Mix thoroughly. Adjust pH
to 7.0–7.5. Gently heat and bring to boiling. Boil for 3 min. Cool while
sparging with 80% N2  + 20% CO2 . Add the solid bicarbonate. Adjust
the pH to 7.8. Dispense  under  80% N2  + 20% CO2  gas atmosphere into
anaerobic vials. Autoclave for 15 min at 15 psi pressure–121°C. Asep-
tically and anoxically add magnesium chloride and calcium chloride
solutions. Adjust final pH of the medium to pH 7.7. Note: It may be
necessary to add 10–20 mg sodium dithionite per liter (e.g., from 5%
(w/v) solution, freshly prepared under N2  and filter sterilized), if the so-
lution is not completely reduced after inoculation.
Clostridium sporogenes Supernatant:
Composition per liter:
Yeast extract...........................................................5.0g
NaHCO3 .................................................................4.0g
Casamino acids..........................................................1.0g
KH2PO4 .................................................................0.4g
NH4Cl ..................................................................0.4g
Resazurin ..............................................................0.5mg
Vitamin solution........................................................10.0mL
Magnesium chloride solution.............................................10.0mL
Calcium chloride solution...............................................10.0mL
Na2S·9H2O solution......................................................10.0mL
Trace element solution SL-10.............................................1.0mL
Selenite-tungstate solution .............................................1.0mL
Clostridium sporogenes................................................Variable
Na2S·9H2O Solution :
Composition per 10.0mL:
Na2S·9H2O...............................................................0.3g
Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Preparation of Clostridium sporogenes Supernatant: Add
components, except bicarbonate, Na2S·9H2O solution, magnesium
chloride and calcium chloride solution, to distilled/deionized water and
bring volume to 980.0mL. Mix thoroughly. Adjust pH to 7.0–7.5. Gen-
tly heat and bring to boiling. Boil for 3 min. Cool while sparging with
80% N2  + 20% CO2 . Add the solid bicarbonate. Adjust the pH to 7.8.
Dispense  under  80% N2  + 20% CO2  gas atmosphere into anaerobic
bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically
and anoxically add Na2S·9H2O solution, magnesium chloride and cal-
cium chloride solutions. Adjust final pH to pH 7.0. Inoculate with
Clostridium sp. DSM 754. Incubate for 5 to 8 days at 37°C. Disrupt
cells of the grown culture by autoclaving for 20 min at 15 psi pressure–
121°C. Centrifuge autoclaved culture at 18,000g for 20 min. Discard
cell pellet. Store the supernatant in screw-capped bottles at −20°C. Be-
fore use sterilize the supernatant by autoclaving under 100% N2  for 15
min at 15 psi pressure–121°C. Cool to room temperature under 100%
N2 .
Preparation of Medium: Aseptically and anoxically combine
650.0mL of solution A with 350.0mL of Clostridum sporogenes super-
natant.
Use:  For the cultivation of Cryptoanaerobacter spp.