E Medium for Anaerobes, Modified

[所属分类:培养基配方] [发布时间:2020-7-1] [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

E Medium for Anaerobes, Modified

培养基配方
Composition per 103.6mL:
L- Cysteine·HCl·H2O.....................................................0.05g
(NH4)2SO4 ................................................................0.05g
Peptone...................................................................0.05g
Yeast extract.............................................................0.05g
Salts solution...........................................................50.0mL
Rumen fluid .............................................................30.0mL
Potassium phosphate buffer (1M, pH 6.5)...................................2.8mL
Hemin solution............................................................1.0mL
Glucose-maltose solution..................................................1.4mL
Starch solution ..........................................................1.4mL
Resazurin (0.025% solution)...............................................0.4mL
Vitamin K 3 solution .....................................................0.2mL
pH 6.5 ± 0.2 at 25°C
Salts Solution:
Composition per liter:
NaHCO3 ....................................................................10.0g
NaCl........................................................................2.0g
K2HPO4 .....................................................................1.0g
KH2PO4 .....................................................................1.0g
CaCl2 , anhydrous...........................................................0.2g
MgSO4 ......................................................................0.2g
Preparation of Salts Solution:  Add CaCl2 and MgSO4 to approx-
imately 300.0mL of distilled/deionized water. Mix thoroughly. Bring
volume to 800.0mL with distilled/deionized water. Add remaining
components. Mix thoroughly. Bring volume to 1.0L with distilled/de-
ionized water. Mix thoroughly. Store at 4°C.
Rumen Fluid:
Composition per 100.0mL:
Rumen fluid .................................................................100.0mL
Preparation of Rumen Fluid:  Obtain the rumen contents from a
cow that has been fed an alfalfa-hay ration. Filter rumen contents
through two layers of cheesecloth. Store under 100% CO 2 in the refrig-
erator. The particulate material will settle out. Use only the supernatant
liquid.
Hemin Solution:
Composition per 100.0mL:
Hemin ......................................................................0.05g
NaOH (1N solution)..........................................................1.0mL
Preparation of Hemin Solution:  Add hemin to 1.0mL of 1N
NaOH solution. Mix thoroughly. Bring volume to 100.0mL with dis-
tilled/deionized water. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to 45°–50°C.
Glucose-Maltose Solution:
Composition per 10.0mL:
Glucose....................................................................0.5g
Maltose ...................................................................0.5g
Preparation of Glucose-Maltose Solution: Add components to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Filter sterilize.
Starch Solution:
Composition per 10.0mL:
Starch, soluble............................................................0.5g
Preparation of Starch Solution: Add starch to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C.
Resazurin Solution:
Composition per 44.0mL:
Resazurin...............................................................0.011g
Preparation of Resazurin Solution:  Add resazurin to distilled/
deionized water and bring volume to 44.0mL. Mix thoroughly.
Vitamin K 3 Solution:
Composition per 25.0mL:
Vitamin K 3 (menadione).................................................0.0125g
Ethanol, absolute.......................................................25.0mL
Preparation of Vitamin K 3 Solution:  Add vitamin K 3 to 99.0mL
of ethanol. Mix thoroughly.
Preparation of Medium: Filter sterilize potassium phosphate buf-
fer. Add components—except  L -cysteine·HCl·H 2 O, vitamin K 3 solu-
tion, potassium phosphate buffer, glucose-maltose solution, and starch
solution—to distilled/deionized water and bring volume to 98.0mL.
Mix thoroughly. Gently heat and bring to boiling. Continue boiling un-
til resazurin turns colorless, indicating reduction. Cool in an ice-water
bath under O2 -free 97% N2 + 3% H2 . Add the  L -cysteine·HCl·H2O and
vitamin K 3 solution. Mix thoroughly. Adjust pH to 6.5 with 8N NaOH
or 5N HCl. Anaerobically distribute into tubes under O2 -free 97% N2
+ 3% H 2 in 7.0mL volumes. Cap tubes with butyl rubber stoppers.
Place tubes in a press. Autoclave for 12 min at 15 psi pressure–121°C
with fast exhaust. Immediately prior to inoculation, aseptically add
0.2mL of filter-sterilized potassium phosphate buffer, 0.1mL of sterile
glucose-maltose solution, and 0.1mL of sterile starch solution to each
tube. Mix thoroughly.
Use:  For the cultivation and maintenance of Bacteroides species, Butyr-
ivibrio fibrisolvens, Clostridium methylpentosum, Eubacterium rumi-
nantium, Lachnospira multipara, Micromonospora ruminantium, Pre-
votella ruminicola, Propionibacterium acidipropionici, Selenomonas
species, Succinivibrio dextrinosolvens, and Treponema species.