RGCA Medium (Rumen Fluid Glucose Cellobiose Agar)

[所属分类:培养基配方] [发布时间:2021-8-3] [发布人:网站管理员2] [阅读次数:] [返回]

RGCA Medium
(Rumen Fluid Glucose Cellobiose Agar)

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

Composition per 300.3mL:

Rumen fluid...........................................................................120.0mL
Solution IV...............................................................................65.0mL
Mineral solution I ....................................................................45.0mL
Mineral solution II...................................................................45.0mL
Na 2 CO 3  solution......................................................................20.0mL
L -Cysteine·HCl·H 2 O solution....................................................5.0mL
Solution III.................................................................................0.3mL
pH 6.6 ± 0.2 at 25°C
Mineral Solution I:
Composition per 100.0mL:
K 2 HPO 4 .........................................................................................0.3g
Preparation of Mineral Solution I: Add K 2 HPO 4 to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Mineral Solution II:
Composition per 100.0mL:
(NH 4 ) 2 SO 4 ....................................................................................0.6g
NaCl..............................................................................................0.6g
KH 2 PO 4 .........................................................................................0.3g
MgSO 4 ........................................................................................0.06g
CaCl 2 ...........................................................................................0.06g
Preparation of Mineral Solution II: Add components to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Solution III:
Composition per 10.0mL:
Resazurin ....................................................................................0.01g
Preparation of Solution III: Add resazurin to 10.0mL of distilled/
deionized water. Mix thoroughly.
Solution IV :
Composition per 65.0mL:
Agar ..............................................................................................4.5g
Glucose .........................................................................................0.6g
Cellobiose .....................................................................................0.6g
Preparation of Solution IV: Add components to distilled/deion-
ized water and bring volume to 65.0mL. Mix thoroughly.
L -Cysteine·HCl·H 2 O Solution:
Composition per 100.0mL:
L -Cysteine·HCl·H 2 O .....................................................................3.0g
Preparation of  L -Cysteine·HCl·H 2 O Solution: Add  L -cyste-
ine·HCl·H 2 O to distilled/deionized water and bring volume to
100.0mL. Mix thoroughly. Filter sterilize.
Na 2 CO 3 Solution:
Composition per 100.0mL:
Na 2 CO 3  .........................................................................................6.0g
Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3  to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.
Rumen Fluid:
Composition per 120.0mL:
Rumen fluid ...........................................................................120.0mL
Preparation of Rumen Fluid: Filter rumen contents, obtained
from a cow on an alfalfa-hay concentrate ration, through two layers of
cheesecloth to remove larger particles. Store under CO 2 in quart milk
bottles in the refrigerator. Much of the particulate matter settles out.
Use the supernatant fluid.
Preparation of Medium: Combine 45.0mL of mineral solution I,
45.0mL of mineral solution II, 0.3mL of solution III, and 65.0mL of so-
lution IV in a 500.0mL flask. Gently heat and bring to boiling. Add
120.0mL of rumen fluid. Gently heat and bring to boiling under 100%
CO 2 . Cap with a rubber stopper and wire the stopper secure. Autoclave
for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Remove stop-
per and gas with 100% CO 2 to eliminate O 2 . Aseptically add 5.0mL of
sterile  L -cysteine·HCl·H 2 O solution and 20.0mL of sterile Na 2 CO 3  so-
lution. Mix thoroughly. Aseptically and anaerobically distribute into
tubes under 100% CO 2 in 6.0mL volumes. Cap with rubber stoppers.
Use:  For the cultivation and maintenance of Ruminococcus albus,
Ruminococcus flavifaciens, and Succinimonas amylolytica.