Hungate’s Habitat-Simulating Medium

[所属分类:培养基配方] [发布时间:2021-4-16] [发布人:李佳颐] [阅读次数:] [返回]

Hungate’s Habitat-Simulating Medium

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

Composition per 1140.2mL:

Rumen fluid ...........................................................................333.0mL

Mineral solution A.................................................................167.0mL

Mineral solution B.................................................................167.0mL
NaHCO 3 solution.....................................................................53.0mL
L -Cysteine·HCl solution ..........................................................10.6mL
Substrate solution.....................................................................10.6mL
Resazurin solution .....................................................................1.0mL
Mineral Solution A:
Composition per liter:
NaCl..............................................................................................6.0g
KH 2 PO 4 .........................................................................................3.0g
(NH 4 ) 2 SO 4 ....................................................................................3.0g
CaCl 2 .............................................................................................0.6g
MgSO 4 ..........................................................................................0.6g
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly.
Mineral Solution B:
K 2 HPO 4 ...........................................................................................3.0
Preparation of Solution B: Add K 2 HPO 4 to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly.
Resazurin Solution:
Composition per 100.0mL:
Resazurin......................................................................................0.1g
Preparation of Resazurin Solution: Add resazurin to distilled/
deionized water and bring volume to 100.0L. Mix thoroughly.
L -Cysteine·HCl Solution:
Composition per 100.0mL:
L -Cysteine·HCl .............................................................................3.0g
Preparation of  L -Cysteine·HCl Solution:  Add  L -cysteine·HCl to
O 2 -free distilled/deionized water and bring volume to 100.0L. Mix
thoroughly. Gently heat and bring to boiling. Continue boiling for 2
min. Cool to 25°C under 100% N 2 . Seal tube with a stopper that is
wired in place. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
25°C.
NaHCO 3 Solution:
Composition per 10.0mL:
NaHCO 3 ........................................................................................1.0g
Preparation of NaHCO 3 Solution: Add NaHCO 3 to O 2 -free dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Filter sterilize. Gas with 100% CO 2 for 15 min.
Substrate Solution:
Composition per 100.0mL:
Sugar...........................................................................................10.0g
Preparation of Substrate Solution: Add sugar to O 2 -free dis-
tilled/deionized water. Mix thoroughly. Gas with 100% N 2 for 15 min.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Preparation of Medium: Add 167.0mL of solution A, 167.0mL of so-
lution B, and 1.0mL of resazurin solution to distilled/deionized water and
bring volume to 733.0mL. Mix thoroughly. Gently heat and bring to boil-
ing. Continue boiling until resazurin turns colorless, indicating reduction.
Bring volume back to 733.0mL (some evaporation will have occurred)
with O 2 -free distilled/deionized water. Cool to 45°–50°C under O 2 -free
100% CO 2 . Anaerobically add rumen fluid. Anaerobically distribute into
tubes in 10.0mL volumes. Cap with butyl rubber stoppers. Place tubes in a
press. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Im-
mediately prior to inoculation, aseptically and anaerobically add 0.1mL of
sterile  L -cysteine·HCl solution, 0.5mL of sterile NaHCO 3 solution, and
0.1mL of substrate solution per 10.0mL of medium in each tube.
Use: For the cultivation of Bacteroides species from rumens.