PY Medium with Serum-Cocarboxylase

[所属分类:培养基配方] [发布时间:2021-7-16] [发布人:网站管理员2] [阅读次数:] [返回]

PY Medium with Serum-Cocarboxylase

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

Composition per liter:

Yeast extract................................................................................10.0g
Peptone .........................................................................................5.0g
Pancreatic digest of casein............................................................5.0g
L -Cysteine·HCl·H 2 O.....................................................................0.5g
Serum-cocarboxylase solution...............................................100.0mL
Salts solution............................................................................40.0mL
Hemin solution.........................................................................10.0mL
Resazurin solution .....................................................................4.0mL
Vitamin K 1 solution...................................................................0.2mL
pH 6.9 ± 0.2 at 25°C
Salts Solution:
Composition per liter:
NaHCO 3 ......................................................................................10.0g
NaCl..............................................................................................2.0g
K 2 HPO 4 .........................................................................................1.0g
KH 2 PO 4 .........................................................................................1.0g
CaCl 2 , anhydrous..........................................................................0.2g
MgSO 4 ..........................................................................................0.2g
Preparation of Salts Solution: Add CaCl 2 and MgSO 4 to 300.0mL
of distilled/deionized water. Mix thoroughly until dissolved. Bring vol-
ume to 800.0mL with distilled/deionized water. Add remaining com-
ponents while stirring. Bring volume to 1.0L. Mix thoroughly. Store at
4°C.
Hemin Solution:
Composition per 100.0mL:
Hemin .......................................................................................0.050g
NaOH (1N solution)...................................................................1.0mL
Preparation of Hemin Solution: Add hemin to NaOH solution.
Mix thoroughly. Adjust volume to 100.0mL with distilled/deionized wa-
ter. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Resazurin Solution:
Composition per 44.0mL:
Resazurin..................................................................................0.044g
Preparation of Resazurin Solution: Add resazurin to distilled/
deionized water and bring volume to 44.0mL. Mix thoroughly.
Vitamin K 1 Solution:
Composition per 30.0mL:
Vitamin K 1 ..................................................................................0.15g
Ethanol (95% solution)............................................................30.0mL
Preparation of Vitamin K 1 Solution:  Add vitamin K 1 to ethanol.
Mix thoroughly. Store in a brown bottle and keep under refrigeration.
Discard after 1 month.
Serum-Cocarboxylase Solution:
Composition per 101.0mL:
Rabbit serum, heat inactivated...............................................100.0mL
Cocarboxylase solution..............................................................1.0mL
Cocarboxylase Solution:
Composition per 100.0mL:
Cocarboxylase...............................................................................5mg
Preparation of Cocarboxylase Solution:  Add cocarboxylase to
distilled/deionized water and bring volume to 100.0mL. Mix thorough-
ly. Filter sterilize.
Preparation of Serum-Cocarboxylase Solution:  Aseptically
combine 100.0mL of sterile, heat-inactivated rabbit serum with 1.0mL
of sterile cocarboxylase solution. Mix thoroughly.
Preparation of Medium: Add components, except serum-cocarbox-
ylase solution,  L -cysteine·HCl·H 2 O, hemin solution, and vitamin K 1 solu-
tion, to distilled/deionized water and bring volume to 889.8mL. Mix
thoroughly. Gently heat and bring to boiling under 80% N 2 + 10% CO 2 +
10% H 2 . Continue boiling until resazurin turns colorless, indicating reduc-
tion. Cool to 45°–50°C. Add the serum-cocarboxylase solution,  L -
cysteine·HCl·H 2 O, hemin solution, and vitamin K 1 solution. Adjust pH to
6.9, if necessary. Anaerobically distribute into tubes under 80% N 2 + 10%
CO 2 + 10% H 2 . Cap the tubes with rubber stoppers. Place tubes in a press.
Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust.
Use: For the cultivation of Treponema phagedenis.