Cultivation Medium for Chlamydiae (DSMZ Medium 1193)

[所属分类:培养基配方] [发布时间:2020-5-28] [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

Cultivation Medium for Chlamydiae
(DSMZ Medium 1193)

培养基配方
Composition per 101.0mL:
IM medium...................................................................90.0mL
Fetal bovine serum..........................................................10.0mL
Amino acids, 100x............................................................1.0mL
pH 7.4 ± 0.2 at 25°C
IM medium:
Composition per 100.0mL:
Pancreatic digest of gelatin.................................................0.05g
Bile salts No.3..............................................................0.05g
Brain heart, solids from infusion ...........................................0.02g
Peptic digest of animal tissue...............................................0.02g
NaCl........................................................................0.017g
Glucose .....................................................................0.01g
Na2HPO4 .....................................................................8.0mg
Earle’s balanced salts solution............................................80.0mL
Fetal bovine serum, heat inactivated (2 hr at 55°C) .......................20.0mL
pH 7.4 ± 0.2 at 25°C
Earle’s Balanced Salts Solution:
Composition per liter:
NaCl..........................................................................6.8g
NaHCO3 .......................................................................2.2g
Glucose ......................................................................1.0g
KCl...........................................................................0.4g
CaCl2 ·2H2O................................................................0.265g
MgSO4 ·7H2O..................................................................0.2g
NaH2PO4 ·H2O................................................................0.14g
Preparation of Earle’s Balanced Salts Solution:  Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Filter sterilize.
Preparation of IM:  Combine components. Mix thoroughly. Filter
sterilize. Store at 4°–10°C.
Preparation of Medium:  Combine components. Mix thoroughly.
Filter sterilize. Store for no longer than 4 weeks at room temperature to
facilitate detection of contamination. Prepare a 25 cm 2 flask and seed
with either cells L929 (ACC 2) or HeLa (ACC 57) cells. Incubate at
37°C plus 5% CO2  . When a confluent layer has formed, infection can
be carried out. Exchange medium with 6.0mL of IM with the addition
of 0.001mg/mL cycloheximide (final concentration)) and add 0.5–
1.0mL of EB stock solution (thawed quickly to 37°C). Centrifuge for
1 h onto the cell layer at 1600 rpm at 20°C. Incubate at 37°C + 5%
CO2 . Control cells daily and look for inclusions. Not all chlamydiae
form well-visible inclusions; ultimately, immunofluorescence or in situ
hybridization techniques are necessary to visualize inclusions.
Use: For the screening for Chlamydia using cell line cultures to test for
infectivity.