1193 .
CULTIVATION MEDIUM FOR CHLAMYDIAE
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* Medium for Chlamydia trachomatis DSM 19102
Plus 5% CO2
Growth conditions: 37 °C
Equipment needed:
Centrifuge
Filter
Final gas composition:
5 % CO2
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Final pH:
n.d.
Select a strain (optional):
ml
| 1 Cultivation of L929 (ACC 2) or HeLa (ACC 57) cells | |||||
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
IMDM-Medium | 45.0 | ml | ||
|
|
Fetal bovine serum | 5.0 | ml | ||
|
|
Aminoacids
(100 x) |
0.5 | ml | ||
| 2 Filter-sterilize (0.2 µm) and keep no longer than 4 weeks. Store at room temperature to facilitate detection of contamination. | |||||
| 3 Prepare a 25 cm2 flask and seed cells according to standard protocols (see DSMZ catalogue for ACC 2 and ACC 57). Incubate at 37°C plus 5% CO2. When a confluent layer has formed, infection can be carried out. | |||||
| 4 Exchange medium with 6 ml of Infection Medium (as above with the addition of 1 µg/ml cycloheximide (final concentration)) and add 500 - 1000 µl of EB stock solution (thawed quickly to 37°C). | |||||
| 5 Centrifuge for 1 h onto the cell layer at 1600 rpm at 20°C. | |||||
| 6 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. | |||||