# 1193: CULTIVATION MEDIUM FOR CHLAMYDIAE


## Main sol. 1193

1. Cultivation of L929 (ACC 2) or HeLa (ACC 57) cells 

45	ml	IMDM-Medium	
5	ml	Fetal bovine serum	
0.5	ml	Aminoacids (100 x)	

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.