Ultramdck

MDCK cells (CCL34; American Type Culture Collection, Manassas, VA) were maintained in serum-free medium (UltraMDCK; Lonza) supplemented with 1% L-glutamine (200 nM; Lonza) and 2% penicillin-streptomycin (PS; 10 000 U penicillin/mL and 10 000 U streptomycin/mL; Lonza).

Buffalo green monkey (BGM) cells in minimal essential medium (MEM, Lonza, Cologne, Germany) with 5% serum were seeded into Trac bottles containing glass coverslips (Bibby Sterilin Ltd., Staffordshire, UK) and incubated at 37°C with 5% CO₂ in a fully humidified cabinet for 4 days. Swabs in 1–2 mL Chlamydia stabilizing medium or PBS buffer were ultrasonicated (ten 0.8-s pulses with 0.2-s pause between each pulse at an amplitude of 80%; Branson 450D Sonifyer) and 30–300 μL of medium or buffer were inoculated into six Trac bottles with confluent grown BGM monolayers. After inoculation, the bottles were centrifuged at 3000 × g and 37° C for 60 min and subsequently incubated for 2 h. The MEM medium was then replaced with serum-free medium UltraMDCK (Lonza Cologne, Germany) containing the antibiotics amphotericin (2.5 μg/mL), gentamicin (10 μg/mL) and vancomycin (25 μg/ mL). The medium was renewed after 18 h. Three days after inoculation, a single coverslip was fixed with methanol, and the monolayer was stained with IMAGEN Chlamydia (Oxoid Ltd., Cambridgeshire, UK). A sample was considered positive when inclusions of typical chlamydial morphology appeared as bright apple-green spots after two passages.