Cultivation and Laboratory Maintenance of Chlamydia pneumoniae

Chlamydiae are Gram-negative obligate intracellular parasites. Chlamydia pneumoniae is a human respiratory pathogen that causes pneumonia, bronchitis, sinusitis, and pharyngitis. C. pneumoniae has also been associated with cardiovascular disease. C. pneumoniae can only be grown in cell culture and is more difficult to isolate from specimens than Chlamydia trachomatis. Commonly used cell lines for isolation of C. trachomatis are not sensitive for C. pneumoniae. The most sensitive cell lines for isolation of C. pneumoniae are HL and HEp-2. Centrifugation of the inoculum onto the monolayer and inclusion of cycloheximide in the medium enhance isolation. Inclusions are smaller than those of other chlamydiae and are visualized by staining with FITC-conjugated genus- or C. pneumoniae–specific monoclonal antibodies. Slow expansion and use of a small inoculum are key to successful culture. Infectious organisms can be purified by use of Hypaque-76 gradients to titers >1 × 108/ml. Curr. Protoc. Microbiol. 12:11B.1.1-11B.1.23. © 2009 by John Wiley & Sons, Inc. Keywords: Chlamydiae; Chlamydia pneumoniae; respiratory pathogen; pneumonia; cell culture; elementary bodies; inclusion staining