Isolation and Propagation of Single Inclusion-Derived Chlamydia Using Laser Microdissection

: Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY® FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the microscope. Individual C. trachomatis-infected cells are harvested into separate wells with a pregrown host cell monolayer. Inclusions in harvested cells maturate, and the released elementary bodies infect the host cell monolayer, thus initiating propagation of single inclusion-derived Chlamydia. The method can be used for generation of microbiological clones of C. trachomatis and recovery of transformants and mutants. Isolated single Chlamydia-infected cells can also be examined by diverse downstream molecular assays to reveal unknown features of the Chlamydia replication at a single inclusion level.