A Novel Flow Cytometric Approach for the Quantification and Quality Control of Chlamydia trachomatis Preparations

is an obligate intracellular pathogenic bacterium with a biphasic developmental cycle manifesting two distinct morphological forms: infectious elementary bodies (EBs) and replicative intracellular reticulate bodies (RBs). Current standard protocols for quantification of the isolates assess infectious particles by titering inclusion-forming units, using permissive cell lines, and analyzing via immunofluorescence. Enumeration of total particle counts is achieved by counting labeled EBs/RBs using a fluorescence microscope. Both methods are time-consuming with a high risk of observer bias. For a better assessment of preparations, we developed a simple and time-saving flow cytometry-based workflow for quantifying small particles, such as EBs with a size of 300 nm. This included optimization of gain and threshold settings with the addition of a neutral density filter for small-particle discrimination. The nucleic acid dye SYBR Green I (SGI) was used together with propidium iodide and 5(6)-carboxyfluorescein diacetate to enumerate and discriminate between live and dead bacteria. We found no significant differences between the direct particle count of SGI-stained preparations measured by microscopy or flow cytometry ( > 0.05). Furthermore, we completed our results by introducing a cell culture-independent viability assay. Our measurements showed very good reproducibility and comparability to the existing state-of-the-art methods, indicating that the evaluation of preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of preparations for downstream applications.