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

Pathogens

Andreas Spittler¹  Alexander Kirschner²  Michael Reiter²  Hannes Stockinger²  Romana Klasinc²  Astrid Digruber³  Waltraud Tschulenk⁴  Ingrid Walter⁴ 

[1] Core Facility Flow Cytometry and Department of Surgery, Research Laboratories, Medical University of Vienna, 1090 Vienna, Austria;Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria;Institute of Microbiology, Department of Pathobiology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria;Institute of Morphology, Department of Pathobiology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria;

关键词: flow cytometry;    Chlamydia trachomatis;    quantification;    viability;    quality control;   

DOI  :  10.3390/pathogens10121617

来源: DOAJ

【 摘 要 】

Chlamydia trachomatis 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 C. trachomatis 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 C. trachomatis preparations measured by microscopy or flow cytometry (p > 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 C. trachomatis preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of C. trachomatis preparations for downstream applications.

【 授权许可】

Unknown