Standardization of flow cytometric techniques to evaluate Corynebacterium pseudotuberculosis murine phagocytic cells

Abstract

The understanding of bioprocesses has been allowed the increase of techniques to study cellular interactions, such as flow cytometry (FC). This technic is an important tool for counting and classifying microscopic particles suspended in flowing liquid medium, analyzing parameters from cells diverse origins, including bacteria. Thus, the aim of this study was to standardize protocols by flow cytometry to evaluate the absolute count and physiological aspects of Corynebacterium pseudotuberculosis in murine phagocytic cells. C. pseudotuberculosis was grown in 0.5% BHI –Tween80 medium, followed by the labeling of this microorganism with fluorescent dyes, at different periods and different physiological stages. In the phagocytosis assay, murine peritoneal cells from two strains were used after sodium thioglycolate-induced peritonitis. After cell adhesion, the challenge with stained bacteria was done and after 1h 30 min the adherent cells were trypsinized, washed and centrifuged for the (FC) assay. A growth period similar to a standard microbial growth curve was observed, and allowed to distinguish among different physiological stages. Data obtained from (FC) show phagocytic cell populations containing bacteria in the cytoplasm. A population with low internal complexity, similar to that presented by lymphocytes, and a population with moderate internal complexity similar to that presented by cells of monocytic origin were observed. The (FC) technique with flowering marking was effective to evaluate the growth and viability of C. pseudotuberculosis, as well as the interaction of bacterial cells with murine peritoneal phagocytic cells. Therefore, FC is an efficient technique to evaluating the growth curve of this bacterium, distinguishing different physiological stages, and may even indicate a mechanism of escape from phagocytosis.