Quantitative assessment of bacterial growth phase utilizing flow cytometry

Abstract Flow cytometry is currently underutilized for bacterial phenotyping and standard microbiological techniques do not provide phenotypic information about the state of the bacterial disease. Pseudomonas aeruginosa is a human pathogen of increased importance in public health due to both the ability to cause chronic diseases and the prevalence of functionally different subsets that can be difficult to treat and diagnose. In the present study, we used flow cytometry to analyze the growth phase of P. aeruginosa. A simple method for single cell quantitative detection of bacterial biofilm and planktonic cells was established with a combination of membrane permeable (SYTO 60) and impermeable (TOTO-1) dyes plus the addition of polystyrene counting beads. The specificity of the dye combination for biofilm detection was determined by comparison with impaired biofilm forming strains of P. aeruginosa LasI/RhlI−/− and ∆PfPhage. Results suggest that flow cytometric bacterial phenotyping serves as an expandable platform that may be useful for enumeration of population level variation in P. aeruginosa studies.