Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester
Fig 3
Metabolic activity sensing of growing and dividing cells.
(A) Non-growing cells can be classified into non-viable non-fluorescent cells and non-growing but metabolically active bacteria, which showed highest mean single cell fluorescence in comparison. Growing bacteria showed moderate to medium fluorescence. (B-C) Mean single cell fluorescence of freshly seeded and heterogeneous sized bacteria is given after a change to minimal medium CGXII + 4% GLC containing CAM (n = 6 cells). (B) Increase in the mean single cell fluorescence of six individual cells of different cell size is shown over time after infusion of CGXII + 4% GLC + 46 μM CAM. (C) The mean single cell fluorescence of the individual cells presented in (B) is given according their size during the short term experiment. Increase of cell size due to growth could was neglectable during the experimental time of 30 min. Mean single cell fluorescence in dependency of cell size revealed marginal differences of the final equilibrium mean fluorescence of individuals. (D-F) Metabolic activity sensing of cells grown in complex medium BHI at pH 6.6 (red), pH 7.0 (green), and pH 7.4 (blue), respectively. (D) Average values of 10 cultivation chambers are presented. No significant difference in calcein fluorescence (solid lines) and growth represented by total cell area (dashed lines) could be observed for cultivation of C. glutamicum at pH 7.0 and pH 7.4. At pH 6.6, however, increased mean calcein fluorescence as well as a reduced total cell area was observed (n = 10 colonies analyzed at each pH). (E) The mean single cell area indicated a tendency of cell size reduction in average over time (n = 10 colonies analyzed at each pH). (F) Mean fluorescence correlates positively with the mean single cell area at all three pH values (n = 10 colonies analyzed at each pH).