Figure 3

CLSM observation of 24 hours-biofilm formed byclinical isolate S. maltophilia OBGTC9 on IB3-1 cell monolayer. A-B. CLSM micrographs of not fixed specimens of unexposed (control; Figure 3A) and OBGTC9-exposed (Figure 3B) IB3-1 cell monolayer stained with Syto-9 (green fluorescence, indicating live cells), propidium iodide (red fluorescence, indcating dead cells), and Con-A (blue fluorescence, indicating extracellular matrix). Image capture was set for visualization of: (a) green fluorescence only; (b) red fluorescence only; (c) blue fluorescence only (3) or; (d) co-localization of all three fluorescence signals. Note the formation of a S. maltophilia microcolony embedded in matrix whose formation is significantly increased in infected vs control IB3-1 cell monolayers. C. CLSM examination of fixed IB3-1 monolayer exposed to S. maltophilia OBGTC9 for 24 hours: three-dimensional representation. Green fluorescence indicates autofluorescence of IB3-1 cytoplasm following exposure to fixation mixture; red fluorescence indicates binding of propidium iodide to nucleic acids of both IB3-1 and S. maltophilia cells. Note the microcolony organization of S. maltophilia on almost all IB3-1 cells. Magnification, ×100.