Fig. 2: Development of L. paracasei ATCC334 biofilm-like microcolonies encapsulated in pectin beads.

a–c Images of CPB_Biofilm obtained by laser scanning confocal microscopy (CLSM) at different incubation times in MRS medium. a CPB is contrasted in the transmitted light channel in gray. Bacterial cells were labeled with Syto 9 green-fluorescent nucleic acid stain (scale bars, 100 μm). b In order to visualize size diversity and microcolony coalescence, fluorescence images obtained at 15 h were transformed into isosurface representations with a coded color associated with microcolony volume (scale bars, 20 μm). c The evolution of the microcolonies’ spatial distribution in a CPB during 48 h of incubation in MRS. The initial homogeneous distribution of fluorescent colonies at 15 h gave way to their peripheral distribution and the emergence of a central hollow void in the beads after 24 and 48 h of incubation (scale bars, 100 μm). d Microcolony organization inside the pectin beads of CPB_Biofilm after an incubation time of 24 hours in MRS medium. Images obtained by CLSM (1 and 2) and cryo-SEM (3, 4, 5, and 6). Black boxes indicate biofilm-like microcolonies (1, 2, 3, and 4) and red boxes highlight the adhesion of bacteria to the matrix of pectin beads (5) and the presence of extracellular polymeric substances in the microcolonies (6). Images 5 and 6 have been colorized. Scale bars, (1) 100 μm, (2) 20 μm, (3) 20 μm, (4) 5 μm, (5) 1 μm and (6) 1 μm.