Figure 1.
Biofilms hybridized with EUB338 and species-specific probes SMIT, SSAN, SORA2, SDOW or ANAES.
EUB338 was labelled with Atto633 (red), species-specific probes were labelled with Cy3 (green). A. Biofilms were dominated by S. mitis SK24. B. Colonies of S. sanguinis SK150, shown in close colocalization with other streptococcal species. C. Cells of A. naeslundii AK6 formed branched spider-like colonies in basal layers of the biofilms. D. Small groups of S. oralis SK248 cells were scattered over the biofilm. E. Cells of S. downei HG594 appeared in particularly long chains intertwined with clusters of other streptococcal cells. Bars = 20 µm.
Table 1.
Pairwise bacterial coaggregation.
Table 2.
Bacterial composition of the model biofilms.
Figure 2.
Extracellular pH in the model biofilms.
Extracellular pH in the bottom layer of the biofilms was determined using the ratiometric probe C-SNARF-4 and confocal microscopy. In each biofilm, 8 different fields of view were monitored for 390 min. Each line graph represents the average pH in one field of view (143 µm2). A and B: When exposed to saliva without glucose, extracellular pH in the biofilms remained continuously above 7. C, D, E and F: Upon addition of saliva containing 0.4% (C and D) or 10% glucose (E and F) (t = 60 min), pH drops occurred in all examined microscopic fields. Decreases in pH varied considerably in different areas of the biofilms, and distinct pH microenvironments persisted for several hours. Error bars indicate standard deviations.
Figure 3.
Extracellular pH gradients in the model biofilms.
A biofilm was exposed to sterile saliva containing 0.4% (w/v) glucose and the ratiometric pH-sensitive dye C-SNARF-4. Bacterial cells were removed from the image, and extracellular pH was determined. False colours have been used to represent the local pH as shown in the scale bar appended to the image. The higher cell density in the top left part of the picture is correlated with lower pH values, as compared to the bottom right part. Bar = 20 µm.
Figure 4.
A. Overnight cultures of S. oralis SK248, A. naeslundii AK6, S. mitis SK24, S. downei HG594, and S. sanguinis SK150 were washed twice and suspended in sterile saliva containing 0.4% (w/v) glucose. pH was measured after 30 min, 4 h and 20 h. All Streptococcus spp. reached terminal pH within four hours. pH drops in suspensions of A. naeslundii occurred more slowly. B. When glucose concentration was raised to 10% (w/v), no differences in terminal pH were observed. After 20 h, pH was lowest for S. sanguinis and S. downei (4.3–4.4) and highest for S. mitis SK24 (4.9–5). In suspensions of A. naeslundii, pH dropped more quickly as compared to 0.4% glucose. Experiments were performed in duplicate and repeated on another day. Error bars indicate standard deviations.