Two representative experiments are shown. Green fluorescence, which is a measure of total biomass, is shown in absolute units. B Biofilm membrane damage, determined using the LIVE/DEAD BacLight HM781-36B Bacterial Viability stain. Green and red fluorescence was measured, and biofilm damage was calculated as reduction of the ratio of green/red fluorescence compared to controls without carolacton. Error values were calculated from the standard deviations of the green/red ratios of control and carolacton treated samples according to the error propagation formula of Gauss. Three representative experiments are shown. Biofilms were grown anaerobically. Mean

and standard deviation are given for triplicate samples. HMPL-504 Membrane damage of the biofilm cells, determined by the LIVE/DEAD BacLight fluorescence staining method by staining with both SYTO9 (green) and propidium iodide (red), was calculated as the reduction of the green/red fluorescence ratio in biofilms grown with carolacton relative to untreated controls and is shown in Figure 5B for three independent experiments. PI3K inhibitor It shows a similar pattern. Biofilm damage

was small during the first 6 h, increased rapidly until about 8.5 or 12.25 h, respectively and then remained stable or increased more slowly till the end of the experiment after 24 hours. The curves for the two concentrations of carolacton tested were very similar, as expected from the concentration range of carolacton Progesterone activity determined previously (Figure 4). The maximum reduction of the relative green/red fluorescence ratio was between 47%

and 69% reflecting the dynamic process of biofilm growth. The pH dropped from pH 7.8 to pH 4.3 (24 h of growth), but there was no difference in controls and carolacton treated cultures. To summarize, the data show that carolacton temporarily reduced the total amount of biofilm cells, indicated by staining with the green fluorescent dye alone, during the period of maximum biofilm growth (Figure 5A). Most importantly, carolacton strongly reduced the viability of cells within the biofilm, determined by the reduction of the relative proportion of green to red fluorescence, throughout 24 h of biofilm development but mainly during the period of maximum biofilm growth and thereafter, while little reduction of viability was observed during the initial hours of biofilm growth (Figure 5B). Investigation of the effect of carolacton on S. mutans biofilms by confocal laser scanning microscopy The effect of carolacton on the spatial distribution, architecture and viability of biofilms of wild-type S. mutans UA159 was investigated by confocal laser scanning microscopy. Figure 6A shows top-down views, flanked by pictures of vertical optical sections after 12 hours of cultivation and Figure 6B represents horizontal sections at a higher magnification.