5 mM for SAL respectively. The formation of the biofilms was observed by determination of total counts on Columbia blood agar (CBA) plates at 5 time points during the incubation time. The final structure,
as well as the thickness of the biofilms at 5 time points during the incubation time, was determined by confocal laser scanning microscopy (CLSM). The experiments confirmed and extended our previous finding  that the composition of the growth medium has a major effect on the development, stability and composition of the biofilms. The iHS medium delayed biofilm formation by 20 h compared to mFUM4 this website (Figure 1). 4 h after inoculation in mFUM4, the discs were densely colonized by cocci. Based on the observation that most of these cocci appeared as chains, they can be assumed to be streptococci. However, after 4 h of incubation in iHS, cocci were observed to appear almost exclusively as dense microcolonies, while rods (morphologically Fusobacterium nucleatum, Prevotella intermedia, or Tannerella forsythia) in low abundance colonized the majority of the disc. Incubation in SAL medium Temsirolimus supplier led to a similar observation as in mFUM4: The disc was colonized mainly by cocci (Figure 2). Figure 1 Time course of biofilm growth comparing
SAL, mFUM4, and iHS as growth media. Total counts determined by plating on CBA agar plates (T. denticola and T. forsythia are not cultivable on CBA). Each box PIK3C2G represents N = 9 independent biofilms from three independent triplicate experiments. The boxes
represent the inter quartile range of the data points, the bar indicates the median. The whiskers cover the data points within the 1.5x inter quartile range. Dots are outliers within 1.5 and 3 box lengths outside the interquartile range. Figure 2 Bacterial attachment to the disc surface under different nutritional conditions 4 h after inoculation. Comparison of the growth media mFUM4 (A), iHS (B) and SAL (C). green: DNA staining using YoPro-1 + Sytox. The disc surface is visualized in grey colour. The images show representative areas of one disc each. Scale bars: 15 μm (A/B) and 10 μm (C). The high concentration of human serum in iHS improved biofilm stability in terms of firm attachment to the disc (less cell loss during dip washing and the FISH staining procedure), and further the average thickness of the biofilms was significantly increased after 64.5 h when compared to biofilms grown in mFUM4, or SAL respectively (Figure 3A). However, the total counts of bacteria per biofilm did not show significant differences between the three growth media (Figure 3B). Figure 3 Thickness (A) and total counts (FISH/IF) (B) of biofilms grown for 64.5 h in SAL, mFUM4, and iHS growth medium. Thickness was determined by CLSM, total counts were calculated from the species specific quantification by visual microscopic counting following FISH- or IF from N=9 independent biofilms from three independent experiments.