(A) cyan: T. forsythia, red: P. intermedia, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). (B) cyan: T. denticola, red: P. gingivalis, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). Figures show a representative area of one disc. Figure 8 Biofilms grown for 64.5 h in iHS Medium. FISH staining of a fixed biofilm; the biofilm base in the side views is directed towards the top view. C. rectus is shown schematically

buy LY2109761 as dots (fluorescence maxima of the cells). (A) red: A. oris, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox), blue: EPS. (B) red: C. rectus, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). The red dots appear yellowish due to the transparency of the green channel. Figures show a representative area of one disc. Scale bars: 20 μm. Discussion This study focused on the importance of the nutritional conditions and the structure of subgingival biofilms generated on HA discs in vitro. The alteration of the growth medium by eliminating

saliva and increasing the concentration of heat-inactivated human serum selleckchem affected the biofilms positively as they developed to higher thickness, were more stable and enabled the extensive proliferation of T. denticola, which were observed only in small numbers using media with low or no heat-inactivated human serum. We were able to locate all the 10 organisms by multiplex FISH https://www.selleckchem.com/products/BI-2536.html in combination with CLSM. The biofilms displayed a stratified structure reminiscent of in vivo subgingival biofilms [13]. However, in contrast to the in vivo situation, F. nucleatum was predominant in the basal layer along with streptococci of the biofilms grown in mFUM4. In biofilms cultured in iHS, F. nucleatum was detected as dispersed cells in the top layer. Earlier experiments showed

that F. nucleatum has a strong dependency on streptococci, and is only able to establish MYO10 along with them (data not shown). This observation is in accordance with the finding of co-aggregation studies that identified the ability of streptococci to attach to components of the pellicle, while F. nucleatum was shown to bind to the streptococci and act as a “bridging organism” for other species to colonize the biofilm [14]. The observed difference that F. nucleatum establishes in the basal layer might very well be due to the fact, that all strains were inoculated simultaneously. If no streptococci were added to the inoculum, but added to the biofilms at a later time point, F. nucleatum did not establish in the basal layer but rather after the addition of the streptococci, forming an intermediate layer. In this case, mainly A. oris was detected as an early colonizer (data not shown). Possibly, it would make sense to add the various strains sequentially, simulating the shift from health to disease. The growth medium affected not only the biofilm composition; it had a strong influence on the rate of biofilm formation as well.