It is known that the obtained
fluorescence intensity, with a few exceptions, is directly correlated with the growth rate of the target bacteria. The accessibility of the targets is controlled mainly by cell wall properties, which again require to get permeabilized by either the fixative or, in case of gram positive cells, lysozyme . As P. intermedia and streptococci were readily stained at the base of the biofilms, a hindered diffusion of the probes or fixatives through the biofilms does not seem to be the problem. The accessibility of the cells can be sorted out as well, as the signal is very clear in the top layer of the biofilm. Careful examination of the images, by enhancing the contrast settings for the general DNA staining in our samples, Selumetinib mouse revealed structures
at the base of the biofilms that very much resembles the well-stained colonies of F. nucleatum observed in less thick biofilms. Combined with the high abundance detected by IF, it seems that F. nucleatum was in fact present in at the base of the biofilms, however, either in a non-viable- or at least non-active state. For future experiments, it might be worth investigating new methods to increase fluorescent signals, in order to obtain a bright staining throughout the whole biofilm. Catalysed reporter deposition (CARD)-FISH , the use of helper oligonucleotides , or designing probes targeting the 23S rRNA  might Alpelisib mw be solutions. Due to the large size of the horseradish peroxidase used with CARD-FISH, it seems unlikely
that this method would be appropriate, and the use of helper oligonucleotides or probes targeting the 23S rRNA seem more promising to reach stronger signals. One of the major differences to the in vivo situation is that the model biofilms Cediranib (AZD2171) grew without the presence of an epithelial cell layer. Some of the observed differences will be caused by the lack of interactions that occur in vivo. A future project will address this circumstance and aims to incorporate an epithelial cell layer into the model system. The main difficulty in maintaining such a co-culture system is that different growth conditions that are needed to cultivate either epithelial cells or biofilms. While the strict anaerobes in the consortium of the biofilms are very sensitive to oxygen, the epithelial cells do require oxygen for growth. Further, biofilms and epithelial cells do have very different nutritional requirements. In our co-culture experiments performed so far, cells and biofilms were cultured separately and incubated as co-culture after the development of both biofilms and epithelial cells . Current experiments showed, that the biofilm consortium is still able to grow on agar plates after 48 h of co-culture, however, the viability of the bacteria was greatly reduced (data not shown).