In the biofilm from disc 013 (biofilm 013 in the following) LGC358a stained clearly two populations of rods that differed in length, whereas LAB759 identified only the shorter of the two morphotypes. The longer and predominant cell type had the probe reactivity profile BTK inhibitor LGC358a+/LAB759-/Lfer466+/Lreu986+/Lcas467- (Figure 2C), whereas

the smaller one was LGC358a+/LAB759+/Lfer466-/Lreu986-/Lcas467+, indicating that the larger rods are L. fermentum and the smaller ones lactobacilli from the casei group. While the total number of L. casei, streptococci or Abiotrophia/Granulicatella seemed not to correlate with the extent of disc demineralization, the high concentration of L. fermentum in the biofilm of the extremely demineralized disc 013 was quite remarkable. Figure 3 Enumeration by FISH of lactic acid producing bacteria in three in situ grown biofilms. Biofilms were harvested from bovine enamel discs, carried in situ for 10 days and nights by three different volunteers. The discs differed greatly in the extent of demineralization indicated in the within legend of the plot. The detection limit (dl) of the FISH assay was approximately 103 bacteria per ml of sample. All other lactobacillus probes gave negative results. Concerning Lsal574 and Lvag222 we found that both

these probes had to be used at much higher stringency conditions (50% formamide) than expected from the in vitro experiments with reference strains to prevent cross-reactivity with other biofilm bacteria. In particular

Temsirolimus cells with the characteristic morphology of Selenomonas were often cross-reactive at conditions of insufficient stringency. Abiotrophia and Granulicatella could be detected in high numbers in all three samples. Both ABI161 and ABI1246 recognized cocci, which in double-labeling experiments stained always negative with the streptococcal probes LGC358c and MIT447 (data not shown). Finally, all samples contained high numbers of streptococci, mostly from the mitis group. S. mutans, however, was found with MUT590 in only one sample at low concentration, and the probes for S. sobrinus and S. constellatus/S. intermedius gave negative results. Identification by FISH of streptococci, http://www.selleck.co.jp/products/erastin.html in particular of the mitis group, is hindered severely by high conservation of the 16S rRNA gene sequence among these taxa [20, 21] and therefore FISH detection of oral streptococci still relies mostly on phylogenic group-specific probes. A surprise finding, confirmed with supragingival plaque samples and scrapings from the dorsum of the tongue, was that both Lactococcus probe LCC1030 and S. constellatus/intermedius probe L-Sco/int172-2 triggered rather strong fluorescence of long filaments with blunted ends (Figure 2D), which could only be suppressed by applying formamide concentrations exceeding 40%. The results were confirmed when probes with exchanged fluorescence labels were used (Cy3 instead of 6-FAM and vice versa).