jensenii UMCG 20557 1096 2-4 + 3-4 + – - – - – - – - – -

– - – * The asterisk indicates that only a small percentage of the cells could be stained by the probe, in spite of enzymatic pretreatment to improve probe penetration. a Fluorescence intensity was graded using an arbitrary five-step scale, where – (no fluorescence above background) and 1+ (very faint fluorescence) were considered negative signals, and 2+ (weak), 3+ (strong) and 4+ (brilliant fluorescence) were considered positive signals. b Probe L-Lcol732-2 CYC202 supplier labeled L. brevis and L. buchneri strains at formamide concentrations below 40%. c L-Lbuc438-2 cross-reacted with certain strains from the L. casei and L. reuteri groups at formamide concentrations of ≤ 45%. d L-Lbre466-2 was positive with L. coleohominis at ≤ 45% formamide in the hybridization buffer. e L. fermentum

was stained with low intensity due to a weak mismatch at position 760. Figure 2 FISH staining of reference strains and biofilm samples check details with LAB probes. (A) L. rhamnosus strain AC 413 stained with Lcas467-Cy3 (40% formamide). (B) L. crispatus ATCC 33820 stained with both Lfer466-Cy3 (plus the corresponding helper probes) and Lgas458-FAM (25% formamide). The strain should be Lfer466-/Lgas458+, the FISH assay identified a previously unnoticed contamination with red-stained Lfer466+ cells, which had to be eliminated by recloning. (C) Identification of L. fermentum in biofilm 013 using probe Lfer466-Cy3 (plus helper probes; 25% formamide). Note the high proportion of L. fermentum in this in situ grown biofilm. (D) Sample from the dorsum of the tongue showing an aggregate of large unidentified filaments stained with the Lactococcus probe LCC1030-Cy3 and the streptococcal probe L-Sco/int172-2-FAM at 30% formamide.

The bacteria are double false positive under these stringency conditions, whereby the detection of the Cy3 fluorescence is hampered by the much stronger FAM fluorescence. almost To prevent such false positive hybridization, the formamide concentration had to be increased to ≥ 40%. Bars: 10 μ m. Enumeration of lactic acid bacteria from in situ formed biofilms The applicability of the probes was tested with three in situ formed biofilm samples. The samples were harvested from bovine enamel discs carried in acrylic GANT61 appliances on the buccal side of the mandibular premolar/molar regions [18] by three volunteers whose discs differed greatly in the extent of demineralization (-3%, -15%, and -32%) generated during the 10 days of intermittent extraoral exposure to a 5% glucose/5% sucrose solution. All samples were positive for lactobacilli as detected by the two broadly reactive Lactobacillus probes LGC358a and LAB759 (Figure 3). Total cell numbers and numbers of lactobacilli were very similar to findings from an earlier study investigating the microbiota associated with the in situ development of caries [19].