FISH staining of reference strains and biofilm samples 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. To prevent such false positive hybridization, the formamide concentration had to be increased to ≥ 40%. Bars: 10 μ m.