9% NaCl as collecting fluid (exact volume determined for each sam

9% NaCl as collecting fluid (exact volume determined for each sample). The samples were frozen at -80 °C and shipped to Zürich on dry ice for further analyses. There, freshly defrosted samples were vortexted for 1 min, sonicated for 5 s, aliquoted and assessed by FISH. Aliquots were also grown at 37 °C anaerobically and in 10% CO2 on LBS agar (Becton Dickinson) with the aim to isolate and type representative strains by partial 16S ARS-1620 concentration rDNA sequencing. Demineralization of discs was determined by quantitative

light-induced fluorescence as described [29]. Preparation of multi-well slides for FISH Overnight cultures of lactobacilli (LBS broth) were washed in 0.9% NaCl, diluted in coating buffer [30], spotted on 18- or 24-well

slides (Cel-Line Associates), air-dried, and fixed in 4% paraformaldehyde/PBS (20 min, 4 °C). Analogously, in situ grown biofilm samples, supragingival plaque samples and tongue scrapings were vortexed at maximum speed for 60 s, diluted in coating buffer and coated to 18- or 24-well slides as described [30]. To improve cell wall permeability PX-478 order each well selected for FISH of lactobacilli was treated individually at room temperature first for 5 min with 9 μl of lysozyme (1 mg ml-1; Sigma-Aldrich L-7651) and achromopeptidase (1 mg ml-1; Sigma-Aldrich A-7550) TGF-beta inhibitor in Tris-HCl (pH 7.5) with 5 mM EDTA, and then for 30 min with 9 μ l of lipase (Sigma-Aldrich L-1754; at 25 mg ml-1 in water the lipase suspension was centrifuged for 5 min at 16’000 × g after which the supernatant was used). Thereafter, to limit unspecific FISH probe binding all wells were covered for 30 min at 37 °C with 9 μ l of PBS containing Denhardt’s solution (Fluka 30915; diluted 1:50) in the presence of protectRNA RNase inhibitor (Sigma-Aldrich R-7397; diluted 1:500) [15, 16, 26, 27]. At the end of the respective incubation periods the solutions were carefully aspirated and the slides briefly washed

in wash-buffer (0.9% NaCl, 0.05% Tween 20, 0.01% NaN3), dipped in water, and air-dried. All solutions were made with water of nano-pure quality. Fluorescent in situ hybridization The 16S rRNA targeted oligonucleotide probes used in this study are S6 Kinase inhibitor listed in Table 1. Custom-synthesized by Microsynth, they were labeled at 5′-end with Cy3 or 6-FAM, or in some cases at both ends with 6-FAM. Probes marked by “”L-”" in front of the probe name, contain one or two LNA to improve in situ hybridization efficiency [16]. Probes were designed as described previously [30] using the ARB software [31] with the SILVA rRNA database [32, 33] and additional rRNA sequence information from ‘The Ribosomal Data Base Project II’ [34, 35] and the ‘National Center for Biotechnology Information’ [36].

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