The aim of the present study was to investigate different fluorescence-based, two-color viability assays for visualization and quantification of initial bacterial adherence and to establish reliable alternatives to the ethidium bromide staining procedure. MATERIALS AND METHODS: Bacterial colonization was attained in situ on bovine enamel slabs (n = 6 subjects). Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (Ba-cLight®), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. Validation was carried out, using the colony-forming units for quantification and the transmission electron microscopy for visualization after staining. RESULTS: The average number of bacteria amounted to 2.9 ± 0.8 × 10(4) cm(-2). Quantification with Syto 9/PI and Calcein AM/Sytox red yielded an almost equal distribution of cells (Syto 9/PI 45 % viable, 55 % avital; Calcein AM/Sytox red 52 % viable, 48 % avital). The live/dead ratio of CFDA/Sytox red and FDA/Sytox red was 3:2. An aberrant dispersal was recorded with FDA/PI (viable 34 %, avital 66 %). The TEM analysis indicated that all staining procedures affect the structural integrity of the bacterial cells considerably. CONCLUSION: The following live/dead assays are reliable techniques for differentiation of viable and avital adherent bacteria: BacLight, FDA/Sytox red, Calcein AM/Sytox red, and CFDA/Sytox red. These fluorescencebased techniques are applicable alternatives to toxic and instable conventional assays, such as the staining procedure based on ethidium bromide. CLINICAL RELEVANCE: Differentiation of viable and avital adherent bacteria offers the possibility for reliable evaluation of different mouth rinses, oral medication, and disinfections.

The purpose of this in vitro study was to analyse the mode of action of self-etching adhesive systems when applied for resin-to-enamel bonding. Transmission electron microscopy was used to investigate the enamel-resin interface after application of non-rinsing self-etching adhesive systems based on phosphoric acid estered methacrylates (Clearfil Liner Bond 2, Clearfil SE Bond, Etch & Prime 3.0 and Resulcin AquaPrime) compared with conventional phosphoric acid etching and bonding (Heliobond). Non-decalcified ultrathin sections from the interface between enamel and self-etching adhesive systems revealed a 1.5-3.2-microm deep enamel surface layer characterized by a less-dense arrangement of enamel crystallites separated from each other by nanometer-sized spaces. A 1.5-3.2-microm wide, netlike resinous structure was observed in corresponding decalcified specimens, indicating that self-etching priming agents dissolve the peripheral and central part of the enamel crystallites, while simultaneously promoting inter- and intra-crystallite monomer infiltration. A similar pattern, but greater depth (6.9 microm) of enamel surface hybridization was found in the phosphoric acid-etched and bonded specimens. The nanoretentive interlocking between enamel crystallites and resin could explain the potential of self-etching adhesive systems in resin-to-enamel bonding despite the less distinct enamel etching pattern observed in scanning electron microscopy investigations.

This study assessed the protective potential of salivary pellicles formed in situ over periods ranging from 2 to 24 h. Pellicles were produced on enamel slabs mounted on the palatal aspect of removable acrylic splints and exposed to the oral environment in three subjects for 2, 6, 12 and 24 h. Enamel specimens with and without pellicles were immersed in citric acid (1%) for 60 s, and the amount of dissolved calcium was measured by atomic absorption spectroscopy. In addition, specimens were processed for transmission electron microscopy (TEM). Mean values (standard deviations) for calcium release (mg/l related to the specimen's surface area of 5 x 5 mm(2)) were: 2-h pellicle 6.94 (1.55); 6-h pellicle 6.69 (2.05); 12-h pellicle 6.57 (2.31); 24-h pellicle 5.71 (2.46); enamel without pellicle 8.95 (1.66). There were no significant differences in calcium release that were dependent on pellicle formation time, but in comparison to enamel specimens without pellicle, significantly less (p <0.05) demineralization of the enamel was observed in pellicle-covered specimens. TEM showed that the pellicle was partly, but not completely dissolved following acid exposure. It is concluded that even a 2-h in-situ-formed pellicle layer protects the enamel surface to a certain extent against demineralization.

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