is mainly used to visualize multiple features in different channels that are spectrally resolved. By means of this imaging procedure, it is possible to analyze the structure, composition, microhabitats, activity, and processes using a variety of speciÞc color probes. Finally, LSM allows the volumetric and structural quantiÞcation of multichannel signals in four dimensions [63]. One of the main disadvantages of LSM, however, is that the information captured from detailed ultrastructure of the bioÞlm is difÞcult. Very recently, this problem of LSM has been overcome with the advent of super-resolution microscopy (SRM). SRM encompasses a suite of cutting-edge microscopy methods able to surpass the resolution limits of common light microscopy [60]. It is foreseen that the application of SRM in combination with rRNA FISH (see below) would allow the tracking of ribosome-associated changes in activity levels and subcellular localization at the single-cell level [2].

rRNA Fluorescence In Situ

Hybridization (FISH)

The combination of FISH with confocal laser scanning microscopy is one of the most powerful tools in modern microbiology as it allows visualization of speciÞc subpopulation of cells while maintaining unaltered the 3D structure of the bioÞlm [1]. This high-throughput microscopy technique allows the speciÞc detection and enumeration of bioÞlm subpopulations in situ in their natural environment without the need for cultivation [1]. Up to date a number of studies have demonstrated the direct use of CLSM-FISH on bioÞlm cultures growing in different surfaces [11, 23]. The most frequent application of FISH is the hybridization of oligonucleotide probes to ribosomal RNA, most often 16S but also 23S rRNA, for identiÞcation of single cells in their natural habitat [2]. Since ribosomes are the protein factories of all cells, their numbers are good proxies of general metabolic activity and of the physiological state of cells. Sequences of oligonucleotide probes targeting 16S rRNA have been developed for speciÞc detection of different bac-

terial species and can be found in online databanks. In endodontics, FISH has been used to visualize and identify bacteria from periapical lesions of asymptomatic root-Þlled teeth [82]. Furthermore, bioÞlm models using CLSM-FISH can be of great advantage to investigate distribution of species in multispecies bioÞlms.

Markers of Cell Viability

Viability of bacteria is conventionally deÞned as the capacity of cells to perform all cell functions necessary for survival under given conditions [62]. The common method to assess bacterial viability is growth on plates, where the number of viable cells approximates the number of colonyforming units. In root canal infections, culture techniques have been the standard method used to assess bacterial viability. Once the living bacterial cells from root canals were isolated after growth on speciÞc substrate, the metabolic properties of these bacterial isolates were then used to infer the potential roles of these and related microorganisms in a clinical context. Under some circumstances, however, such methods may underrepresent the number of viable bacteria for a variety of reasons, such as cases where slightly damaged organisms are present [4], the laboratory growth media employed are deÞcient for one or more essential nutrients required for the growth of some bacteria in the sample [93], or viable cells are present that have lost their ability to form colonies [95]. Furthermore, if the bacteria exist in a bioÞlm, they may assume a status of low metabolic activity similar to stationary-phase planktonic growth for the majority of time [65]. The bacteria in such low active states may be undetectable by regular culture techniques. The extent of this problem is reßected in the indiscriminate use of terms that are used to assess nonviable states, such as dead, moribund, starved, dormant, resting, quiescent, viable but not culturable, injured, sublethally damaged, inhibited, and resuscitable [62]. Many of these terms are used conceptually and do not reßect the actual knowledge of the exact viability state of the organism in question.

A number of viability indicators that can be assessed at the single-cell level without culturing cells have gained increased popularity in the latest years. These indicators are based mostly on ßuorescent molecules, which can be detected with epißuorescence microscopy or laser scanning microscopy.

The LIVE/DEAD kit tests the integrity of the cell membrane by applying two nucleic acid stains, SYTO-9 and propidium iodide (PI), which can simultaneously detect dead/injured (ßuorescent red by stain with PI) and intact cells (ßuorescent green by staining with SYTO-9) [5]. This ßuorescent probe has been used to assess the viability of root canal strains ex vivo [10] and to determine the autoaggregation and coaggregation of bacteria isolated from teeth with acute endodontic infections [44].

Alternative ßuorescent probes to test bacterial viability are those that target speciÞc cell metabolic functions, such as the tetrazolium salts 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). The tetrazolium salts INT and CTC are often used as markers of bacterial respiratory activity, as well as viability [20]. With these relatively simple methods, a good correlation between the number of INT/ CTC-positive cells and the CFU count can be obtained.

In Vivo Models for Biofilm Testing

To better understand the pathogenesis of human polybacterial diseases, such as oral infections including apical periodontitis, there is a great need of experimental models that will closely mimic in vivo features of the disease. However, modeling polybacterial infections presents speciÞc challenges such as establishing a mixed infection and, in some cases, managing the effects of the native microbiota.

Oral infections including periodontitis and endodontic infections have been modeled in the oral cavity of antibiotic-treated rats or in mouse skin wound infections [56, 84, 89]. Although the

former model is a closer representation of the disease, the wound infection model is easier to administer and monitor. It is also easier to exclude other bacteria in this model. Both models have been useful in revealing some of the interbacterial interactions that inßuence oral diseases [43]. Advances in in vivo models will make it possible in the future to observe the events of human infections in detail. It is likely that these in vivo bioÞlm models will help improve the resolution of our understanding of chronic infections and will bridge the gap from the lab to the clinic.

Antibiofilm Strategies

Along the years, different therapeutic strategies have been developed to prevent bioÞlm formation and to eliminate established bioÞlm-related infections. Most of these strategies are summarized in Fig. 1.5. Although the majority of these antibioÞlm approaches arise from basic science research, most of them have been developed with the prospective view for them to be applied to Þght root canal bioÞlms. Up until now, the most common and efÞcient antibioÞlm strategy used in root canal therapy is the mechanical removal with instrumentation and irrigation. BioÞlm basic research that focuses to test novel antibioÞlm strategies allows the characterization and effect of antimicrobials on speciÞc bioÞlm properties. The validation of these new strategies will likely require efÞcient translational collaborations between basic research and clinical practice before these strategies can be included in future clinical measures.

Surface Coating

A reasonable approach to prevent or reduce secondary bioÞlm formation in root canals is to replace the conditioning Þlm with repelling substances that will alter the chemical composition of the substrates [36]. Once a surface has been artiÞcially conditioned, its properties become permanently altered, so that the afÞnity of an