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Endodontic Topics 2012, 22, 5878 All rights reserved

2012 John Wiley & Sons A/S ENDODONTIC TOPICS 2012

1601-1538

Biolm models and methods of biolm assessment

ANIL KISHEN & MARKUS HAAPASALO
Endodontic microbes dwell within the infected root canal system as surface-adherent biolm structures. In order to simulate this in vivo situation, a variety of in vitro biolm models are currently used in Endodontics for different microbiological experiments. Unfortunately a cogent selection of the best models to be used for a particular research application has not been obtained. This article outlines various factors to be considered while developing biolm models in Endodontics. Different in vitro endodontic biolm models, devices used to generate biolms, and biolm assays used to analyze these biolm structures qualitatively and quantitatively are also presented in this article. Received 9 December 2011; accepted 3 March 2012.

Introduction: changing paradigm in endodontic infection

Biolm is a mode of microbial growth where dynamic communities of interacting sessile cells are irreversibly attached to a solid substratum, as well as to each other, and are embedded in a matrix of extracellular polymeric substances (EPS) (1). While endodontic microbial ora is established to be less diverse compared to the oral microbial ora, the task of disinfecting a root canal system is one of the most prominent challenges in Dentistry. Endodontic microbes dwell within the entire root canal anatomy as surface-adherent biolm (intraradicular biolm). The endodontic bacterial activities that are usually conned to the intracanal spaces may, under certain conditions, form biolm on locations beyond the apical foramen (extraradicular biolm) (24). The geometrical and anatomical complexity in the root canal system tends to shelter the bacterial biolm from root canal disinfectants and instrumentation procedures (2). Furthermore, the progression of endodontic infection alters the nutritional and environmental status of the root canal system, apparently rendering it more anaerobic and depleting it of nutrients. This yields a tough ecological niche for the surviving microorganisms (5). The biolm mode of growth allows the resident bacteria to survive unfavorable environmental and nutritional conditions (6,7). On the above basis, it is vital to

consider bacterial biolm models as essential models for in vitro microbiological investigations and the assessment of different disinfectants and disinfection strategies in Endodontics.

Biolm model systems: factors to be considered

Bacterial biolms are developed in order to study the microbial interactions within the root canal space or between bacteria and host immune cells (8,9). Currently, they are more commonly grown to test the efcacy of different irrigants/medicaments and irrigation procedures in Endodontics. It should be noted that the antimicrobial resistance observed in biolm bacteria is not generally due to classic genetic mechanisms; instead, this arises due to certain peculiarities of biolm growth. The types of resident bacterial species, nature of bacterial adherence to substrate, physico-chemical characteristics of the substrate, thickness of the biolm, bacterial cell density, amount of EPS, and phenotypical/genotypical modication of the resident bacteria are all factors that could contribute to antimicrobial resistance in biolm bacteria (6,10,11). Typically, different antimicrobial resistance mechanisms may act concurrently or synergistically in a biolm structure, and understanding some of these mechanisms is the key to developing biolm model systems for different application in Endodontics.

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Broadly, factors associated with bacterial adherence, bacteriasubstrate interaction, and biolm ultrastructure should be standardized to develop useful biolm models for in vitro experiments (6). Currently, there is no universally accepted in vitro model that reproduces biolm infection in Endodontics. adherence to a substrate is mediated by the bacterial surface structures such as mbrae, pili, agella, and EPS (PHASE 2: initial non-specic microbialsubstrate adherence phase). The bacterial surface structures form bridges between the bacteria and the conditioning lm (11). Porphyromonas gingivalis, Streptococcus mitis, Streptococcus salivarius, Prevotella intermedia, Prevotella nigrescens, Streptococcus mutans, and Actinomyces naeslundii are some of the oral bacteria possessing surface structures (11,12). The bridges formed between bacteria and substrate are a combination of electrostatic attraction and covalent/hydrogen bonding. Initially the bonds between bacteria and substrate may not be strong. However, with time these bonds gain in strength, making the bacterial attachment irreversible. In the nal stage, a more specic bacterial adhesion to the substrate is established via polysaccharide adhesin or ligand formation (PHASE 3: specic microbialsubstrate adherence phase). In this phase, adhesin or ligand molecules on the bacterial cell surface will bind to receptors on the substrate. Specic bacterial adhesion is less affected by environmental factors (13,14). It is critical to realize that these phases involved in bacterial adherence to a substrate are a dynamic process which occurs as a function of time. The reversible and irreversible steps in phase 1 of the bacterial adherence occur in a few seconds to minutes, while phase 2 and 3 interaction take a few hours to days to occur, depending upon the bacteria and the environment conditions (Fig. 1). Therefore, while developing in vitro models, it is important to provide sufcient bacteriasubstrate interaction time and optimum environmental conditions. The development and maturation of biolm structure occurs subsequent to bacterial adherence.

Bacterial adherence and bacteriasubstrate interaction

The earliest stage in the formation of most oral biolms involves the adsorption of macromolecules from tissue uids such as saliva onto a biomaterial (natural or synthetic) surface, leading to the formation of a conditioning layer. The conditioning uid will form a layer of adsorbed inorganic and organic molecules on the solid surface, and alters the physical/chemical properties of the surface. The conditioning layer, formed prior to the inux of microorganisms, will selectively promote the adhesion of microbial cells to the surface. It may also serve as a source of nutrition for adherent bacteria. Bacteria can generally form biolms on any surface that is conditioned with such conditioning uids (11). The next step in the development of a biolm is the adhesion of microbial cells to the substrate surface. The adhesive potential of microbes to natural (e.g. dentin) or synthetic (e.g. restorative/endodontic material) biomaterials is considered to be a vital ecologic and pathogenic determinant in biolm-mediated infection. Bacterial adherence to a surface is inuenced by (i) the environmental conditions such as pH, temperature, uid ow rate, nutrient availability, etc.; (ii) bacteria-associated factors such as type of bacteria (species/strain), growth phase of bacteria (log or stationary phase), type and charge of the surface molecules, etc.; and (iii) substrate-associated factors such as physical and chemical characteristics of the substrate. It is crucial to standardize these parameters in order to develop clinically realistic biolm model systems for in vitro experiments (12,13). The initial phase of the bacteriasubstrate interaction is determined by the physical and chemical properties (e.g. surface energy and charge density) of the bacteria and substrate (PHASE 1 of bacterial adherence: transport of microbe to substrate surface). This reversible interaction is followed by the molecular-level nonspecic interactions between the bacterial surface structures and the substrate. This phase of microbial

Biolm ultrastructure
During the development of a biolm, the resident bacterial cells proliferate, leading to expansion of the biolm structure. In this stage, the mono-layer of microbes (primary colonizers) attracts the secondary colonizers, forming micro-colonies, and the collection of micro-colonies gives rise to the nal structure of the biolm. Ultrastructurally, a biolm consists of a population of bacterial cells attached irreversibly to a substrate and encased in a hydrated, polyanionic matrix of EPS, proteins, polysaccharides, and nucleic acids (15,16). Bacteria themselves account for a variable

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Fig. 1. Schematic diagram showing the stages in the development of a biolm.

fraction of the total biolm volume (typically 535%) (17). A mature biolm will be a metabolically active community of microorganisms where individuals share duties and benets (16). For instance, some microorganisms help in adhering to the solid support while some others (such as Fusobacterium nucleatum) create bridges between different species. This signies the relevance of a polymicrobial biolm over a mono-species biolm. The physiological characteristics of the resident microorganisms in a biolm also offer an inherent resistance to antimicrobial agents (17,18). Bacterial species such as staphylococci, enterococci, Klebsiella pneumoniae, Pseudomonas spp., etc., are inherently resistant to many antimicrobials (1719). Interestingly, it has been reported that the biolms formed in pure cultures of bacteria under laboratory conditions and the mixed-species biolms formed in natural ecosystems show a similar basic organization in which cells grow in matrix-enclosed micro-colonies separated by a network of open-water channels (19). The thickness of the EPS will inuence the biolm permeability and consequently provide a certain degree of protection or barrier effect against physical and chemical threats. Each step in the development of biolm, from the adherence of bacteria to a substrate to the nal formation of a matured biolm structure, as well as protein expression/slime production, is modulated by a large number of variables, including type of bacterial species, environmental conditions, and age of the biolm. Previous studies on endodontic biolm models have shown that mature biolms and biolms with limited nutrient supply are more resistant to irrigants such as chlorhexidine and Light Activated Disinfection than early (immature) biolms under normal

nutrient conditions. Studies have emphasized that the age or degree of maturation, nutritional condition of the biolm, and interactions between resident bacterial species are some of the major confounding factors in designing in vitro biolm models to test the efcacy of various endodontic disinfectants (2026). On the above basis, it is important to use relevant bacteria (primary colonizers), and provide ideal environmental conditions (substrate, uid conditioning, nutritional conditions, and temperature) and optimum bacteria substrate interaction time (matured biolm) in order to achieve a standardized endodontic biolm model for in vitro applications. Two types of microbial interactions occur at the cellular level during the formation of biolm. One is the process of recognition between a suspended cell and a cell already attached to substratum. This type of interaction is termed co-adhesion. In the second type of interaction, genetically distinct cells in suspension recognize each other and clump together. This type of interaction is called co-aggregation. This association is highly specic and occurs between co-aggregating partners only. Interestingly, most oral bacteria recognize each other as co-aggregating partners. Fusobacterium nucleatum, a Gram-negative lamentous anaerobe, can co-aggregate with all oral bacteria tested, and can act as a bridging bacterium that binds together even non-aggregating bacteria (6). The association of long-lamentous bacteria and surface-adsorbed spherical-shaped cocci produce the characteristic corncob structure of oral biolms (24). The attachment of cocci to lamentous bacteria is said to be mediated via mbriae of the oral streptococci. Although the genetic makeup of the bacteria is the main determinant of co-aggregation, the physico-chemical characteristics

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of the environment also play a crucial role (24,26). It was observed that bacteria in a co-aggregated suspension were signicantly more resistant to antimicrobials when compared to planktonic suspension, while bacteria in a biolm mode showed the most resistance to antimicrobials (25,26). Consequently, antimicrobial agents selected on the basis of traditional susceptibility methods (such as broth-based Minimal Inhibitory Concentration [MIC]) may not be very appropriate to eliminate co-aggregated or biolm bacteria.

Bacterial biolm models: in vitro development

Laboratory models are conventionally used to mimic natural biolms for different experimental purposes. These in vitro biolms are easy to control and useful in obtaining standardized biolm models with predictable structure and behavior. Conventional biolm models range from monocultures in static growth conditions to diverse mixed cultures in dynamic growth conditions. The static biolm models use different substrates (e.g. glass, polycarbonate, silicon, hydroxyl apatite, nitrocellulose, enamel, dentin) to grow biolms while the dynamic biolm models use reactor or fermenting systems to grow biolms on a particular substrate. Both aerobic and anaerobic environments can be employed for in vitro biolm development. Figures 26 show different in vitro biolms grown on different substrates. Given that the in vivo conditions are commonly dynamic, studies evaluating biolm formation under static conditions might be somewhat misleading depending upon the research question. These in vitro bacterial biolm models are routinely applied to: (i) examine the adherence of specic bacterial species to any biomaterial surface (27); (ii) study the nature and pattern of early microbial biolm formation on a particular substrate (28); (iii) study the interaction between different biolm bacteria and host immune cells (29); and (iv) test the efcacy of antimicrobial agents or antimicrobial treatment strategies (30,31). Currently in Endodontics, most in vitro biolms are developed and utilized for testing antimicrobials and irrigation strategies (Table 1). Presently, published results on the activity of disinfectants show noticeable discrepancies between experiments, and this may be attributed to the diversity of the microbial growth phase, biolm models, and procedures/assays utilized for the analysis. Obviously,

Fig. 2. (a) Photograph of a biolm grown in vitro for three weeks on a collagen-coated hydroxyapatite disc. The biolm is coated with a palladiumgold mixture for SEM. (b) SEM image of mixed bacterial (multi-species) biolm grown anaerobically for seven days in BHI broth on collagen-coated hydroxyapatite disc. Low magnication. (c) SEM image of six-month-old biolm grown anaerobically in BHI broth on collagen-coated hydroxyapatite disc. Several bacterial morphotypes including coiled spirochetes can be seen.

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Fig. 3. (a) SEM of three-day-old biolm grown anaerobically in BHI broth on a nitrocellulose lter. Cocci and long rod-shaped bacteria dominate in the specimen. Low magnication. (b) Three-day-old biolm grown anaerobically in BHI broth on a nitrocellulose lter. Cocci and long rod-shaped bacteria dominate in the specimen. High magnication.

the number of parameters needs to be considered in the design of a representative biolm model for applications in Endodontics (Fig. 7). In recent years, it has become evident that a number of parameters may be important when performing adherence assays, including the microbial concentration in the inoculum, incubation time, growth conditions, and substrate properties. If laboratory strains are used for adherence assays, then it is important that they are representative of clinical isolates. In addition, assays that do not take into account the presence of saliva may be unsuitable for the study of adhesion and early biolm formation (27). Findings from experiments with planktonic and biolm bacteria (50) have revealed large differences in the dynamics of killing

Fig. 4. (a) SEM image of six-week-old biolm grown aerobically in BHI broth on a dentin disc. (b) Low magnication SEM image of the previous sample. Cocci and long rods dominate in the sample. (c) Mixture of bacteria grown aerobically for one month on dentin has failed to grow a mature biolm. Smeared dentin can be seen between the bacteria.

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Fig. 5. (a) Mixture of bacteria grown anaerobically for one month on dentin. (b) Mixed bacterial biolm grown anaerobically in BHI broth for one month on dentin. (c) Mixed bacteria biolm grown anaerobically for one month in BHI on root canal wall dentin. (d) High magnication image of the previous SEM picture.

between planktonic and biolm bacteria. There is no doubt that the results from planktonic killing studies must be interpreted with caution and direct extrapolation as to the efcacy of the agent in complex in vivo systems is not possible. A comparison of the planktonic and biolm tests in a study indicated that planktonic killing tests may be useful for preliminary screening of new disinfectants before proceeding onto more complex biolm designs (50).

Biolm devices: ow cells and fermentors

There are several in vitro devices that are used to develop biolms. Some of them produce biolms irrigated with fresh culture medium; in this case, the biolms experience a continuous ow of medium supplemented with fresh nutrients. These in vitro devices are used to grow dynamic biolm models. The ow cell system is one of the most utilized dynamic models. It consists of a transparent chamber of xed depth through which the growth medium ows. The

inlet tubing supplies growth medium and the outlet tubing drains the medium to a waste reservoir. The growth medium is passed through the cell with the aid of a peristaltic pump, which controls the ow rate of the medium. Pre-fabricated ow cell systems are available commercially or they can be custom-made based on any particular application. In conjunction with a microscope, charge-coupled-device (CCD) camera, or Confocal Laser Scanning Microscopy (CLSM), this method can be used to observe the early events in biolm formation in real time (55). Chemostats are also used to grow dynamic biolms of microbes on experimental substrates submerged within the chemostat. One of the most important features of chemostats is that microbial biolms can be grown at a constant rate and under constant culture conditions (temperature, pH). Similar to chemostat, there is another category of reactors in which biolms are formed on thin lter membranes in a physiological steady state. These systems permit evaluation of growth rate dependence and cell-cycle specicity of

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Fig. 6. (a) Multi-species biolm grown anaerobically for three weeks on a glass coverslip conditioned with media for 24 hrs. The biolm was sparse and not uniform. (b) High magnication image of the previous SEM picture showing mixed bacterial ora. (c) The biolms were approximately 20 microns thick at certain areas. (d) High magnication image of biolm with abundant extracellular matrix.

antibacterial agents. Finally, there are constant depth reactors in which surface growth is periodically removed to maintain a constant geometry of the biolm. In these reactors, microorganisms can be grown in a physiological steady state with all culture parameters constant. The system can generate large numbers of biolms with comparable and reproducible data (55). The static biolm system generates biolms that have exhausted important nutrient components at the end of an overnight incubation. The key features of this system are that numerous biolms can be

handled at any given time, and it does not require time-consuming sterilization and set-up procedures, allowing it to be used as a high-throughput system for biolm analysis (31). This system provides a basis for the rapid screening of biolm mutants (56), biomass development, and biolm-forming capacity (57), as well as extracellular matrix composition (58). Essentially, detection of any microbial phenotype that can be processed by a microtiter plate/reader can be used for the approach. However, this system is incompatible with CLSM, which is the preferred methodology for studying the structure of biolms.

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Table 1: List of literature of in vitro biolm models for different endodontic applications
Authors Shabahang & Torabinejad, 2003 (32) Type of Model In vitro E. faecalis Purpose Preparation of Biolm

The antimicrobial effect of MTAD: an in Extracted human teeth contaminated with vitro investigation E. faecalis for 4 weeks Dentinal shavings and CFU-based method was used for the analysis Biolm formation of oral and endodontic 96-well plates for 24 hours E. faecalis Crystal violet assay used for assessment (optical density at 570 nm)

Duggan & In vitro Sedgley, 2007 E. faecalis strains (33) recovered from root canals, oral cavity, and non-oral sources George & Kishen, 2007 (34)

Two-day-old biolms in multi-well plates Methylene blue dissolved in different In vitro (polystyrene) formulations: water, 70% glycerol, 70% E. faecalis Four-day-old biolms in human teeth polyethylene glycol, and a mixture of (Gram-positive), glycerol:ethanol:water (30:20:50) was CFU-based method Actinomycetes tested actinomycetemcomitans (Gram-negative) In vitro E. faecalis This study aimed to investigate the effect Human teeth (10-week-old biolm) CFU-based method of including an oxidizer and oxygen carrier in photosensitization formulation to disinfect a mature endodontic biolm by light activated disinfection The efcacy of dynamic irrigation using a A collagen-based bio-molecular lm commercially available system formed on extracted human teeth (RinsEndo) Digital image analysis of the canal surfaces (ipWin4) DIAGNOdent laser uorescence assessment of endodontic infection Extracted teeth with endodontic pathology Fluorescence emissions in the near-infrared range were measured Collagen-coated hydroxyapatite (CHA) and uncoated hydroxyapatite (HA) discs Confocal laser scanning microscopy was used for the analysis of dead versus viable cells

George & Kishen, 2008 (35)

McGill et al., 2008 (36)

In vitro E. faecalis

Sainsbury et al., 2009 (37)

Ex vivo

Shen et al., 2009 (38)

In vitro Multi-species

Evaluation of the effect of two chlorhexidine preparations on biolm bacteria in vitro: a three-dimensional quantitative analysis

Williamson et al., 2009 (39)

In vitro E. faecalis (clinical isolate)

Glass substrate Antimicrobial susceptibility of CFU-based method monoculture biolms to 6% NaOCl, 2% CHX, <6% NaOCl with surface modiers (Chlor-XTRA), and 2% CHX with surface modiers (CHX-Plus) The efcacy of an improved light activated disinfection technique utilizing a specic photosensitizer formulation, liquid optical-conduit, oxygen-carrier, and light energy of appropriate wavelength were tested The study of the effect of hydrogen peroxide on the antibacterial effect of chlorhexidine Two different biolms were tested: (i) Four-day-old (immature) (ii) Four-week-old (mature) Human teeth CFU-based method Dentin tubes prepared from maxillary central and lateral incisors CFU-based method was used

Lim et al., 2009 In vitro biolms (20) E. faecalis

Shahriari et al., 2010 (40)

In vitro E. faecalis

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Table 1: Continued Authors Kishen et al., 2010 (41) Type of Model In vitro E. faecalis Purpose Efux pump inhibitor potentiates antimicrobial photodynamic inactivation of Enterococcus faecalis biolm Antimicrobial efcacy of 3.8% silver diamine uoride Nanoparticulates for anti-biolm treatment and effect of aging on its antibacterial activity Biolm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite The effects of antimicrobials on endodontic biolm bacteria Preparation of Biolm Microwell plates CFU-based method Confocal laser scanning microscopy Membrane lters CFU-based method Microwell plates/saliva CFU-based method Confocal laser scanning microscopy Human dentin and polystyrene blocks CFU-based method SEM 24-hour biolm within a mini-ow cell system Confocal microscopy and image analysis

Hiraishi et al., 2010 (42) Shrestha et al., 2010 (43) Liu et al., 2010 (44)

In vitro E. faecalis In vitro E. faecalis In vitro E. faecalis

Chavez de Paz et al., 2010 (45)

In vitro (clinical isolates) E. faecalis, L. paracasei, S. anginosus, S. gordonii In vivo

Su et al., 2010 (46)

This study explored the effect of surgical endodontic treatment of refractory periapical periodontitis with extraradicular biolm Effectiveness of chemomechanical preparation with alternating use of sodium hypochlorite and EDTA in eliminating intracanal Enterococcus faecalis biolm

Resected root-end samples

Soares et al., 2010 (47)

In vitro E. faecalis

Human teeth (21-day-old biolm) SEM CFU-based method

Bhuva et al., 2010 (48)

In vitro E. faecalis

Human teeth The effectiveness of passive ultrasonic irrigation on intraradicular Enterococcus SEM-based image analysis faecalis biolms in extracted single-rooted human teeth The synergistic antimicrobial effect by mechanical agitation and two chlorhexidine preparations To investigate the antibacterial effect of Tetraclean, MTAD, and ve experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biolm model The aim of this study was to enumerate viable bacteria at different growth stages of a multi-species oral biolm and to compare results obtained with the LIVE/DEAD BacLight Kit with those from culturing and plate counting Collagen-coated hydroxyapatite (CHA) discs (3 weeks old) Confocal laser scanning microscopy Collagen-coated hydroxyapatite (CHA) discs (2 weeks old) Confocal laser scanning microscopy

Shen et al., 2010 (49)

In vitro Multi-species biolm (subgingivial plaque) In vitro Multi-species biolm (subgingivial plaque)

Pappen et al., 2010 (50)

Shen et al., 2010 (51)

In vitro Multi-species biolm (subgingivial plaque)

Collagen-coated hydroxyapatite (CHA) discs Confocal laser scanning microscopy CFU-based method

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Table 1: Continued Authors Lundstrom et al., 2010 (52) Type of Model In vitro (multi-species) Streptococcus sanguinis, Actinomyces viscosus, Fusobacterium nucleatum, Peptostreptococcus micros, and Prevotella nigrescens In vitro E. faecalis In vitro E. faecalis and P. aeruginosa Purpose Bactericidal activity of stabilized chlorine dioxide as an endodontic irrigant in a polymicrobial biolm tooth model system Preparation of Biolm Permanent bovine incisors coated with mucin and inoculated with standardized suspensions of bacteria (anaerobically for 14 days) CFU-based method

Hope et al., 2010 (53) Upadya & Kishen, 2010 (9)

A direct comparison between extracted tooth and lter-membrane biolm models of endodontic irrigation To evaluate the efcacy of light-activated disinfection (LAD) using Methylene blue (33) and a non-coherent light source on Gram-positive and Gram-negative bacteria in different growth modes. The inuence of different photosensitizer (PS) formulations in the MB-mediated LAD of biolms was also evaluated. This study examined the biolm-forming capacity of E. faecalis on gutta-percha points under different nutrient status and surface conditioning with saliva and serum A laboratory evaluation of the antibacterial and cytotoxic effect of liquorice when used as root canal medicament

Human teeth CFU-based method Mono-species biolms in 24-well polystyrene plates (4 days) CFU-based method Confocal laser scanning microscopy

George et al., 2010 (28)

In vitro E. faecalis

Gutta-percha Conditioned with saliva or serum (2, 4, and 12-weeks) Biolm growth for 2 weeks CFU-based method SEM Grown on cellulose nitrate membrane lters CFU-based method

Badr et al., 2011 (54)

In vitro E. faecalis

Shen et al., 2011 (21)

In vitro Multi-species biolm (subgingivial plaque)

The aim of this study was to examine the Collagen-coated hydroxyapatite (CHA) discs (2 days to months) susceptibility of multi-species biolms Confocal laser scanning microscopy at different phases of growth to root CFU-based method canal irrigants (2% chlorhexidine [CHX] or CHX-Plus)

Fig. 7. Schematic diagram showing different factors that would inuence the structure and development of in vitro biolms.

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Fig. 8. Basic outline of the experimental methods for assessing antimicrobial efcacy using in vitro biolm.

Structural evaluation of biolm requires the use of irrigated biolm systems. An irrigated and owthrough cell system allows for the study of the development of biolm over time. These analyses include non-destructive evaluation of temporal and spatial expression of selected genes and the complete lifecycle of biolm formation and dispersal. Several ndings on the unique behavioral responses of biolm cells that cannot be obtained using static microtiterbased systems were observed with the aid of irrigated biolm ow systems (59).

Microbiological culture techniques

The biolm formed on a substrate can be quantied by directly enumerating the Colony Forming Units (CFU) of the bacteria adhering to the surface. The CFU measurement will provide information on the amount of viable bacteria adherent to the substrate or growing within the biolm structure. However, the CFU may only detect bacteria that are able to initiate cell division at a sufcient rate to form colonies and whose growth requirements are supported by the culture medium used. Several protocols recommend the removal of biolm bacteria from the substrate by a sonication or centrifugation process. In such cases, the CFU is usually determined from the supernatant obtained after the sonication/centrifugation procedure. The recovery of microorganisms after treatment with disinfectants remains an important point in these experiments. The bacteria can be sensitive to these procedures and changing growth conditions and, in that case, there may be a 24-hour to more than a week lag phase in the bacterial response. A recent study showed that in older, starved biolms the bacteria are viable based on the green staining pattern as observed by CLSM, but over 99% of these bacteria could not be grown when removed from the biolm and grown in a culture media (49). Ultrasonic vibrations and enzymes are used to remove bacterial biolm before quantication. However, it is imperative to use an

Biolm assays
Biolm assays are used to characterize factors such as (i) number and type of microorganism, (ii) vitality (dead/living cells) of the resident microbial population, (iii) age, (iv) thickness (mono-layered or multilayered), (v) structure (homogeneous, irregular, dense, porous), and (vi) surface topography (peaks and valleys) of biolms. Different techniques such as (i) microbiological culture techniques, (ii) colorimetric techniques, (iii) microscopic techniques, (iv) physical methods, (v) biochemical methods, and (vi) molecular methods are applied to biolm assays. The basic outline of the experimental methods to assess antibacterial or antibiolm efcacy of irrigants or irrigation strategies is shown in Figure 8.

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Fig. 10. Fluorescent microscope image of bacteria stained with Live/Dead uorescent stain: green, live cells; red, dead cells.

Fig. 9. Multi-well plate showing E. faecalis biolm growth that is quantied using a colorimetric (crystal violet) assay.

is not a true indicator of the EPS in the biolm structure. Although glass tubes and microtiter plates have traditionally been used to grow in vitro biolms, inconsistency and lack of standardization in the developing biolm has been a growing concern. These assays usually work well for strains which are strong biolm-producers, however they may not be very useful in differentiating weak biolm-producers from biolm-negative strains (26).

Microscopic techniques
appropriate energy level and concentration since few studies have highlighted the possible lethal effects on bacterial cells (60). Light microscopy is the fundamental technique used for biolm assays, either directly on in vitro samples or on histological sections. It is a relatively inexpensive, simple to use, rapid, and readily available method. Different microscopic methods have been used to assess adherence of bacteria to substrates, structure of biolms, and distribution/type/viability of bacteria in a biolm structure (Figs. 10 and 11). In the microscopic method, the bacterial biolm is stained with a suitable dye that is uorescent (e.g. propidium iodide) or non-uorescent in nature (e.g. safranin). Most high resolution light microscopy will enable the counting of bacterial cells on a substrate surface. The biolm slime may be stained with Alcian blue, a phthalocyanine dye that stains acidic mucopolysaccharides and glycosaminoglycans in the EPS (61). The stained portions will appear as a blue to bluish-green color. Bacteria cells may also be visualized under uorescent microscopes

Colorimetric techniques
The colorimetric assay is a semi-quantitative method based on dye uptake by the bacterial cells in a biolm. In this assay, after the bacterial biolm is stained with a dye (e.g. crystal violet), it is disrupted using a known quantity of alcohol or a surfactant (sodium dodecyl sulfate) and the intensity of the eluted dye is measured using a spectrophotometer (Fig. 9). This is an easy assay that allows the rapid quantication of biolm bacteria. However, this test may sometimes be difcult to interpret because the absorbance/optical density measured is a reection of the number of bacteria and

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Fig. 11. Fluorescent microscope image showing E. faecalis cells adhering to type 1 collagen (100 , oil immersion). (a) Control. (b) After EDTA treatment.

without using uorescent probes by using plasmidencoded green uorescent protein (GFP). These transformed E. coli O157:H7 have been used to study their attachment onto a surface (62,63). The viability of these cells may be determined by staining the transformed cells with membrane-impermeable uorescent dye (63). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) have been effective workhorses in biolm analyses for many years. Highresolution electron microscopy has been employed for the morphological and structural characterization of microbial biolms. The main disadvantage with these techniques is the need for extensive sample preparation steps such as xation, dehydration, freeze- or critical point-drying, and sputtering. These treatments can deeply affect the original biolm morphology (Fig. 12). Environmental SEM (ESEM) is a relatively new technique that represents a powerful alternative to conventional SEM (high vacuum) as it allows the imaging of biological samples in their original hydrated condition at relatively high resolution (64). Structural modications in microbial biolm architecture, particularly an overall loss of matrix volume, were appreciable when comparing conventional high-vacuum SEM to ESEM images. Sutton et al. (65) compared different dehydration techniques and showed that freeze-dried samples presented signicant detachment of microbial biolm from the substrate, while more complex dehydration procedures such as critical point

Fig. 12. SEM images of (a) E. faecalis cells adhering to root canal dentin and (b) multi-layered mono-species biolm of E. faecalis on root canal dentin.

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drying caused an almost complete disappearance of the EPS matrix. Although proper xation processes were applied, the collapse of the biolm structure upon dehydration procedures is mainly due to the lack of a self-sustaining scaffold in the EPS matrix. Nevertheless, bacteria cells maintained their shape and dimensions in vacuum after xation and could clearly be identied by SEM. In the ESEM mode, the semitransparent appearance of the EPS and the low signalto-noise ratio at high pressures results in a limited image resolution. In brief, ESEM represents an effective technique for detecting highly hydrated bacterial biolms by preserving the substantial EPS component. On the contrary, conventional high-vacuum SEM allows a detailed examination of the cellular components and favors the detection of the three-dimensional hollow structures, but fails to show the actual biolm architecture consisting of a large volume of EPS matrix surrounding the cells. The combined use of conventional SEM and ESEM techniques can therefore provide complementary information on different biolm components, bacterial cells, and the extracellular matrix (65). Epiuorescent microscopy has been used to study bacterial biolm microstructure. Biolms grown on biomaterial surfaces are usually stained with a uorescent dye and viewed under an epiuorescent microscope. In a study, binary species biolms were stained using two different uorescent probes for each organism and observed under an epiuorescent microscope using two excitation wavelengths. Two different images and the background biolm were captured with appropriate wavelengths. The images were then combined to construct a new image that simultaneously showed both organisms (66). Epiuorescent microscopy is used to determine viable cells, biolm cell arrangement, micro-colony formation, biolm pH, and distribution of chemicals in a biolm structure (67). CLSM is a particularly important biolm analysis technique that is restricted to 50200-mm-thick biolm structures. CLSM has overcome some of the limitations exhibited by most of the earlier microscopic techniques such as epiuorescence, SEM, and TEM. Together with improvements in the molecular techniques for bacteria, CLSM has become an important tool for studying biolms. Green uorescent protein (GFP) tagging of certain bacterial strains such as Pseudomonas aeruginosa is utilized to study biolm formation. This method uses a uorescent imagingbased analysis or CLSM to quantify the biolm structures. The preferred method of tagging has been to construct chromosomal insertions in order to ensure a stable gene dosage of the tag sequence (68,69). Recently introduced, time-lapse CLSM together with the gfp reporter system has been used to study the role of agr in biolm formation and has given an interesting insight into gene regulation during the course of biolm development (70). This technique is likely to be an important tool for future studies on the regulators and genes involved in biolm development. CLSM creates a thin (~0.3 mm) plane of focus (optical sections) in which out-of-focus light will be blocked, either conventionally by optical barriers or by applying the physics of light absorption as equipped in multi-photon microscopy (71). These optical sections can then be stacked by software to generate a three-dimensional reconstructed image of the entire biolm. The CLSM images can be used to determine the thickness and distribution of cells in a biolm structure. CLSM can also be used to determine the pH gradients in biolms. The interior pH of biolms is measured by a uorescent lifetime imaging technique using uorescein as a pH indicator. Currently, the use of a uorescent dye combination (LIVE/DEAD BAC light) with CLSM has become a routine practice for in vitro biolm analysis. The LIVE/DEAD Bacterial Viability kit (Molecular Probes, Eugene, OR) contains separate vials of the two component dyes (SYTO 9 and propidium iodide). The dyes are used in a 1:1 mixture for staining the biolm bacteria following the manufacturers instructions. The dead cells emit red light and the viable cells emit green light under CLSM examination (Fig. 13). In a recent in vitro study, it was shown that bacteria in the multi-species anaerobic biolm grown under nutrient deprivation changed into the viable-but-non-cultural (VBNC) state but could be returned to the normal physiological state and cultured by re-establishing the supply of nutrients while they were still in the biolm. The results from this study indicated that viability staining was a better reection of the true viability of the biolm bacteria than the culturing method during starvation. This nding needs to be taken into account when assessing results from cultural studies employed to determine in vivo root canal biolms (51). A uorescence in situ hybridization (FISH) technique using probes to target specic 16S rRNA

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Fig. 13. Three-dimensional confocal laser scanning microscopy reconstruction of E. faecalis biolm (inlet shows sagittal section) (60 ). Left: the biolm has received no treatment. Right: the biolm has been subjected to Light Activated Disinfection with Methylene blue and laser (660 nm).

sequences in bacteria is applied for the simultaneous analysis of the spatial distribution of both Grampositive and Gram-negative bacteria in biolms. FISH is a recognized tool for the specic and sensitive identication of target organisms within complex microbial communities. Visualization of FISHlabeled cells in biolms can be carried out by uorescent microscopy and LSCM (68,69). However, CLSM is preferred in a biolm analysis because it allows a three-dimensional non-invasive visualization of cells and the computational reconstruction of mature biolms without distortion of their structure (25,70).

Physical methods: thickness, weight, area, and density measurements

Basic physical parameters such as biolm thickness, area, weight (wet and dry), and density estimates are used to quantify biolm growth. A thickness measurement by light microscopy is usually effective in thin biolms but may not work with thick biolms. In this method, the biolm is placed on the stage of a microscope that has calibration scales on the ne control and the objective is lowered until the biolm surface is in focus and the ne adjustment dial setting of the microscope is recorded (71). The microscope objective is then focused on the substrate surface, preferably in an area with no biolm. The difference in the ne adjustment settings can be used to calculate the thickness. A

simple manual-gauge needle method (72) and an electronic probe to measure biolm thickness (73) have also been described. A properly prepared SEM sample or cryosection enables the estimation of biolm thickness and also reveals layering of embedded bacterial cells (74). Biolm wet-weight is a useful measure of the biomass, especially on tared substrates. This is a very simple and quick procedure. The substrate can be weighed before biolm growth (with the assumption that no substrate solubilization occurred during biolm formation) and then cleaned, dried, and weighed again in order to record the dry biolm weight. If both wet and dry weight measurements on the same biolm sample are performed, the approximate density may be determined by assuming that the volume of the biolm sample is the same as the water volume estimated as the wet weight minus the dry weight. Routinely, for comparative purposes, physical parameters such as biolm density and weight can be calculated per unit of substratum.

Biochemical methods: biomass and extracellular matrix (ECM)

Microbial biomass denotes the total number of microbes in a given area. The measurement of microbial biomass is considered to be a rapid method and includes measurements of the wet or dry weight of the entire biolm, measurements of the cell contents, measurements of the cellular activities or viable cells,

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etc. One method that is used for the early detection of viable bacteria is based on metabolic activity. Adenosine triphosphate (ATP) bioluminescence is widely used to determine the metabolic activity of a bacterial population. This technique requires a cell lysis step to release ATP, which is determined by a luciferine luciferase reaction (75). However, it should be noted that the rate of lysis and the ATP content vary depending on the microorganism. Hence, the ATP assay cannot be correlated with the initial number of microbial cells. Test strains genetically modied (containing genes for bioluminescence) have also been developed and used for in vitro analysis (76). All of these methods present advantages and disadvantages. Except for the microscopy-based techniques, most other methods require a good number of viable cells or the ability of bacteria to multiply to a signicant number in a normal period of time. This will facilitate the detection of even a minimum microbial population level. Revival of bacteria is also a concern because it is unknown how to determine the time needed before physical or biochemical measurements are made (77 81). Currently there is no standardized rapid method that can replace conventional biochemical assays. Further research is required before employing rapid methods more routinely for the detection of viable bacteria in biolm assays. groups, there is a possibility of data disparity between laboratories and in vivo situations. Enzyme-linked immunosorbent assay (ELISA) is a very sensitive method used to detect the presence of antigens or antibodies of interest in a sample. ELISA can be used for quantitative analysis when used in conjunction with standard curves. ELISA is typically performed using one of two detection methods: the direct or indirect assay. In direct ELISA, an enzymelinked (labeled) antibody is used to directly detect the captured antigen or antibody of interest. In the more common indirect ELISA, a detection or primary antibody is bound to the sample antigen/antibody and then a secondary labeled antibody (antiglobulin) is used to detect the primary antibody. For any ELISA procedure, the sample antigen/antibodies of interest are concentrated and solublized in an appropriate buffer. ELISA has been used as an alternative method to quantify biomass within biolms and even protein production in biolms (82,83). ELISA may be used to quantify the population of a particular bacterium in a mixed biolm. An ELISA-based approach can circumvent errors due to cell clumping and EPS production, which can lead to signicant errors in bacterial quantication. The disadvantages of ELISA are similar to all antibody-based methods and are related to crossreactivity and non-specic signal production. This method is also poorly suited for low concentrations of antigens (59). The detection of differential gene expression may also aid in capitalizing on the novel high-resolution and specic assays to understand differential gene expressions in biolm communities. However, current assays may only depict the average signal or response from all of the cells in the biolm. This measurement would not provide signals from the specic cell population in the biolm that responded to specic environmental/treatment-mediated changes. These localized cells responses would be useful for the identication and design of interfering therapeutic measures. Furthermore, there are several factors in an in vivo environment that may inuence bacterial adhesion and biolm formation. The shear forces, salivary/ plasma protein binding of biomaterials/device, and the immune response are some of the in vivo factors that are difcult to reproduce in vitro. Therefore, it is questionable whether the assessment of gene expression in vitro is actually indicative of gene expression in vivo.

Molecular biological methods

Molecular biological techniques have provided a great deal of genetic information on biolm bacteria. The primary goal of most of these methods is to develop standard assays to study factors affecting bacterial adherence and biolm formation. Microarray analysis and the use of dened regulatory mutants have been important tools for studying biolm development. In addition, cloning and expression of bacterial virulence factors in less pathogenic organisms is another important tool for assessing the role of bacterial factors in biolm-mediated infections (78). It is important to realize that, when molecular-based analyses of bacterial adherence and biolm formation are performed, the bacteria are grown under appropriate laboratory conditions. These conditions might not be a standard protocol or a clinically realistic condition for the resident bacterial cells. Although such experiments can be used for a relative comparison between experimental

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Fig. 14. Atomic force microscope images showing the details of bacterial cell surfaces in the nanometric range.

Polymerase chain reaction (PCR) is a method that allows exponential amplication of short DNA sequences. The method relies on thermal cycling and enzymatic replication of the DNA. Primers, which consist of short DNA fragments/sequences complementary to the target region and a DNA polymerase, are key components to enable selective and repeated amplication. As PCR progresses, the DNA generated is itself used as a template for replication, setting up a chain reaction in which the DNA template is exponentially amplied. This method of analysis is mostly used as a qualitative tool for detecting the presence or absence of a particular bacterial DNA. A real-time polymerase chain reaction, also called quantitative real-time polymerase chain reaction (Q-PCR) is based on PCR and is employed to amplify and simultaneously quantify a targeted DNA molecule. RT-PCR enables both detection and quantication of one or more specic sequences in a DNA sample. The key feature in RT-PCR is that the amplied DNA is detected as the reaction progresses in real time (84). This is a new approach compared to standard PCR where the product of the reaction is detected at the end. Two common methods for the detection of products in real-time PCR are (i) non-specic uorescent dyes that intercalate with any double-stranded DNA, and (ii) sequence-specic DNA probes consisting of oligonucleotides that are labeled with a uorescent reporter, which permits detection only after hybridization of the probe with its complementary DNA target. RT-PCR can be used to estimate the number of copies of a target gene in a sample and is reported to be more sensitive than conventional quali-

tative PCR. This method has been used to detect and quantify bacterial populations in a biolm. Often, the RT-PCR is combined with reverse transcription to quantify messenger RNA and non-coding RNA in cells or tissues. Quantitative reverse transcriptase realtime PCR (qRT-PCR) can be used effectively to quantify the number of RNA transcripts of specic genes from bacteria growing in biolms. qRT-PCR has a large dynamic range and may be used to verify gene expression data obtained from microarrays. In addition, qRT-PCR is sensitive and therefore may be used to quantify gene expression from biolm samples where only a small amount of biological material is available (8588).

Miscellaneous advanced techniques

Atomic force microscopy (AFM) has been applied recently to study the forces of interaction between bacteria cells and between bacteria cells and substrates (8992) (Fig. 14). In order to use AFM to determine bacteriasubstrate interaction, the bacteria cell or substrate particle is attached onto an AFM tip and the forces of interaction between bacterial cells and between the bacterial cell and substrate are determined. Briey, as the AFM tip approaches the substrate and the gap between the two interacting bodies closes to the nanometer range, the interacting forces developed are registered by the AFM tip (90). The AFM force curves can be used to estimate the duration of interaction and adhesion events in the interaction between the bacteria and the substrate. AFM has also become an accepted tool to measure interaction

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forces between bacteria and substrates (92). In this analysis, positively charged polymers, such as polyethyleneimine and poly-L-lysine, are necessary to securely attach bacteria onto the cantilever tips. The physical attachment of bacterial cells using positively charged polymers might promote structural rearrangements in bacterial cell surface structures, which in turn may affect the value of the forces measured. Based on this concept, an investigation aimed to study the effects of endodontic irrigants on the adherence of E. faecalis to dentin (93). The ndings from this study highlighted that chemicals which altered the physicochemical properties of dentin might inuence the nature of bacterial adherence and adhesion forces to dentin that are factors in biolm formation. Recently, mechanical tools such as micromanipulators have been used to sample individual cells or biolm compartments. However, the sensitivity of such tools is too low to allow any analysis of a population of cells (ideally less than 1,000 cells). Laser-based optical tweezers are non-invasive and non-contact tools that can probe the interaction between microscopic objects such as bacteria and collagen with sub-pN sensitivity. The optical tweezers technique gives more quantitative information about the forces of interaction between bacteria and substrate (93). Fourier Transform Infra-Red (FTIR) spectroscopy has been applied to characterize the chemical composition of mature biolm structures. In an FTIR spectroscopic analysis, infrared radiation is interacted with a test sample. During this interaction, some of the infrared radiation is absorbed by the sample and some of it is transmitted through the sample. The resulting spectrum represents the molecular level absorption and transmission, which is a molecular ngerprint of the sample. FTIR spectroscopy can be used for the qualitative and quantitative analysis of the chemical constituents on a biolm structure (70). On a similar line, biophysical techniques such as solid-state nuclear magnetic resonance (NMR) are powerful analytical tools and have been applied to study the constituents of bacterial biolm. NMR spectroscopy techniques have been used as a non-invasive method to obtain metabolic information of viable prokaryotic cell suspensions, eukaryotic cells, and tissue samples (95). NMR spectroscopy techniques are also useful to obtain metabolic information in planktonic cells, adherent bacterial cells, and in situ biolm bacteria (94,95).

Conclusion
A variety of biolm models are used for different experimental purposes in Endodontics today. One of the main issues for researchers is making a rational choice regarding the best model to use for their particular research problem. Generally, systems that closely reproduce in vivo conditions should be chosen when the aim is solely to reproduce natural biolms under laboratory conditions. However, there is no single, ideal biolm model for all applications. Direct, non-destructive visualization of biolms is advantageous in monitoring changes in biolm bacteria and structures. Recent advances in CLSM, ow cytometry, micromanipulator-assisted analysis, GFP tagging, and FISH have made biolm characterization very comprehensive. In spite of the amount of work carried out and the multitude of available methods, the quantication of bacterial biolm and the evaluation of the disinfectant activity remain a major challenge in Endodontics. Efforts are warranted to standardize the type of biolm models, test methods, parameters used in the analysis, sample collection, and analysis of results.

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