biofilm models and methods of

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Biofilm models and methods of

biofilm assessmentANIL KISHEN & MARKUS HAAPASALO

Endodontic microbes dwell within the infected root canal system as surface-adherent biofilm structures. In order to

simulate thisin vivosituation, a variety ofin vitrobiofilm models are currently used in Endodontics for different

microbiological experiments. Unfortunately a cogent selectionof 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 biofilm

models in Endodontics. Different in vitroendodontic biofilm models, devices used to generate biofilms, and biofilm

assays used to analyze these biofilm structures qualitatively and quantitatively are also presented in this article.

Received 9 December 2011; accepted 3 March 2012.

Introduction: changing paradigm inendodontic infection

Biofilm 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 poly-

meric substances (EPS) (1). While endodontic micro-

bial flora is established to be less diverse compared tothe oral microbial flora, 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 biofilm

(intraradicular biofilm). The endodontic bacterial

activities that are usually confined to the intracanal

spaces may, under certain conditions, form biofilm on

locations beyond the apical foramen (extraradicular

biofilm) (24). The geometrical and anatomical com-

plexity in the root canal system tends to shelter the

bacterial biofilm from root canal disinfectants andinstrumentation procedures (2). Furthermore, the

progression of endodontic infection alters the nutri-

tional 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

biofilm 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 biofilm models as essential models

for in vitro microbiological investigations and the

assessment of different disinfectants and disinfection

strategies in Endodontics.

Biofilm model systems: factors tobe considered

Bacterial biofilms are developed in order to study themicrobial interactions within the root canal space or

between bacteria and host immune cells (8,9). Cur-

rently, they are more commonly grown to test the

efficacy of different irrigants/medicaments and irriga-

tion procedures in Endodontics. It should be noted

that the antimicrobial resistance observed in biofilm

bacteria is not generally due to classic genetic mech-

anisms; instead, this arises due to certain peculiarities

of biofilm growth. The types of resident bacterial

species, nature of bacterial adherence to substrate,

physico-chemical characteristics of the substrate, thick-ness of the biofilm, bacterial cell density, amount of

EPS, and phenotypical/genotypical modification of

the resident bacteria are all factors that could contrib-

ute to antimicrobial resistance in biofilm bacteria

(6,10,11). Typically, different antimicrobial resistance

mechanisms may act concurrently or synergistically in

a biofilm structure, and understanding some of these

mechanisms is the key to developing biofilm model

systems for different application in Endodontics.

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Broadly, factors associated with bacterial adherence,

bacteriasubstrate interaction, and biofilm ultrastruc-

ture should be standardized to develop useful biofilm

models forin vitroexperiments (6). Currently, there is

no universally acceptedin vitromodel that reproduces

biofilm infection in Endodontics.

Bacterial adherence andbacteriasubstrate interaction

The earliest stage in the formation of most oral bio-

films involves the adsorption of macromolecules from

tissue fluids such as saliva onto a biomaterial (natural

or synthetic) surface, leading to the formation of a

conditioning layer. The conditioning fluid 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 influx 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 bio-

films on any surface that is conditioned with such

conditioning fluids (11). The next step in the devel-

opment of a biofilm 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 pathogenicdeterminant in biofilm-mediated infection. Bacterial

adherence to a surface is influenced by (i) the environ-

mental conditions such as pH, temperature, fluid flow

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 biofilm model systems for in vitroexperiments (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: trans-

port of microbe to substrate surface). This reversible

interaction is followed by the molecular-level non-

specific interactions between the bacterial surface

structures and the substrate. This phase of microbial

adherence to a substrate is mediated by the bacterial

surface structures such as fimbrae, pili, flagella, and

EPS (PHASE 2: initial non-specific microbialsubstrate

adherence phase). The bacterial surface structures form

bridges between the bacteria and the conditioning film

(11). Porphyromonas gingivalis, Streptococcus mitis,

Streptococcus salivarius, Prevotella intermedia, Prevo-tella nigrescens, Streptococcus mutans, andActinomyces

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 sub-

strate may not be strong. However, with time these

bonds gain in strength, making the bacterial attach-

ment irreversible. In the final stage, a more specific

bacterial adhesion to the substrate is established via

polysaccharide adhesin or ligand formation (PHASE 3:specific microbialsubstrate adherence phase). In this

phase, adhesin or ligand molecules on the bacterial cell

surface will bind to receptors on the substrate. Specific

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 theenvironment conditions (Fig. 1). Therefore, while

developingin vitromodels, it is important to provide

sufficient bacteriasubstrate interaction time and

optimum environmental conditions. The development

and maturation of biofilm structure occurs subsequent

to bacterial adherence.

Biofilm ultrastructure

During the development of a biofilm, the resident

bacterial cells proliferate, leading to expansion of thebiofilm 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 final structure of the

biofilm. Ultrastructurally, a biofilm consists of a popu-

lation of bacterial cells attached irreversibly to a sub-

strate and encased in a hydrated, polyanionic matrix of

EPS, proteins, polysaccharides, and nucleic acids

(15,16). Bacteria themselves account for a variable

Biofilm models and methods of biofilm assessment

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fraction of the total biofilm volume (typically 535%)

(17). A mature biofilm will be a metabolically active

community of microorganisms where individuals share

duties and benefits (16). For instance, some microor-ganisms help in adhering to the solid support while

some others (such asFusobacterium nucleatum) create

bridges between different species. This signifies

the relevance of a polymicrobial biofilm over a

mono-species biofilm. The physiological characteris-

tics of the resident microorganisms in a biofilm also

offer an inherent resistance to antimicrobial agents

(17,18). Bacterial species such as staphylococci,

enterococci,Klebsiella pneumoniae,Pseudomonasspp.,

etc., are inherently resistant to many antimicrobials

(1719).Interestingly, it has been reported that the biofilms

formed in pure cultures of bacteria under laboratory

conditions and the mixed-species biofilms 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 influence the biofilm

permeability and consequently provide a certain degree

of protection or barrier effect against physical and

chemical threats. Each step in the development of

biofilm, from the adherence of bacteria to a substrate tothe final formation of a matured biofilm structure, as

well as protein expression/slime production, is modu-

lated by a large number of variables, including type of

bacterial species, environmental conditions, and age of

the biofilm. Previous studies on endodontic biofilm

models have shown that mature biofilms and biofilms

with limited nutrient supply are more resistant to irri-

gants such as chlorhexidine and Light Activated Dis-

infection than early (immature) biofilms under normal

nutrient conditions. Studies have emphasized that the

age or degree of maturation, nutritional condition of

the biofilm, and interactions between resident bacterial

species are some of the major confounding factors indesigningin vitrobiofilm models to test the efficacy of

various endodontic disinfectants (2026). On the

above basis, it is important to use relevant bacteria

(primary colonizers), and provide ideal environmental

conditions (substrate, fluid conditioning, nutritional

conditions, and temperature) and optimum bacteria

substrate interaction time (matured biofilm) in order

to achieve a standardized endodontic biofilm model for

in vitroapplications.

Two types of microbial interactions occur at the

cellular level during the formation of biofilm. One is theprocess of recognition between a suspended cell and a

cell already attached to substratum. This type of inter-

action is termed co-adhesion. In the second type of

interaction, genetically distinct cells in suspension rec-

ognize each other and clump together. This type of

interaction is called co-aggregation. This association is

highly specific and occurs between co-aggregating

partners only. Interestingly, most oral bacteria recog-

nize each other as co-aggregating partners. Fuso-

bacterium nucleatum, a Gram-negative filamentous

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-filamentous bacteria and surface-adsorbed

spherical-shaped cocci produce the characteristic

corncob structure of oral biofilms (24). The attachment

of cocci to filamentous bacteria is said to be mediated

via fimbriae of the oral streptococci. Although the

genetic makeup of the bacteria is the main determinant

of co-aggregation, the physico-chemical characteristics

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

Kishen & Haapasalo

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of the environment also play a crucial role (24,26). It

was observed that bacteria in a co-aggregated suspen-

sion were significantly more resistant to antimicrobials

when compared to planktonic suspension, while bacte-

ria in a biofilm mode showed the most resistance to

antimicrobials (25,26). Consequently, antimicrobial

agents selected on the basis of traditional susceptibilitymethods (such as broth-based Minimal Inhibitory

Concentration [MIC]) may not be very appropriate to

eliminate co-aggregated or biofilm bacteria.

Bacterial biofilm models:in vitrodevelopment

Laboratory models are conventionally used to mimic

natural biofilms for different experimental purposes.

Thesein vitrobiofilms are easy to control and useful in

obtaining standardized biofilm models with predict-able structure and behavior. Conventional biofilm

models range from monocultures in static growth con-

ditions to diverse mixed cultures in dynamic growth

conditions. The static biofilm models use different sub-

strates (e.g. glass, polycarbonate, silicon, hydroxyl

apatite, nitrocellulose, enamel, dentin) to grow bio-

films while the dynamic biofilm models use reactor or

fermenting systems to grow biofilms on a particular

substrate. Both aerobic and anaerobic environments

can be employed for in vitro biofilm development.

Figures 26 show differentin vitrobiofilms grown ondifferent substrates. Given that thein vivoconditions

are commonly dynamic, studies evaluating biofilm for-

mation under static conditions might be somewhat

misleading depending upon the research question.

These in vitrobacterial biofilm models are routinely

applied to: (i) examine the adherence of specific bac-

terial species to any biomaterial surface (27); (ii) study

the nature and pattern of early microbial biofilm for-

mation on a particular substrate (28); (iii) study the

interaction between different biofilm bacteria and host

immune cells (29); and (iv) test the efficacy of antimi-crobial agents or antimicrobial treatment strategies

(30,31). Currently in Endodontics, most in vitro

biofilms are developed and utilized for testing anti-

microbials and irrigation strategies (Table 1). Pres-

ently, 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, biofilm models, and

procedures/assays utilized for the analysis. Obviously,

Fig. 2. (a) Photograph of a biofilm grown in vitro forthree weeks on a collagen-coated hydroxyapatite disc.The biofilm is coated with a palladiumgold mixture forSEM. (b) SEM image of mixed bacterial (multi-species)biofilm grown anaerobically for seven days in BHI brothon collagen-coated hydroxyapatite disc. Low magnifica-tion. (c) SEM image of six-month-old biofilm grownanaerobically in BHI broth on collagen-coated hydrox-

yapatite disc. Several bacterial morphotypes includingcoiled spirochetes can be seen.


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the number of parameters needs to be considered in

the design of a representative biofilm model for appli-

cations 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 concentra-tion in the inoculum, incubation time, growth condi-

tions, 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 biofilm formation (27). Findings from experi-

ments with planktonic and biofilm bacteria (50) have

revealed large differences in the dynamics of killing

Fig. 3. (a) SEM of three-day-old biofilm grown anaero-bically in BHI broth on a nitrocellulose filter. Cocci and

long rod-shaped bacteria dominate in the specimen. Lowmagnification. (b) Three-day-old biofilm grown anaero-bically in BHI broth on a nitrocellulose filter. Cocci andlong rod-shaped bacteria dominate in the specimen.High magnification.

Fig. 4. (a) SEM image of six-week-old biofilm grownaerobically in BHI broth on a dentin disc. (b) Lowmagnification SEM image of the previous sample. Cocciand long rods dominate in the sample. (c) Mixture ofbacteria grown aerobically for one month on dentin hasfailed to grow a mature biofilm. Smeared dentin can beseen between the bacteria.

Kishen & Haapasalo

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between planktonic and biofilm bacteria. There is nodoubt that the results from planktonic killing studies

must be interpreted with caution and direct extrap-

olation as to the efficacy of the agent in complex

in vivo systems is not possible. A comparison of the

planktonic and biofilm tests in a study indicated that

planktonic killing tests may be useful for preliminary

screening of new disinfectants before proceeding onto

more complex biofilm designs (50).

Biofilm devices: flow cells and fermentorsThere are several in vitro devices that are used to

develop biofilms. Some of them produce biofilms irri-

gated with fresh culture medium; in this case, the

biofilms experience a continuous flow of medium

supplemented with fresh nutrients. These in vitro

devices are used to grow dynamic biofilm models. The

flow cell system is one of the most utilized dynamic

models. It consists of a transparent chamber of fixed

depth through which the growth medium flows. The

inlet tubing supplies growth medium and the outlettubing 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 flow rate of

the medium. Pre-fabricated flow cell systems are avail-

able 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

biofilm formation in real time (55).

Chemostats are also used to grow dynamic biofilmsof microbes on experimental substrates submerged

within the chemostat. One of the most important

features of chemostats is that microbial biofilms 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 biofilms

are formed on thin filter membranes in a physiological

steady state. These systems permit evaluation of

growth rate dependence and cell-cycle specificity of

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


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antibacterial agents. Finally, there are constant depth

reactors in which surface growth is periodically

removed to maintain a constant geometry of thebiofilm. In these reactors, microorganisms can be

grown in a physiological steady state with all culture

parameters constant. The system can generate large

numbers of biofilms with comparable and repro-

ducible data (55).

The static biofilm system generates biofilms that

have exhausted important nutrient components at

the end of an overnight incubation. The key features

of this system are that numerous biofilms 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 systemfor biofilm analysis (31). This system provides a basis

for the rapid screening of biofilm mutants (56),

biomass development, and biofilm-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 biofilms.

Fig. 6. (a) Multi-species biofilm grown anaerobically for three weeks on a glass coverslip conditioned with media for24 hrs. The biofilm was sparse and not uniform. (b) High magnification image of the previous SEM picture showingmixed bacterial flora. (c) The biofilms were approximately 20 microns thick at certain areas. (d) High magnificationimage of biofilm with abundant extracellular matrix.

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Table 1: List of literature of in vitrobiofilm models for different endodontic applications

Authors Type of Model Purpose Preparation of Biofilm

Shabahang &

Torabinejad,

2003 (32)

In vitro

E. faecalis

The antimicrobial effect of MTAD: anin

vitroinvestigation

Extracted human teeth contaminated with

E. faecalisfor 4 weeks

Dentinal shavings and CFU-based

method was used for the analysis

Duggan &

Sedgley, 2007

(33)

In vitro

E. faecalisstrains

recovered from root

canals, oral cavity,

and non-oral

sources

Biofilm formation of oral and endodontic

E. faecalis

96-well plates for 24 hours

Crystal violet assay used for assessment

(optical density at 570 nm)

George &

Kishen, 2007

(34)

In vitro

E. faecalis

(Gram-positive),

Actinomycetes

actinomycetemcomitans

(Gram-negative)

Methylene blue dissolved in different

formulations: water, 70% glycerol, 70%

polyethylene glycol, and a mixture of

glycerol:ethanol:water (30:20:50) was

tested

Two-day-old biofilms in multi-well plates

(polystyrene)

Four-day-old biofilms in human teeth

CFU-based method

George &

Kishen, 2008

(35)

In vitro

E. faecalis

This study aimed to investigate the effect

of including an oxidizer and oxygen

carrier in photosensitization

formulation to disinfect a mature

endodontic biofilm by light activated

disinfection

Human teeth (10-week-old biofilm)

CFU-based method

McGill et al.,

2008 (36)

In vitro

E. faecalis

The efficacy of dynamic irrigation using a

commercially available system

(RinsEndo)

A collagen-based bio-molecular film

formed on extracted human teeth

Digital image analysis of the canal surfaces

(ipWin4)

Sainsbury et al.,

2009 (37)

Ex vivo DIAGNOdent laser fluorescence

assessment of endodontic infection

Extracted teeth with endodontic

pathology

Fluorescence emissions in thenear-infrared range were measured

Shen et al.,

2009 (38)

In vitro

Multi-species

Evaluation of the effect of two

chlorhexidine preparations on biofilm

bacteria in vitro: a three-dimensional

quantitative analysis

Collagen-coated hydroxyapatite (CHA)

and uncoated hydroxyapatite (HA)

discs

Confocal laser scanning microscopy was

used for the analysis of dead versus

viable cells

Williamson

et al., 2009

(39)

In vitro

E. faecalis

(clinical isolate)

Antimicrobial susceptibility of

monoculture biofilms to 6% NaOCl,

2% CHX,


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Table 1: Continued


Kishen et al.,

2010 (41)

In vitro

E. faecalis

Efflux pump inhibitor potentiates

antimicrobial photodynamic

inactivation ofEnterococcus faecalis

biofilm

Microwell plates

CFU-based method

Confocal laser scanning microscopy

Hiraishi et al.,

2010 (42)

In vitro

E. faecalis

Antimicrobial efficacy of 3.8% silver

diamine fluoride

Membrane filters

CFU-based method

Shrestha et al.,

2010 (43)

In vitro

E. faecalis

Nanoparticulates for anti-biofilm

treatment and effect of aging on its

antibacterial activity

Microwell plates/saliva

CFU-based method


Liu et al., 2010

(44)

In vitro

E. faecalis

Biofilm formation capability of

Enterococcus faecaliscells in starvation

phase and its susceptibility to sodium

hypochlorite

Human dentin and polystyrene blocks

CFU-based method

SEM

Chavez de Paz

et al., 2010

(45)

In vitro(clinical

isolates)

E. faecalis,L. paracasei,

S. anginosus,

S. gordonii

The effects of antimicrobials on

endodontic biofilm bacteria

24-hour biofilm within a mini-flow cell

system

Confocal microscopy and image analysis

Su et al., 2010

(46)

In vivo This study explored the effect of surgical

endodontic treatment of refractory

periapical periodontitis with

extraradicular biofilm

Resected root-end samples

Soares et al.,

2010 (47)

In vitro

E. faecalis

Effectiveness of chemomechanical

preparation with alternating use of

sodium hypochlorite and EDTA in

eliminating intracanalEnterococcus

faecalisbiofilm

Human teeth (21-day-old biofilm)

SEM

CFU-based method

Bhuva et al.,

2010 (48)

In vitro

E. faecalis

The effectiveness of passive ultrasonic

irrigation on intraradicularEnterococcus

faecalisbiofilms in extracted

single-rooted human teeth

Human teeth

SEM-based image analysis

Shen et al.,

2010 (49)

In vitro

Multi-species biofilm

(subgingivial

plaque)

The synergistic antimicrobial effect by

mechanical agitation and two

chlorhexidine preparations


discs

(3 weeks old)


Pappen et al.,

2010 (50)

In vitro


(subgingivial

plaque)

To investigate the antibacterial effect

of Tetraclean, MTAD, and five

experimental irrigants using both

direct exposure test with planktonic

cultures and mixed-species in vitro

biofilm model


discs

(2 weeks old)


Shen et al.,

2010 (51)

In vitro


(subgingivial

plaque)

The aim of this study was to enumerate

viable bacteria at different growth

stages of a multi-species oral biofilm

and to compare results obtained with

the LIVE/DEAD BacLight Kit with

those from culturing and plate

counting


discs


CFU-based method

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Table 1: Continued


Lundstrom

et al., 2010

(52)

In vitro(multi-species)

Streptococcus sanguinis,

Actinomyces viscosus,

Fusobacterium

nucleatum,Peptostreptococcus

micros,and

Prevotella nigrescens

Bactericidal activity of stabilized chlorine

dioxide as an endodontic irrigant in a

polymicrobial biofilm tooth model

system

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)

In vitro

E. faecalis

A direct comparison between extracted

tooth and filter-membrane biofilm

models of endodontic irrigation

Human teeth

CFU-based method

Upadya &

Kishen, 2010

(9)

In vitro

E. faecalisand

P. aeruginosa

To evaluate the efficacy 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 influence ofdifferent photosensitizer (PS)

formulations in the MB-mediated

LAD of biofilms was also evaluated.

Mono-species biofilms in 24-well

polystyrene plates (4 days)

CFU-based method


George et al.,

2010 (28)

In vitro

E. faecalis

This study examined the biofilm-forming

capacity ofE. faecalison gutta-percha

points under different nutrient status

and surface conditioning with saliva

and serum

Gutta-percha

Conditioned with saliva or serum (2, 4,

and 12-weeks)

Biofilm growth for 2 weeks

CFU-based method

SEM

Badr et al.,

2011 (54)

In vitro

E. faecalis

A laboratory evaluation of the

antibacterial and cytotoxic effect of

liquorice when used as root canalmedicament

Grown on cellulose nitrate membrane

filters

CFU-based method

Shen et al.,

2011 (21)

In vitro


(subgingivial

plaque)

The aim of this study was to examine the

susceptibility of multi-species biofilms

at different phases of growth to root

canal irrigants (2% chlorhexidine

[CHX] or CHX-Plus)


discs (2 days to months)


CFU-based method

Fig. 7. Schematic diagram showing different factors that would influence the structure and development ofin vitrobiofilms.


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Structural evaluation of biofilm requires the use of

irrigated biofilm systems. An irrigated and flow-

through cell system allows for the study of the devel-

opment of biofilm over time. These analyses include

non-destructive evaluation of temporal and spatial

expression of selected genes and the complete life-

cycle of biofilm formation and dispersal. Several find-

ings on the unique behavioral responses of biofilm

cells that cannot be obtained using static microtiter-

based systems were observed with the aid of irrigated

biofilm flow systems (59).

Biofilm assays

Biofilm assays are used to characterize factors such

as (i) number and type of microorganism, (ii) vitality

(dead/living cells) of the resident microbial popula-

tion, (iii) age, (iv) thickness (mono-layered or multi-

layered), (v) structure (homogeneous, irregular,dense, porous), and (vi) surface topography (peaks

and valleys) of biofilms. Different techniques such as

(i) microbiological culture techniques, (ii) colori-

metric techniques, (iii) microscopic techniques, (iv)

physical methods, (v) biochemical methods, and (vi)

molecular methods are applied to biofilm assays. The

basic outline of the experimental methods to assess

antibacterial or antibiofilm efficacy of irrigants or irri-

gation strategies is shown in Figure 8.

Microbiological culture techniques

The biofilm formed on a substrate can be quantified 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 biofilm structure. However, the

CFU may only detect bacteria that are able to initiatecell division at a sufficient rate to form colonies and

whose growth requirements are supported by the

culture medium used. Several protocols recommend

the removal of biofilm 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 proce-

dure. 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, inthat 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 biofilms 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 biofilm and grown in

a culture media (49). Ultrasonic vibrations and

enzymes are used to remove bacterial biofilm before

quantification. However, it is imperative to use an

Fig. 8. Basic outline of the experimental methods for assessing antimicrobial efficacy using in vitrobiofilm.

Kishen & Haapasalo

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without using fluorescent probes by using plasmid-

encoded green fluorescent protein (GFP). These

transformedE. coliO157:H7 have been used to study

their attachment onto a surface (62,63). The viability

of these cells may be determined by staining the trans-

formed cells with membrane-impermeable fluorescent

dye (63).

Scanning Electron Microscopy (SEM) and Transmis-

sion Electron Microscopy (TEM) have been effective

workhorses in biofilm analyses for many years. High-resolution electron microscopy has been employed for

the morphological and structural characterization of

microbial biofilms. The main disadvantage with these

techniques is the need for extensive sample preparation

steps such as fixation, dehydration, freeze- or critical

point-drying, and sputtering. These treatments can

deeply affect the original biofilm 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 hydratedcondition at relatively high resolution (64). Structural

modifications in microbial biofilm architecture, par-

ticularly an overall loss of matrix volume, were appre-

ciable when comparing conventional high-vacuum

SEM to ESEM images. Sutton et al. (65) compared

different dehydration techniques and showed that

freeze-dried samples presented significant detachment

of microbial biofilm from the substrate, while more

complex dehydration procedures such as critical point

Fig. 11. Fluorescent microscope image showingE. faecaliscells adhering to type 1 collagen (100 , oil immersion).(a) Control. (b) After EDTA treatment.

Fig. 12. SEM images of (a) E. faecalis cells adhering toroot canal dentin and (b) multi-layered mono-speciesbiofilm ofE. faecalison root canal dentin.

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drying caused an almost complete disappearance of the

EPS matrix. Although proper fixation processes were

applied, the collapse of the biofilm structure upon

dehydration procedures is mainly due to the lack of a

self-sustaining scaffold in the EPS matrix. Neverthe-

less, bacteria cells maintained their shape and dimen-

sions in vacuum after fixation and could clearly beidentified by SEM. In the ESEM mode, the semi-

transparent appearance of the EPS and the low signal-

to-noise ratio at high pressures results in a limited

image resolution. In brief, ESEM represents an effec-

tive technique for detecting highly hydrated bacterial

biofilms by preserving the substantial EPS component.

On the contrary, conventional high-vacuum SEM

allows a detailed examination of the cellular compo-

nents and favors the detection of the three-dimensional

hollow structures, but fails to show the actual biofilm

architecture consisting of a large volume of EPS matrixsurrounding the cells. The combined use of conven-

tional SEM and ESEM techniques can therefore

provide complementary information on different

biofilm components, bacterial cells, and the extracellu-

lar matrix (65).

Epifluorescent microscopy has been used to study

bacterial biofilm microstructure. Biofilms grown on

biomaterial surfaces are usually stained with a fluores-

cent dye and viewed under an epifluorescent micro-

scope. In a study, binary species biofilms were stained

using two different fluorescent probes for each organ-ism and observed under an epifluorescent microscope

using two excitation wavelengths. Two different images

and the background biofilm were captured with appro-

priate wavelengths. The images were then combined to

construct a new image that simultaneously showed

both organisms (66). Epifluorescentmicroscopy is used

to determine viable cells, biofilm cell arrangement,

micro-colony formation, biofilm pH, and distribution

of chemicals in a biofilm structure (67).

CLSM is a particularly important biofilm analysis

technique that is restricted to 50200-mm-thickbiofilm structures. CLSM has overcome some of the

limitations exhibited by most of the earlier micro-

scopic techniques such as epifluorescence, SEM, and

TEM. Together with improvements in the molecular

techniques for bacteria, CLSM has become an impor-

tant tool for studying biofilms. Green fluorescent

protein (GFP) tagging of certain bacterial strains such

as Pseudomonas aeruginosa is utilized to study biofilm

formation. This method uses a fluorescent imaging-

based analysis or CLSM to quantify the biofilm struc-

tures. 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

thegfpreporter system has been used to study the role

ofagrin biofilm formation and has given an interest-ing insight into gene regulation during the course of

biofilm development (70). This technique is likely to

be an important tool for future studies on the regula-

tors and genes involved in biofilm 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 ofthe entire biofilm. The CLSM images can be used to

determine the thickness and distribution of cells in a

biofilm structure. CLSM can also be used to deter-

mine the pH gradients in biofilms. The interior pH of

biofilms is measured by a fluorescent lifetime imaging

technique using fluorescein as a pH indicator. Cur-

rently, the use of a fluorescent dye combination

(LIVE/DEAD BAC light) with CLSM has become a

routine practice for in vitro biofilm analysis. The

LIVE/DEAD Bacterial Viability kit (Molecular

Probes, Eugene, OR) contains separate vials of thetwo component dyes (SYTO 9 and propidium

iodide). The dyes are used in a 1:1 mixture for stain-

ing the biofilm bacteria following the manufacturers

instructions. The dead cells emit red light and the

viable cells emit green light under CLSM examina-

tion (Fig. 13). In a recent in vitrostudy, it was shown

that bacteria in the multi-species anaerobic biofilm

grown under nutrient deprivation changed into the

viable-but-non-cultural (VBNC) state but could be

returned to the normal physiological state and cul-

tured by re-establishing the supply of nutrients whilethey were still in the biofilm. The results from this

study indicated that viability staining was a better

reflection of the true viability of the biofilm bacte-

ria than the culturing method during starvation. This

finding needs to be taken into account when assessing

results from cultural studies employed to determine

in vivoroot canal biofilms (51).

A fluorescence in situ hybridization (FISH) tech-

nique using probes to target specific 16S rRNA


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sequences in bacteria is applied for the simultaneous

analysis of the spatial distribution of both Gram-

positive and Gram-negative bacteria in biofilms.

FISH is a recognized tool for the specific and sensi-

tive identification of target organisms within complex

microbial communities. Visualization of FISH-

labeled cells in biofilms can be carried out by fluo-

rescent microscopy and LSCM (68,69). However,

CLSM is preferred in a biofilm analysis because it

allows a three-dimensional non-invasive visualizationof cells and the computational reconstruction of

mature biofilms without distortion of their structure

(25,70).

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

Basic physical parameters such as biofilm thickness,

area, weight (wet and dry), and density estimates are

used to quantify biofilm growth. A thickness measure-

ment by light microscopy is usually effective in thinbiofilms but may not work with thick biofilms. In this

method, the biofilm is placed on the stage of a micro-

scope that has calibration scales on the fine control and

the objective is lowered until the biofilm surface is in

focus and the fine adjustment dial setting of the micro-

scope is recorded (71). The microscope objective is

then focused on the substrate surface, preferably in an

area with no biofilm. The difference in the fine adjust-

ment settings can be used to calculate the thickness. A

simple manual-gauge needle method (72) and an elec-

tronic probe to measure biofilm thickness (73) have

also been described. A properly prepared SEM sample

or cryosection enables the estimation of biofilm thick-

ness and also reveals layering of embedded bacterial

cells (74). Biofilm 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 biofilm growth (with the assumption

that no substrate solubilization occurred duringbiofilm formation) and then cleaned, dried, and

weighed again in order to record the dry biofilm

weight. If both wet and dry weight measurements on

the same biofilm sample are performed, the approxi-

mate density may be determined by assuming that the

volume of the biofilm 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 biofilm density and weight can be

calculated per unit of substratum.

Biochemical methods: biomass andextracellular matrix (ECM)

Microbial biomass denotes the total number of

microbes in a given area. The measurement of micro-

bial biomass is considered to be a rapid method and

includes measurements of the wet or dry weight of the

entire biofilm, measurements of the cell contents,

measurements of the cellular activities or viable cells,

Fig. 13. Three-dimensional confocal laser scanning microscopy reconstruction of E. faecalis biofilm (inlet showssagittal section) (60 ). Left: the biofilm has received no treatment. Right: the biofilm has been subjected to Light

Activated Disinfection with Methylene blue and laser (660 nm).

Kishen & Haapasalo

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etc. One method that is used for the early detection of

viable bacteria is based on metabolic activity. Adeno-

sine 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 notedthat the rate of lysis and the ATP content vary depend-

ing on the microorganism. Hence, the ATP assay

cannot be correlated with the initial number of micro-

bial cells. Test strains genetically modified (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 significant

number in a normal period of time. This will facilitatethe 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 biofilm assays.

Molecular biological methods

Molecular biological techniques have provided a great

deal of genetic information on biofilm bacteria. The

primary goal of most of these methods is to develop

standard assays to study factors affecting bacterial

adherence and biofilm formation. Microarray analysis

and the use of defined regulatory mutants have been

important tools for studying biofilm development. In

addition, cloning and expression of bacterial virulence

factors in less pathogenic organisms is another impor-tant tool for assessing the role of bacterial factors in

biofilm-mediated infections (78). It is important to

realize that, when molecular-based analyses of bacterial

adherence and biofilm formation are performed, the

bacteria are grown under appropriate laboratory con-

ditions. These conditions might not be a standard

protocol or a clinically realistic condition for the resi-

dent bacterial cells. Although such experiments can be

used for a relative comparison between experimental

groups, there is a possibility of data disparity between

laboratories andin vivosituations.

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 typicallyperformed using one of two detection methods: the

direct or indirect assay. In direct ELISA, an enzyme-

linked (labeled) antibody is used to directly detect the

captured antigen or antibody of interest. In the more

common indirect ELISA, a detection or primary anti-

body 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 methodto quantify biomass within biofilms and even protein

production in biofilms (82,83). ELISA may be used to

quantify the population of a particular bacterium in a

mixed biofilm. An ELISA-based approach can circum-

vent errors due to cell clumping and EPS production,

which can lead to significant errors in bacterial quan-

tification. The disadvantages of ELISA are similar to all

antibody-based methods and are related to cross-

reactivity and non-specific 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 specific assays to understand differential gene

expressions in biofilm communities. However, current

assays may only depict the average signal or response

from all of the cells in the biofilm. This measurement

would not provide signals from the specific cell popu-

lation in the biofilm that responded to specific

environmental/treatment-mediated changes. These

localized cells responses would be useful for the iden-

tification and design of interfering therapeutic meas-ures. Furthermore, there are several factors in an

in vivoenvironment that may influence bacterial adhe-

sion and biofilm formation. The shear forces, salivary/

plasma protein binding of biomaterials/device, and

the immune response are some of the in vivo factors

that are difficult to reproduce in vitro. Therefore, it is

questionable whether the assessment of gene expres-

sion in vitro is actually indicative of gene expression

in vivo.


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Polymerase chain reaction (PCR) is a method that

allows exponential amplification of short DNAsequences. The method relies on thermal cycling and

enzymatic replication of the DNA. Primers, which

consist of short DNA fragments/sequences comple-

mentary to the target region and a DNA polymerase,

are key components to enable selective and repeated

amplification. 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 expo-

nentially amplified. This method of analysis is mostly

used as a qualitative tool for detecting the presence or

absence of a particular bacterial DNA. A real-timepolymerase chain reaction, also called quantitative

real-time polymerase chain reaction(Q-PCR) is based

on PCR and is employed to amplify and simulta-

neously quantify a targeted DNA molecule. RT-PCR

enables both detection and quantification of one or

more specific sequences in a DNA sample. The key

feature in RT-PCR is that the amplified 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 ofproducts in real-time PCR are (i) non-specific fluores-

cent dyes that intercalate with any double-stranded

DNA, and (ii) sequence-specific DNA probes consist-

ing of oligonucleotides that are labeled with a fluo-

rescent 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 biofilm. Often, theRT-PCR is combined with reverse transcription to

quantify messenger RNA and non-coding RNA in

cells or tissues. Quantitative reverse transcriptase real-

time PCR (qRT-PCR) can be used effectively to quan-

tify the number of RNA transcripts of specific genes

from bacteria growing in biofilms. qRT-PCR has a

large dynamic range and may be used to verify gene

expression data obtained from microarrays. In addi-

tion, qRT-PCR is sensitive and therefore may be used

to quantify gene expression from biofilm samples

where only a small amount of biological material isavailable (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 andbetween the bacterial cell and substrate are deter-

mined. Briefly, as the AFM tip approaches the sub-

strate 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 cur ves 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

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

Kishen & Haapasalo

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forces between bacteria and substrates (92). In this

analysis, positively charged polymers, such as poly-

ethyleneimine 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 rearrange-

ments in bacterial cell surface structures, which in turnmay affect the value of the forces measured. Based on

this concept, an investigation aimed to study the

effects of endodontic irrigants on the adherence ofE.

faecalis to dentin (93). The findings from this study

highlighted that chemicals which altered the physico-

chemical properties of dentin might influence the

nature of bacterial adherence and adhesion forces to

dentin that are factors in biofilm formation. Recently,

mechanical tools such as micromanipulators have been

used to sample individual cells or biofilm compart-

ments. However, the sensitivity of such tools is too lowto 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 bac-

teria and collagen with sub-pN sensitivity. The optical

tweezers technique gives more quantitative informa-

tion about the forces of interaction between bacteria

and substrate (93).

Fourier Transform Infra-Red (FTIR) spectroscopy

has been applied to characterize the chemical compo-

sition of mature biofilm structures. In an FTIR spec-troscopic 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 fingerprint of

the sample. FTIR spectroscopy can be used for the

qualitative and quantitative analysis of the chemical

constituents on a biofilm structure (70). On a similar

line, biophysical techniques such as solid-state nuclear

magnetic resonance (NMR) are powerful analyticaltools and have been applied to study the constituents

of bacterial biofilm. NMR spectroscopy techniques

have been used as a non-invasive method to obtain

metabolic information of viable prokaryotic cell sus-

pensions, 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 biofilm bacteria

(94,95).

Conclusion

A variety of biofilm models are used for different experi-

mental purposes in Endodontics today. One of the main

issues for researchers is making a rational choice regard-

ing the best model to use for their particular research

problem. Generally, systems that closely reproduce invivoconditions should be chosen when the aim is solely

to reproduce natural biofilms under laboratory condi-

tions. However, there is no single, ideal biofilm model

for all applications. Direct, non-destructive visualiza-

tion of biofilms is advantageous in monitoring changes

in biofilm bacteria and structures. Recent advances in

CLSM, flow cytometry, micromanipulator-assisted

analysis, GFP tagging, and FISH have made biofilm

characterization very comprehensive. In spite of the

amount of work carried out and the multitude of

available methods, the quantification of bacterialbiofilm and the evaluation of the disinfectant activity

remain a major challenge in Endodontics. Efforts are

warranted to standardize the type of biofilm models,

test methods, parameters used in the analysis, sample

collection, and analysis of results.

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