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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Endodontic Topics 2012, 22, 5878
All rights reserved
2012 John Wiley & Sons A/S
ENDODONTIC TOPICS 20121601-1538
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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
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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.
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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.
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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
Authors Type of Model Purpose Preparation of Biofilm
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
Confocal laser scanning microscopy
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
Collagen-coated hydroxyapatite (CHA)
discs
(3 weeks old)
Confocal laser scanning microscopy
Pappen et al.,
2010 (50)
In vitro
Multi-species biofilm
(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
Collagen-coated hydroxyapatite (CHA)
discs
(2 weeks old)
Confocal laser scanning microscopy
Shen et al.,
2010 (51)
In vitro
Multi-species biofilm
(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
Collagen-coated hydroxyapatite (CHA)
discs
Confocal laser scanning microscopy
CFU-based method
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Table 1: Continued
Authors Type of Model Purpose Preparation of Biofilm
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
Confocal laser scanning microscopy
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
Multi-species biofilm
(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)
Collagen-coated hydroxyapatite (CHA)
discs (2 days to months)
Confocal laser scanning microscopy
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.
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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).
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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.
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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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