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Endodontic Topics 2012, 22, 58–78 2012 © John Wiley & Sons A/S
All rights reserved ENDODONTIC TOPICS 2012
1601-1538

Biofilm models and methods of

biofilm assessment
ANIL KISHEN & MARKUS HAAPASALO

Endodontic microbes dwell within the infected root canal system as surface-adherent biofilm structures. In order to
simulate this in vivo situation, a variety of in vitro biofilm models are currently used in Endodontics for different
microbiological experiments. Unfortunately a cogent selection of the best models to be used for a particular research
application has not been obtained. This article outlines various factors to be considered while developing biofilm
models in Endodontics. Different in vitro endodontic 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 in consider bacterial biofilm models as essential models

endodontic 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 to
the 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) (2–4). The geometrical and anatomical com-
plexity in the root canal system tends to shelter the
bacterial biofilm from root canal disinfectants and
instrumentation 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
for in vitro microbiological investigations and the
assessment of different disinfectants and disinfection
strategies in Endodontics.
Biofilm model systems: factors to
be considered
Bacterial biofilms are developed in order to study the
microbial 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.

58

Broadly, factors associated with bacterial adherence,
bacteria–substrate interaction, and biofilm ultrastruc-
ture should be standardized to develop useful biofilm
models for in vitro experiments (6). Currently, there is
no universally accepted in vitro model that reproduces
biofilm infection in Endodontics.
Bacterial adherence and
bacteria–substrate 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 pathogenic
determinant 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 vitro
experiments (12,13).
The initial phase of the bacteria–substrate 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
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Biofilm models and methods of biofilm assessment
adherence to a substrate is mediated by the bacterial
surface structures such as fimbrae, pili, flagella, and
EPS (PHASE 2: initial non-specific microbial–substrate
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, and Actinomyces
naeslundii are some of the oral bacteria possessing
surface structures (11,12). The bridges formed
between bacteria and substrate are a combination
of electrostatic attraction and covalent/hydrogen
bonding. Initially the bonds between bacteria and 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 microbial–substrate 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 the
environment conditions (Fig. 1). Therefore, while
developing in vitro models, it is important to provide
sufficient bacteria–substrate 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 the
biofilm 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
59

Kishen & Haapasalo
Fig. 1. Schematic diagram showing the stages in the development of a biofilm.
fraction of the total biofilm volume (typically 5–35%)
(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 as Fusobacterium 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, Pseudomonas spp.,
etc., are inherently resistant to many antimicrobials
(17–19).
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 to
the 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
60
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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 in
designing in vitro biofilm models to test the efficacy of
various endodontic disinfectants (20–26). 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 vitro applications.
Two types of microbial interactions occur at the
cellular level during the formation of biofilm. One is the
process 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
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Biofilm models and methods of biofilm assessment

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 susceptibility
methods (such as broth-based Minimal Inhibitory
Concentration [MIC]) may not be very appropriate to
eliminate co-aggregated or biofilm bacteria.

Bacterial biofilm models:

in vitro development
Laboratory models are conventionally used to mimic
natural biofilms for different experimental purposes.
These in vitro biofilms 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 2–6 show different in vitro biofilms grown on
different substrates. Given that the in vivo conditions
are commonly dynamic, studies evaluating biofilm for-
mation under static conditions might be somewhat
misleading depending upon the research question.
These in vitro bacterial 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
Fig. 2. (a) Photograph of a biofilm grown in vitro for
immune cells (29); and (iv) test the efficacy of antimi- three weeks on a collagen-coated hydroxyapatite disc.
crobial agents or antimicrobial treatment strategies The biofilm is coated with a palladium–gold mixture for
(30,31). Currently in Endodontics, most in vitro SEM. (b) SEM image of mixed bacterial (multi-species)
biofilms are developed and utilized for testing anti- biofilm grown anaerobically for seven days in BHI broth
on collagen-coated hydroxyapatite disc. Low magnifica-
microbials and irrigation strategies (Table 1). Pres- tion. (c) SEM image of six-month-old biofilm grown
ently, published results on the activity of disinfectants anaerobically in BHI broth on collagen-coated hydrox-
show noticeable discrepancies between experiments, yapatite disc. Several bacterial morphotypes including
and this may be attributed to the diversity of the coiled spirochetes can be seen.
microbial growth phase, biofilm models, and
procedures/assays utilized for the analysis. Obviously,

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Kishen & Haapasalo

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

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- Fig. 4. (a) SEM image of six-week-old biofilm grown
tion in the inoculum, incubation time, growth condi- aerobically in BHI broth on a dentin disc. (b) Low
tions, and substrate properties. If laboratory strains are magnification SEM image of the previous sample. Cocci
used for adherence assays, then it is important that and long rods dominate in the sample. (c) Mixture of
bacteria grown aerobically for one month on dentin has
they are representative of clinical isolates. In addition, failed to grow a mature biofilm. Smeared dentin can be
assays that do not take into account the presence of seen between the bacteria.
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

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Biofilm models and methods of biofilm assessment

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

between planktonic and biofilm bacteria. There is no
doubt 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 fermentors
There 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 outlet
tubing drains the medium to a waste reservoir. The
growth medium is passed through the cell with the aid
of a peristaltic pump, which controls the 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 biofilms
of 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

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Kishen & Haapasalo

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

antibacterial agents. Finally, there are constant depth
reactors in which surface growth is periodically
removed to maintain a constant geometry of the
biofilm. 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 system
for 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.

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Biofilm models and methods of biofilm assessment

Table 1: List of literature of in vitro biofilm models for different endodontic applications
Authors Type of Model Purpose Preparation of Biofilm

Shabahang & In vitro The antimicrobial effect of MTAD: an in Extracted human teeth contaminated with
Torabinejad, E. faecalis vitro investigation E. faecalis for 4 weeks
2003 (32) Dentinal shavings and CFU-based
method was used for the analysis

Duggan & In vitro Biofilm formation of oral and endodontic 96-well plates for 24 hours
Sedgley, 2007 E. faecalis strains E. faecalis Crystal violet assay used for assessment
(33) recovered from root (optical density at 570 nm)
canals, oral cavity,
and non-oral
sources

George & In vitro Methylene blue dissolved in different Two-day-old biofilms in multi-well plates
Kishen, 2007 E. faecalis formulations: water, 70% glycerol, 70% (polystyrene)
(34) (Gram-positive), polyethylene glycol, and a mixture of Four-day-old biofilms in human teeth
Actinomycetes glycerol:ethanol:water (30:20:50) was CFU-based method
actinomycetemcomitans tested
(Gram-negative)

George & In vitro This study aimed to investigate the effect Human teeth (10-week-old biofilm)
Kishen, 2008 E. faecalis of including an oxidizer and oxygen CFU-based method
(35) carrier in photosensitization
formulation to disinfect a mature
endodontic biofilm by light activated
disinfection

McGill et al., In vitro The efficacy of dynamic irrigation using a A collagen-based “bio-molecular film”
2008 (36) E. faecalis commercially available system formed on extracted human teeth
(RinsEndo®) Digital image analysis of the canal surfaces
(ipWin4)

Sainsbury et al., Ex vivo DIAGNOdent laser fluorescence Extracted teeth with endodontic
2009 (37) assessment of endodontic infection pathology
Fluorescence emissions in the
near-infrared range were measured

Shen et al., In vitro Evaluation of the effect of two Collagen-coated hydroxyapatite (CHA)
2009 (38) Multi-species chlorhexidine preparations on biofilm and uncoated hydroxyapatite (HA)
bacteria in vitro: a three-dimensional discs
quantitative analysis Confocal laser scanning microscopy was
used for the analysis of dead versus
viable cells

Williamson In vitro Antimicrobial susceptibility of Glass substrate

et al., 2009 E. faecalis monoculture biofilms to 6% NaOCl, CFU-based method
(39) (clinical isolate) 2% CHX, <6% NaOCl with surface
modifiers (Chlor-XTRA), and 2% CHX
with surface modifiers (CHX-Plus)

Lim et al., 2009 In vitro biofilms The efficacy of an improved light
(20) E. faecalis activated disinfection technique
utilizing a specific photosensitizer
formulation, liquid optical-conduit,
oxygen-carrier, and light energy of
appropriate wavelength were tested
Two different biofilms were tested:
(i) Four-day-old (immature)
(ii) Four-week-old (mature)
Human teeth
CFU-based method

Shahriari et al., In vitro The study of the effect of hydrogen Dentin tubes prepared from maxillary
2010 (40) E. faecalis peroxide on the antibacterial effect of central and lateral incisors
chlorhexidine CFU-based method was used

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Kishen & Haapasalo

Table 1: Continued

Authors Type of Model Purpose Preparation of Biofilm

Kishen et al., In vitro Efflux pump inhibitor potentiates Microwell plates

2010 (41) E. faecalis antimicrobial photodynamic CFU-based method
inactivation of Enterococcus faecalis Confocal laser scanning microscopy
biofilm

Hiraishi et al., In vitro Antimicrobial efficacy of 3.8% silver Membrane filters

2010 (42) E. faecalis diamine fluoride CFU-based method

Shrestha et al., In vitro Nanoparticulates for anti-biofilm Microwell plates/saliva

2010 (43) E. faecalis treatment and effect of aging on its CFU-based method
antibacterial activity Confocal laser scanning microscopy

Liu et al., 2010 In vitro Biofilm formation capability of Human dentin and polystyrene blocks
(44) E. faecalis Enterococcus faecalis cells in starvation CFU-based method
phase and its susceptibility to sodium SEM
hypochlorite

Chavez de Paz In vitro (clinical The effects of antimicrobials on 24-hour biofilm within a mini-flow cell
et al., 2010 isolates) endodontic biofilm bacteria system
(45) E. faecalis, Confocal microscopy and image analysis
L. paracasei,
S. anginosus,
S. gordonii

Su et al., 2010 In vivo This study explored the effect of surgical Resected root-end samples
(46) endodontic treatment of refractory
periapical periodontitis with
extraradicular biofilm

Soares et al., In vitro Effectiveness of chemomechanical Human teeth (21-day-old biofilm)

2010 (47) E. faecalis preparation with alternating use of SEM
sodium hypochlorite and EDTA in CFU-based method
eliminating intracanal Enterococcus
faecalis biofilm

Bhuva et al., In vitro The effectiveness of passive ultrasonic Human teeth

2010 (48) E. faecalis irrigation on intraradicular Enterococcus SEM-based image analysis
faecalis biofilms in extracted
single-rooted human teeth

Shen et al., In vitro The synergistic antimicrobial effect by Collagen-coated hydroxyapatite (CHA)
2010 (49) Multi-species biofilm mechanical agitation and two discs
(subgingivial chlorhexidine preparations (3 weeks old)
plaque) Confocal laser scanning microscopy

Pappen et al., In vitro To investigate the antibacterial effect Collagen-coated hydroxyapatite (CHA)
2010 (50) Multi-species biofilm of Tetraclean, MTAD, and five discs
(subgingivial experimental irrigants using both (2 weeks old)
plaque) direct exposure test with planktonic Confocal laser scanning microscopy
cultures and mixed-species in vitro
biofilm model

Shen et al., In vitro The aim of this study was to enumerate Collagen-coated hydroxyapatite (CHA)
2010 (51) Multi-species biofilm viable bacteria at different growth discs
(subgingivial stages of a multi-species oral biofilm Confocal laser scanning microscopy
plaque) and to compare results obtained with CFU-based method
the LIVE/DEAD BacLight Kit with
those from culturing and plate
counting

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Biofilm models and methods of biofilm assessment

Table 1: Continued

Authors Type of Model Purpose Preparation of Biofilm

Lundstrom In vitro (multi-species) Bactericidal activity of stabilized chlorine Permanent bovine incisors coated with
et al., 2010 Streptococcus sanguinis, dioxide as an endodontic irrigant in a mucin and inoculated with standardized
(52) Actinomyces viscosus, polymicrobial biofilm tooth model suspensions of bacteria (anaerobically
Fusobacterium system for 14 days)
nucleatum, CFU-based method
Peptostreptococcus
micros, and
Prevotella nigrescens

Hope et al., In vitro A direct comparison between extracted Human teeth

2010 (53) E. faecalis tooth and filter-membrane biofilm CFU-based method
models of endodontic irrigation

Upadya & In vitro To evaluate the efficacy of light-activated Mono-species biofilms in 24-well
Kishen, 2010 E. faecalis and disinfection (LAD) using Methylene polystyrene plates (4 days)
(9) P. aeruginosa blue (33) and a non-coherent light CFU-based method
source on Gram-positive and Confocal laser scanning microscopy
Gram-negative bacteria in different
growth modes. The influence of
different photosensitizer (PS)
formulations in the MB-mediated
LAD of biofilms was also evaluated.

George et al., In vitro This study examined the biofilm-forming Gutta-percha

2010 (28) E. faecalis capacity of E. faecalis on gutta-percha Conditioned with saliva or serum (2, 4,
points under different nutrient status and 12-weeks)
and surface conditioning with saliva Biofilm growth for 2 weeks
and serum CFU-based method
SEM

Badr et al., In vitro A laboratory evaluation of the Grown on cellulose nitrate membrane
2011 (54) E. faecalis antibacterial and cytotoxic effect of filters
liquorice when used as root canal CFU-based method
medicament

Shen et al., In vitro The aim of this study was to examine the Collagen-coated hydroxyapatite (CHA)
2011 (21) Multi-species biofilm susceptibility of multi-species biofilms discs (2 days to months)
(subgingivial at different phases of growth to root Confocal laser scanning microscopy
plaque) canal irrigants (2% chlorhexidine CFU-based method
[CHX] or CHX-Plus)

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

67

Kishen & Haapasalo
Fig. 8. Basic outline of the experimental methods for assessing antimicrobial efficacy using in vitro biofilm.
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.
68
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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 initiate
cell 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, in
that case, there may be a 24-hour to more than a week
lag phase in the bacterial response. A recent study
showed that in older, starved 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. 9. Multi-well plate showing E. faecalis biofilm
growth that is quantified using a colorimetric (crystal
violet) assay.
appropriate energy level and concentration since few
studies have highlighted the possible lethal effects on
bacterial cells (60).
Colorimetric techniques
The colorimetric assay is a semi-quantitative method
based on dye uptake by the bacterial cells in a biofilm.
In this assay, after the bacterial biofilm is stained with
a dye (e.g. crystal violet), it is disrupted using a known
quantity of alcohol or a surfactant (sodium dodecyl
sulfate) and the intensity of the eluted dye is measured
using a spectrophotometer (Fig. 9). This is an easy
assay that allows the rapid quantification of biofilm
bacteria. However, this test may sometimes be difficult
to interpret because the absorbance/optical density
measured is a reflection of the number of bacteria and
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Biofilm models and methods of biofilm assessment
Fig. 10. Fluorescent microscope image of bacteria
stained with Live/Dead fluorescent stain: green, live
cells; red, dead cells.
is not a true indicator of the EPS in the biofilm struc-
ture. Although glass tubes and microtiter plates have
traditionally been used to grow in vitro biofilms,
inconsistency and lack of standardization in the devel-
oping biofilm has been a growing concern. These
assays usually work well for strains which are strong
biofilm-producers, however they may not be very
useful in differentiating weak biofilm-producers from
biofilm-negative strains (26).
Microscopic techniques
Light microscopy is the fundamental technique used
for biofilm assays, either directly on in vitro samples or
on histological sections. It is a relatively inexpensive,
simple to use, rapid, and readily available method.
Different microscopic methods have been used to
assess adherence of bacteria to substrates, structure of
biofilms, and distribution/type/viability of bacteria in
a biofilm structure (Figs. 10 and 11). In the micro-
scopic method, the bacterial biofilm is stained with a
suitable dye that is fluorescent (e.g. propidium iodide)
or non-fluorescent in nature (e.g. safranin). Most high
resolution light microscopy will enable the counting of
bacterial cells on a substrate surface. The biofilm slime
may be stained with Alcian blue, a phthalocyanine dye
that stains acidic mucopolysaccharides and glycosami-
noglycans in the EPS (61). The stained portions will
appear as a blue to bluish-green color. Bacteria cells
may also be visualized under fluorescent microscopes
69
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Kishen & Haapasalo

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

without using fluorescent probes by using plasmid-

encoded green fluorescent protein (GFP). These
transformed E. coli O157:H7 have been used to study
their attachment onto a surface (62,63). The viability
of these cells may be determined by staining the 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 hydrated
condition 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 Fig. 12. SEM images of (a) E. faecalis cells adhering to
of microbial biofilm from the substrate, while more root canal dentin and (b) multi-layered mono-species
complex dehydration procedures such as critical point biofilm of E. faecalis on root canal dentin.

70

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 be
identified 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 matrix
surrounding 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). Epifluorescent microscopy 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 50–200-mm-thick
biofilm 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-
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Biofilm models and methods of biofilm assessment
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
the gfp reporter system has been used to study the role
of agr in 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 of
the 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 the
two component dyes (SYTO 9 and propidium
iodide). The dyes are used in a 1:1 mixture for stain-
ing the biofilm bacteria following the manufacturer’s
instructions. The dead cells emit red light and the
viable cells emit green light under CLSM examina-
tion (Fig. 13). In a recent in vitro study, 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 while
they 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 vivo root canal biofilms (51).
A fluorescence in situ hybridization (FISH) tech-
nique using probes to target specific 16S rRNA
71

Kishen & Haapasalo
Fig. 13. Three-dimensional confocal laser scanning microscopy reconstruction of E. faecalis biofilm (inlet shows
sagittal 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).
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 visualization
of 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 thin
biofilms 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
72
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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 during
biofilm 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 and
extracellular 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,

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 noted
that 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 facilitate
the detection of even a minimum microbial population
level. Revival of bacteria is also a concern because it is
unknown how to determine the time needed before
physical or biochemical measurements are made (77–
81). Currently there is no standardized rapid method
that can replace conventional biochemical assays.
Further research is required before employing rapid
methods more routinely for the detection of viable
bacteria in 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
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Biofilm models and methods of biofilm assessment
groups, there is a possibility of data disparity between
laboratories and in vivo situations.
Enzyme-linked immunosorbent assay (ELISA) is a
very sensitive method used to detect the presence of
antigens or antibodies of interest in a sample. ELISA
can be used for quantitative analysis when used in
conjunction with standard curves. ELISA is typically
performed using one of two detection methods: the
direct or indirect assay. In direct ELISA, an 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 method
to 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 vivo environment 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.
73

Kishen & Haapasalo
Fig. 14. Atomic force microscope images showing the details of bacterial cell surfaces in the nanometric range.
Polymerase chain reaction (PCR) is a method that
allows exponential amplification of short DNA
sequences. 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-time
polymerase 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 of
products 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-
74
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tative PCR. This method has been used to detect and
quantify bacterial populations in a biofilm. Often, the
RT-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 is
available (85–88).
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
(89–92) (Fig. 14). In order to use AFM to determine
bacteria–substrate interaction, the bacteria cell or
substrate particle is attached onto an AFM tip and the
forces of interaction between bacterial cells and
between the bacterial cell and substrate are 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 curves can be used to estimate the duration
of interaction and adhesion events in the interaction
between the bacteria and the substrate. AFM has
also become an accepted tool to measure interaction

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 turn
may affect the value of the forces measured. Based on
this concept, an investigation aimed to study the
effects of endodontic irrigants on the adherence of E.
faecalis to dentin (93). The 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 low
to allow any analysis of a population of cells (ideally
less than 1,000 cells). Laser-based optical tweezers are
non-invasive and non-contact tools that can probe the
interaction between microscopic objects such as 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 analytical
tools 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).
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Biofilm models and methods of biofilm assessment
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 in
vivo conditions 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 bacterial
biofilm 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.
References
1. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D,
Korber D, James G. Biofilms, the customized micro-
niche. J Bacteriol 1994: 176: 2137–2142.
2. Nair PNR, Sjögren U, Krey G, Kahnberg K-E, Sund-
qvist G. Intraradicular bacteria and fungi in root-filled,
asymptomatic human teeth with therapy-resistant peri-
apical lesions: a long-term light and electron micro-
scopic follow-up study. J Endod 1990: 16: 580–588.
3. Nair PNR. Light and electron microscopic studies of
root canal flora and periapical lesions. J Endod 1987: 13:
29–39.
4. Nair PNR. On the causes of persistent apical periodon-
titis: a review. Int Endod J 2006: 39: 249–281.
5. Sundqvist G, Figdor D. Life as an endodontic pathogen:
ecological differences between the untreated and root-
filled root canals. Endod Topics 2003: 6: 3–28.
6. Baumgartner JC, Siqueira JR JF, Sedgley CM, Kishen
A. Microbiology of endodontic disease. In: Ingle JI,
Bakland LK, Baumgartner JC, eds. Ingle’s Endodontics,
6th edn. Hamilton: BC Decker, 2008: 221–222.
7. Grenier D, Mayrand D. Nutritional relationships
between oral bacteria. Infect Immun 1986: 53: 616–
620.
8. Sundqvist G. Ecology of the root canal flora. J Endod
1992: 18: 427–430.
9. Casadevall A, Pirofski LA. Host–pathogen interactions:
redefining the basic concepts of virulence and pathoge-
nicity. Infect Immun 1999: 67: 3703–3713.
75

Kishen & Haapasalo
10. Chavez de Paz LE. Redefining the persistent infection
in root canals: possible role of biofilm communities. J
Endod 2007: 33: 652–662.
11. Miron J, Ben-Ghedalia D, Morrison M. Invited review:
adhesion mechanisms of rumen cellulolytic bacteria. J
Dairy Sci 2001: 84: 1294–309.
12. Cowan, MM, Taylor KG, Doyle RJ. Energetics of the
initial phase of adhesion of Streptococcus sanguis to
hydroxyapatite. J Bacteriol 1987: 169: 2995–3000.
13. Costerton JW, Stewart PS, Greenberg EP. Bacterial
biofilm: a common cause of persistent infections. Science
1999: 284: 1318–1322.
14. Costerton JW, Lewandowski Z. The biofilm lifestyle.
Adv Dent Res 1997: 11: 192–195.
15. del Pozo JL, Patel R. The challenge of treating biofilm-
associated bacterial infections. Clin Pharmacol Ther
2007: 82: 204–209.
16. Huang R, Li M, Gregory RL. Bacterial interactions in
dental biofilm. Virulence 2011: 2: 435–444.
17. Costerton JW, Stewart PS. Battling biofilms. Sci Am
2001: 285: 74–81.
18. Sun D, Accavitti MA, Bryers JD. Inhibition of biofilm
formation by monoclonal antibodies against Staphy-
lococcus epidermidis RP62A accumulation-associated
protein. Clin Diagn Lab Immunol 2005: 12: 93–100.
19. Stoodley P, Sauer K, Davies DG, Costerton JW. Bio-
films as complex differentiated communities. Annual
Rev Microbiol 2002: 56:187–209.
20. Lim Z, Cheng JL, Lim TW, Teo EG, Wong J, George S,
Kishen A. Light activated disinfection: an alternative
endodontic disinfection strategy. Aust Dent J 2009: 54:
108–114.
21. Shen Y, Stojicic S, Haapasalo M. Antimicrobial efficacy
of chlorhexidine against bacteria in biofilms at different
stages of development. J Endod 2011: 37: 657–661.
22. Kolenbrander PE, Parrish KD, Andersen RN, Green-
berg EP. Intergeneric coaggregation of oral Treponema
spp. with Fusobacterium spp. and intrageneric coaggre-
gation among Fusobacterium spp. Infect Immun 1995:
63: 4584–4588.
23. Jones SJ. A special relationship between spherical and
filamentous microorganisms in mature human dental
plaque. Arch Oral Biol 1972: 17: 613–616.
24. Rosan B, Correeia FF, DiRienzo JM. Corncobs: a
model for oral microbial biofilms. In: Busscher HJ,
Evans LV, eds. Oral Biofilms and Plaque Control: Con-
cepts in Dental Plaque Formation. Amsterdam:
Harwood Academic Publishers, 1998: 145–162.
25. Upadya MH, Kishen A. Influence of bacterial growth
modes on the susceptibility to light-activated disinfec-
tion. Int Endod J 2010: 43: 978–987.
26. McBain AJ. Chapter 4: In vitro biofilm models: an
overview. Adv Appl Microbiol 2009: 69: 99–132.
27. Kishen A, Sum CP, Mathew S, Lim CT. Influence of
irrigation regimens on the adherence of Enterococcus
faecalis to root canal dentin. J Endod 2008: 34: 850–
854.
28. George S, Basrani B, Kishen A. Possibilities of
gutta-percha-centered infection in endodontically
76
16011546, 2010, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1601-1546.2012.00285.x by University of Hong Kong, Wiley Online Library on [14/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
treated teeth: an in vitro study. J Endod 2010: 36:
1241–1244.
29. Mathew S, Yaw-Chyn L, Kishen A. Immunogenic
potential of Enterococcus faecalis biofilm under
simulated growth conditions. J Endod 2010: 36: 832–
836.
30. Pratten J, Ready D. Use of biofilm model systems to
study antimicrobial susceptibility. Methods Mol Biol
2010: 642: 203–215.
31. Merritt JH, Kadouri DE, O’Toole GA. Growing and
analyzing static biofilms. Curr Protoc Microbiol 2011:
22: 1B.1.1–1B.1.18.
32. Shabahang S, Torabinejad M. Effect of MTAD on
Enterococcus faecalis-contaminated root canals of
extracted human teeth. J Endod 2003: 29: 576–579.
33. Duggan JM, Sedgley CM. Biofilm formation of oral and
endodontic Enterococcus faecalis. J Endod 2007: 33:
815–818.
34. George S, Kishen A. Photophysical, photochemical, and
photobiological characterization of methylene blue for-
mulations for light-activated root canal disinfection. J
Biomed Opt 2007: 12: 034029.
35. George S, Kishen A. Augmenting the antibiofilm effi-
cacy of advanced noninvasive light activated disinfection
with emulsified oxidizer and oxygen carrier. J Endod
2008: 34: 1119–1123.
36. McGill S, Gulabivala K, Mordan N, Ng YL. The efficacy
of dynamic irrigation using a commercially available
system (RinsEndo) determined by removal of a collagen
‘bio-molecular film’ from an ex vivo model. Int Endod J
2008: 41: 602–608.
37. Sainsbury AL, Bird PS, Walsh LJ. DIAGNOdent laser
fluorescence assessment of endodontic infection. J
Endod 2009: 35: 1404–1407.
38. Shen Y, Qian W, Chung C, Olsen I, Haapasalo M.
Evaluation of the effect of two chlorhexidine prepara-
tions on biofilm bacteria in vitro: a three-dimensional
quantitative analysis. J Endod 2009: 35: 981–985.
39. Williamson AE, Cardon JW, Drake DR. Antimicrobial
susceptibility of monoculture biofilms of a clinical
isolate of Enterococcus faecalis. J Endod 2009: 35:
95–97.
40. Shahriari S, Mohammadi Z, Mokhtari MM, Yousefi R.
Effect of hydrogen peroxide on the antibacterial
substantivity of chlorhexidine. Int J Dent 2010: 2010:
946384.
41. Kishen A, Upadya M, Tegos GP, Hamblin MR. Efflux
pump inhibitor potentiates antimicrobial photodynamic
inactivation of Enterococcus faecalis biofilm. Photochem
Photobiol 2010: 86: 1343–1349.
42. Hiraishi N, Yiu CK, King NM, Tagami J, Tay FR.
Antimicrobial efficacy of 3.8% silver diamine fluoride
and its effect on root dentin. J Endod 2010: 36: 1026–
1029.
43. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticu-
lates for antibiofilm treatment and effect of aging on its
antibacterial activity. J Endod 2010: 36: 1030–1035.
44. Liu H, Wei X, Ling J, Wang W, Huang X. Biofilm
formation capability of Enterococcus faecalis cells in

starvation phase and its susceptibility to sodium
hypochlorite. J Endod 2010: 36: 630–635.
45. Chávez de Paz LE, Bergenholtz G, Svensäter G. The
effects of antimicrobials on endodontic biofilm bacteria.
J Endod 2010: 36: 70–77.
46. Su L, Gao Y, Yu C, Wang H, Yu Q. Surgical endodontic
treatment of refractory periapical periodontitis with
extraradicular biofilm. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2010: 110: e40–44.
47. Soares JA, Roque de Carvalho MA, Cunha Santos SM,
Mendonça RM, Ribeiro-Sobrinho AP, Brito-Júnior M,
Magalhães PP, Santos MH, de Macêdo Farias L. Effec-
tiveness of chemomechanical preparation with alter-
nating use of sodium hypochlorite and EDTA in
eliminating intracanal Enterococcus faecalis biofilm.
J Endod 2010: 36: 894–898.
48. Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Man-
nocci F. The effectiveness of passive ultrasonic irrigation
on intraradicular Enterococcus faecalis biofilms in
extracted single-rooted human teeth. Int Endod J 2010:
43: 241–250.
49. Shen Y, Stojicic S, Qian W, Olsen I, Haapasalo M. The
synergistic antimicrobial effect by mechanical agitation
and two chlorhexidine preparations on biofilm bacteria.
J Endod 2010: 36: 100–104.
50. Pappen FG, Shen Y, Qian W, Leonardo MR, Giardino
L, Haapasalo M. In vitro antibacterial action of Tetra-
clean, MTAD and five experimental irrigation solutions.
Int Endod J 2010: 43: 528–535.
51. Shen Y, Stojicic S, Haapasalo M. Bacterial viability in
starved and revitalized biofilms: comparison of viability
staining and direct culture. J Endod 2010: 36: 1820–
1823.
52. Lundstrom JR, Williamson AE, Villhauer AL, Dawson
DV, Drake DR. Bactericidal activity of stabilized chlo-
rine dioxide as an endodontic irrigant in a polymicrobial
biofilm tooth model system. J Endod 2010: 36: 1874–
1878.
53. Hope CK, Garton SG, Wang Q, Burnside G, Farrelly
PJ. A direct comparison between extracted tooth and
filter-membrane biofilm models of endodontic irriga-
tion using Enterococcus faecalis. Arch Microbiol 2010:
192: 775–781.
54. Badr AE, Omar N, Badria FA. A laboratory evaluation
of the antibacterial and cytotoxic effect of Liquorice
when used as root canal medicament. Int Endod J 2011:
44: 51–58.
55. Pavarina AC, Dovigo LN, Sanitá PV, Machado AL,
Giampaolo ET, Vergani CE. Dynamic models for
in vitro biofilm formation. In: Bailey WC, ed. Biofilms:
Formation, Development and Properties. Hauppauge,
NY: Nova Science Publishers, Inc., 2011: 125–162.
56. Kulasekara HD, Ventre I, Kulasekara BR, Lazdunski A,
Filloux A, Lory S. A novel two-component system con-
trols the expression of Pseudomonas aeruginosa fimbrial
cup genes. Mol Microbiol 2005: 55: 368–380.
57. Watnick PI, Kolter R. Steps in the development of a
Vibrio cholerae El Tor biofilm. Mol Microbiol 1999: 34:
586–595.
16011546, 2010, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1601-1546.2012.00285.x by University of Hong Kong, Wiley Online Library on [14/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Biofilm models and methods of biofilm assessment
58. Friedman L, Kolter R. Genes involved in matrix forma-
tion in Pseudomonas aeruginosa PA14 biofilms. Mol
Microbiol 2004: 51: 675–690.
59. Davey ME, O’toole GA. Microbial biofilms: from
ecology to molecular genetics. Microbiol Mol Biol Rev
2000: 64: 847–867.
60. Johansen C, Falholt P, Gram L. Enzymatic removal and
disinfection of bacterial biofilms. Appl Environ Microbiol
1997: 63: 3724–3728.
61. Di Bonaventura G, Pompilio A, Picciani C, Iezzi M,
D′Antonio D, Piccolomini R. Biofilm formation by the
emerging fungal pathogen Trichosporon asahii: develop-
ment, architecture, and antifungal resistance. Anti-
microb Agents Chemother 2006: 50: 3269–3276.
62. Burnett SL, Chen J, Beuchat LR. Attachment of
Escherichia coli O157:H7 to the surfaces and internal
structures of apples as detected by confocal scanning
laser microscopy. Appl Environ Microbiol 2000: 66:
4679–4687.
63. Takeuchi K, Frank JF. Expression of red-shifted green
fluorescent protein by Escherichia coli O157:H7 as a
marker for the detection of cells on fresh produce. J
Food Prot 2001: 64: 298–304.
64. McKinlay KJ, Allison FJ, Scotchford CA, Grant DM,
Oliver JM, King JR, Wood JV, Brown PD. Comparison
of environmental scanning electron microscopy with
high vacuum scanning electron microscopy as applied to
the assessment of cell morphology. J Biomed Mater Res
A 2004: 69: 359–366.
65. Sutton NA, Hughes N, Handley PS. A comparison of
conventional SEM techniques, low temperature SEM
and the electroscan wet scanning electron microscope to
study the structure of a biofilm of Streptococcus crista
CR3. J Appl Bacteriol 1994: 76: 448–454.
66. Trachoo N, Frank JF. Effectiveness of chemical sani-
tizers against Campylobacter jejuni-containing biofilms.
J Food Prot 2002: 65: 1117–1121.
67. Wolfaardt GM, Lawrence JR, Hendry MJ, Robarts RD,
Caldwell DE. Development of steady-state diffusion
gradients for the cultivation of degradative microbial
consortia. Appl Environ Microbiol 1993: 59: 2388–
2396.
68. Harraghy N, Seiler S, Jacobs K, Hannig M, Menger
MD, Herrmann M. Advances in in vitro and in vivo
models for studying the staphylococcal factors involved
in implant infections. Int J Artif Organs 2006: 29:
368–378.
69. Sheppard, CRJ, Shotton, DM. Confocal Laser Scanning
Microscopy. Oxford, UK: BIOS Scientific Publishers
Ltd., 1997.
70. Kishen A, George S, Kumar R. Enterococcus faecalis-
mediated biomineralized biofilm formation on root
canal dentine in vitro. J Biomed Mater Res A 2006: 77:
406–415.
71. Bakke R, Olsson PQ. Biofilm thickness measurements
by light microscopy. J Microbiol Methods 1986: 5:
93–98.
72. Peyton BM, Characklis WG. A statistical analysis of the
effect of substrate utilization and shear stress on the
77

Kishen & Haapasalo
kinetics of biofilm detachment. Biotechnol Bioeng 1993:
41: 728–735.
73. Main C, Geddes DA, McNee SG, Collins WJ, Smith
DC, Weetman DA. Instrumentation for measurement
of dental plaque thickness in situ. J Biomed Eng 1984: 6:
151–154.
74. Mattila-Sandholm T, Wirtanen G. Biofilm formation
in the industry: a review. Food Rev Int 1992: 8:
573–603.
75. Stewart GS. In vivo bioluminescence: new potentials for
microbiology. Lett Appl Microbiol 1990: 10: 1–8.
76. Walker AJ, Stewart GS, Sheppard F, Bloomfield SF,
Holah JT, Denyer SP. Bioluminescence imaging as a
tool for studying biocide challenge upon planktonic and
surface attached bacteria. Bin Comp Microbiol 1994: 6:
16–17.
77. Marshall KC, Stout R, Mitchell R. Mechanisms of the
initial events in the sorption of marine bacteria to solid
surfaces. J Gen Microbiol 1971: 68: 337–348.
78. Marshall KC. Colonization, adhesion and biofilms. In:
Hurst CJ, Knudsen GR, McInerney MJ, Stetzenbach
LD, eds. Manual of Environmental Microbiology.
Washington, DC: American Society for Microbiology
Press, 1997: 358–65.
79. DeBeer D, Stoodley P, Roe F, Lewandowski Z.
Effects of biofilm structures on oxygen distribution and
mass transport. Biotechnol Bioeng 1994: 43: 1131–
1138.
80. McAllister TA, Bae HD, Jones GA, Cheng KJ. Micro-
bial attachment and feed digestion in the rumen. J
Anim Sci 1994: 72: 3004–3018.
81. Millsap KW, Reid G, Van Der Mei HC, Busscher HJ.
Adhesion of Lactobacillus species in urine and phos-
phate buffer to silicone rubber and glass under flow.
Biomaterials 1997: 18: 87–91.
82. Lee HA, Wyatt GM, Bramham S, Morgan MR.
Enzyme-linked immunosorbent assay for Salmonella
typhimurium in food: feasibility of 1-day Salmonella
detection. Appl Environ Microbiol 1990: 56: 1541–
1546.
83. Bauer-Kreisel P, Eisenbeis M, Scholz-Muramatsu H.
Quantification of Dehalospirillum multivorans in mixed-
culture biofilms with an enzyme-linked immunosorbent
assay. Appl Environ Microbiol 1996: 62: 3050–
3052.
84. Pérez-Osorio AC, Franklin MJ. qRT-PCR of microbial
biofilms. CSH Protoc 2008: 2008: pdb.prot5066.
78
16011546, 2010, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1601-1546.2012.00285.x by University of Hong Kong, Wiley Online Library on [14/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
85. Kindaichi T, Kawano Y, Ito T, Satoh H, Okabe S.
Population dynamics and in situ kinetics of nitrifying
bacteria in autotrophic nitrifying biofilms as determined
by real-time quantitative PCR. Biotechnol Bioeng 2006:
94: 1111–1121.
86. Heydorn A, Ersbøll B, Kato J, Hentzer M, Parsek MR,
Tolker-Nielsen T, Givskov M, Molin S. Statistical analy-
sis of Pseudomonas aeruginosa biofilm development:
impact of mutations in genes involved in twitching
motility, cell-to-cell signaling, and stationary-phase
sigma factor expression. Appl Environ Microbiol 2002:
68: 2008–2017.
87. Wimpenny J, Manz W, Szewzyk U. Heterogeneity in
biofilms. FEMS Microbiol Rev 2000: 24: 661–671.
88. Thurnheer T, Gmür R, Guggenheim B. Multiplex FISH
analysis of a six-species bacterial biofilm. J Microbiol
Methods 2004: 56: 37–47.
89. Postollec F, Norde W, de Vries J, Busscher HJ, van der
Mei HC. Interactive forces between co-aggregating and
non-co-aggregating oral bacterial pairs. J Dent Res
2006: 85: 231–234.
90. Razatos A, Ong YL, Sharma MM, Georgiou G. Molecu-
lar determinants of bacterial adhesion monitored by
atomic force microscopy. Proc Natl Acad Sci USA
1998: 95: 11059–11064.
91. Gaboriaud F, Dufrêne YF. Atomic force microscopy of
microbial cells: application to nanomechanical proper-
ties, surface forces and molecular recognition forces.
Colloids Surf B Biointerfaces 2007: 54: 10–19.
92. Vadillo-Rodríguez V, Busscher HJ, Norde W, De Vries
J, Dijkstra RJ, Stokroos I, Van Der Mei HC. Compari-
son of atomic force microscopy interaction forces
between bacteria and silicon nitride substrata for three
commonly used immobilization methods. Appl Environ
Microbiol 2004: 70: 5441–5446.
93. Sum C, Mohanty S, Gupta PK, Kishen A. Influence of
endodontic chemical treatment on Enterococcus faecalis
adherence to collagen studied with laser scanning con-
focal microscopy and optical tweezers: a preliminary
study. J Biomed Opt 2008: 13: 044017.
94. Grivet JP, Delort AM, Portais JC. NMR and micro-
biology: from physiology to metabolomics. Biochimie
2003: 85: 823–840.
95. Majors PD, McLean JS, Pinchuk GE, Fredrickson JK,
Gorby YA, Minard KR, Wind RA. NMR methods for in
situ biofilm metabolism studies. J Microbiol Methods
2005: 62: 337–344.