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4 CE credits
This course was
written for dentists,
dental hygienists,
and assistants.

Biofilm Formation,
Identification
and Removal
A Peer-Reviewed Publication
Written by Fiona M. Collins, BDS, MBA, MA

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Educational Objectives
Upon completion of this course, the clinician will be able to
do the following:
1. Understand the components that make up the structure
of biofilm and its biochemistry
2. Understand the process resulting in biofilm formation
and maturation, including the role of adhesion
3. Understand the process of identification, whether
unaided or chemically-aided, and, as a result, understand
the differences in plaque-disclosing agents available for
use in this step
4. Assess the appropriate type of disclosing agent and
method by which to efficiently remove plaque
5. Understand how to educate patients and what
information is most needed to help prevent and fight the
development of biofilm in the intraoral environment
Abstract
Dental caries and periodontal disease are among the most
prevalent diseases known to man. Both are associated with
the bacteria contained in dental biofilm. Dental biofilm is
complex, with a well-organized structure. Up to 500 bacterial
species have been identified in dental biofilm. Studies have
shown that plaque accumulates rapidly on clinically plaque-
free teeth. For oral and systemic health, the development and
maturation of dental biofilm should be impeded and the den-
tal biofilm needs to be regularly and meticulously removed.
Removal and reduction of biofilm can be by mechanical
means or mechanical and chemical means, and disclosing
agents enable visual identification of plaque. Current che-
motherapeutics in general do not effectively penetrate thick
biofilm, underscoring the importance of the identification
and rigorous mechanical removal of the dental biofilm.
Introduction
Dental caries and periodontal disease are the most common
intraoral diseases and among the most prevalent disorders
known to man. Both are associated with bacteria contained
in dental biofilms. Studies, including one that compared
sucrose-restricted and sucrose-supplemented diets, have
shown that plaque accumulates rapidly on clinically plaque-
free teeth. In the absence of any oral hygiene measures,
plaque accumulated on 52 percent to 73 percent of surfaces.1
While poor oral hygiene is causally related to the presence of
dental biofilm and gingivitis, there is not a strong correlation
between poor oral hygiene and severe periodontitis, nor does
oral hygiene have significant influence on the subgingival
microflora within deep periodontal pockets.2 Plaque removal
from subgingival pockets should be accompanied by profes-
sional prophylaxis, scaling, and root planing to halt clinical at-
tachment loss. Supragingival microbes are known to migrate
subgingivally, and it is also known that microbes will migrate
from one periodontal site to another or to an implant site.3
Regular and diligent removal of supragingival dental biofilm
2 www.ineedce.com
is essential to prevent a large microbial load from developing
and to prevent the development of subgingival plaque and
periodontal pockets.
Biofilm Structure and Biochemistry
Prior to the development of dental biofilm, the salivary or
acquired pellicle forms. This occurs through the adsorption
of proteins from saliva onto the clean tooth surface. One
study found different patterns in the adsorption of salivary
proteins onto various components of orthodontic appliances
— modules, brackets, springs, and elastics — and found
differing levels of bacterial accumulation.4 Acquired pellicle
formation provides oral bacteria with binding sites, resulting
in bacterial adhesion,5 the first step in the formation of dental
biofilm. Surface modification can inhibit the development of
the acquired pellicle and dental biofilm. When ceramic crown
surfaces were treated in vivo with hydrophobic coatings, using
disclosing agents it was found that almost no plaque formed
on the hydrophobic crown surface and there was almost no
detectable pellicle, even after seven days.6
The Role of Adhesion
Dental biofilms accumulate on hard and soft surfaces in the
oral cavity, including nonbiological surfaces such as implant
surfaces, orthodontic appliances and amalgams, as well as on
other bacteria. Bacterial adherence is essential for formation
of dental biofilm. Following adhesion, the bacteria divide,
grow, and accumulate. The bacteria contain a factor called
an “adhesin” that meets its counterpart receptor at the site of
adhesion, resulting in the adhesion of the bacteria to the sur-
face. Different receptors and adhesins are present on different
bacteria and surfaces, such as fimbrae that act as receptors on
subgingival bacteria, or collagen and residues of sialic acid and
galactosyl that serve a similar function on tissue surfaces.7
Adhesion by one species of bacteria may limit the
amount of adhesion by another species in the same space.
When the adhesion of streptococci is selectively reduced by
preventing the build-up of sialic acid, more P. gingivalis and
P. intermedia adhere in the space. It is believed that this is
due to galactosyl residues acting as receptors for the Porphy-
romonas species.8
For a subgingival biofilm to exist, the bacteria must be able
to adhere to one or more subgingival surfaces. This is enabled
by an early inflammatory process that creates a pseudopocket
at the gingival margin, allowing for the development of the
anaerobic species associated with periodontal disease and the
subsequent development of true periodontal pockets, which
in turn provide extra space for the anaerobes9 and an oxygen-
poor environment. Unfortunately, the presence of subgingival
calculus provides an excellent adhesion site for bacteria and
for the retention of subgingival plaque. Bacterial adhesion to
soft tissue within the periodontal pocket may also play a role
in the deeper invasion of periodontal tissues. Adhesion to the
basement membrane and collagen has been found to occur.
Biofilm Formation and Maturation
Biofilm development involves selection and adaptation by
bacteria already present in the intraoral environment. Acid-
producing Strep. mutans and lactobacilli are selected in a low-
pH environment, leading to acid production by the bacteria in
the subsequent biofilm and the initiation of the caries process.
Similarly, anaerobic microorganisms are selected when there
is an increase in gingival crevicular fluid, nutrients, and pH, all
of which contribute to the establishment of periodontopatho-
gens and periodontal disease.10 In addition to the selection of
specific bacterial species in a dental biofilm, the bacteria adapt
to their environment.
In vivo dental biofilm is colonized by up to 500 species.
The bacteria are tightly bound to each other and to a solid
substrate, interspersed with fluid-filled channels. The major-
ity of these species are present in health — the host response
and susceptibility determine disease. Only 20 percent of peri-
odontal disease is attributed to bacterial variances, with host
response being the key factor for most periodontal disease.11
Within periodontal pockets, between 30 and 100 species can
be isolated from one periodontal site, and over 300 species
have been identified by culturing samples from human peri-
odontal pockets.12
Young dental biofilm, assessed for 40 strains of bacte-
ria, has been found to consist mainly of Actinomyces until
between two hours and six hours after formation begins. At
this point, the numbers of Streptococci increase relative to
Actinomyces, especially S. mitis and S. oralis.13 Periodontal
pathogens are found at extremely low levels in biofilm up
to six hours old.14 Until day three, mostly gram-positive
streptococci and rods are present. The next phase of biofilm
maturation involves the appearance of filamentous bacteria
on the biofilm surface. These then invade the body of the
biofilm after day seven.15
CLSM combined with fluorescence of plaque harvested
from in situ enamel blocks worn for five days has demon-
strated that bacterial vitality increases with the depth of the
biofilm and the depth of the bacteria within. CLSM has
also demonstrated voids together with layers of live bacteria
packed with nonvital materials of bacterial origin.16 The
Biofilm Formation
0 hours
Aquired Bacterial
Pellicle Adhesion
Formation
www.ineedce.com 3
composition of the biofilm depends upon its location and its
degree of maturity.
Between three and 12 weeks after supragingival plaque
starts to form, subgingival biofilm is well differentiated with
predominantly gram-negative anaerobic bacteria. Porphy-
romonas gingivalis — strongly associated with periodontal
disease — and Treponema denticola are often found together
in dental biofilms.
Research using CLSM and blot analyses to assess gene
expression has shown that P. gingivalis forms a synergistic
biofilm with T. denticola, and has concluded that other bac-
teria present in dental biofilm may interact synergistically.17
Other bacterial species have been shown to engage in, in
effect, chemical warfare by producing substances that either
prevent adhesion by another species or are bactericidal for a
second species. Hydrogen peroxide produced by S. sanguis
inhibits A. actinomycetemcomitans; conversely, A. actino-
mycetemcomitans produces a bactericide for S. sanguis.18
The composition of subgingival plaque has been ex-
tensively researched. Socransky and Haffajee defined five
complexes as bacterial complexes in subgingival plaque.
In addition, specific complexes were found to have an as-
sociation with other specific complexes within the group,
demonstrating the presence of relationships between
bacterial species. While specific bacteria are known to be
pathogens, it is also now recognized that the presence of
periodontopathogens does not predict long-term progress
of periodontitis.19 Recent research has identified clonal sub-
groups within species of bacteria,20 the relevance of which is
not yet fully understood. It has been hypothesized that the
virulence within a particular bacterial species may depend
upon the clonal subtypes present.
Subgingival plaque is not subject to the same assaults
as supragingival plaque and is relatively protected from
saliva and mastication forces. A further factor is the mor-
phology of bacteria, both intraspecies and interspecies.
In vitro evaluation of biofilm formation with A. actino-
mycetemcomitans has found that variants with a rougher
morphology resulted in greater biofilm formation than in
vitro smooth morphology variants. It was also concluded
6 hours Day 7 12 weeks
Well-Differentiated
Subgingival
Supragingival Plaque Plaque
Plaque Maturation
Actinomyces Filamentous Predominantly
Steptococci Bacteria Gram-Negative Anaerobes
that a rough morphology in the biofilm in vivo may help
protect bacteria from assault.21
The role of Candida albicans in dental biofilm is of
particular interest given the increase in opportunistic C.
albicans infections in immunocompromised patients. The
growth and survival of C. albicans in dental biofilms has been
shown to be influenced by the concentration of aerobic and
anaerobic bacteria present. Research by Samaranayake et al.
has shown that culturing various bacteria together with C.
albicans results in varying biofilm formation and that higher
concentrations of Actinomyces israelii, Prevotella nigrescens
or Pseudomonas aeruginosa result in lower concentrations of
C. albicans. A statistically significant negative correlation was
found between concentrations of P. gingivalis or E. coli, and
C. albicans. This effect was not seen with Strep. Intermedius,
Strep. Mutans, or L. acidophilus.22
Biofilm Identification
The meticulous and regular removal of dental biofilm is
important for oral and systemic health, given the strong
associations between the presence of periodontal disease
and cardiovascular disease,23 diabetes, respiratory disease,
disseminated infections and other conditions. Meticulous
removal of biofilm is hindered by its relative lack of visibility,
mechanical or physical difficulties, and by its chemical resis-
tance. The use of current antimicrobial agents is known to be
effective in the outer surface where a thick dental biofilm is
present24 with poor penetration into the inner layers of the
dental biofilm, which underscores the importance of frequent
and adequate plaque removal.
By removing supragingival plaque in the early phase of
biofilm maturation, the microbial load can be maintained
at a relatively low level and colonization by anaerobic
species can be contained. In the absence of adequate oral
hygiene, subgingival plaque will develop, at which point
elimination and effective control of the bacterial environ-
ment is considerably more difficult. One of the difficulties
for both dental professionals and patients in eliminating
plaque is its identification.
Unaided Detection
Plaque is not always visible even supragingivally. This is
a factor in incomplete removal of supragingival plaque by
patients and clinicians alike. One study compared the efficacy
of removal of 48-hour-old plaque by three groups of dental
hygienists with three different combinations of instruments.
The first group used an ultrasonic cleaner and prophy cups,
the second added dental floss to the armamentarium, and the
third group used Gracey curettes and prophy cups. Regard-
less of the method employed, use of fluorescein and UV light
afterwards revealed that more residual plaque remained inter-
proximally than buccally or lingually and that not one person
in any group was able to remove 100 percent of supra-gingival
plaque in a patient in the absence of a disclosing agent.25
4 www.ineedce.com
Chemically Aided Detection — Disclosing Agents
Chemically aided visual identification of plaque through
the use of disclosing agents is a useful patient educator and
motivator, and, as demonstrated by Checchi et al., a useful
tool for dental hygienists. Disclosing agents have been in use
since the early 20th century, starting with the introduction of
iodine by Skinner in 1914.26 More recent plaque disclosing
agents are based upon approved food colorants.27 Basic fuch-
sin and erythrosin, a coal-tar derivative,28 were used as early
as the 1960s. Recent online research suggests that about half
of dental hygienists use disclosants.29 The majority of disclos-
ing agents in current use are red dye disclosants. Disclosing
agent vehicles include solutions used as rinses, tablets that
are chewed to mix with saliva moved around the teeth, and
solutions applied using an applicator.
Erythrosin and basic fuchsin both stain dental plaque
red. A further variant stains 24-hour plaque red and 48-hour
plaque a purple/blue color. Erythrosin is a simple, inexpen-
sive and readily available home care disclosing agent. It lacks
specificity and will stain the gingivae, pellicle, calculus and
oral mucosa in addition to dental plaque — thereby making
it harder to identify the plaque around the gingival margin
because dye penetration is similar on plaque and gingival
tissue.30 This could result in particularly motivated patients
brushing too hard to remove what they perceive to be dental
plaque, instead creating a soft tissue abrasion at the gingival
margin. The non-specificity of staining may also be estheti-
cally displeasing to patients.
An alternative disclosing agent, fluorescein is safe and
effective in disclosing plaque. It is specific and will not stain
the soft tissue or pellicle. Fluorescein disclosing systems, such
as Plak-Check® (Sunstar Butler), use liquid fluorescein and a
blue light. After the liquid fluorescein has been applied, the
fluorescein is not visible to the naked eye. Using the blue light
gives the plaque a yellowish tinge after fluorescein application,
making it visible to patients and clinicians. While erythrosin
was found in one study to stain twice as well as fluorescein,31
fluorescein is specific for the plaque and does not stain the
gingival margin or oral mucosa, reducing the risk of abrasion
by overzealous cleaning by the patient. Fluorescein has been
shown to be more effective in disclosing interproximal plaque
than other disclosing agents.32
Plaque disclosing methods include the use of a plaque
probe. Access to interdental areas can be difficult, and the use
of a plaque probe with a contrasting color at the tip may aid
access and enable plaque to be more easily detected.33
Plaque disclosing agents containing red dye may stain
tooth-colored restorative materials. A recent in situ study
found that a single use of a basic fuchsin solution notice-
ably changed the color of resin-modified glass ionomers in
children. In contrast, there were no statistically significant
changes when a fluorescent dye was used.34 While the com-
posite resin changes were not significant with either disclosing
method according to the methodology on visually acceptable
values,35 the study concluded that basic fuchsin should be Removal of Biofilm
used with caution in the presence of both resin-modified glass Daily removal of dental plaque is essential in the prevention
ionomers and composites, and that the use of fluorescent dye of oral disease as well as in the continued rehabilitation and
was preferable. maintenance of patients with preexisting periodontal disease.
Staining of clothing has on occasion been an issue. Most Reducing the biofilm mass and/or or selectively reducing
disclosing agents can be removed completely from clothing, the numbers and proportion of pathogenic species will help
although silk shows more resistance. In this regard, the least prevent caries and periodontal disease.
residual stain was found with the use of fluorescein36 — an
anticipated result since the dye is not normally visible and the Mechanical Removal
clothing was not tested under ultraviolet light. In industrialized countries, patients brush for less than one
minute on average.41 The education and motivation of patients
Dental Biofilm Identification to mechanically remove as much plaque as possible is a prior-
Erythrosin Fluorescein Plaque Probe ity.42 The use of soft-bristled brushes, interdental brushes,
Useful for floss, toothpicks, and rubber tips have all been recommended
Yes Yes Yes for the removal of plaque from the various surfaces of the
Dental Office
Useful for teeth during home care.
Yes Yes No Toothbrushes and toothbrushing techniques have evolved
Patients
over time. Use of a soft-bristled brush will achieve the best
Yellowish
Stain Visible red when exposed No stain results for plaque removal and will help prevent abrasion
to blue light associated with use of hard-bristled brushes or overzealous
Specificity Differentiates brushing. While a straight horizontal brushing motion may
Nonspecific N/A remove plaque, the Bass technique is well accepted as opti-
of Stain plaque
Esthetics mal. By brushing at a 45 degree angle, the brush is better able
Negative Neutral N/A to reach under the gingival margin into the gingival crevice
of Stain
Visible Stain to remove plaque in this area. Without this care, inflamma-
Possible No No tion will occur in the gingival crevice with the formation of
Restorations
Stain the pseudopocket, making it a more attractive environment
Yes No No for the development of anaerobic colonization and the forma-
Materials
tion of a true periodontal pocket. Toothbrushes are available
Rationale for Use and Results with ergonomically-designed handles to help patients brush
Using Disclosing Agents (Cross-Action®, Oral-B®; Colgate Wave, Colgate-Palmol-
Plaque disclosing agents are used in both children and adults ive), and some have handle designs with thumb grips that are
to help patients see the areas of plaque coverage and educate positioned to tilt the bristles at a 45 degree angle to the sulcus
them on plaque removal. Disclosing plaque during a dental (GUM® Technique Toothbrush, Sunstar Butler).
hygiene appointment helps the clinician motivate and educate Electric toothbrushes are a further option. Clinical
patients. These agents are useful visual feedback tools, show- research into the effectiveness of these toothbrushes has
ing patients the areas they have missed while brushing during produced widely varying and often contradictory results.
home care. Some studies suggest that the improvement is due Selection is based upon preference and the ability and will-
to oral hygiene motivation and not due to the use of disclosing ingness of patients to brush manually. Where a dexterity
agents;37 however, other studies have suggested otherwise. problem exists, the use of an electric toothbrush may be easier
One study compared the use of a toothbrush and an or more effective.
erythrosin plaque disclosing tablet (Signal® Integral, Unile- Interdental brushes, floss, and toothpicks are all avail-
ver®), with the use of a toothbrush, and the effect of visual able as cleaning aids. Floss requires less interdental space;
feedback. The use of the disclosing tablets increased brushing however, its use requires more control/dexterity than
time by over 20 percent for supragingival plaque removal.38 interdental brushes to clean on either side of the interdental
A disadvantage of red dye disclosants is that patients may be papillae, and tight contact points may make flossing difficult.
reluctant to have them applied or to apply them themselves One-handed flossing devices (Eez-Thru Flossers, Sunstar
at home, due to cosmetic concerns. Another study con- Butler; Glide Flosser, Crest) may help solve the dexterity
cluded that using a fluorescent disclosing system “results in a problems for some patients. Flossing is often inadequately
healthier mouth” and reduces periodontal disease progress.39 and infrequently performed. A survey of dental hygienists
In a further study in 10-year-old children, knowledge of oral found that none reported all of their patients flossing daily,
hygiene and use of disclosing tablets resulted in a 20 percent and 54 percent reported that only 20 percent to 30 percent of
reduction in plaque over a four-month period compared to their patients flossed daily.43 Many patients return for main-
the control group.40 tenance appointments — such as for implants — without ever

www.ineedce.com 5
flossing.44 When used properly, interdental brushes placed
below the contact point are effective interdentally and even
subgingivally, provided sufficient space is available. Smaller-
head interdental brushes are easier to use than large ones
and facilitate access. Both flossing and interdental brushing
are effective with an appropriate technique. The choice will
depend upon clinician and patient preference, the likelihood
of the patient complying with the method chosen, the space
available, and the patient’s dexterity.
Studies have substantiated that subgingival plaque in
deeper pockets is not responsive to home-care oral hygiene
measures alone. Professional treatment (e.g., root plan-
ing and scaling, professional prophylaxis) and home care
(toothbrushing and either flossing or an interdental brush, at
a minimum) combined are required to treat and remove sub-
gingival plaque. In the absence of subgingival plaque control
and therapy, supragingival plaque control does not prevent
the progression of periodontal disease.45
Chemotherapeutic Reduction
and Removal of Biofilm
Chemotherapeutics can be used to reduce the number of bac-
teria in the dental biofilm, to reduce the numbers of specific
pathogenic species, and to inhibit adhesion of microbes to the
tooth surface.
Essential oils, fluoride, chlorhexidine, zinc citrate, and
triclosan have all been used as antimicrobial rinses and den-
tifrices. At low concentrations, fluoride starts to affect acid
production, and at high concentrations has been shown to
be bactericidal.46 Triclosan copolymer dentifrice (Colgate®
Total®) has been shown to significantly reduce plaque and
gingivitis and inhibit the progress of periodontal disease.47
The effectiveness of chlorhexidine used as a chemothera-
peutic is well documented. Twice-daily use of CHX for 21
days in the absence of any other oral hygiene measures has
been shown to completely prevent plaque and gingivitis from
developing.48 Sekino et al. found that twice-daily 60-second
rinsing with 0.2 percent CHX as well as gargling and using 1
percent CHX gel on the tongue resulted in significant reduc-
tion in the microbial load over a four-day period in the absence
of any mechanical oral hygiene measures. Some microorgan-
isms are influenced more than others and the presence of
Actinomyces is substantially less following CHX use. Once
rinsing ceases, the microbial flora composition in individuals
can revert to the pre-CHX flora within four days.49 The use
of a single CHX one-minute rinse was recently studied to
assess antimicrobial activity in situ. Compared to the control
group, a single rinse of CHX was found to be significant at
six hours. However, in 48-hour-old plaque, a single rinse only
had a significant effect in the outer layer.50 Studies have been
conducted on both 0.2% and 0.12% CHX rinses. When the
rinsing time and dosage are taken into consideration, these
two formulations are equivalent.51 In the U.S., CHX rinse
is available as 0.12 percent (Peridex®, Zila Pharmaceuticals;
6 www.ineedce.com
Periogard®, Colgate Oral Pharmaceuticals) and recently an
alcohol-free CHX was introduced (GUM®, Sunstar Butler).
Promising advances are being made in the development of
antimicrobial agents, including research into agents that alter
the surface of the tooth or the acquired pellicle to prevent the
adhesion of bacteria and alter formation of the biofilm. Other
areas of research include the use of bacterial macrophages,
bacterial inhibitors, vaccines, and antimicrobial peptides.52
Summary
Dental biofilm is complex, with a well-organized structure.
Up to 500 bacterial species have been identified in dental
biofilm, including cariogenic as well as periodontopathic bac-
teria. Young dental plaque consists mainly of gram-positive
bacteria, and as the plaque matures and a subgingival plaque
develops, an increasing number of gram-negative microor-
ganisms are seen. For oral and systemic health, the develop-
ment and maturation of dental biofilm should be impeded by
regular and meticulous removal. This can be accomplished
mechanically, chemically, or via a combination of the two.
Disclosing agents provide clinicians and patients alike with
the ability to visually identify plaque, and a variety of tooth-
brushes and interdental aids are available to assist with its re-
moval. Adjunctive chemotherapeutics such as chlorhexidine
and triclosan can be used to reduce and inhibit the plaque
burden. Recent promising advances in the development of
antibacterial therapy include altering the surface of the tooth
or acquired pellicle to prevent bacterial adhesion. Generally,
current chemotherapeutics do not effectively penetrate thick
biofilm, underscoring the importance of identifying and rig-
orously removing the dental biofilm.
References
1. Lim LP, Tay FB, Waite IM, Cornick DE. A comparison of 4 techniques for clinical
detection of early plaque formed during different dietary regimes. J Clin Periodontol.
1986 Aug;13(7):658–65.
2. Westfelt E. Rationale of mechanical plaque control. J Clin Periodontol 1996;23(3
pt.2):263–267.
3. Quirynen M, de Soete M, van Steenberghe D. Infectious risks for oral implants: a
review of the literature. Clin Oral Impl Res 2002;13:1–19.
4. Steinberg D, Eyal S. Initial biofilm formation of Streptococcus sobrinus on various
orthodontics appliances. J Oral Rehabil. 2004 Nov;31(11):1041–5.
5. Scheie AA, Petersen FC. The biofilm concept: consequences for future prophylaxis of
oral diseases? Crit Rev Oral Biol Med 2004;15(1):4–12.
6. Olsson J, van der Heijde Y, Holmberg K. Plaque formation in vivo and bacterial
attachment in vitro on permanently hydrophobic and hydrophilic surfaces. Caries
Res. 1992;26(6):428–33.
7. Socransky SS, Haffajee AD. Microbial mechanisms in the pathogenesis of destructive
periodontal diseases: a critical assessment. J Perio Res 1991;26: 195–212.
8. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease.
J NZ Soc Periodontol 2004;87:7–21.
9. Listgarten MA. The structure of dental plaque. Periodontol 2000;5:52–62.10.
Auschill TM, Arweiler NB, Brecx M, Reich E, Sculean A, Netuschil L. The
effect of dental restorative materials on dental biofilm. Eur J Oral Sci. 2002
Feb;110(1):48–53.
11. Page RC, Offenbacher S, et al. Advances in the pathogenesis of periodontitis:
Summary of developments, clinical implications and future directions. Periodontol
2000 1997;14:216–248.
12. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease.
J NZ Soc Periodontol 2004;87:7–21.
13. Li J, Helmerhorst EJ, Leone CW, Troxler RF, Yaskell T, Haffajee AD, Socransky SS,
Oppenheim FG. Identification of early microbial colonizers in human dental biofilm.
J Appl Microbiol. 2004;97(6):1311–8.
14. Li J, Helmerhorst EJ, Leone CW, Troxler RF, Yaskell T, Haffajee AD, Socransky SS,
Oppenheim FG. Identification of early microbial colonizers in human dental biofilm.
J Appl Microbiol. 2004;97(6):1311–8.
15. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease.
J NZ Soc Periodontol 2004;87:7–21.
16. Auschill TM, Arweiler NB, Netuschil L, Brecx M, Reich E, Sculean A. Spatial
distribution of vital and dead microorganisms in dental biofilms. Arch Oral Biol
2001;46(5):471–476.
17. Kuramitsu HK, Chen W, Ikegami A. Biofilm formation by the periodontopathic
bacteria Treponema denticola and Porphyromonas gingivalis. J Periodontol. 2005
Nov;76(11 Suppl):2047–51.
18. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease.
J NZ Soc Periodontol 2004;87:7–21.
19. Epidemiology of Periodontal Diseases. Position Paper, Academy of
Periodontology, 2005.
20. Page RC, Offenbacher S, et al. Advances in the pathogenesis of periodontitis:
Summary of developments, clinical implications and future directions. Periodontol
2000 1997;14:216–248.
21. Haase EM, Bonstein T, Palmer RJ Jr, Scannapieco FA. Environmental influences on
Actinobacillus actinomycetemcomitans biofilm formation. Arch Oral Biol. 2006
Apr;51(4):299–314. Epub 2005 Oct 13.
22. Thein ZM, Samaranayake YH, Samaranayake LP. Effect of oral bacteria on growth
and survival of Candida albicans biofilms. Arch Oral Biol. 2006 Apr 16; [Epub ahead
of print]
23. Kuramitsu HK, Qi M, Kang IC, Chen W. Role for periodontal bacteria in cardiovascular
diseases. Ann Periodontol. 2001 Dec;6(1):41–7.
24. Zaura-Arite E, van Marle J, ten Cate JM. Confocal microscopy study of undisturbed
and chlorhexidine-treated dental biofilm. J Dent Res 2001;80:1436–1440.
25. Checchi L, Forteleoni G, Pelliccioni GA, Loriga G. Plaque removal with variable
instrumentation. J Clin Periodontol 1997;24:71–717.
26. Skinner FH. The prevention of pyorrhea and dental caries by oral prophylaxis. D
Cosmos 1914;56:299.
27. Hunt DR, Makinson OF. Removal of plaque disclosing stains from clothing. Aus Dent
J 1984;29(1):5–9.
28. Ibid.
29. www.hygienetown.com. July 2005.
30. Squillaro RC, Cohen WD, Laster L. A comparison of microbial plaque
disclosants after personal oral hygiene instruction and prophylaxis. J Prev Dent
1975;2(2):3–7.
31. Lang NP, Ostergaard E, Loe H. A fluorescent plaque disclosing agent. J Periodont Res
1972;7:59–67.
32. Gillings B, Harvey B. A comparison of erythrosin and fluorescein as plaque
disclosants. J Dent Res 1975;54(3) (Abstract).
33. Lim LP, Tay FB, Waite IM, Cornick DE. A comparison of 4 techniques for clinical
detection of early plaque formed during different dietary regimes. J Clin Periodontol.
1986 Aug;13(7):658–65.
34. Hino DM, Mendes FM, de Figueiredo JL, Gomide KL, Imparato JC. Effects of plaque
disclosing agents on esthetic restorative materials used in pediatric dentistry. J Clin
Pediatr Dent. 2005 Winter;29(2):143–6.
35. Ruyter IE, Nilner K, Moller B. Color stability of dental composite resin materials for
crown and bridge veneers. Dent Mater 1987;3:246–251.
36. Hunt DR, Makinson OF. Removal of plaque disclosing stains from clothing. Aus Dent
J 1984;29(1):5–9.
37. Tan AE, Wade AB. The role of visual feedback by a disclosing agent in plaque control.
J Clin Periodontol. 1980 Apr;7(2):140–8.
38. Schafer F, Nicholson JA, Gerritsen N, Wright RL, Gillam DG, Hall C. The effect of oral
care feed-back devices on plaque removal and attitudes towards oral care. Int Dent
J. 2003 Dec;53(6 Suppl 1):404–8.
39. Squillaro RC, Cohen WD, Laster L. A comparison of microbial plaque
disclosants after personal oral hygiene instruction and prophylaxis. J Prev Dent
1975;2(2):3–7.
40. Worthington HV, et al. A cluster randomized controlled trial of a dental health
education program for 10-year-old children. J Public Health Dent. 2001
www.ineedce.com 7
Winter;61(1):22–7.
41. Baehni PC, Takeuchi Y. Anti-plaque agents in the prevention of biofilm-associated
oral disease. Oral Diseases 2003;9 (suppl):23–29.
42. Westfelt E. Rationale of mechanical plaque control. J Clin Periodontol. 1996
Mar;23(3 Pt 2):263–7.
43. www.docere.com/Hygienetown/Article Accessed June 2006.
44. Clarizio, L.F. Peri-implant infections. Oral and Maxillofacial Infections
2000;8(1):35–54.
45. Westfelt E, Rylander H, Dahlen G, Lindhe J. The effect of supragingival plaque
control on the progression of advanced periodontal disease. J Clin Periodontol. 1998
Jul;25(7):536–41.
46. Van Leuveren. J. Dent. Research 1990 (Spec Issue); 69.
47. Panagakos FS, Volpe AR, et al. Advanced oral antibacterial/anti-inflammatory
technology: A comprehensive review of the clinical benefits of a triclosan/
copolymer/fluoride dentifrice. J Clin Dent. 2005;16 Suppl:S1–19.
48. Loe H, Schiot, CR. The effect of mouthrinses and topical application of chlorhexidine
on the development of dental plaque and gingivitis in man. J Periodont Res
1970;5:79–83.
49. Sekino S, Ramberg P, Uzel NG, Socransky S, Lindhe J. The effect of a
chlorhexidine regimen on de novo plaque formation. J Clin Periodontol. 2004
Aug;31(8):609–614.
50. Zaura-Arite E, van Marle J, ten Cate JM. Confocal microscopy study of undisturbed
and chlorhexidine-treated dental biofilm. J Dent Res. 2001 May;80(5):1436–40.
51. Scheie AA, Petersen FC. The biofilm concept: consequences for future prophylaxis of
oral diseases? Crit Rev Oral Biol Med 2004;15(1):4–12.
52. Van Strydonck DA, Timmerman MF, van der Velden U, van der Weijden, GA. Plaque
inhibition of two commercially available chlorhexidine mouthrinses. J Periodontol
2003;74(2):214–218.
Author Profile
Dr. Fiona M. Collins has over
20 years of clinical, marketing,
education and training, and pro-
fessional relations experience. She
has practiced as a general dentist
for 13 years, written and given CE
courses to dental professionals and
students, and conducted market
research projects. Dr. Collins is a
past member of the Academy of General Dentistry Health
Foundation Strategy Board and has been a member of the
British Dental Association, the Dutch Dental Association,
and the American Dental Association. Dr. Collins earned
her dental degree from Glasgow University and holds an
MBA and MA from Boston University.
Acknowledgement
Cover image courtesy of Dr. Gary Carr, Pacific Endodontic
Research Foundation
Disclaimer
The author of this course has no commercial ties with the
sponsors or the providers of the unrestricted educational
grant for this course.
Reader Feedback
We encourage your comments on this or any PennWell course.
For your convenience, an online feedback form is available at
www.ineedce.com.
 Questions
1. To prevent the development of a large 10. In in vivo dental biofilm, the bacteria
microbial load, what is essential? are loosely bound to each other and
a. Development of subgingival plaque a solid substrate, interspersed with
b. Development of periodontal pockets fluid-filled channels.
c. Regular and diligent removal of supragingival a. True
dental biofilm b. False
d. Subgingival migration of
supragingival microbes 11. Within periodontal pockets, how
many species can be isolated from
2. Prior to the development of one periodontal site?
dental biofilm: a. Between 30 and 40
a. The salivary or acquired pellicle forms b. 40
b. A subgingival pellicle forms c. Between 30 and 100
c. Bacteria bind to the tooth, followed by d. 300
a pellicle
d. None of the above 12. Mostly gram-positive streptococci
and rods are present until day:
3. The development of bacterial a. Two
adhesion, resulting from provision b. Three
of binding sites for oral bacteria by c. Five
acquired pellicle formation, is: d. Seven
a. The first step in the formation of 13. The composition of the biofilm
dental biofilm depends upon its location and its
b. The second step in the formation of degree of maturity.
dental biofilm a. True
c. The third step in the formation of b. False
dental biofilm
d. Not a crucial step in the formation of 14. Subgingival plaque is subject to the
dental biofilm same assaults as supragingival and is
relatively protected from saliva and
4. The hard and soft tissue surfaces mastication forces.
on which dental biofilms a. True
accumulate include: b. False
a. Implant surfaces
b. Other bacteria 15. The use of current antimicrobial
c. Amalgams agents is known to be effective:
d. All of the above a. In the outer surface where a thick dental
biofilm is present
5. Following adhesion, the bacteria b. In the inner surface where a thick dental
divide, grow, and accumulate. biofilm is present
a. True c. In conjunction with mechanical cleaning
b. False d. a and c
6. An example of different receptors and 16. In the absence of adequate
adhesions present on different bacteria oral hygiene:
and surfaces is: a. Subgingival plaque will develop
a. Residues of sialic acid and collagen b. Elimination of bacteria is more difficult
and galactosyl c. Effective control of the bacterial environment
is more difficult
b. Fimbrae on subgingival bacteria
c. a and b d. All of the above
d. None of the above
17. In dental biofilm identification,
7. Bacterial adhesion to soft tissue within which of the following has the capabil-
the periodontal pocket may also play ity to visibly stain restorations?
a. Erythrosin
a role in:
a. Influencing dental biofilm in conjunction with b. Fluorescein
restorative materials c. Plaque probe
d. None of the above
b. Deeper invasion of periodontal tissues
c. Providing extra space for anaerobes 18. Plaque is not always visible
d. Creating an oxygen-poor environment even supragingivally.
a. True
8. Biofilm development involves:
a. Selection and adaptation by bacteria already b. False
present in the intraoral environment 19. Disclosing agents have been in
b. Maturation of Strep. mutans and lactobacilli in use since:
a low-pH environment a. 1900
c. Decrease in acid production b. 1914
d. a and b c. The 1960s
d. Late 20th century
9. Anaerobic microorganisms are
selected when there is an increase in: 20. Disclosing agent vehicles include:
a. Nutrients a. Solutions used as rinses
b. Gingival crevicular fluid b. Tablets that are chewed
c. pH c. Solutions applied using an applicator
d. All of the above d. All of the above
8 www.ineedce.com
21. _____ is a simple, inexpensive,
and readily available home-care
disclosing agent.
a. Plaque probe
b. Fluorescein
c. Erythrosin
d. UV light
22. From a clinical research perspective,
erythrosin is:
a. Antibacterial
b. Indicated for longitudinal plaque studies
c. Less likely to stain than fluorescein
d. All of the above
23. With the use of plaque disclosing
agents, the staining of clothing has on
occasion been an issue.
a. True
b. False
24. Disclosing plaque during a dental
hygiene appointment helps the clini-
cian motivate and educate the patient.
a. True
b. False
25. Prevention of caries and periodontal
disease is aided by:
a. Reduction in the numbers and proportion of
pathogenic species
b. Reduction in biofilm mass
c. Selective increase in the proportion of
pathogenic species
d. a and b
26. Which of the following have been
recommended for plaque removal from
various teeth surfaces during home care?
a. Rubber tips
b. Toothpicks
c. Interdental brushes
d. All of the above
27. While floss requires less interdental
space, its use requires:
a. Less control
b. Dexterity
c. Loose contact points
d. Infrequent performance
28. Studies have substantiated that
subgingival plaque in deeper pockets
is responsive solely to home-care oral
hygiene measures.
a. True
b. False
29. At low concentrations, fluoride
______, and at high concentrations
fluoride ______.
a. Starts to affect acid production;
becomes bactericidal
b. Becomes bactericidal; starts to affect
acid production
c. Reduces plaque and gingivitis; inhibits the
progress of periodontal disease
d. Inhibits the progress of periodontal disease;
reduces plaque and gingivitis
30. Promising advances currently taking
place include research into agents that:
a. Prevent changes in the surface of the tooth
b. Prevent the formation of biofilm
c. Alter the acquired pellicle to prevent adhesion
of bacteria
d. Do not stain clothing
 ANSWER SHEET

Biofilm Formation, Identification and Removal

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Requirements for successful completion of the course and to obtain dental continuing education credits: 1) Read the entire course. 2) Complete all
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Mail completed answer sheet to

Educational Objectives Academy of Dental Therapeutics and Stomatology,
1. Understand the components that make up the structure of biofilm and its biochemistry A Division of PennWell Corp.
2. Understand the process resulting in biofilm formation and maturation, including the role of adhesion P.O. Box 116, Chesterland, OH 44026
or fax to: (440) 845-3447
3. Understand the process of identification, whether unaided or chemically-aided, and, as a result, understand the
differences in plaque-disclosing agents available for use in this step
For immediate results, go to www.ineedce.com
4. Assess the appropriate type of disclosing agent and method by which to efficiently remove plaque and click on the button “Take Tests Online.” Answer
5. Understand how to educate patients and what information is most needed to help prevent and fight the development of sheets can be faxed with credit card payment to
(440) 845-3447, (216) 398-7922, or (216) 255-6619.
biofilm in the intraoral environment
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