This document provides an overview of biofilms in dentistry. It defines biofilms and discusses their importance, describing the basic structure and characteristics of biofilms including the extracellular polymeric substance matrix, microcolonies, and fluid channels. The document outlines the stages of biofilm development and characteristics such as resistance to antimicrobials. It discusses the role of biofilms in dental diseases like caries and endodontic infections. It also reviews current hypotheses around the role of biofilms in caries development and prevention strategies.

1. DR RENJITH RAJ C V
Biofilms in Dentisry

2. Contents
• Biofilm - Definition
• Introduction
• Ultrastructure of Biofilm
• Development of Biofilm
• Characteristics of Biofilm
• Significance of Biofilms
• Cariogenic biofilms
• Ultrastructural changes in enamel related to the biofilm
• Current hypotheses to explain the role of plaque bacteria in the etiology of dental caries
• Endodontic biofilms
• Types of Endodontic Biofilms
• Biofilms in Dental-Unit Waterlines
• Methods to eradicate biofilms
• Journal Review
• Conclusion

3. Introduction

4. • The vast majority of micro organisms in nature
invariably grow and function as members of
metabolically integrated communities , or biofilms.
• Biofilm infections account for 65-80% of bacterial
infections affecting humans in the developing world.
• Given its importance, there has been a high level of
interest in the study of biofilm properties, not only in
medical microbiology, but also in different sectors of
industrial and environmental microbiology.

5. • HISTORY
1684- Antony van leewenhoek remarked on vast accumulation of
microorganisms in dental plaque.
1940- Claude zobel described many fundamental characters of
attatched microbial communities.
1977- Earliest use of term “biofilm” in swedish journal “vatten”.
1990- US national science foundation funded centre for biofilm
engineering at montana state university
Since that time, the field of biofilm research has exploded. Several
countries other than US also study biofilms namely Denmark,
england, germany, australia and singapore.

6. It is a mode of microbial growth where dynamic communities of
interacting sessile cells are irreversibly attached to a solid
substratum, as well as each other, and are embedded in a self made
matrix of extra cellular polymeric substances.
(Ingle’s Endodontics, 6th edn)
Defined as a community of microcolonies of microoorganisms in an
acqueos solution that is surrounded by a matrix made of glycocalyx,
which also attatches the bacterial cells to a solid
substratum.(grossman’s endodontic practice , 12th edn)
Biofilm

7. Basic criteria for biofilm formation
Microbial biofilm is considered a community and
the microorganisms living in the community must
possess :-
 Ability to self organize ( autopoeisis)
 Resist environmental perturbations (homeostasis)
 Must be more effective in association than in isolation
(synergy)
 Respond to environmental changes as a unit rather than
single individuals (communality)

8. Significance of biofilms
• It is one of the basic survival strategy employed by microorganisms
in all natural and industrial ecosystems in response to starvation.
• The sessile microbial cells in a biofilm state differ greatly from their
planktonic counterparts.
• Inside a biofilm, the bacterial cells exhibit altered phenotypic
properties and are protected from antimicrobial environmental
stresses, bacteriophage and phagocytic amoebe.
• Responsible for most of the chronic infections and almost all
recalcitrant infections in human beings, as bacteria in biofilm are
resistant to both antimicrobials and host defense mechanisms.

10. Ultrastructure of biofilm
A fully developed biofilm is described as a heterogenous
arrangement of microbial cells on a solid surface.
Basic structural unit
• microcolonies or cell clusters formed by surface adherent
bacterial cells.
• Microcolonies are discrete units of densely packed
bacterial cells aggregates.
Structure of biofilm

11. Three factors essential for
biofilm are
Microorganisms
Solid substrate
Fluid channels

12. • A fresh biofilm is made up of biopolymers such as
polysaccharides, proteins, nucleic acids and salts
Matrix material 85% vol.+ 15% microcolonies
• Glycocalyx matrix
 Made up of EPS
 Surrounds the microcolonies
 Anchors the bacterial cells to the substrate.
Composition of biofilm

13. Importance of matrix
• Part of scaffold that determine biofilm structure.
• Biologically active-retains nutrients, water and
essential enzymes within the biofilm
• Can protect biofilm community from exogenous
threats.
• Participate in adherence to the surface.

14. Shape of biofilm
• Typically, a viable fully hydrated biofilm appears as
“tower” or “mushroom shaped structure”.
• The overall shape of biofilm structure is determined
by the shear forces generated by flushing of fluid
media.

15. The water filled channels
• Primitive circulatory system
• Intersect the structure of biofilm
• Establish connections between the
microbial colonies.
• Facilitates efficient exchange of
materials between bacterial cells and
bulk fluid.
• Coordinate functions in a biofilm
community.

16. Microcolonies
• Arise from surface colonization by planktonic
bacterial cells.
• During early stages, bacteria bind to many host
proteins and coaggregate with other bacteria.
• These interactions lead to changes in growth rate,
gene expression , and protein production.

17. Microorganisms can form biofilm on any surface that is bathed in
nutrient containing fluid.
Stages in the development of biofilm:
• Formation of the conditioning layer
• Adhesion of microbial cells to this layer
Development of biofilm
•pH
• temperature
• surface energy of substrate
• nutrient availability
•length of the time the bacteria is in
contact with the surface
• bacterial cell surface charge
•bacterial surface structure s
• surface hydrophobicity

18. • Adhesion -3 phases:-
• Transport of the microbe to substrate surface and its
attachment. Adherence factors includes :-
fimbriae, pili, flagella, EPS(glycocalyx).
• Microbial and substrate adherence phase to form bridge.
These bridges which are formed in a combination of
electrostatic attraction, covalent and hydrogen bonding
,dipole interaction and hydrophobic interaction.
• specific microbial –substrate adherence phase which
involves bonding of adhesin or ligand on the bacterial cell
surface

19. • Formation of bridges between the bacteria and conditioning layer
• Combination of electrostatic attractions, covalent and hydrogen
bonding and dipole interaction
Weak bond
Irreversible bond
A specific bacterial adhesion with a substrate
(polysaccharide adhesion)

20. Bacterial growth and biofilm expansion
• the monolayer of microbes attracts secondary colonizers
forming microcolonies, collectively forming the final structure
of biofilm.
• A mature biofilm will be metabolically active community of
microorganisms where individuals share duties and benefits.

21. Detachment of microcolonies
Two types of Detachment
Erosion
Sloughing
Erosion : continual detachment of single cell and small
portions of biofilm
Sloughing: rapid massive loss of biofilm
• Has important role in shaping the morphological
characteristics.
• “active dispersal mechanism” ( “Seeding dispersal”)

24. The bacterial cells in a biofilm will exhibit considerable variation in its
genetic and biochemical constitutions compared to its planktonic
counterparts.

25. • Coadhesion: process of recognition between a suspended cell
and a cell already attached to substrate
• Coaggregation: process where genetic distinct cells in
suspension recognize each other and clump together.
Microbial interactions in a biofilm

26. • Bacteria in a biofilm shows distinct capacity to survive tough
growth and environmental conditions.
• This unique capacity of bacteria in biofilm is due to following
features:
 Biofilm structure protects the residing bacteria from
environmental threats.
 Structure of biofilm permit trapping of nutrients and
metabolic co- operativity between resident cells of same
species and different species.
 Biofilm structure display organized internal
compartmentalization
 Bacterial cells in biofilm may communicate and
exchange genetic materials to acquire new traits.
Characteristics of biofilm

27. Protection of biofilm bacteria from environmental threats:
• protection and homeostasis
• capable of producing polysaccharides either as a cell surface
structure (capsules) or as extracellular excretions(EPS)
EPS
↓
covers biofilm communities
↓
Microniche
↓
Long term survival and functioning of bacterial communities
↓
Protects biofilm bacteria from environmental threat
(uv radiation, pH shift, osmotic shock and desiccation)

28. Nutrient trapping and establishment of metabolic
co-operativity in a biofilm
• Ability to concentrate trace elements and nutrients by physical
trapping or by electrostatic interaction.
• Outer fluid → water channels → interior of biofilm
↓
Nutrient availability to microbial communities
• For eg. Cells located near the center of a microcolony are more
likely to experience low oxygen tensions compared to cells located
near the surface. Moreover, due to juxtapostioning of different
cells, cross feeding and metabolic co-operativity between different
species of microorganisms are seen in a biofilm.

29. Organized internal compartmentalization
• Environmental niches that support the physiological
requirements of different species are available in a
biofilm.
• A mature biofilm displays gradients in the
distribution of nutrients, pH, oxygen, metabolic
products and signaling molecules within a biofilm.
• These gradients are influenced by types of nutrients
and physiological requirements of the resting
microorganisms.
• In a multispecies biofilm- both aerobic and anaerobic
microorganisms exists.
• Oxygen is consumed by aerobic and facultative
anaerobic bacteria and creates environment rich in
CO2 which supports the obligatory anaerobic species.

30. Bacterial cells residing in a biofilm communicate and
exchange genetic materials and acquire new traits
“Quorum Sensing”
• Communication through signaling molecules
• Mediated by low molecular wt molecules
• In sufficient concentration, can alter the
metabolic activity of neighboring cells
• Coordinate the function of resident bacterial
cells within a biofilm.
• Cross the cell membranes and trigger changes
• Helps in exchange of genetic materials
• Evolution of microbial communities with
different traits.

31. Resistance to microbes in biofilm to antimicrobials:
• Nature and physiological characterstics of biofilm provides
inherent resistance to antimicrobial agents –antibiotics,
disinfectants and germicides.
• Resistance amplify more than 1000 times for microbes in a
biofilm compared to planktonic cells.
• mechanisms responsible:-
 Resistance associated with extracellular polymeric substance
 Growth rate and nutrient availability
 Adoption of resistant phenotype (metabolic alterations and
genetic alterations).

32. Resistance associated with EPS
• Inactivation of antimicrobials by EPS- observed
antimicrobial resistance in a biofilm.
• EPS forms the biofilm matrix- modify the response of
biofilm bacteria to antimicrobial treatment through its
actons as a diffusion barrier and reactor sink(neutralizer).
• highly charged and interwoven structure deters the
penetration of antimicrobials by ionic or electrostatic
interactions.
• constituents of biofilm matrix polymers may react
chemically and directly neutralize antimicrobials such as
iodine, chlorine and peroxides.

33. Resistance associated with growth rate and
nutrient availability
• Localized high cell density within a biofilm exposes
the deep lying cells to less nutrients and redox
potential.
• resistance to antimicrobials increase in thicker and
matured biofilms due to limited oxygen.

34. Resistance associated with adoption of
resistant phenotypes
• Long term survival of biofilm communities results in the
adoption or clonal expansions of a more resistant
phenotypes
• Nutrient limitation cause diminished growth, increased
expression of stress response gene, increased production
of shock proteins and activation of multi drug efflux
pump.
• Biofilm –enriched with Persistor cells-specialized survivor
cells which survive treatment procedures and proliferate
in the post-treatment phase.

35. • A community of organisms is formed rather than a haphazard collection of
bacteria.
• The bacteria in the biofilm are always metabolically active, causing
fluctuations in pH.
• These fluctuations may cause a loss of mineral from the tooth when the
pH is dropping or a gain of mineral when the pH is increasing (Manji et al.,
1991).
• The cumulative result - a net loss of mineral, leading to dissolution of the
dental hard tissues and the formation of a caries lesion.
• The biofilm tends to form and mature in certain locations on the tooth-
occlusal surface, approximal surface cervical to contact, gingival margin.
• these are the sites where caries lesions may become visible.
Caries and biofilms

36.  After one week
• Clinically : no changes in the enamel.
• Ultrastructural level: signs of direct dissolution of the outer enamel
surface. This was seen as an enlargement of the intercrystalline spaces due
to partial dissolution of the individual crystal peripheries.
 After two weeks
• Clinically "white spot" lesion
 After three and four weeks :
• Clinically :white spot lesion :opaque with a matte surface.
• Ultrastructurally : complete dissolution of the thin perikymata
overlappings; marked dissolution corresponding to developmental
irregularities such as tomes' processes, pits, and focal holes; and
continued enlargement of the intercrystalline spaces.
Ultrastructural changes in enamel
related to the biofolm

37. Specific Plaque Hypothesis
• The "Specific Plaque Hypothesis" proposed that, out of the
diverse collection of organisms comprising the resident plaque
microflora, only a few species are actively involved in disease.
This proposal focused on controlling disease by targeting
preventive measures and treatment against a limited number
of organisms.
• "Non-Specific Plaque Hypothesis" considered that disease is
the outcome of the overall activity of the total plaque
microflora. In this way, a heterogeneous mixture of
microorganisms could play a role in disease.
Current hypotheses to explain the role of plaque
bacteria in the etiology of dental caries:

38. • More recently, an alternative hypothesis has been
proposed (the "Ecological Plaque Hypothesis") that
reconciles the key elements of the earlier two
hypotheses.
• Any environmental change that favours increasing
colonization by potential pathogenic bacteria would
cause disease.

39. Key features of this hypothesis
(a) the selection of "pathogenic" bacteria is directly coupled to
changes in the environment and
(b) diseases need not have a specific etiology; any species with
relevant traits can contribute to the disease process.

40. • A key element of the ecological plaque hypothesis is that
disease can be prevented not only by targeting the
putative pathogens directly, e.g. by antimicrobial or anti-
adhesive strategies, but also by interfering with the
selection pressures responsible for their enrichment.
• In dental caries, regular conditions of sugar/low pH or
reduction in saliva flow appear to be primary
mechanisms that disrupt microbial homeostasis.

41. Strategies that are consistent with the prevention of disease via the principles of the
ecological plaque hypothesis include the following:
(a) Inhibition of plaque acid production, e.g. by fluoride-containing products or other
metabolic inhibitors. Fluoride not only improves enamel chemistry but also inhibits
several key enzymes, especially those involved in glycolysis and in maintaining
intracellular pH.
• Fluoride can reduce the pH fall following sugar metabolism in plaque biofilms,
and in so doing, prevent the establishment of conditions that favor growth of acid-
tolerating cariogenic species.
(b) avoidance between main meals of foods and drinks containing fermentable sugars
and/or the consumption of foods/drinks that contain non-fermentable sugar
substitutes such as aspartame or polyols, thereby reducing repeated conditions of
low pH in plaque.
(c) the stimulation of saliva flow after main meals, e.g. by sugar-free gum. Saliva will
introduce components of the host response, increase buffering capacity, remove
fermentable substrates, promote re-mineralization, and more quickly return the
pH of plaque to resting levels.

42. Endodontic biofilms

43. • less diverse compared to oral microbiota.
• This transition in the microbial population is more
conspicuous with the progression of infections.
Progression of infection
↓ alters
Nutritional and environmental status within the root canal
↓
Root canal environment apparently becomes more anaerobic
and nutrient level will be depleted
↓
Tough ecological niche for the surviving microorganisms
Endodontic microbiota

44. • complete disinfection of the root canal system is very
difficult to achieve
 root canal system complexities-deltas, isthumuses ,
lateral canals in apical portions .
 Advantages of biofilm mode of microbial growth

45. Endodontic biofilms are of four types:
– intracanal biofilm
– extraradicular biofilm
– Periapical biofilm
– biomaterial centerd biofilms

46. • Microbial biofilms that are formed on root canal dentine of
an endodontically infected teeth.
• Exist as both loose microbial cells and biofilm structures,
• Made up of cocci, rods, and filamentous bacteria.
• It is monolayered or multilayered in structure.
Intracanal biofilms :

47. • Studies have revealed the ability of E. faecalis to resist
starvation and develop biofilms under different
environmental and nutrient conditions ( aerobic,
anaerobic, nutrient rich and nutrient deprived
conditions)
• A recent investigation has highlighted the ability of
E.faecalis to coaggregate with F.nucleatum.
• The coaggregation provides the ability of these
microorganisms to coexist in microbial community and
contribute to endodontic infection.
• Resistance to bactericidal action
• ability to form biofilm under tough environmental
conditions & nutritional conditions

49. Stages in the development of E.faecalis
• Stage I: E.faecalis adhered and formed microcolonies
on dentin surface
• Stage II: E.faecalis induces bacterial mediated
dissolution of mineral fraction from dentin substrate-
increases calcium and phosphates ions and promote
calcifications of E.faecalis biofilms.(stage III)

50. • These biofilms are also known as “ root surface
biofilms”
• These biofilms are microbial films formed on the root
(cementum)surface adjacent to root apex of
endodontically infected teeth.
Extraradicular biofilms

51. • These types of biofilms are reported in teeth with
asymptomatic periapical periodontitis and teeth with
chronic apical abscess associated with sinus tracts.
• They consist of cocci and rods and filamentous
species, with cocci attached to tooth substrate.
• Mostly multi-species in nature, contained varying
degrees of extracellular matrix materials(Tronstad et
al)

52. Periapical microbial biofilms
• These are isolated biofilms found in periapical region of an
endodontically infected teeth.
• The microbiota in majority of teeth associated with apical
periodontitis is restricted to root canal as most of the
microbial species that infect the root canal are opportunistic
pathogens that don’t have the ability to survive the host
defense mechanisms in periapical tissues.
• Rarely, microbial species or even strains within species may
possess strategies to survive and thus infect periapical tissues.

54. • The members of genus Actinomyces and
P.propionicum have been demonstrated in
asymptomatic periapical lesions refractory to
endodontic treatment.
• These microorganisms have the ability to overcome
host defense mechanisms, thrived in inflamed
periapical tissue and subsequently induce a
periapical infection

55. Biomaterial centered biofilm
• It is caused when microorganisms adheres to an
artificial biomaterial surface and forms biofilms
structures.
• Presence of biomaterials in close proximity to host
immune systems can increase the susceptibility to
Biomaterial centered infections
• Biomaterial centered infection is one of the major
complications associated with prosthesis and or
implant related infections

56. • Furthermore, biofilms are extremely resistant to host
defense mechanisms and antibiotic treatment, BCI
are rarely resolved and often the only solution to an
infected biomaterial is the surgical removal.
• species like Staphylococcus aureus, Enterococcus
fecalis, streptococci and Pseudomonas aeruginosa
and fungi ( Candida albicans) are most commonly
isolated species.

57. • Biomaterial centered biofilms would form on root
canal obturating materials and can be intraradicular
or extraradicular.
Biofilm on extruded gutta percha

58. • presence of biofilms in dental-unit waterlines -known
since 1963.
• discovery that biofilms contribute to the microbial
contamination of dentalunit waterlines has made the
need for cleansing systems apparent, to minimize the
potential danger of infection and cross contamination.
• In dental-unit waterlines, biofilms measured to be
30-to-50 micrometers thick.
BIOFILMS IN DENTAL- UNIT WATER LINES

59. • Microbial populations found in dental-unit waterlines - most
common opportunistic pathogens linked to hospital-related
waterborne infections; e.g., Pseudomonas, Legionella, and
non-tuberculous Mycobacterium.
• -Predominant early colonizers - Pseudomonas spp.,
Pasteurella, Moraxella, Ochrobactrum, with Aeromonas spp.,
Flavobacterium, and Acinetobacter spp. being observed later.
Many of these organisms are opportunistic pathogens.

60. • American Dental Association and the Centers for Disease
Control and Prevention endorse flushing water lines for
several minutes prior to the first patient visit and for 20 to 30
seconds between patients.
• Flushing between patients will most likely reduce levels of oral
flora, which do not typically colonize upstream tubing.
• quality of dental-unit water is of considerable importance
since patients and dental staff are regularly exposed to water
and aerosols generated from the dental-unit.

61. • Average living microbial counts in water from handpieces and
air-water syringes are in the range of 300,000 to 400,000 CFU
per ml .
• Most plastic dental tubing has an inside diameter of 1/16-to-
1/8 inch and thus has a very large surface area to volume
ratio.
• hydrophobic surface of waterline plastics promotes the
attachment and colonization of biofilm organisms.
• The layered structure of biofilms (limiting diffusion) combined
with the low flow conditions renders these microbial colonies
intrinsically resistant to many biocides and cleansing schemes

62. • Schemes to reduce microbial counts in dental treatment
water fall into four broad categories:
• (i) use of water systems that are independent of public
systems, including those designed to deliver sterile water
• (ii) chemical treatments that are provided either
continuously or intermittently;
• (iii) filters placed inline just before the point of use (i.e.
handpiece, three-way syringe, ultrasonic scaler);
• (iv) devices to create turbulent and/or high energy flow
conditions to cleanse fine tubing.

63. Microbial control techniques for DUWs
• Peracetic acid
• Tetra-sodium EDTA
• 1-2%NaOCl +<0.2%Na-p-tolusulfonechloramide & 1-5%EDTA (Alpron BRS)
• Filtration (Aquasafe)
• CHX 0.12%, glycerol 0.12% & alcohol base (Bioblue)
• NaOCl (Bleach)
• 1% H2O2,0.014%Ag ions(Dentasept)
• UV for syringe & handpiece(AXCS system)
• 0.4% H 2O2 (Oxigenal)
• Chlorine dioxide (Dioxiclear)
• Ozone 2100ppm (HealOzone Unit)
• 5% Alkaline peroxide (Sterilex Ultra)
• Iodine cartridge (DentaPure)
• 2% or 5% superoxidised water (Sterilox)

64. Methods to eradicate biofilms
• Sodium hypochlorite -effective irrigant to destroy
all forms of Enterococcus faecalis including its biofilm
forms.
• Chlorhexidine 2% gel or liquid form- effective to
eliminate Enterococcus faecalis from the superficial
layers of dentinal tubules up to 100 micrometer.
• New techniques- ultrasonic irrigation, ozone, plasma
dental probe, photoactivated disinfection with low-
energy laser.

66. • 1-minute use of ultrasonically activated irrigation,
followed by root canal cleaning and shaping -shown
to improve canal and isthmus cleanliness in terms of
necrotic debris/biofilm removal.
• High concentrated gaseous and aqueous ozone is
strain, dose and time dependently effective against
the tested microorganisms in suspension and biofilm
test model.

67. Plasma dental probe
• effective for tooth disinfection.
• SEM shows complete destruction of endodontic biofilms for a
depth of 1 mm inside a root canal after plasma treatment for 5 min.
Er:YAG laser
• produced excellent results due to its capacity for ablating hard
tissue with very less thermal effects.
• considered to be effective tool for the removal of apical biofilm.

68. Photodynamic therapy/ Light Activated Therapy
• latest method used to destruct endodontic biofilm.
• involves the killing of microorganisms when a photo
sensitizer selectively accumulated in the target is
activated by a visible light of appropriate wavelength.
PAD is a unique combination of a photosensitizer solution
and low-power laser light. The photosensitizer, which is
mostly colored, adheres to or gets absorbed by microbial
cells. The low-power laser will destruct the target area
and inactivate the microbial invaders

69. CONCLUSION
Biofilm biology has become an expanding field of research in
human, industrial and environmental ecosystems. The
knowledge accumulated suggests that organisms growing in
biofilms develop properties different to those dwelling in the
planktonic state.
When assessing treatment options, an appreciation of the
ecology of the oral cavity will enable the enlightened clinician
to take a more holistic approach and consider the nutrition,
physiology, host defenses, and general well-being of the
patient, as these will affect the balance and activity of the
resident oral microflora.
Identification of such critical control points can lead to the
selection of appropriate preventive strategies that are
tailored to the needs of individual patients.

70. Screening, characterisation , in vitro biofilm
formation and antifungal resistance of candida
species
• Vinodini, latha et al :King saudi university journal of
dental sciences, july 2013
Aim:-This study determined the presence of Candida spp. in dental plaques of
both males and females. Pooled samples of dental plaque were collected
from 25 males and 55 females aged between 25 and 50 years.
Methods:-Colony growth was verified and 30 Candida isolates were chosen
for the screening. The identification of biofilm forming Candida was
confirmed by performing several screening techniques (Microtiter plate
method, Test tube method.

71. • The biofilm formation ofCandida spp. on catheter was evaluated using
scanning electron microscopic analysis. The extra cellular polysaccharide
(EPS) quantity was measured with the effect of different carbon sources,
adherence time and biofilm forming time. The above isolates were
screened for antifungal resistance against six clinically important
antifungal agents such as Amphotericin B, Ketaconazole, Fluconazole,
Itraconazole, Nystatin and Clotrimazole (10 μg/ml)
• Results:-The biofilm forming isolates were significantly resistant to the
antifungal drugs in comparison with non-biofilm forming Candida isolates.
The present study reveals the presence of Candida biofilm on human
dental surface and indicates the magnitude of antibiotic resistance.

72. Difference in initial biofilm accumulation during
night and day, Trene dige, sebastian et
al,Scandinavian dental journal..dec 2012
• Objective. The study of initial microbial colonization on dental surfaces is a
field of intensive research because of the aetiological role of biofilms in
oral diseases. Most previous studies of de novo accumulation and
composition of dental biofilms in vivo do not differentiate between
biofilms formed during day and night. This study hypothesized that there is
a diurnal variation in the rate of accumulation of bacteria on solid surfaces
in the oral cavity.
• Materials and methods. In situ biofilm from healthy individuals was
collected for 12 h during day and night, respectively, subjected to
fluorescent in situ hybridization and visualized using confocal laser
scanning microscopy

73. • Results. Analysis of the biofilms using stereological methods and digital
image analysis revealed a consistent statistically significant difference
between both the total number of bacteria and the biovolume with the
highest accumulation of bacteria during daytime .
• Conclusion. The data provide firm evidence that initial biofilm formation
decreases during the night, which may reflect differences in the availability
of salivary nutrients. This finding is of significant importance when
studying population dynamics during experimental dental biofilm
formation

74. Anti-biofilm dentin primer with quaternary
ammonium and silver nanoparticles
• Cheng L, Zhang K, Melo MA, j Dental research june
2012
• The objectives of this study were to develop novel antibacterial dentin
primers containing quaternary ammonium dimethacrylate (QADM) and
nanoparticles of silver (NAg), and to investigate the effects on dentin bond
strength and dental plaque microcosm biofilms for the first time.
• Methods:- Scotchbond Multi-Purpose ("SBMP") bonding agent was used.
QADM and NAg were incorporated into SBMP primer, yielding 4 primers:
SBMP primer (control), control + 10% QADM (mass), control + 0.05% NAg,
and control + 10% QADM + 0.05% NAg. Human saliva was collected to
grow microcosm biofilms.

75. • Results:-QADM-NAg-containing primer increased the bacteria inhibition
zone by 9-fold, compared with control primer (p < 0.05). QADM-NAg-
containing primer reduced lactic acid production and colony-forming units
of total micro-organisms, total streptococci, and mutans streptococci by an
order of magnitude. In conclusion, novel QADM-NAg-containing primers
were strongly antibacterial without compromising dentin bond strength,
and hence are promising to inhibit biofilms and secondary caries

76. Antimicrobial efficacy of non-thermal plasma in comparison to
chlorhexidine against dental biofilms on titanium discs in vitro
• Koban I, Holtfreter B, Hübner NO, archives of oral
biology ,Aug 2012
• AIM:
In this study, the effect of three different plasma devices on the reduction
of Streptococcus mutans (S. mutans) and multispecies human saliva
biofilms was evaluated.
• MATERIAL AND METHODS:
• assessed the efficacy of three different non-thermal atmospheric pressure
plasma devices against biofilms of S. mutans and saliva multispecies grown
on titanium discs in vitro in comparison with a chlorhexidine digluconate
(CHX) rinse. Efficacy of plasma treatment was determined by the number
of colony forming units (CFU) and by scanning electron microscopy. The
results were reported as reduction of CFU (CFU(untreated) -CFU(treated) ).

77. • The application of plasma was much more effective than CHX against
biofilms. The maximum reduction of CHX was 3.36 for S. mutans biofilm
and 1.50 for saliva biofilm, whereas the colony forming units (CFU)
reduction of the volume dielectric barrier discharge argon plasma was
5.38 for S. mutans biofilm and 5.67 for saliva biofilm.
• CONCLUSIONS:
• Treatment of single- and multispecies dental biofilms on titanium discs
with non-thermal atmospheric pressure plasma was more efficient than
CHX application in vitro.

78. REFERENCES
• INGLE’S ENDODONTICS-6TH EDITION
• COHEN’S PATHWAYS OF THE PULP-10TH EDITION
• GROSSMAN’s Endodontic practice, 12TH EDITION
• Biofilm in endodontics- Review by Usha H.L,
Anjali Kaiwar, Deepak Mehta.