Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2018; 63: 208–216

doi: 10.1111/adj.12580

Comparative efficacy of endodontic medicaments and

sodium hypochlorite against Enterococcus faecalis biofilms
B Plutzer,* P Zilm,* J Ratnayake,† P Cathro*†
*School of Dentistry, Faculty of Health Science, The University of Adelaide, Adelaide, South Australia, Australia.
†University of Otago, Faculty of Dentistry, Dunedin, Otago, New Zealand.

ABSTRACT
Background: Many studies have investigated the effectiveness of root canal irrigants and medicaments against Enterococ-
cus faecalis. The aim of this study was to compare the efficacy of commonly used medicaments against E. faecalis cul-
tured as a biofilm on dentine substrate.
Methods: An E. faecalis biofilm was established on human dentine slices using a continuous flow cell. Each test medica-
ment (Ledermix, Ca(OH)2, Odontopaste, 0.2% chlorhexidine and 50:50 combinations of Ledermix/Ca(OH)2 and Odon-
topaste/Ca(OH)2) was introduced into the flow cell and biofilms were harvested and quantitated by determining cellular
protein. Cellular viability was determined using serial plating and the number of colony-forming units was normalized
against cellular protein to allow treatment protocols to be compared. Qualitative scanning electron microscopy analyses
of the biofilm were performed after a 48-h exposure to each test agent.
Results: Sodium hypochlorite achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect
on the E. faecalis biofilm. Ca(OH)2 and 50:50 combinations of Ca(OH)2/Ledermix or Ca(OH)2/Odontopaste reduced
the viability by more than 99% while 0.2% chlorhexidine reduced bacterial numbers by 97%.
Conclusions: Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. However,
Ca(OH)2 in isolation and combined with Ledermix, and Odontopaste was highly effective in reducing bacterial viability.
Keywords: Biofilm, calcium hydroxide, Enterococcus faecalis, Ledermix, Odontopaste.
Abbreviations and acronyms: ATCC = American Type Culture Collection; CFU = colony-forming units; PBS = phosphate-buffered sal-
ine; SEM = scanning electron microscopy.
(Accepted for publication 21 November 2017.)

medicaments clinically, and in vitro studies which

INTRODUCTION
The microbial flora of root-filled canals with persist-
ing periapical radiolucencies differs markedly from
that of untreated necrotic dental pulps, with a very
limited assortment of microorganisms able to survive
or be recovered. Generally, only one or a small num-
ber of species are recovered, with a predominance of
gram-positive microorganisms and facultative anaer-
obes.1–4 Enterococcus faecalis is the most commonly
recovered species in failed root canal treatment, which
is nine times higher than in primary endodontic infec-
tions.3,5,6 Clinically isolated E. faecalis species possess
many of the characteristics of biofilm growth, includ-
ing increased adhering capacity, increased virulence
factors and increased resistance to antimicrobials.7,8
The biofilm structure may go some way to explain the
frequent discrepancy observed in studies testing the
antimicrobial effectiveness of endodontic irrigants and
208
often use planktonic cultures.
Sodium hypochlorite is the most widely used irrigat-
ing solution due to its antimicrobial effect and its
unique ability to dissolve necrotic tissue and organic
components of the smear layer. However, using
sodium hypochlorite as an endodontic irrigant is
problematic as it is highly toxic at high concentra-
tions, and tends to induce tissue irritation on con-
tact.9,10 Currently, calcium hydroxide is recognized as
one of the most effective antimicrobial dressings avail-
able for the purposes of endodontic treatment due to
its high alkalinity.11,12 However, E. faecalis can with-
stand high pH levels (pH >11.5) and thus has the
capacity to resist the antibacterial effect of calcium
hydroxide.13–15 Chlorhexidine is a synthetic cationic
bisbiguanide and is highly efficacious against several
gram-positive and gram-negative oral bacterial spe-
cies.16 Although several in vitro and ex vivo studies
© 2017 Australian Dental Association

have shown that chlorhexidine has antibacterial
effects, one of the major disadvantages associated
with chlorhexidine is that it has no tissue solvent
activity.17 Ledermixâ (Aspen Pharma, Dandenong
South, Vic., Australia) paste is a corticosteroid vehicle
aimed at controlling pain and inflammation in conser-
vative pulp therapy and is an effective intracanal
medicament for the control of postoperative pain
associated with acute apical periodontitis and conser-
vative pulp therapy.18,19 These benefits need to be bal-
anced with the clinical concern of possible
discolouration because of its use and the antibacterial
effect being limited to a small spectrum of bacte-
ria.20,21 On the other hand, Odontopasteâ (Australian
Dental Manufacturing, Brisbane, Qld, Australia) con-
tains clindamycin as the antimicrobial component
which has proven effective against a comprehensive
spectrum of microbes associated with endodontic
infections and has the advantage of not causing denti-
nal discolouration as seen with Ledermix.
Until present, no research has been conducted on
the antimicrobial efficacy of Ledermix or Odontopaste
in the context of a microbial biofilm. Combinations
of these medicaments with calcium hydroxide are also
yet to be tested using a biofilm model, while calcium
hydroxide on its own has only been evaluated by a
handful of biofilm studies.7,22 The aim of this study
was to compare the efficacy of sodium hypochlorite
and commonly used endodontic medicaments against
E. faecalis cultured as a biofilm on dentine substrate.
The medicaments tested were Ledermix paste, calcium
hydroxide, Odontopaste, 0.2% chlorhexidine and
50:50 combinations of Ledermix/calcium hydroxide
and Odontopaste/calcium hydroxide.
(a)
Fig. 1 (a) Experimental setup consisting the flow cell, nutrient reservoir, peristaltic pump and waste vessel. (b) The flow cell showing coupons and den-
tine slices in situ.
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E. faecalis and endodontic medicaments
METHODS
Organism
Enterococcus faecalis was stored as frozen stock cul-
tures in 40% v/v glycerol at 80°C. Aliquots of
100 lL were used to inoculate 20 mL of Todd Hewitt
Broth (Oxoid, Scoresby, Vic., Australia). After over-
night growth at 37°C, 10 mL of the broth was used
to inoculate the flow cells. Two strains of E. faecalis
were used: a tetracycline-resistant strain, American
Type Culture Collection (ATCC) 51299; and a tetra-
cycline-sensitive strain, ATCC 29212. Purity of cul-
tures was monitored by periodic plating onto Bile
Aesculin Agar (Oxoid).
Dentine sample preparation
Dentine samples were produced from extracted teeth
stored in thymol, which were decoronated and sec-
tioned longitudinally to expose the inner walls of the
pulp chamber and canals. Following sectioning, the
smear layer and pulpal tissue remnants were removed
by treating the specimens with 17% w/v ethylenedi-
aminetetraacetic acid, followed by 1% v/v sodium
hypochlorite for 4 min. Samples were attached to
scanning electron microscopy (SEM, Selleys, Auck-
land, New Zealand) stainless steel coupons using Ara-
ldite© adhesive.
Biofilm growth
A biofilm grown on dentine was established using a
continuous flow cell model based on that described
(b)
209

B Plutzer et al.
by Dunavant et al. (Fig. 1).23 The flow cell, with
the dentine slices in situ, was sterilized using ethy-
lene oxide. The nutrient reservoir, silicone tubing
and waste vessel were sterilized by autoclaving. The
flow cell temperature was maintained at 37°C using
a slide dryer. After sterilization, the flow cell was
filled with Todd Hewitt Broth and sterility was con-
firmed before inoculation. An overnight broth cul-
ture of E. faecalis (10 mL) was introduced into the
flow cell through a syringe injection port upstream
of the flow cell. Batch culture was established over
24 h before culture medium was pumped through
the flow cell at 20 mL/h using a peristaltic pump
giving a dilution rate of 0.4 h 1. Following a series
of preliminary experiments, biofilm growth was
established over 4 weeks.
Scanning electron microscopy
After removal from the flow cell, dentine slices were
stored overnight in fixative (4% paraformaldehyde/
1.25% glutaraldehyde in phosphate-buffered saline
(PBS) + 4% sucrose). They were then washed in
PBS + 4% sucrose and dehydrated in ascending con-
centrations of ethanol. Following critical point dry-
ing, the samples were coated with platinum and
analysed under an SEM (Philips XL30 field emission
scanning electron microscope; Philips, Andover, MA,
USA). The biofilm was assessed for confluence, bac-
terial density and tubular penetration of the dentine
substrate.
Test agents
Sodium hypochlorite (4%; Asis Scientific, Adelaide, SA,
Australia), Ledermix, Odontopaste, Calxylâ calcium
hydroxide (OCO Pr€ aparate, Otto & Co, Frankfurt,
Germany), Ledermix/calcium hydroxide, Odontopaste/
calcium hydroxide and 0.2% chlorhexidine gel (Profes-
sional Dentist Supplies, Victoria, Australia) was used
as the test agents. Each test agent in paste or gel for-
mulation was applied to cover each dentine surface,
while the control samples were removed from the flow
cell and placed into sterile PBS for corresponding time
periods. The dentine samples were exposed to each
medicament for either 24 or 48 h. For sodium
hypochlorite treatment, samples were removed from
the flow cell and submerged in sodium hypochlorite
solution for either 1, 10, 30 or 60 min. Each protocol
was performed in duplicate.
Biofilm harvesting
Following exposure to the test agent or the control
solution, each coupon was washed three times with
210
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sterile PBS. The biofilm was detached by sonication
with Soniprobe (Dawe Instruments, London, UK) at a
setting of 2 A for 60 s into 2 mL of sterile PBS.
Cellular viability and protein measurement
Cellular viability was determined using serial plating
onto Todd Hewitt agar plates (Oxoid). Viable counts
were performed in duplicate and incubated aerobi-
cally at 37°C for 48 h. Cellular protein levels were
measured to quantitate the amount of biofilm har-
vested and normalize the viable count measurements.
Sodium hydroxide (100 lL of 1 M) was added to
900 lL of suspension and placed in a boiling water
bath for 30 min. A measure of 150 lL of each sam-
ple was then pipetted into microtitre plate wells in
triplicate before the addition of 150 lL of Coomassie
Plus protein assay reagent (Pierce Biotechnology,
Rockford, IL, USA). The microtitre plate was then
shaken for 5 min, and the absorbance was read at
595 nm using a microplate reader (Bio-Tek Instru-
ments, Winooski, VT, USA). The concentration of the
samples was standardized against known dilutions of
bovine serum albumin, which were assayed in parallel
with the samples. The colony-forming units (CFU)/
mL per mg of protein was then calculated by dividing
the CFU/mL by the cellular protein levels per mL of
suspension.
Tooth crushing protocol
Dentine samples were crushed after placing them into
an apparatus consisting of a brass piston fitting clo-
sely into a brass cylinder which was struck by a ham-
mer. Sterile saline (2 mL of 0.9%) was added and the
contents poured into a sterile tube and vortexed for
30 s. Serial dilutions were made for each sample in
0.9% sterile saline, and 100-lL aliquots of each dilu-
tion were plated in duplicate onto Todd Hewitt Broth
agar plates. Plates were incubated at 37°C for 24 h,
and the number of CFU per mL were calculated. Ten
flow cells provided the basis for the results, with each
one containing duplicate samples for each time of
exposure to the test or control agent.
Statistical analysis
Statistical analysis was performed using the GraphPad
Prism 6 software (GraphPad Software, La Jolla, CA,
USA) software. Bar graphs were reported as stan-
dard error of the mean. Statistically significant differ-
ences were determined using one-way ANOVA. If
differences were detected, multiple comparisons were
made using Tukey’s multiple comparison tests at a
confidence level of 95% (P < 0.05).
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E. faecalis and endodontic medicaments

Fig. 2 Scanning electron microscopy images showing biofilm after (a) 1 week, (b) 2 weeks, (c) 3 weeks and (d) 4 weeks.

adherent biofilms. At week 1, the cells appeared quite

RESULTS
dispersed, with few dense aggregates. Interconnecting
networks of polymer-like strands were observed. At
Biofilm growth
2 weeks, denser cellular aggregates became apparent,
Preliminary experiments using SEM were conducted to but the coverage was not confluent. Three weeks post-
determine an appropriate time to produce confluent inoculation showed a more confluent biofilm, with

Fig. 3 Effect of 4% NaOCl on Enterococcus faecalis biofilm viability. Viable cells expressed as colony-forming units per microgram of protein.
© 2017 Australian Dental Association 211

B Plutzer et al.
Fig. 4 Dentine surface allowing a 10 min exposure to 4% sodium
hypochlorite (original magnification 9800).
thick bacterial growth covering the dentine surface,
leaving small areas of loosely organized cells. At
4 weeks, cells were present in dense, multilayered, con-
fluent aggregations, generally arranged in short chains.
Uncolonized sections of dentine were absent (Fig. 2).
Sodium hypochlorite
Exposure to 4% sodium hypochlorite produced a
95% drop in bacterial viability at 1 min, and this
increased to 99.99% at 10 min of exposure (Fig. 3).
No viable bacteria could be cultured after 30 min,
60 min or 6 h (Fig. 3). SEM analysis of the E. fae-
calis biofilm after exposure to hypochlorite for 10,
30, and 60 min showed dentine surfaces free of
microbes and debris. The dentinal tubules appeared
patent (Fig. 4).
Ledermix and Odontopaste
Ledermix and Odontopaste had no observed effect on
the cellular viability in a 4-week E. faecalis biofilm
Fig. 5 Effect of calcium hydroxide, calcium hydroxide combined with
Ledermix and calcium hydroxide combined with Odontopaste on Entero-
coccus faecalis biofilm viability. The data represents the averages of
duplicate samples taken from three flow cells.
212
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after 48 h exposure to each medicament (data not
shown). SEM analysis of the biofilm following expo-
sure to Ledermix and Odontopaste showed an appar-
ently undisturbed biofilm covering the entirety of the
dentine surface. Identical results were obtained when
a tetracycline-sensitive strain, ATCC 29212, was used
to repeat the experiments.
Calcium hydroxide and 50:50 combinations of
calcium hydroxide with either Ledermix or
Odontopaste
Exposure to calcium hydroxide for 24 h and 48 h
reduced the viability of E. faecalis by more than 99.9%
Compared with the controls, calcium hydroxide used in
combination with either Ledermix or Odontopaste
reduced viability of E. faecalis by more than 99.9%.
However, addition of Odontopaste or Ledermix did not
significantly increase the antimicrobial effect of calcium
hydroxide (Fig 5). SEM images of the dentine surface
following 48 h exposure to either calcium hydroxide,
Ledermix/calcium hydroxide or to Odontopaste/calcium
hydroxide showed a disturbed biofilm with residual cells
clearly visible. The images show E. faecalis cells residing
within dentinal tubules (Fig. 6).
Chlorhexidine gel
Compared with the controls, 0.2% chlorhexidine gel
reduced bacterial numbers by 97% after 24 and
48 h of exposure (Fig. 7). SEM images of the den-
tine surface following 48 h exposure showed a dis-
turbed biofilm, but with residual cells clearly visible
(Fig. 8).
DISCUSSION
The results of the present study indicate that antibi-
otic-based medicaments alone have little quantitative
effect on reducing the viability of E. faecalis. Expo-
sure to either Ledermix paste or Odontopaste for up
to 48 h did not produce any significant decrease in
microbial numbers where Ledermix paste and Odon-
topaste eliminated bacteria by 35.5% and 71.1%,
respectively, which suggests that Ledermix failed to
eliminate E. faecalis from the root canal. A statisti-
cally significant difference (P < 0.05) was found
between Ledermix and Odontopaste which suggests
that Odontopaste is superior to Ledermix (Fig 9).
Scanning electron microscopy analyses also showed
a complete inability of the medicaments to remove the
biofilm. This was consistent with recent findings by
Norrington et al. who used a scanning electron micro-
scope to qualitatively examine mixed biofilms exposed
to a variety of antibiotics (amoxicillin, clindamycin,
metronidazole, doxycycline and azithromycin) for 3
© 2017 Australian Dental Association
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E. faecalis and endodontic medicaments

Fig. 6 Dentine surfaces after 48 h exposure to (a) Ledermix paste, (b) Odontopaste, (c) 50:50 Ledermix and calcium hydroxide, (d) 50:50 Odontopaste
and calcium hydroxide, (e) calcium hydroxide and (f) 0.2% chlorhexidine

and 8 days.24 A major reason for the lack of effective-
ness of either Ledermix paste or Odontopaste is the
intrinsic resistance of E. faecalis to several commonly
used antibiotics and perhaps more importantly, its
ability to acquire resistance to all currently available
© 2017 Australian Dental Association
antibiotics, either by mutation or by horizontal gene
transfer.25
A significant proportion of enterococcal isolates
demonstrate acquired resistance to tetracyclines and
intrinsic resistance to clindamycin. The proximity of
213

B Plutzer et al.
Fig. 7 Effect of 0.2% chlorhexidine gel on Enterococcus faecalis bio-
film viability. Viable cells expressed as colony-forming units per micro-
gram of protein.
Fig. 8 Dentine surface following 48 h of exposure to 0.2% chlorhexi-
dine gel (original magnification 910 000).
bacterial cells within the biofilm readily allows the
exchange of genetic information, including the transfer
of antibiotic resistance. Two strains of E. faecalis with
reportedly different levels of tetracycline sensitivity
were used in this experiment to test the efficacy of
Ledermix paste. No significant reductions in bacterial
viability could be noted for either strain and this was
supported by the lack of susceptibility to Ledermix
paste by the two strains using the plate diffusion
method. This may well be an indication that the level
of tetracycline in Ledermix paste is too low to affect
antibiosis. It was difficult to source a strain of E. fae-
calis susceptible to clindamycin, an indication of how
widespread resistance to this antibiotic is among ente-
rococcal species. In addition to the intrinsic or
acquired antibiotic resistance of E. faecalis, the organi-
zation of cells in a biofilm could be a further factor
contributing to the lack of antimicrobial efficacy of
Ledermix paste and Odontopaste. Cells growing in
biofilms have been shown to be up to 1000 times more
resistant to antimicrobial agents than their planktoni-
cally growing counterparts.26,27 Laboratory and clini-
cal research is urgently needed to assess the properties
of Odontopaste. Its clinical benefit is likely to be
214
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Fig. 9 Percentage kill versus different treatment endodontic medica-
ments. Error bars represent  standard error of the mean after one-way
ANOVA with Tukey’s multiple comparison test, *P < 0.05,
***P < 0.001, n = 2.
derived from the potential anti-resorptive and sedative
properties of the steroid component, rather than the
antibacterial effects of the antibiotic, but this is yet to
be supported by evidence, as are the manufacturer’s
claims that it causes no dentinal discolouration.
The results of this study showed that calcium
hydroxide was able to achieve reductions in microbial
viability by 99% at 24 h and 48 h of exposure. This
finding was at variance with reports showing that the
antimicrobial effectiveness of calcium hydroxide was
abolished in the presence of dentine.9,28 This inconsis-
tency in findings is most likely a reflection of method-
ological differences, with the present study involving
sections of dentine rather than dentine powder, and
E. faecalis cells in a biofilm rather than a planktonic
state. The present study is consistent with other
reports showing that calcium hydroxide was unable to
achieve total elimination of E. faecalis. The persistence
of a small proportion of bacteria was confirmed
through SEM. Many of the persisting cells were seen
to be present in the protective environment of the
dentinal tubules. Similar findings were noted when cal-
cium hydroxide was combined with either Ledermix
or Odontopaste. The combining of antibiotic-based
medicaments with calcium hydroxide did not appear
to elicit any synergistic action, neither did it diminish
the effectiveness of calcium hydroxide when used.
Recent research has shown that calcium hydroxide
is highly effective in the context of a microbial bio-
film.7 A possible explanation for its efficacy against
microbial biofilms is that calcium hydroxide is capable
© 2017 Australian Dental Association

of dissolving organic tissue, and is therefore less inhib-
ited by dense cellular aggregations and exopolysaccha-
ride matrix. Tissue dissolution is a property that is
neglected in investigations of antimicrobial efficacy,
probably because it is redundant in study designs
incorporating agar diffusion or bacterial suspension
methods. In clinical endodontics, however, the capac-
ity of a medicament to physically dissolve necrotic pul-
pal tissue or microbial colonies is critical to achieving
disinfection. Despite its ability to drastically reduce
bacterial numbers, calcium hydroxide failed to achieve
sterilization of the dentine surface. Bystrom et al.
showed that the pH needs to reach levels of 11.5 or
higher to exert a bactericidal effect on E. faecalis.13
Investigations of the mechanisms involved in the resis-
tance of E. faecalis to calcium hydroxide identified
that its survival at high pH was related to the effective
function of its proton pump. Furthermore, the buffer-
ing effects of dentine may not allow a sufficiently high
pH to be achieved in the dentinal tubules.29 The bio-
film-forming ability of E. faecalis constitutes another
important survival strategy, while its proficiency at
invading dentinal tubules affords the organism physi-
cal protection from chemo-mechanical root canal
preparation and intracanal dressings.29,30
Chlorhexidine gel was able to eliminate 97% of bac-
teria at 24 and 48 h of exposure, making it less effec-
tive than calcium hydroxide (99.9%), calcium
hydroxide combined with Ledermix (99.9%) and cal-
cium hydroxide combined with Odontopaste (99.9%).
Figure 9 clearly illustrates that NaClO, Ca(OH)2,
Odontopaste/ Ca(OH)2, Ledermix/Ca(OH)2 and
chlorhexidine were effective in killing E. faecalis. A
statistically significant difference (P < 0.05) was found
between Ledermix and the medicaments mentioned
above. However, there was no statistical significant
difference between Odontopaste and these medica-
ments. There was no statistically significant difference
between Odontopaste and chlorhexidine, although
chlorhexidine appears to be more effective in eliminat-
ing E. faecalis (Fig 9). SEM images of dentine disks
exposed to chlorhexidine showed residual bacteria and
exopolymeric material persisting on the dentine sur-
face. This is consistent with findings by Clegg et al.,
who noted a virtually intact biofilm after exposure to
chlorhexidine.31 The activity of chlorhexidine is pH
dependent, and is greatly reduced in the presence of
organic matter.32 Its inability to dissolve organic tis-
sues is therefore a major shortcoming and responsible
for the reduced antimicrobial effectiveness of chlorhex-
idine when challenged by an increased bioburden.
Sodium hypochlorite, on the other hand, acts as a
solvent in the presence of organic tissue, releasing
chlorine which combines with protein amino groups
forming chloramines.33 This tissue-dissolving ability is
one of the features that makes sodium hypochlorite
© 2017 Australian Dental Association
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E. faecalis and endodontic medicaments
such an effective endodontic irrigant. In this study,
4% sodium hypochlorite was the only agent capable
of eliminating the E. faecalis biofilm. Its antimicrobial
efficacy was time-dependant, with a 95% drop in bac-
terial viability noted at 1 min, 99.9% at 10 min, and
no detectable bacteria cultured at 30 or 60 min.
Crushing the dentine revealed viable cells after
30 min of sodium hypochlorite exposure, even though
surface sonication failed to produce any CFU from
the same dentine slice. This highlights the limitation
of surface sampling methods in assessing disinfection,
such as the use of paper point sampling in clinical
endodontics. A negative culture result is merely an
indication that microbial levels are below the point of
detection, rather than a guarantee of sterility.
Scanning electron microscopy supported the quanti-
tative findings, with small numbers of E. faecalis cells
and polymeric matrix substance visible after 1 min.
Longer exposure times produced dentine walls free of
microbial cells or debris, with patent dentinal tubules.
Variable degrees of surface erosion were noted, and
this appeared to be a function of time. Clinically, this
indicates that sodium hypochlorite is capable of both
eliminating infecting microbes and physically remov-
ing them, but its action is time-dependant. Continuous
replenishing with fresh solution and ultrasonic
activation are mechanisms that may speed up the
antimicrobial and tissue-dissolving action of sodium
hypochlorite, although this is yet to be shown against
a bacterial biofilm.34,35 Consequently, the time
required to exert its effect in the root canal may be
reduced, limiting the structural damage to dentine.
CONCLUSIONS
When used in isolation, antibiotic-containing medica-
ments had no appreciable effect on the viability
of E. faecalis. The microorganism’s tetracycline sensi-
tivity did not influence the antimicrobial effectiveness
of Ledermix paste. Calcium hydroxide in isolation and
when combined with Ledermix or Odontopaste
achieved a 99.9% reduction in E. faecalis viability
compared with 97% achieved with chlorhexidine gel.
The antimicrobial effectiveness of sodium hypochlorite
increased exponentially with increased exposure time.
Total bacterial elimination was predictably achieved at
30 min of exposure. A comparison of sampling meth-
ods showed that crushing the dentine sample was able
to identity viable bacteria in specimens deemed sterile
through surface sampling.
ACKNOWLEDGEMENTS
We are grateful for the financial funding support of
the Australian Dental Research Foundation and the
Australian Society of Endodontology.
215

B Plutzer et al.
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Address for correspondence:
Associate Professor Peter Cathro
Department of Oral Rehabilitation
Faculty of Dentistry
PO Box 56
Dunedin
Otago 9054
New Zealand
Email: peter.cathro@otago.ac.nz
© 2017 Australian Dental Association