Antimicrobial efficacy of Irrigants,

Intra-canal medicaments &

combinations against Enterococcus
Faecalis- An In-Vitro comparative
microbiological study.
Introduction:
One of the goals of endodontic treatment is the complete debridement of the
root canal system to eliminate all bacteria, bacterial byproducts, and tissue
debris from the root canal system. Micro-organisms play a fundamental role in
the aetiology of pulp and periapical diseases and their control and elimination
are important during endodontic treatment. Chemomechanical cleaning and
shaping of the root canal can greatly reduce the number of bacteria, although it
has been shown that it is impossible to obtain predictable disinfection in all
cases. The use of intracanal medications to disinfect the root canal system has
been advocated to enhance the success of root canal treatment. The chances of a
favourable outcome with root canal treatment are significantly higher if
infection is eradicated effectively before the root canal system is obturated.
However, if microorganisms persist in the root canal at the time of root filling or
if they penetrate into the canal after filling, there is a higher risk that the
treatment will fail.

Need For Study:

Enterococcus faecalis, a facultative anaerobic gram-positive coccus, is the most
common Enterococcus sp. cultured from nonhealing endodontic cases. This
microorganism can even survive in an environment with scant available
nutrients and in which commensality with other bacteria is minimal.
Enterococcus faecalis is a microorganism commonly detected in asymptomatic,
persistent endodontic infections. Its prevalence in such infections ranges from
24% to 77%. The inherent ability of E. faecalis to tolerate starvation, extremes
of pH, salt concentration, biofilm formation, dentin tubular invasion, and
emergence of antibiotic resistant strains has made their eradication challenging
in endodontically treated teeth

Aims & Objectives:

1. To compare the efficacy of Ca(OH)2 , Nisin & Propolis (Intra-canal
medicaments)
2. To compare the efficacy of NaOCl , CHX-Plus & Smear OFF 2-in-1
(EDTA+CHX) (Irrigants)
3. To compare the efficacy of combination of NaOCl with different intracanal medicaments

Materials & Method:

SOURCE OF DATA:
One hundred extracted single rooted teeth will be collected from theDepartment
of Oral and Maxillofacial Surgery.
INCLUSION CRITERIA:
Non-carious human Single rooted Teeth extracted for Orthodontic purpose.
EXCLUSION CRITERIA:
Teeth with complicated root canal anatomy, curved root canals and immature
root canals, calcification, root fractures.

METHODOLOGY:
100 extracted human single rooted teeth will be selected. All teeth will be
thoroughly cleaned and washed and preserved in thymol solution until further
use. All teeth will be analysed by digital radiography in the buccal and proximal
directions to confirm noncomplicated root canal anatomy, single straight root
canals, and mature root formation. The crowns will be sectioned with a diamond
disk and the root length will be standardized to approximately 16mm.
Root canals will be instrumented 0.5mm beyond the apical foramen up to size
30 or size 40 k-files depending on the experimental group. Irrigation will be
perform with 2ml of physiologic saline solution. The root surface will be then
coated with an epoxy resin except the cervical access and apical foramen. After
setting of epoxy resin, the canals will be fill with EDTA for 3 min in order to
remove the smear layer. This will be followed by 5ml of physiologic saline

solution. Subsequently, the roots will be sterilized in autoclave at 121c for

20min.
Enterococcus faecalis grown overnight in brain heart infusion broth and
adjusted to 0.5 turbidity reading of the McFarland scale will be injected into
each canal using disposable syringe. The specimens will be incubated at 370C
to the experimental time period.After contamination the cervical seal will be
remove and the 100 samples will be randomly assigned in 10 groups (10 teeth
each) according to irrigants, intra-canal medicaments, combination & control as
below.

SUBGROUP 1- NaOCl
SUBGROUP 2- Smear OFF 2-in-1
(EDTA+CHX)
SUBGROUP 3- CHX-Plus

GROUP 1
Irrigants
GROUP 2
Intra-canal
Medicaments

SUBGROUP 4- Ca(OH)2
SUBGROUP 5- Nisin
SUBGROUP 6- Propolis

GROUP 3
Combination

SUBGROUP 7- NaOCl + Ca(OH)2

SUBGROUP 8- NaOCl + Nisin
SUBGROUP 9- NaOCl + Propolis

GROUP 4
Control

SUBGROUP 10- No Irrigants & No

Intracanal
medicaments

 Group 1 ( Irrigants): instrumentation and irrigation with saline up to

size 30 k-file, smear layer removal with EDTA for 3 min, sterilization,
contamination for 7 days, instrumentation up to a size 40 k-file and
irrigation according to subgroups 1, 2, 3.
Group 2 ( Intra-canal medicaments): instrumentation and irrigation
with saline up to size 40 k-file, smear layer removal with EDTA for 3
min, sterilization, contamination for 7 days, re-instrumentation up to a
size 40 k-file and irrigation with saline, filled with intra-canal
medicaments according to subgroups 4, 5, 6.
Group 3 ( Combinations): instrumentation and irrigation with saline up
to size 30 k-file, smear layer removal with EDTA for 3 min, sterilization,
contamination for 7 days, instrumentation up to a size 40 k-file and
irrigation with NaOCl and filled with intra-canal medicaments according
to subgroups 7, 8, 9.
Group 4 ( Control): instrumentation and irrigation with saline up to size
40 k-file, smear layer removal with EDTA for 3 min, sterilization,
contamination for 7 days, re-instrumentation up to a size 40 k-file and
irrigation with saline without intra-canal medicaments.
After 15 Days, the cervical & apical seals will be remove and the canals will be
instrumented with a size 40 k-file and irrigated with 5ml of saline solution to
remove the intracanal medicaments, irrigants & combination.
When the canals filled with sterile saline, the microbiological sampling will be
carried out using a sterile paper point for 60 seconds.Paper points will be placed
into test tubes containing 1.0ml of stable physiological saline solution. Test
tubes containing microbiological samples will be shaken vigorously for 60
seconds. Serial ten fold dilutions will be made (upto 1:105) in physiological
saline solution. From the serial dilutions, 0.1ml transferred and plated on blood
agar plates. The plates will be incubated in anaerobic chamber for 24hrs at
37C. The bacterial growth will be detected and colony count will be
performed.

STATISTICAL ANALYSIS:
The amount of extruded debris will be analysed using different statistical
methods.

REVIEW OF LITERATURE:
Menezes, Valera et al (2004) conducted An in vitro study evaluated the
effectiveness of irrigants and intracanal medicaments on micro-organisms
within root canals. This study revealed that Ca(OH)2 + CMCP was the
most effective intracanal medicament. 2% CHX solution was more
effective than 2.5% NaOCl against E. Faecalis.
Edgar, Klaus et al. (2005) conducted An in-vitro study compared the
antimicobial efficacy of CHX and Two Ca(OH)2 formulations against E.
Faecalis. This study showed that CHX was significantly more effective
than Ca(OH)2 paste or a mixture of CHX + Ca(OH)2 paste .
Oncag O, Dilash, Uzel et al.(2006) conductedAn in-vitro study compared
the antibacterial efficacy of three commonly used intracanal medicaments
calcium hydroxide, chlorhexidine gel and Vitapex with propolis against
Enterococcus faecalis. This study revealed that propolis had better
antibacterial activity against E.faecalis in the root canals suggesting that
it would be used as an alternative medicament.
Zhongchun, Lin et al (2011) conductedAn in vitro study evaluated the
antibacterial activities of MTAD in combination with Nisin against E.
Faecalis. This study revealed that MTAD in conjunction with Nisin
inhibits E. Faecalis better than MTAD alone.
Suneel, Ashok et al. (2011)conductedAn in vitro study investigated the
newer intracanal medicament Nisin on E. Faecalis in comparison with
CHX and Ca(OH)2. this study showed that Nisin was more effective in
eradicating E. Faecalis cell in pure culture and root canal dentin.
Manavalan, Narasimhan et al. (2011) conductedAn in vitro study
evaluated Propolis and Triantibiotic Mixture as an intracanal
medicaments against E. Faecalis. This study showed that Propolis was
more effective than TAM against E. Faecalis at a 2 day time period and
both were equally effective at 7 days.

REFERENCE:
1. Menezes, Valera et al. An in vitro evaluation the effectiveness of
irrigants and intracanal medicaments on micro-organisms within root
canals. IEJ 37, 311-319, 2004
2. Edgar, Klaus et al. Antimicobial efficacy of CHX and Two Ca(OH)2
formulations against E. Faecalis. JOE Vol31, No.1 Jan 2005
3. Oncag O, Dilash, Uzel A. Efficacy of propolis as an intracanal
medicament against Enterococcus faecalis. Gen dent 2006;10:319-322
4. Zhongchun, Lin et al. An in vitro evaluation the antibacterial activities
of MTAD in combination with Nisin against E. Faecalis. JOE- Vol37,
no.8 , Aug 2011
5. Suneel, Ashok et al. An in vitro investigation the newer intracanal
medicament Nisin on E. Faecalis in comparison with CHX and Ca(OH)2.
Journal of the International Clinical Dental Research Organization/ JanApr 2011, vol 3, Issue 1.
6. Manavalan, Narasimhan et al. Comparativeevaluation of Propolis and
Triantibiotic Mixture as an intracanal medicaments against E. Faecalis.
JOE-Vol 37, No. 9, Sep 2011