International Medical Journal Vol. 28, Supplement No. 1, pp.

3 - 5 , June 2021 3
DENTISTRY

Immediate and Delay Antimicrobial Activity of Three

Disinfection Solutions on Gutta-Percha Cones as
Routine Chair-Side Procedure

Ahmed I Al-Jobory1), Anas F Mahdee2), Raghad A Alhashimi2), Safwan A Suliman3)

ABSTRACT
Objective: The antimicrobial efficacy of three disinfection solutions: 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine
(CHX) and Listerine mouthwash were investigated as routine chair-side gutta-percha (GP) disinfection reagents.
Design: four groups of gutta percha points were contaminated with E. faecalis bacteria then disinfected by immersion in dif-
ferent solutions (5.25% sodium hypochlorite, 2% chlorhexidine gluconate, Listerine mouth wash and distilled water as control)
after 1 and 7 days culturing periods. The antibacterial efficacy of these disinfection solutions was evaluated by using colonies
per units (CPU)
Methods: Forty GP cones (F3 Dentsply) were sterilized with ethylene oxide gas before immersed contamination within broth
media containing E. faecalis for 20 min. Cones then divided them equally into four groups (n = 10) based on the treated disinfec-
tion solution, whilst one group received no disinfection treatment (control group). Each cone was then immersed into broth
media within separated Eppendorf tubes for 3 min before 100 μl was taken to be cultured in an agar plate for 24h. The remain-
ing broth media tubes were incubated for 7 days at 37℃ before taking another 100 μl to be cultured in new agar plates. CPU for
E. faecalis growth were calculated for the two period cultured plates: immediately after GP disinfection (day 0) and after 7 days
incubation.
Results: All groups except control showed low E. faecalis colonies growth. The colony-forming units (CFU) of E. faecalis on
day 0 was zero growth in NaOCl and CHX disinfected groups and much higher (14) within Listerine group. On day 7, only
NaOCl group still have complete inhibition for E. faecalis colonies growth. CHX antimicrobial potency was decreased with time,
while CFU for Listerine group decreased after 7 days incubation.
Conclusion: The NaOCl was the best disinfection for gutta-percha cones, followed by CHX and Listerine mouthwash.

KEY WORDS
gutta percha, Listerine, sodium hypochlorite, chlorhexidine

INTRODUCTION
The endodontic treatment is basically considered as a chemo-me-
chanical preparation of the root canal system which should include
mechanical cleaning and shaping, in addition to the microbial disinfec-
tion1). This should be followed by a complete three dimensional filling
of the cleaned root canal space with fluid tight seal endodontic filling
materials2). These materials include small quantity of endodontic sealer
to seal the spaces between the core of the filling and the root canal
walls. This core is mainly consist of the gutta percha (GP) which comes
into different forms, but still the GP cones are the most popular type3).
This is due to that these cones have several advantages including good
dimensional stability, radiopacity, thermoplasticity and biocompatibili-
ty; which make them the most desirable and first choice for root canal
filling4).
Despite the manufacturing of the GP cones is produced under asep-
tic condition and packed in well-sealed containers, they can be easily
contaminated either due to improper storage process or through han-
dling of the cones before obturation procedure. Additionally, healing
delay or inhibition due to contaminated GP cones have been previously
reported5). Therefore, sterilization of the GP cones should be performed
before usage6).
Because of the thermoplasticized property of GP, the heat steriliza-
tion cannot be achieved6). Cold sterilization by disinfectant chemical
solutions is a good alternative, fast acting and simple method used
during dental work. Different disinfectant solutions can be employed for
this purpose, such as ethyl alcohol, povidine iodine, chlorhexidine, sodi-
um hypochlorite, glutaraldehyde1).
Although, several investigations identify the presence of
Staphylococcus bacteria within the contaminated GP cones especially
during their handling with gloves6,7). The Enterococcus faecalis (E. fae-
calis) is still considered the gold standard microorganism to be chosen
in the antimicrobial researches, because of its superior virulence proper-

Received on March 31, 2021 and accepted on April 30, 2021

1) Department of Esthetic and Conservative Dentistry, College of Dentistry, Tikrit University
Tikrit, Iraq
2) Department of Restorative and Aesthetic Dentistry, College of Dentistry, University of Baghdad
Baghdad, Iraq
3) Department of Prosthodontic and Maxillofacial Prosthesis, College of Dentistry, Tikrit University
Tikrit, Iraq
Correspondence to Anas F Mahdee
(e-mail: a.f.mahde@codental.uobaghdad.edu.iq)

C 2021 Japan University of Health Sciences

& Japan International Cultural Exchange Foundation
4 Al-Jobory A. I. et al.
Table 1: Colony-forming units (CFU) for E. faecalis on day 0
and day 7 for all groups of the study.
Group No. Reagent Day 0 Day 7
I Control Complete growth Complete growth
II NaOCl 0 0
III CHX 0 3
IV Listerine 14 4
ty as the most resistance root canal microorganisms8).
Among different antimicrobial solutions, the sodium hypochlorite
(NaOCl) (5.25%) is still the cheapest, fastest, most effective, and most
popular one GP cones disinfectant6). Although there were no reported
chemical or physical changes on the tested GP cones have been previ-
ously reported after their disinfection with NaOCL9), this solution
should be finally rinsed out from the surface of the cones before using
then in the canal obturation. This is to remove the chlorine crystals that
were reported to be formed on the surface of GP cones, which may
interfere with the final root canal treatment outcomes10,11).
Another important disinfection solution is Chlorhexidine gluconate
(CHX) (2%) as an effective antimicrobial reagent for GP cones. It is a
broad spectrum antimicrobial agent with cationic molecular structure
that can be absorbed by the anionic bacterial cell membrane and causes
leakage of intracellular components12). At lower concentration (0.2%) it
action is bacteriostatic as in its use in the mouth washes. But at a higher
concentration (2%) it acts as an active bactericidal agent in root canal
irrigation and disinfectant of the GP cones13). However, the effective of
CHX has clear side effects like brown discoloration of the teeth, oral
mucosal erosion, and bitter taste12).
Listerine is also another type of mouth washes that has been effec-
tively used in the reduction of bacterial counts in the oral cavity and
dental plaque14,15). Additionally, there are no side effects present within
its use which makes it proper antimicrobial disinfectant solution14).
There were several reports about different antimicrobial disinfectant
solutions and protocols used for GP cones disinfections6,15,16). However,
only few studies comparing the short and long term effect of these anti-
microbial solutions in preventing the growth of vulnerable bacterial
types on the GP cones. Thus, the purpose of this in vitro study was to use
short and long term incubation period of contaminated GP cones after
treating them with three disinfectant solutions (5.25% sodium hypochlo-
rite (NaOCl), 2% chlorhexidine (CHx) and Listerine mouth wash).
MATERIAL AND METHODS
Three different disinfection solutions were used in this study includ-
ing: NaOH 5.25% (Cerkamed, Stalowa Wola, Poland), CHx 2%
(Cerkamed, Stalowa Wola, Poland) and Listerine antiseptic mouthwash
(Johnson & Johnson, Rome, Italy). Forty GP cones (Size F3, Dentsply
Maillefer, Ballaigues, Switzerland) from freshly opened boxes were
used in this study. These cones were sterilized with ethylene oxide gas
before leaving them for 1 minute on a dry gaze to let the vaporization of
ethylene oxide gas17). This is followed by the procedure of GP cones
contamination which was adopted from Kulkarni and Desai (18). This
procedure included the total immersion of GP cones in a broth media
containing E. faecalis (ATCC 29212) for 20 min before dividing them
equally into 4 groups (n = 10) as followed:
Group I (control) in which the contaminated GP cones received no
disinfection solution.
Group II: each GP cone was immersed completely in 5.25% NaOCl
solution for 1 minute within a sterile plastic tube.
Group III: the same as group 2 but the immersion solution was 2%
CHX solution.
Group IV: the same as group 2 but the immersion solution was
Listerine mouth wash.
After that, each sample was washed with 1 mL distilled water to
eliminate the excess of the disinfectant solution, before leaving them on
a sterile gauze for dryness at room temperature for one hour. Each cone
was individually transferred into a sterile Eppendorf tube containing 1.5
ml of nutrient broth for standardization purpose. All tubes were incubat-
ed at 37℃ for 7 days. The nutrient broth was prepared according to
Jyothsna, Kumar (19), in which 30 grams nutrient broth powder were
dissolved in 1 L of distilled water to prepare 30 g/L nutrient broth solu-
Figure 1: E. faecalis colonies with different groups of this study
at different time intervals. (Note: the number of groups
presented on the images of the agar plates is not repre-
senting the number of the groups determined in the
present study)
tion. The control samples follow the same procedures as the test sam-
ples. Before incubation time started and after 3 min from soaking GP
cones within broth media tubes, 100 μl of the nutrient broth was taken
from each tube and transferred into individual petri dishes containing
nutrient agar to identify the presence of viable bacteria immediately
after disinfection. The broth solution was spread in a thin layer above
the agar plate by using a sterile cotton swap. The plates were then incu-
bated at 37℃ in an aerobic condition for 24h. The colony count was
then counted and this was noted as day 0 measurements. The procedure
was repeated on the 7th day, where another 100 μl was pipetted from
each of the stored Eppendorf tubes and spread on new nutrient agar
plates to be incubated at 37℃ for 48h. The colony-forming units (CFU)
were then counted to be noted as day 7 measurements18).
RESULTS
The antimicrobial efficacy of the different disinfectant solution was
evaluated by the CFU numbers of the incubated E. faecalis bacteria at
two time intervals: day 0 (immediately after disinfection) and day 7 (after
incubation) (See fig 1). The data of CFU for all groups are presented in
Table 1. In the control group I, it is clearly obvious that the E. faecalis has
complete growth at day 0 and 7, when the GP cones were not treated with
any disinfectant solution. Inversely, NaOCl group II show complete inhi-
bition of the bacterial growth with (CFU = 0) at day 0 and 7 time inter-
vals. Similar data was obtained for CHX group III at the first time (0
day). However the CFU number of E. faecalis colonies for this group
increased to 3 units after 7 days. Interesting findings were obtained in the
Listerine group IV which shows the highest rate of growth at day 0
obtained within this study (CFU = 14) (as shown in table 1). A marked
decrease in CFU at day 7 was obtained reaching to 4 units only.
DISCUSSION
Despite the overarching aim for a successful endodontic treatment
is the complete debridement and asepsis of the root canal space, the bio-
logical complexity and limited accessibility may stand beyond this tar-
get. However, working in aseptic field, using sterile instruments, irrigat-
ing root canals with disinfectant solutions and obturation with a sterile
root filling material increase the rate of therapy success. Therefore, pre-
vention of the access of exogenous microorganism through disinfection
of the GP cones is essential to promote healing and prevent secondary
root pathosis18,19).
It's easy and possible that new GP cones may get contamination
with different microorganisms such as rods, cocci and yeast during
exposure to the dental environment though they were kept in a new ster-
ile sealed package6). Research also reported the presence of microbial
contamination between 5-19% of the newly opened gutta-percha packs.
Although, the number of these microorganisms were comparatively low
at the time of packaging, sometimes it is routinely for dentists to use
 Immediate and Delay Antimicrobial Activity of Three Disinfection Solutions on Gutta-percha Cones as Routine
Chair-side Procedure
gutta-percha points "straight from the box" without sterilization16). Also
the strong virulence of E. faecalis as the most resistant bacterial within
infected root canal against antimicrobial reagents6-8), was behind choos-
ing of E. faecalis to be the tested bacteria in this investigation.
Within the limitation of this study, all of the used disinfectant solu-
tions showed marked inhibition and /or prevention in E. faecalis colo-
nies formation within the two incubation periods of the study, in com-
parison to the control samples which had complete colonies growth.
This is in agreement with previous studies6,9,18), which either tested simi-
lar or different bacterial species.
The NaOCl (5.25% concentration) is the only tested solution within
the current study that showed complete prevention of E. faecalis colo-
nies growth after immediate or delay exposure times. Different concen-
trations of NaOCl (0.5%, 1%, 2.5% and 5.25%) were investigated6). The
5.25% concentration of NaOCl required from 15 second to 1 minute to
kill all microorganisms on the GP cones6). This agrees with the results of
this study which have shown an immediate killing of E. faecalis bacteria
after immersion of GP cones in 5.25% NaOCl for 1 min.
In the same way, this study also evaluate long incubation time of the
disinfected GP cones (7 days) within a broth media. This was to allow a
good time for any viable bacteria, if present, after GP cones disinfection
to multiply, to identify the efficacy of the disinfectant solutions18). Most
of the previous investigations used shorter incubation periods up to 48
h20,21), which could be insufficient for the accomplishment for the growth
cycle of the microorganisms. NaOCl (5.25%) used in this study demon-
strate complete E. faecalis colonies inhibition after the second incuba-
tion time (7 days). The potency of NaOCl as an efficient antimicrobial
and organic dissolving solution is related to the formation of active
chlorines. This anion may inhibit the germination and outgrowth of veg-
etative bacteria and spores, which makes NaOCl potent bactericidal and
veridical solution6). Chlorine precipitates was also reported on the sur-
face of GP cones after disinfection with NaOCl. These precipitates were
recommended to be washed out with distilled water to prevent any
future intervention with tissue healing process10,11).
The antimicrobial property of CHX was clearly obvious during the
immediate exposure which showed complete growth inhibition of E.
faecalis. This agrees with previous reports which found fast antimicro-
bial activity of CHX solution (within 15 sec --- 5 min) against the vege-
tative bacteria6,9,18,19). CHX is water soluble, relatively low toxicity, and
broad spectrum antimicrobial solution which have different application
in the field of endodontic6). It comes in different forms either solution or
gel and can be used as root canal irrigation, intracanal medication or GP
disinfectant. The antimicrobial action of CHX could be on disrupting
the bacterial cell membrane that inducing cytoplasmic precipitation22).
However, according to the results of the current study, after long incuba-
tion of the disinfected GP cones, the antimicrobial action of CHX solu-
tion was slightly reduced with time (CFU = 3). Although this reduction
is low, it should be considered for future studies to investigate the effect
of extended incubation time on the antimicrobial efficacy of different
GP disinfection materials.
Listerine is a well-known antimicrobial mouth wash composed of
eucalyptol oil, thymol, and synthetic derivatives of menthol and methyl
salicylate dissolved in ethanol alcohol23). The undiluted Listerine is an
efficient bactericidal mouth wash inhibits most of the viable oral and
plaque bacterial within 30 sec of exposure. However, clinically its
countable effect can be detected within continuous 2 weeks exposure24).
The current investigation was the first that used Listerine as a disinfec-
tion solution for GP cones. The results have shown that Listerine had
less immediate inhibition of E. faecalis colonies growth in comparison
to the other tested antimicrobial solution. This could be similar to the
action of Listerine mouth wash against oral bacteria which has lower
potency in comparison to CHX24). However, after 7 days incubation the
preventive efficacy of Listerine against E. faecalis colonization has been
raised (CPU = 4). This could be due to the antimicrobial action of
Listerine against E. faecalis may require an extended time to increase its
antibacterial activity. Additionally, because of the lack information
about the possible cause for such antibacterial behavior of Listerine, fur-
ther investigations can be suggested.
Finally, the GP cones contamination is more likely to be poly-mi-
crobial rather than mono-microbial. Therefore, the antimicrobial effect
of different disinfectant solutions on different types of microorganisms
such as other bacterial species, virus, yeast or fungi can be considered
for future investigations.
CONCLUSIONS
According to the findings of the present study, as routine chair side
5
disinfection of gutta percha cones, the best antimicrobial (anti-E. faeca-
lis) solution was NaOCl followed by CHX. Listerine showed more
potent results after extended incubation time.
ACKNOWLEDGEMENT
The Authors would like to thank Prof. Hadeel M Younus for her
technical contribution to this study.
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