Journal of Indian Society of Pedodontics and Preventive Dentistry | Apr-Jun 2014 | Vol 32| Issue 2 | 91

ABSTRACT
Background: The removal of plaque is utmost
important to control dental caries. But in
children, factors like lack of dexterity, individual
motivation and monitoring limit the effectiveness
of tooth brushing. This necessitates the use of
chemotherapeutic agents for control of plaque.
Aims: To compare the antimicrobial efcacy of
0.2% chlorhexidine mouth rinse and mouth rinse
containing 0.03% triclosan, 0.05% sodium uoride,
and 5% xylitol in reducing the Mutans streptococcus
count in plaque. Materials and Methods: Thirty
healthy children aged 8-10 years with dmft (decay
component) of three or four were selected. They
were divided randomly into two groups: The control
or chlorhexidine group and the study group or
combination mouth rinse. Both the groups practiced
rinsing with respective mouth wash for 1 min for 15
d twice a day. The plaque samples were collected
and after incubation Mutans streptococcus count
was estimated on the strips from the Dentocult
SM kit and evaluated using manufactures chart.
Statistical Analysis Used: Wilcoxon matched pairs
signed ranks test and MannWhitney U test were
used to analyze the ndings. Results: Statistically
signicant reduction in the Mutans streptococci
count in the plaque was seen in the control and
study group from baseline level. But when both the
groups were compared, the antimicrobial effect of
chlorhexidine was more.
KEYWORDS: Dental plaque, chlorhexidine,
combination mouth rinse, triclosan, xylitol, Mutans
streptococci, Dentocult SM strip
Comparison of antimicrobial efcacy of chlorhexidine
and combination mouth rinse in reducing the Mutans
streptococcus count in plaque
Laxmi S. Lakade, Preetam Shah
1
, Dayanand Shirol
2
Assistant Professor,
1
Professor, Pediatric and Preventive Dentistry, Bharati Vidyapeeth Deemed University Dental College and Hospital,
Pune,
2
Professor, Pediatric and Preventive Dentistry, M. A. Rangoonwala Dental College and Hospital, Pune, Maharashtra, India
Introduction
Modern concepts consider caries as an interaction
between genetic and environmental factors in which
social, behavioral, psychological, and biological factors
are expressed in a highly complex interactive manner.
[1]

But the important part in the understanding of the caries
process is that it does not occur in the absence of dental
plaque or dietary fermentable carbohydrate hence, is
considered a dietobacterial disease.
[2]
The role played by bacteria in initiation of dental caries and
periodontal diseases
[3]
is well established. The removal of
plaque is utmost important to control dental caries that
is commonly maintained by mechanical methods. But
in children, factors like lack of dexterity and individual
motivation and monitoring limit the effectiveness of tooth
brushing. They also experience difculty in maintaining
adequate plaque control, particularly at interproximal
sites which necessitates the use of chemotherapeutic
agents for control of plaque.
[4]
Among the chemotherapeutic agents used in
mouthwashes, chlorhexidine is the gold-standard or
positive control for comparison with other substances
due to its proven efciency.
[5,6]
Though effective, it
has certain side effects like brown discoloration of the
teeth, oral mucosal erosion, and bitter taste. Hence,
there is need of an alternative mouth rinse that could
negate all the side effects of chlorhexidine, but yet
effective equivalent to it.
Address for correspondence:
Dr. Laxmi S Lakade,
Patang plaza tower building phase 6, Flat no 1, Opp- Pune
Institute of Computer Technology College,
Katraj, Pune - 411 046, Maharashtra, India.
E-mail: pedolax@gmail.com
Original Article
Access this article online
Quick response code
Website:
www.jisppd.com
DOI:
10.4103/0970-4388.130780
PMID:
******
Lakade, et al.: Efcacy of Chlorhexidine vs Combination mouth rinse
Journal of Indian Society of Pedodontics and Preventive Dentistry | Apr-Jun 2014 | Vol 32| Issue 2 | 92
Therefore, this study was carried out to evaluate the
antimicrobial efcacy of 0.2% chlorhexidine mouth
rinse and mouth rinse containing 0.03% triclosan,
0.05% sodium uoride, and 5% xylitol in reducing the
Mutans streptococci count in plaque.
Materials and Methods
The randomized, controlled, and double-blind study
was carried out in the 30 healthy children aged 8-10 years
from the Department of Pedodontics and Preventive
Dentistry, Bharati Vidyapeeth Dental College, Pune.
Children with high caries risk experience of dmft of
three or four (decay component) were selected.
Exclusion criteria were children with physical
limitations, which might preclude the normal tooth
brushing and mouth rinsing, intraoral soft tissue
pathology, medically compromised patients, and
subjects with history of taking antibiotics three
months before or during the course of study. Subjects
undergoing orthodontic treatment or with extensive
intraoral prosthesis, children who had previously
restored/crowned teeth and children having teeth with
periapical pathology were excluded from the study.
The study was explained to parents and consent
was obtained. Childs personal details, details of
past medical history including any recent antibiotic
exposure, past dental history including recent uoride
treatment, frequency of brushing, sweets/snacks intake
and consumption of sugared/energy drinks, and the
brand of toothpaste (to determine uoride content)
were obtained from parents through questionnaire.
The Mutans streptococci count in plaque was
determined by using the chairside method (Dentocult
SM Strip mutans Orion Diagnostica, Espoo, Finland)
[Figure 1]. This test is based on the principle of use of
a selective culture broth, the adherence and growth of
Mutans streptococcus bacteria on the test strip.
The procedure of using this test was in accordance to
the manufacturer. Before collecting the plaque samples,
the vials were brought down to room temperature 1
h before use and shaken gently. Using a forceps, two
bacitracin discs were placed in the selective culture
broth about 15 min before sampling [Figure 2].
Isolation with cotton rolls was done. Plaque samples
were collected using different toothpicks from the
four sites enamel buccal surface of the maxillary
right molar, labial surface of the maxillary incisor,
labial surface of the mandibular incisor, and lingual
surface of the mandibular left molar. These samples
were spread thoroughly but gently on the four sites
of the rough surface of the strip [Figure 3]. The
selective culture vial was gently shaken for even
distribution of bacitracin, and the strip was placed
in the selective culture broth. The vials were then
labeled with the numbers and incubated in an
upright position at 37C for 48 h with one quarter
of the cap turned open to allow growth of the
organisms. Following incubation, the presence of
Mutans streptococcus was conrmed by dark-blue
to light-blue raised colonies [Figure 4].
Inspection of growth was done sideways against light
or with a magnifying glass to look for raised colonies.
Colonies suspended in the culture broth were excluded
from the evaluation as suggested by manufacturer
of Dentocult SM Strip mutans. The results were
interpreted according to the manufactures model
density chart and classied as
Class 0: < 10,000 CFU/ml*
Class 1: < 100,000 CFU/ml
Class 2: 100,000-1,000,000 CFU/ml
Class 3: > 1,000,000 CFU/ml
*CFU/ml colony forming unit/milliliter
Inspection of the growth was done with the strip held
sideways against light and magnifying glass. The
presence of epithelial cells on the strip surface can be
differentiated from the Streptococcus mutans colonies by
Figure 1: Dentocult SM strip mutans kit Figure 2: Bacitracin disc placement
Lakade, et al.: Efcacy of Chlorhexidine vs Combination mouth rinse
Journal of Indian Society of Pedodontics and Preventive Dentistry | Apr-Jun 2014 | Vol 32| Issue 2 | 93
passing a gloved nger along the strip: The epithelial cells
are smooth, whereas the S. mutans colonies are rough.
After the collection of baseline status of plaque samples,
the subjects were randomly divided into two groups,
15 children in each group. The control group was
advised to rinse with 10 ml of 0.2% chlorhexidine
gluconate (Hexidine, ICPA products Ltd) in the morning
and in the night after brushing for l min

for 15 d under
parents supervision. The study group was advised to
rinse with 5 ml of combination mouth rinse containing
0.03% triclosan, 0.05% sodium uoride, and 5% xylitol
(Kidodent, Warren) in the morning and in the night after
brushing for l min for 15 d under parents supervision.
After rinsing, the children were advised not to eat or drink
for 30 min. During the course of the study, the children
were asked to use the non-uoridated tooth paste and the
new brush (Colgate Zig Zig Junior) that was provided
to them to prevent microbial contamination. The subjects
in both the groups were blinded about the division
of group.
After 15 d of using mouth rinse, the plaque samples
were again collected, incubated, and interpreted in
similar manner as taken for baseline status to assess
change in count level of Mutans streptococcus. The
data obtained from the study were tabulated and
analyzed statistically with Wilcoxon matched pairs
signed ranks test and MannWhitney U tests.
Results
There was no difference in total number of Mutans
streptococci at baseline level in both the groups as seen
in Table 1.
Table 2 shows statistically signicant reduction (P = 0.001)
in mean Mutans streptococci count after rinsing with
0.2% chlorhexidine when compared with baseline count.
Table 3 shows statistical signicant reduction
(P = 0.002) in mean Mutans streptococci count after
rinsing with combination mouth rinse when compared
with baseline count.
Table 4 when both the groups were compared 0.2%
chlorhexidine reduced more number of Mutans
streptococcus then the other group. Therefore, 0.2%
chlorhexidine was more effective antimicrobial agent.
Discussion
Many children have inadequate oral and general
health because of active and uncontrolled dental
caries. It is the single most common chronic childhood
disease. Owing to its non-life-threatening nature and
ubiquitousness has minimized its signicance in
Figure 3: Spreading plaque sample on strip
Figure 4: Mutans streptococci growth on four sites of strip
Table 1: Mutans streptococci level at baseline in
30 subjects
Mutans streptococciin
30 children
Mean score SD mean (Prerinse)
2.540.585
SD = Standard deviation
Table 2: Comparison of mutans streptococcus
score in plaque in chlorhexidine group with
respect to prerinse and postrinse
Chlorhexidine mouth rinse Mean score SD mean P-value*
Prerinse (0 d) 2.680.121 0.001
Postrinse (15
th
d) 1.610.140
*Wilcoxon matched pairs signed ranks test was used to calculate the
P-value; SD = Standard deviation
Table 3: Comparison of mutans streptococcus
score in plaque in combination mouth rinse
group with respect to prerinse and postrinse
Combination mouth rinse Mean score SD mean P-value*
Prerinse (0 d) 2.40.163 0.002
Postrinse (15
th
d) 1.410.128
*Wilcoxon matched pairs signed ranks test was used to calculate the
P-value; SD = Standard deviation
Lakade, et al.: Efcacy of Chlorhexidine vs Combination mouth rinse
Journal of Indian Society of Pedodontics and Preventive Dentistry | Apr-Jun 2014 | Vol 32| Issue 2 | 94
overall human health. Initiation of dental caries and
the microbial composition of plaque have generally
involved either S. mutans or Lactobacilli. Children with
high dmft have increased S. mutans count. As a result
variety of anti-plaque agents has been examined for
their ability to control S. mutans.
Among the chemotherapeutic agents used in
mouthwashes, chlorhexidine is the gold-standard or
positive control for comparison with other substances
due to its proven efciency.
[5,6]
Along with antibacterial
effect, its main advantage is substantivity. But due to
its known disadvantages, there is need for ideal anti-
plaque agent, which is not yet available. Hence, this
study evaluated the efcacy of two mouth rinses to
reduce S. mutans. An approach to increase the efcacy
of anti-plaque agent and to reduce the adverse effects
may be to combine two or more agents. Triclosan is
broad spectrum antimicrobial activity and is effective
against S. mutans at low concentration.
[7]
The effects
of uoride on bacterial metabolism are well-known.
Fluorides inhibit several essential enzymes in oral
bacteria as stated by Hamiton and Bowden in 1988.
[8]
Mellberg and Ripa
[9]
suggested that low potency-high
frequency rinsing may be more benecial. Xylitol is
non-sugar sweetener permitted for use in food.
[10]

Hence, combination mouth rinse of 0.03% triclosan,
0.05% sodium uoride, and xylitol was used.
In this study, a chairside test the Strip mutans
test was used that was developed by Jensen and
Bratthall.
[11]
The conventional technique
[12-15]
involves
laborious laboratory steps. Hence, it lacks widespread
use in routine dental practice. The sensitivity, specicity,
and accuracy of the Dentocult SM was found to be
better than those of conventional methods.
[1]
In their
study, the plaque test surpassed the salivary strip test
in terms of sensitivity and accuracy when both were
compared. Therefore, plaque collection was preferred
over saliva collection in the present study.
There was no difference in total number of Mutans
streptococci at baseline level in both the groups as seen
in Table 2. Similar nding was observed by Neeraja
et al.,
[16]
This can be explained by the fact that dmft
(decay component) was standardized.
[16]
A total of 0.2% chlorhexidine signicantly reduced
[Table 3] Mutans streptococci count. Its action is due
to adsorption of chlorhexidine onto the cell wall of the
microorganism, resulting in a leakage of intracellular
components.
[17]
At low concentration, chlorhexidine a
small molecular weight substance such as potassium
and phosphorus, will leach out, exerting a bacteriostatic
effect. At higher concentrations, chlorhexidine is
bactericidal because of precipitation or coagulation
of the cytoplasm, probably caused by protein cross-
linking.
[18]
But bactericidal effect is thought to be less
important than the bacteriostatic effect provided by a
slow release of chlorhexidine.
[19]
This observation adds to earlier studies carried by
Happonen et al.,
[20]
Hef and Huber
[21]
Kulkarni and
Damle
[22]
Neeraja et al.,
[16]
noted immediate signicant
reduction in Mutans streptococci in chlorhexidine
group, but after 15 d therapy there was an increase in
Mutans streptococcus count. But this increase was less
in chlorhexidine as compared with povidone-iodine.
Whereas Kulkarni and Damle
[22]
found the signicant
reduction in Mutans streptococcus in chlorhexidine
and triclosan group than control group after 2 weeks
of rinsing. Many studies have shown that it is not
possible to devoid the mouth completely of S. mutans
for an extended period, no matter how rigorous or
extended the application.
[23-25]
So, complete elimination
of Mutans streptococcus (or Class 0) was not seen in
any children at the end of 15 d in our study also.
The combination mouth rinse [Table 4] also showed
signicant reduction in Mutans streptococcus count
due to effects of its components. Triclosan has the
antimicrobial action. Owing to its hydrophobic and
lipophilic nature, it adsorbs to lipid portion of the
bacterial cell membrane and in low concentrations it
interferes with vital transport mechanism.
[26]
Fluoride,
which is another component, is a powerful inhibitor
of acid formation by plaque microorganisms.
[27]

The current evidence indicates that uoride has a
multitude of direct and indirect effects on the bacterial
cell, which have a signicant inuence on those
organisms in dental plaque.
[28]
Enzyme enolase in
glycolytic pathway of the carbohydrate metabolism is
uoride sensitive.
[28]
Guha-Chowdhury et al.,
[29]
found
that it inhibits the streptococcal enolases also.

Maguire
and Rugg-Gunn
[30]
described the action of xyiltol as
non-fermentability and non-cariogenicity as passive
effects, whereas active caries prevention effects as
bacteriostatic and cariostatic.
However, in this study 0.2% chlorhexidine showed a
greater reduction of Mutans streptococcus count than
combination mouth rinse [Table 4]. Similarly Sharma
et al.,
[31]
found 0.2% chlorhexidine most effective than
combination mouthwash containing 0.03% triclosan
and 0.05% sodium uoride.
The high efcacy of chlorhexidine could be due to
its immediate bactericidal action during the time of
application followed by a prolonged bacteriostatic
Table 4: Comparison of postrinse mutans
streptococcus score with respect to chlorhexidine
group and combination mouth rinse group
Postrinse (median rank)
Chlorhexidine Combination
mouth rinse
P-value*
Total microbe
score
1.5 2 0.023
*MannWhitney U test used to calculate the P-value
Lakade, et al.: Efcacy of Chlorhexidine vs Combination mouth rinse
Journal of Indian Society of Pedodontics and Preventive Dentistry | Apr-Jun 2014 | Vol 32| Issue 2 | 95
action due to adsorption at the tooth surface.
[32]
The
adsorbed (substantivity) chlorhexidine is gradually
released for up to 24 h.
[33]
A total of 0.2% chlorhexidine
inhibits acid production for 24 h after sucrose applied
to dental plaque in vivo.
[34]
Studies
[35-37]
showed that chlorhexidine signicantly
reduced the bacterial numbers upto 7 h, and effect
persists for many hours.
[38]
Perhaps more importantly
there was no evidence of bacterial regrowth following
chlorhexidine as indicated by negative increments.
Such was not the case for triclosan, the incremental
values were positive from 60 min indicating recovery
of bacterial counts.
[35]
Triclosan has plaque inhibitory
effects mediated by an antimicrobial action, which
would not be much different from that of sodium
lauryl sulfate.
[39]
Therefore, triclosan itself has
moderate plaque-inhibitory effect.
[7]
Limited potential
for absorption and reduced substantivity of triclosan
compared with chlorhexidine.
[32,40]
There is evidence
indicating that the ingredients, vehicle, and other active
substances may inuence its antimicrobial activity and
consequently its efciency of triclosan.
[41]
Effects of
uoride are well established as anticaries process. But
certain studies state that uoride preparations (except
SnF
2
and amine

uorides) seem to have little effect
on the quantity of plaque and thereby antimicrobial
effect.
[7]
Therefore, more extensive studies with larger samples
and varied time periods should be carried out to
establish the efcacy of combination mouth rinse.
Summary and conclusions
Apparently, the ideal anti-plaque agent is not available.
Any chemical agent that affects microbial cells may be
expected to have some adverse effects against host
cells, unless the target structure or metabolic pathway
is unique to the microbial cell. But plaque is a complex
aggregation of various bacterial species. Thus, no
single agent can be effective in complete elimination
of plaque. Combining two or more agents will
compliment the modes of action resulting in additive
or synergistic effects, with minimal adverse effects.
For effective anti-plaque agents to be developed, it is
crucial for the target structures to be dened, and the
exact modes of action of the potential agents be known.
Conclusions from this study:
A total of 0.2% chlorhexidine gluconate was a more
effective antimicrobial agent than mouth rinse
containing 0.03% triclosan, 0.05% sodium uoride,
and 5% xylitol in reducing the Mutans streptococci
count in plaque.
Although the chemical anti-plaque agents are effective
in reducing the microbial count in plaque but are only
as an adjunct to mechanical modes of plaque control
and not the substitutes.
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How to cite this article: Lakade LS, Shah P, Shirol D.
Comparison of antimicrobial efcacy of chlorhexidine and
combination mouth rinse in reducing the Mutans streptococcus
count in plaque. J Indian Soc Pedod Prev Dent 2014;32:91-6.
Source of Support: Nil, Conflict of Interest: Nil.
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