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Jundishapur Journal of Microbiology (2010); 3(1): 15-27 15

Original article

The antimicrobial potential of ten often used mouthwashes against four

dental caries pathogens
Kamal Rai Aneja, Radhika Joshi, Chetan Sharma
Department of Microbiology, Kurukshetra University, Kurukshetra-136119, Haryana, India

How to cite this article:

Aneja KR, Joshi R, Sharma C. The antimicrobial potential of ten often used mouthwashes against four dental
caries pathogens. Jundishapur J Microbiol. 2010; 3(1): 15-27.

Received: November 2009 Accepted: January 2010

Abstract
Introduction and objective: Increasing number of people are using mouthwashes for
general and oral health care. Few of these mouthwashes, however, have undergone rigorous
testing, as evidenced by the limited amount of information on their safety and efficacy in the
literature. The aim of this study was to determine the antimicrobial properties of ten
commonly available mouthwashes against four oral pathogens related to caries and to oral
fungal infections, to verify the claims made by the manufacturers to provide information to
dental professionals about the efficacy of their products in vitro and to use these
mouthwashes as a base for the evaluation of antimicrobial plant products.
Materials and methods: The authors used two different techniques: microbial growth in
nutrient broth by turbidity measurement and an agar well diffusion method to evaluate the
antimicrobial effectiveness of ten often used mouthwashes against four microorganisms:
Streptococcus mutans and Staphylococcus aureus (bacteria), Candida albicans and
Saccharomyces cerevisiae (fungi). Nutrient broth without mouthwash and sterile distilled
water served as the control respectively in the two techniques.
Results: Hexidine mouthwash emerged as the most effective mouthwash [maximum mean
diameter of inhibition zone against S. aureus (28.3mm to 33.9mm) followed by S. mutans
(23.6mm to 26mm), S. cerevisiae (20.6mm to 26.3mm) and minimum against C. albicans
(11.9mm to 22.9mm)] followed by Chlohex and Triguard, all of which had excellent level of
activity. Following Triguard were Zytee, Chlohexplus, Hexnor and Chlorhexidine that
showed good antimicrobial activity and finally, displaying very little antimicrobial activity
was Listerine while Toss-K and Senquel-AD totally lacked antimicrobial activity.
Conclusion: Hexidine mouthwash (ICPA Health Products Ltd., Ankleshwar, India) showed
excellent antimicrobial activity against the four dental caries causing microorganisms in
vitro. The six mouthwashes found to be effective against all the four tested microorganisms
at all the four concentrations, comprising of Chlorhexidine gluconate as the basic
constituent, presented different antimicrobial activities.

Key words: Dental caries, Antimicrobial activity, Zone of inhibition, Microbial growth
inhibition, Mouthwashes, Chlorhexidine gluconate

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Introduction the majority of the newer mouthwashes also

Despite great improvements in the global claim to have antiseptic properties [5,13,15-
oral health status, dental caries still remains 17]. While many manufacturers claim that
one of the most prevalent diseases [1]. The their mouthwashes have antimicrobial
early stage of dental caries is characterized properties, the aim of this study was to
by a destruction of superficial dental determine the antimicrobial properties of
structures caused by acids which are by- ten commonly available mouthwashes
products of carbohydrate metabolism by against four oral pathogens related to caries
Streptococcus mutans, a cariogenic and to oral fungal infections {since these
bacterium [2]. Colonization of teeth by organisms have now gained more
cariogenic bacteria is one of the most importance due to the increased incidence
important risk factors in the development of of AIDS/HIV [18]}, to verify the claims
dental diseases [2]. S. mutans and Candida made by the manufacturers to provide
albicans are the two microbes often information to dental professionals about
implicated in oral diseases, C. albicans is the efficacy of their products in vitro and to
the most common yeast isolated from the use these mouthwashes as a base for the
oral cavity and a common cause of oral evaluation of antimicrobial plant products.
thrush, endocarditis, septicemia, vaginitis
and infection of skin, nails and lungs [3-5]. Materials and methods
It is by far the fungal species most Collection of mouthwashes
commonly isolated from infected root Ten often used mouthwash products (Table
canals, showing resistance to intercanal 1) were purchased from the drug stores of
medication [6,7]. Staphylococcus aureus is Kurukshetra and Gurgaon, Haryana, India.
a major human pathogen, responsible for a
number of hospital-acquired infections, Test microorganisms
initially colonizes several locations in the The test microorganisms S. mutans (MTCC
human body, but the mouth and hands are *497), S. aureus (MTCC 740), C. albicans
the main reservoirs for propagation of this (MTCC 227) and Saccharomyces cerevisiae
pathogen in the hospital environment [8- (MTCC 170) were procured from MTCC,
10]. IMTECH, Chandigarh. These were
Individuals heavily colonized by subcultured on specific media, procured
cariogenic bacteria are considered to be at from HiMedia Laboratory Pvt. Ltd.,
high risk for dental caries. Hence Bombay, India, recommended for different
eradication of these microorganisms is microorganisms such as Brain Heart
important for dental treatment [11]. Infusion Agar, BHI (S. mutans), Nutrient
Prevention of oral diseases is easier than a Agar (S. aureus) and Malt Yeast Agar (C.
cure. The widespread use of mouthwashes albicans and S. cerevisiae) and incubated
as an aid to oral hygiene is a relatively aerobically at 37oC. The identification of all
recent phenomenon in the developing the microbes was confirmed by standard
countries of the world. Development work biochemical and staining methods [19-21].
on the mouthwashes has been done mostly
by the manufacturers, and the little work Screening for antimicrobial activity
that has been done relates to the individual Antimicrobial effectiveness of various
ingredients they contain rather than to their mouthwashes was assessed by using two
complete formulations [12-14]. techniques:
While their primary appeal is as an aid Turbidity measurement by spectrophoto-
to breath freshness and cleansing the mouth, meter: One percent of nutrient broth

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(HiMedia Ltd.) was prepared containing a optical density was measured by

10% concentration of the mouthwashes. spectrophotometer at a wavelength of
After autoclaving, the broth and 490nm as a guide to microbial growth. The
mouthwashes were inoculated with 100µl of experiments were performed in triplicates
the microbial inoculums adjusted equal to for each mouthwash and the mean for each
106cfu/ml (with turbidity equating to a test microorganism was calculated. Broth
McFarland standard of 0.5) and were without mouthwash was used as control
incubated aerobically at 37°C for 24h. The [13].
inoculated broths were suspended and their

Table 1: Ingredients of various mouthwashes tested for antimicrobial potential

Name Batch Expiry date Manufacturer Ingredients as listed on packages

number
Listerine (cool 7208 December Pfizer Limited, Kolhapur, Thymol 0.06%, Eucalyptol
mint) 2010 India 0.09%, Menthol 0.04%, Ethanol
21.6%v/v.
Chlorhexidine ZM-701 March Blue Cross Laboratories Chlorhexidine gluconate 0.2%w/v
2010 Ltd., Nasik, India in pleasantly flavored aqueous
base
Toss-K TK-4316 March Ind-Swift Limited, Potassium nitrate 3%w/v, sodium
2010 Chandigarh, India fluoride 0.2%w/v in pleasantly
flavored aqueous base
Zytee W6021 December Raptakos, Brett and Co. Clove oil 1%, Mentha oil 1%,
2010 Ltd., Mumbai, India Menthol 1%, Chamomile oil
0.015%, Sodium benzoate 2%,
Ethanol 31%v/v
Hexnor EHN-001 December Dynor Pharmaceuticals Chlorhexidine gluconate
2010 Pvt. Ltd., Delhi, India 0.2%w/v, Sodium
fluoride0.05%w/v, Zinc chloride
0.09%w/v
Senquel-AD BSD 6028 December Dr. Reddy’s Laboratories Potassium nitrate 3%w/v, Sodium
2012 Ltd., Hyderabad, India fluoride 0.2%w/v in pleasantly
flavored aqueous base
Hexidine AU8059 December ICPA Health Products Chlorhexidine gluconate 0.2%w/v
2012 Ltd., Ankleshwar, India in pleasantly flavored aqueous
base
Chlohex BCX 6023 March Dr. Reddy’s Laboratories Chlorhexidine gluconate 0.2%w/v
2011 Ltd., Hyderabad, India in pleasantly flavored aqueous
base
Chlohex plus BCP 7008 March Dr. Reddy’s Laboratories Chlorhexidine gluconate 0.2%
2011 Ltd., Hyderabad, India w/v, Sodium fluoride 0.05%w/v,
Zinc vhloride 0.09%w/v in
pleasantly flavored aqueous base
Triguard RKR8022 December FDC Ltd., Aurangabad, Chlorhexidine gluconate 0.2%,
2010 India Sodium fluoride 0.05%, Zinc
chloride 0.09% in pleasantly
flavored base

Agar well diffusion method: The sterile distilled water as the diluent, using
mouthwashes were tested at four different agar well diffusion method or cup plate
concentrations: 1:4(25%), 1:1(50%), method [22-23]. In this method, pure isolate
3:4(75%) and full strength (100%), taking of each microbe was subcultured on the

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recommended specific media for each deviation were calculated. Nutrient broth
microorganism at 37οC for 24h. From each without mouthwash in turbidity measure-
inoculated agar plate, a minimum of four ment method and sterile distilled water in
colonies were touched with a sterile loop agar well diffusion method were used as
and transferred into a tube containing negative control.
normal saline (0.85%) and density of each
microbial suspension was adjusted equal to Results
that of 106 cfu/ml (standardized by The mouthwashes were measured at 10%
0.5McFarland standard) and was used as the concentration for turbidity by spectrophoto-
inoculum [23-27]. meter, Hexidine showed no turbidity at all
A 100µl volume of each mouthwash thus having excellent activity, while
concentration (full strength, 3:4, 1:1, 1:4) Chlohex and Triguard showed very little
and the control was propelled directly into turbidity thus having equally good activity
the wells (in triplicates) of the inoculated against all the microorganisms. Zytee and
specific media agar plates for each test Chlohex plus possessed comparatively
organism. The plates were allowed to stand lesser potential while Hexnor,
for ten minutes for diffusion of the Chlorhexidine and Listerine showed
mouthwash to take place and incubated at average ability to inhibit the microbial
37οC for 24h, 48h and 72h [28-29]. The growth. Toss-K and Senquel-AD showed
antimicrobial activity, indicated by an even more turbidity than the control, thus
inhibition zone surrounding the well showing total inability to control the dental
containing the mouthwash, was recorded if caries pathogens. Figure 1 shows the
the zone of inhibition was greater than 8mm antimicrobial activity of ten mouthwashes
[30]. The experiments were performed in against S. mutans in BHI broth by
triplicates and the mean values of the measuring the optical density.
diameter of inhibition zones with ± standard

Fig. 1: Microbial growth of S. mutans in Brain heart infusion broth with different mouthwash
substitutes: summary of optical density data (three sets of experiments)

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Although few negligible changes in the and Chlohex plus (Dr. Reddy’s
inhibition zones were observed in some Laboratories Ltd., Hyderabad, India) and
mouthwashes after 48h and 72h, most of the Triguard (FDC Ltd., Aurangabad, India)
antimicrobial activity was observed, in all produced consistent antimicrobial activity
the ten mouthwashes tested, during the against all the four test organisms i.e. S.
initial 24h of incubation when tested by mutans, S. aureus, S. cerevisiae and C.
agar well diffusion method. Six test albicans at all the four test concentrations-
mouthwashes, namely Chlorhexidine (Blue full strength, 3:4, 1:1 and 1:4 (Fig. 2). At
Cross Laboratories Ltd., Nasik, India), 3:4, 1:1 and 1:4 dilutions, the differences
Hexnor (Dynor Pharmaceuticals Pvt. Ltd., among the ten test mouthwashes shown to
Mumbai, India), Hexidine (ICPA Health inhibit the growth of microorganisms at full
Products Ltd., Ankleshwar, India), Chlohex strength became less evident (Table 2).

40
Mean diameter of inhibition zones (mm)

35

30

25 Streptococcus mutans
Staphylococcus aureus
20
Candida albicans
15 Saccharomyces cerevisiae

10

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Mouthwashes (Full strength)

Fig. 2: Mean diameter and standard deviation of zones of microbial inhibition exhibited by ten
mouthwashes after 24h at full strength (100%concentration) against four microorganisms (Bar
indicates standard deviation)

Out of these, Hexidine showed the highest tested mouthwashes, Zytee (Raptakos, Brett
antimicrobial activity against all the four and Co. Ltd., Mumbai, India) showed
microorganisms, the maximum inhibition inhibition of S. aureus at all the four
zone produced against S. aureus (28.3mm concentrations ranging between 14mm and
to 33.9mm) followed by S. mutans (23.6mm 31.3mm at 24h which gradually reduced to
to 26mm), S. cerevisiae (20.6mm to 25.9mm at 48h and further 20.6mm at 72h,
26.3mm) and minimum against C. albicans thus showing the bacteriostatic nature of
(11.9mm to 22.9mm), at different this mouthwash. Zytee showed inhibitory
concentrations with the maximum activity against S. mutans at 1:1, 3:4 and
inhibition zone being produced at full full strength ranging between 10.3mm and
strength (Fig. 3(a, b, c, d)). One of the 13.9mm but had no inhibitory effect on the

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two fungi C. albicans and S. cerevisiae. inhibitory effect against any of the four
Another tested mouthwash Listerine cool microorganisms at any of the four tested
mint (Pfizer Ltd., Kolhapur, India) showed concentrations. The inhibitory activity of
inhibitory zones against the two bacteria S. Hexnor against S. aureus was found to
mutans and S. aureus ranging increase from 27.3mm to 30.3mm at 3:4
between12.6mm and 18.6mm at all the four concentration and from 29.9mm to 33.0mm
concentrations. It produced zones of at full strength when measured after 72h of
inhibition ranging between 10.3mm and incubation. Rest all mouthwashes (except
12.6mm against C. albicans and S. Zytee) showed almost the same zone
cerevisiae at 1:1, 3:4 and full strength but diameter after 24h, 48h or 72h, while the
no zone of inhibition against the two yeasts zone diameter increased slightly in all the
at 1:4 concentrations (Table 2). mouthwashes when moving from 1:4
Two of the ten tested mouthwashes, concentration to full strength showing that
Toss-K (Ind-Swift Ltd., Chandigarh, India) full strength is the most effective
and Senquel-AD (Dr. Reddy’s Laboratories concentration against all the tested
Ltd., Hyderabad, India) did not show any microorganisms.

(a) (b)

(c) (d)
Fig. 3: Zones of inhibition produced by Hexidine mouthwash at 24h against the four tested
microorganisms at four different concentrations and control (a) S. mutans, (b) S. aureus, (c) S.
cerevisiae and (d) C. albicans, (C= Control, F.S. = Full Strength)

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Table 2: Antimicrobial activity of ten mouthwashes against four dental caries pathogens (bacteria and yeasts) determined by agar well diffusion method

Mouthwash Mean diameter of growth of inhibition zones (mm)

Concentratio- S. mutans S. aureus C. albicans S. cerevisiae
ns tested 24h 48h 72h 24h 48h 72h 24h 48h 72h 24h 48h 72h
Control - - - - - - - - - - - -
Listerine 1:4 12.6a ± 12.6 ± 12.5 ± 15.6 ± 15.6 ± 15.6 ± - - - - - -
(cool mint) 0.57b 0.57 0.57 0.57 0.57 0.57
1:1 12.6 ± 12.6 ± 12.6 ± 16.2 ± 16.2 ± 15.9 ± 10.3 ± 10.3 ± 10.3 ± 10.6 ± 10.6 ± 10.6 ±
0.57 0.57 0.57 1.52 1.52 1 0.57 0.57 0.57 0.57 0.57 0.57
3:4 13.6 ± 13.6 ± 13.6 ± 16.3 ± 16.3 ± 16.3 ± 10.6 ± 10.6 ± 10.6 ± 11.9 ± 11.9 ± 11.9 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 1 1 1
F.S. 14.3 ± 14.3 ± 14.3 ± 18.6 ± 18.6 ± 18.6 ± 11.6 ± 11.6 ± 11.6 ± 12.6 ± 12.6 ± 12.6 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 1 1 1
Chlorhexidine 1:4 18.3 ± 18.3 ± 17.9 ± 23.6 ± 23.6 ± 23.6 ± 13.3 ± 13.3 ± 13.3 ± 15.9 ± 15.9 ± 16.0 ±
0.57 0.57 1 0.57 0.57 0.57 0.57 0.57 0.57 1 1 0
1:1 19.6 ± 19.6 ± 19.6 ± 25.3 ± 25.3 ± 25.3 ± 16.3 ± 15.9 ± 15.9 ± 18.3 ± 18.3 ± 18.3 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 1 1 0.57 0.57 0.57
3:4 20.3 ± 20.3 ± 20.3 26.3 ± 26.9 ± 27.3 ± 18.0 ± 18.0 ± 17.9 ± 20.6 ± 20.6 ± 20.6 ±
0.57 0.57 ±0.57 0.57 1 0.57 0 0 0.57 0.57 0.57 0.57
F.S. 20.3 ± 20.3 ± 20.3 ± 28.6 ± 28.6 ± 28.6 ± 20.0 ± 20.0 ± 19.6 ± 21.3 ± 21.3 ± 21.3 ±
0.57 0.57 0.57 0.57 0.57 0.57 0 0 0.57 0.57 0.57 0.57
Toss-K 1:4 - - - - - - - - - - - -
1:1 - - - - - - - - - - - -
3:4 - - - - - - - - - - - -
F.S. - - - - - - - - - - - -
Zytee 1:4 - - - 14.0 ± 14.0 ± 14.0 ± - - - - - -
0 0 0
1:1 10.3 ± 10.3 ± 10.3 ± 17.0 ± 16.9 ± 16.3 ± - - - - - -
0.57 0.57 0.57 0 1 0.57
3:4 12.6 ± 12.6 ± 12.6 ± 19.6 ± 19.6 ± 19.6 ± - - - - - -
0.57 0.57 0.57 0.57 0.57 0.57
F.S. 14.0 ± 0 14.0 ± 13.9 ± 31.3 ± 25.9 ± 20.6 ± - - - - - -
0 1 0.57 1 0.57
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Table 2 (continued)

Mouthwash Mean diameter of growth of inhibition zones (mm)

Concentratio- S. mutans S. aureus C. albicans S. cerevisiae
ns tested 24h 48h 72h 24h 48h 72h 24h 48h 72h 24h 48h 72h

Hexnor 1:4 21.3 ± 21.3 ± 21.3 ± 24.0 ± 24.0 ± 24.0 ± 21.6 ± 21.6 ± 21.6 ± 19.3 ± 19.3 ± 19.4 ±
0.57 0.57 0.57 0 0 0 1.15 0.57 0.57 0.57 0.57 0.57
1:1 21.6 ± 21.6 ± 21.6 ± 25.6 ± 25.6 ± 25.6 ± 23.3 ± 23.3 ± 23.3 ± 22.6 ± 22.6 ± 23.0 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0
3:4 22.6 ± 22.6 ± 22.6 ± 27.3 ± 28.0 ± 30.3 ± 25.9 ± 25.9 ± 25.9 ± 23.3 ± 23.3 ± 23.3 ±
0.57 0.57 0.57 0.57 0 0.57 1 1 1 0.57 0.57 0.57
F.S. 22.6 ± 22.6 ± 22.6 ± 29.9 ± 30.3 ± 33.0 ± 26.6 ± 26.6 ± 27.3 ± 24.0 ± 23.9 ± 23.9 ±
0.57 0.57 0.57 1 0.57 0 0.57 0.57 0.57 0 1 1
Senquel-AD 1:4 - - - - - - - - - - - -
1:1 - - - - - - - - - - - -
3:4 - - - - - - - - - - - -
F.S. - - - - - - - - - - - -
Hexidine 1:4 23.6 ± 23.6 ± 23.6 ± 28.3 ± 28.3 ± 28.3 ± 11.9 ± 12.6 ± 13.0 ± 20.6 ± 20.6 ± 20.6 ±
0.57 0.57 0.57 0.57 0.57 0.57 1 0.57 1 0.57 0.57 0.57
1:1 25.3 ± 25.3 ± 25.3 ± 32.6 ± 32.6 ± 32.6 ± 20.3 ± 20.3 ± 20.3 ± 23.6 ± 23.6 ± 23.6 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57
3:4 25.6 25.6 ± 26.0 ± 33.6 ± 33.6 ± 33.6 ± 23.6 ± 23.6 ± 22.9 ± 25.0 ± 25.0 ± 25.0 ±
±0.57 0.57 0 0.57 0.57 0.57 0.57 0.57 1 0 0 0
F.S. 25.9 ± 1 25.9 ± 26.0 ± 33.9 ± 33.9 ± 33.9 ± 23.0 ± 23.0 ± 22.9 ± 26.3 ± 26.3 ± 26.3 ±
1 0 1 1 1 0 0 1 0.57 0.57 0.57
Chlohex 1:4 18.0 ± 0 18.0 ± 18.3 ± 26.3 ± 26.3 ± 26.3 ± 16.3 ± 16.3 ± 15.9 ± 20.0 ± 20.0 ± 20.0 ±
0 0.57 0.57 0.57 0.57 0.57 0.57 1 0 0 0
1:1 20.6 ± 20.6 ± 20.6 ± 29.3 ± 29.3 ± 29.3 ± 19.3 ± 19.3 ± 18.9 ± 23.3 ± 23.3 ± 23.3 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 1 0.57 0.57 0.57
3:4 21.3 ± 21.3 ± 21.3 ± 30.6 ± 30.6 ± 30.6 ± 20.0 ± 20.0 ± 20.0 ± 25.3 ± 25.3 ± 25.3 ±
0.57 0.57 0.57 0.57 0.57 0.57 0 0 0 0.57 0.57 0.57
F.S. 22.6 ± 22.6 ± 22.6 ± 32.6 ± 32.5 ± 33.0 ± 22.3 ± 22.3 ± 22.3 ± 26.0 ± 26.0 ± 26.0 ±
0.57 0.57 0.57 0.57 0.57 0 0.57 0.57 0.57 0 0 0
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Table 2 (continued)

Mouthwash Mean diameter of growth of inhibition zones (mm)

Concentratio- S. mutans S. aureus C. albicans S. cerevisiae
ns tested 24h 48h 72h 24h 48h 72h 24h 48h 72h 24h 48h 72h
Chlohex plus 1:4 17.9 ± 1 17.9 ± 17.9 ± 23.3 ± 23.3 ± 23.3 ± 13.3 ± 12.9 ± 10.6 ± 17.6 ± 17.6 ± 18.3 ±
1 1 0.57 0.57 0.57 0.57 1 0.57 0.57 0.57 0.57
1:1 18.3 ± 18.3 ± 18.3 ± 27.6 ± 27.6 ± 28.0 ± 15.3 ± 14.9 ± 14.6 ± 20.0 ± 20.0 ± 21.3 ±
0.57 0.57 0.57 0.57 0.57 0 0.57 1 0.57 0 0 0.57
3:4 19.9 ± 1 19.9 ± 20.0 ± 27.9 ± 27.9 ± 28.0 ± 17.6 ± 17.6 ± 16.9 ± 21.3 ± 21.3 ± 22.6 ±
1 0 1 1 0 0.57 0.57 1 0.57 0.57 0.57
F.S. 20.6 ± 20.6 ± 20.6 ± 28.6 ± 29.3 ± 30.0 ± 19.0 ± 19.0 ± 18.9 ± 21.6 ± 21.6 ± 21.5 ±
0.57 0.57 0.57 0.57 0.57 0 0 0 1 0.57 0.57 0.57
Triguard 1:4 16.3 ± 16.3 ± 16.3 ± 27.6 ± 27.6 ± 27.6 ± 14.3 ± 14.3 ± 14.3 ± 18.0 ± 18.0 ± 18.0 ±
0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0 0 0
1:1 19.6 ± 19.6 ± 19.6 ± 28.0 ± 28.0 ± 28.0 ± 17.3 ± 17.3 ± 17.3 ± 22.3 ± 22.3 ± 22.3 ±
0.57 0.57 0.57 0 0 0 0.57 0.57 0.57 0.57 0.57 0.57
3:4 19.6 ± 19.6 ± 20.0 ± 30.3 ± 30.3 ± 30.3 ± 19.0 ± 19.0 ± 19.0 ± 22.6 ± 22.6 ± 22.6 ±
0.57 0.57 0 0.57 0.57 0.57 0 0 0 0.57 0.57 0.57
F.S. 19.9 ± 1 20.0 ± 20.0 ± 31.6 ± 31.6 ± 31.6 ± 20.9 ± 20.9 ± 20.9 ± 22.6 ± 22.6 ± 22.6 ±
0 0 0.57 0.57 0.57 1 1 1 0.57 0.57 0.57

F.S. = Full Strength, (-) = No Zone,a Values including diameter of the well (8mm) are means of three replicates, b± Standard deviation.
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Discussion glands [36]. Chlorhexidine formulations are

Following the completion of the two considered to be the “gold standard”
different techniques to assess the antiplaque mouthrinses due to their
antimicrobial potential of the mouthwashes, prolonged broad spectrum antimicrobial
statistical ranking procedures were used to activity and plaque inhibitory potential [16,
place the ten different mouthwashes in 17].
order of antimicrobial effectiveness. The It is known that a balance exists in a
results reveal wide variations in their person’s oral microbial population. If this
effectiveness against the four tested balance is lost, opportunistic microorg-
microorganisms. Of the ten mouthwashes anisms can proliferate, enabling the
tested, Hexidine mouthwash emerged as the initiation of disease processes. Therefore,
most effective antimicrobial mouthwash, the mouthwash identified as having the
based on the optical density in liquid largest microbial inhibition zone-and thus
nutrient media and the mean diameter of the probably the strongest antibacterial and
zones of microbial inhibition produced by antifungal properties-may not be necessarily
the mouthwashes in agar well diffusion superior to those found to have smaller
method, against all the four tested diameter inhibition zones. Because a
microorganisms followed by Chlohex and mouthwash used in vivo likely is diluted by
Triguard, all of which showed excellent saliva, the level to which antimicrobial
level of activity. Following Triguard were properties are buffered or lost in dilution in
Zytee, Chlohexplus, Hexnor and vitro is of interest [37]. In addition, dentists
Chlorhexidine that showed good should keep in mind that the mean average
antimicrobial activity and finally, inhibition zone of one mouthwash may not
displaying very little antimicrobial activity be directly comparable with that of another
was Listerine while Toss-K and Senquel- mouthwash because different mouthwashes
AD totally lacked antimicrobial activity. are constituted of different active
Interestingly, all the three mouthwashes ingredients and may diffuse at different
that showed excellent antimicrobial rates.
activities had Chlorhexidine gluconate as This testing method also functioned as a
the active ingredient. Chlorhexidine screening method, and it may not have been
gluconate is a cationic biguanide with able to detect the effects of a chemical that
broad-spectrum antimicrobial action, whose does not diffuse through the agar matrix.
effectiveness in decreasing the formation of More importantly, the test was conducted in
dental biofilm (plaque) and gingivitis has vitro, so it cannot be assumed that the
been demonstrated in several clinical results of antimicrobial efficacy could be
studies [31-34]. Its mechanism of action is proportional or transferable to the oral
that the cationic molecule binds to the cavity and translated into clinical
negatively-charged cell walls of the effectiveness. Studies have demonstrated
microbes, destabilising their osmotic the effectiveness of rinsing with an
balance [16,35]. antimicrobial mouthrinse in significantly
Its substantivity, the ability of an agent reducing both salivary [38-40] and mucosal
to be retained in particular surroundings, is [41-42] levels of bacteria. Thus, from the
due to its ability to bind to the carboxyl overall results obtained, it is evident that
groups of the mucin that covers the oral various mouthwashes listing Chlorhexidine
mucus and be steadily released from these gluconate as the active ingredient presented
areas in an active form, displaced by the different antimicrobial activities.
calcium ions segregated by the salivary

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This is probably due to the different 3) Agbelusi GA, Odukoya OA, Otegbeye AF.
formulations in different mouthwashes in In vitro screening of chewing stick extracts
association with other ingredients. The and sap on oral pathogens: immune
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4) Bagg J. Essentials of microbiology for
with other constituents, in addition to dental students. New York, Oxford
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responsible for different effects. The result 5) Lee SS, Zhang W, Li Y. The antimicrobial
justifies the antimicrobial claims of the potential of 14 natural herbal dentifrices:
mouthwashes, made by earlier workers [13, Results of an in vitro diffusion method
43-44]. study. J Am Dent Assoc. 2004; 135: 1133-
41.
Conclusion 6) Odds FC. Candida and candidosis: a review
Hexidine mouthwash (ICPA Health and bibliography. 2nd ed. London, Bailiere
Products Ltd., Ankleshwar, India) showed Tindall, 1988; 252-78.
excellent antimicrobial activity against the 7) Oztan MD, Kiyan M, Gerceker D.
Antimicrobial effect, in vitro, of gutta-
four dental caries causing microorganisms percha points containing root canal
in vitro. The six mouthwashes found to be medications against yeasts and
effective against all the four tested Enterococcus faecalis. Oral Surg Oral Med
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tions, comprising of Chlorhexidine glucon- 410-6.
ate as the basic constituent, presented 8) Knighton HT. Study of bacteriophage types
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product concentration and its interaction members. J Dent Res. 1960; 39: 906-11.
with other constituents, in addition to 9) Lowy FD. Staphylococcus aureus
infections. N Engl J Med. 1998; 339(8):
differences in the formulations, would be
520-32.
responsible for different effects. 10) Piochi BJ, Zelante F. Contribution to the
study of Staphylococcus isolated in the
Acknowledgement mouth. III. Staphylococcus isolated from
We would like to thank Dr. Tapan dental plaque. Rev Fac Odontol Sao Paulo.
Chakrabarti, Institute of Microbial 1975; 13(1): 91-7.
Technology, Chandigarh, for providing the 11) Rodis OM, Shimono T, Matsumura S,
microbial cultures and the Chairperson of Hatomoto K, Matsuo K, Kariya N.
the Department of Microbiology for Cariogenic bacteria and caries risk in
providing laboratory facilities. elderly Japanese aged 80 and older with at
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Jundishapur Journal of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
www.SID.ir
Iran, Phone: +98611 3330074; Fax: +98611 3332036; URL: http://jjm.ajums.ac.ir; E-mail: editorial office: [email protected]
Archive of SID
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www.SID.ir
Iran, Phone: +98611 3330074; Fax: +98611 3332036; URL: http://jjm.ajums.ac.ir; E-mail: editorial office: [email protected]
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93-5. Email: [email protected]

Jundishapur Journal of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
www.SID.ir
Iran, Phone: +98611 3330074; Fax: +98611 3332036; URL: http://jjm.ajums.ac.ir; E-mail: editorial office: [email protected]