Efficacy of Two Pre-Procedural Rinses at Two Different

Journal of the International Academy of Periodontology 2017 19/4: 138–144
Efficacy of Two Pre-Procedural Rinses at Two
Different Temperatures in Reducing Aerosol
Contamination Produced During Ultrasonic
Scaling in a Dental Set-up - A Microbiological
Amruta Arun Joshi, Ashvini Mukul Padhye and Himani Swatantrakumar Gupta
Department of Periodontics, MGM Dental College and Hospital, Navi Mumbai, India
Aerosol has been considered one of the main concerns in the dental community because of
possible risk of infection transmission. Antiseptics used in the form of pre-procedural rinses
can reduce aerosol contamination during dental procedures. The aim of this study is to evaluate and compare the efficacy of 0.05% cetylpyridinium chloride and 0.2% chlorhexidine
pre-procedural rinses at 47ºC and 18ºC in reducing aerosol contamination during ultrasonic
scaling procedures. Forty subjects were divided randomly and equally into four groups: A1
and A2 to receive cetylpyridinium chloride and B1 and B2 to receive chlorhexidine as a preprocedural rinses. Aerosol produced during the ultrasonic scaling procedure was collected
on blood agar plates at three different locations, which were incubated at 37ºC for 48 hours
and analysed for bacterial colony forming units (CFU). Cetylpyridinium chloride (0.05%) as
a pre-procedural rinse was found to be equally effective in reducing aerosol contamination
when compared with 0.2% chlorhexidine rinse (p > 0.05). Also, greater reduction of CFU was
found with the use of tempered rinses at 47ºC with a highly statistically significant difference
(p < 0.001). Cetylpyridinium chloride (0.05%) can be considered as a promising alternative
to the gold standard 0.2% chlorhexidine, with tempering the rinse showing the definite edge.
Keywords: Aerosol, pre-procedural mouthrinse, cetylpyridinium chloride,
chlorhexidine, ultrasonic scaling, temperature
Aerosols emanating from human fluids and medical
procedures are solid or liquid particles, ranging in size
from sub- to multi-micrometre, that are suspended in
a gas (Occupation Safety and Health Administration).
Harrel et al. (1998), Harrel (2004) and Saini (2015)
stated that dental aerosols produced with the use of
high speed hand pieces, ultrasonic scalers, air polishing
devices and abrasion units, are complex and dynamic,
wherein some particles are projected onto surfaces,
Correspondence to: Amruta Arun Joshi, Department of Periodontics, MGM Dental College and Hospital, Junction of NH4
and Sion-Panvel Highway, Sector 1, Kamothe – 410209, Navi
Mumbai, Maharashtra, India. Phone: +919619140326; E-mail:
© International Academy of Periodontology
some settle due to gravity, and others can remain suspended in the air for long periods of time. Elevated
levels of these contaminants present in the aerosol
have been found during ultrasonic scaling procedures
that may get inhaled and transported to alveoli. This
process could result in respiratory problems and increase the risk of transmission of tuberculosis, severe
acute respiratory syndrome, avian flu and herpetic infections from patients to health care workers (Barbeau
2000; Harrel, 2004).
Worrall et al. (1987), Harrel (1996) and Gupta et al.
(2014) inferred that use of a pre-procedural antimicrobial mouthrinse may decrease the microbial aerosol
contamination to a great extent. According to Lyle
(2000), chlorhexidine (CHX) is considered the gold
standard rinse due to its broad-spectrum antimicrobial
activity and high substantivity. However, CHX presents
side effects such as temporary loss of taste, staining of
teeth, dryness and soreness of mucosa, and bitter taste.
Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling
Therefore, a need arises for evaluation of other
equally effective antimicrobial rinses. Albert-Kiszely
et al. (2007) and Silva et al. (2009) found that cetylpyridinium chloride (CPC), a member of the quaternary
ammonium compound family, is an effective anti-plaque
and anti-gingivitis agent. It is monocationic at oral pH
that permits dual retention in the oral environment, as
both surfactant chains and cationic charges may adsorb
to intraoral surfaces, which are lipophilic and anionic.
CPC acts primarily by penetrating the bacterial cell membrane, causing leakage of cell components, disruption
of bacterial metabolism, inhibition of cell growth and
finally cell death (Quirynen et al., 2005)
Complementarily, Konig (2002) demonstrated that
temperature appears to be a key determinant in altering
the kinetics of the active ingredient in a mouthrinse.
Tempering 0.2% CHX rinse to 47ºC demonstrated significantly more intensive anti-plaque effect as compared
to the solution cooled at 18ºC. In a previous study by
Reddy et al. (2012), tempered 0.2% CHX demonstrated
reduced bacterial counts in dental aerosols when compared to that of non-tempered CHX and sterile water.
A vista that remains to be explored is modulating the
temperature/concentration of CPC for enhancing its
antimicrobial activity when used as a pre-procedural
rinse. Hence, this study attempts to evaluate and compare the efficacy of two pre-procedural mouth rinses
containing 0.05% CPC and 0.2% CHX at two different
temperatures of 47ºC and 18ºC in reducing aerosol
contamination during ultrasonic scaling procedure.
Materials and methods
This single-centre, double-masked, randomized, prospective, four-group parallel designed study was conducted
over a period of 60 days. From September 2015 to October 2015, 40 patients (28 males and 12 females, mean age
32.5 years) were recruited. The patients were included if
they had a minimum of 20 natural teeth present, excluding
third molars, diagnosed with chronic gingivitis having a
sulcus probing depth of ≤ 3 mm, modified gingival index
≥ 1 (Lobene et al., 1986) and gingival bleeding index >
30% of the sites examined (Ainamo and Bay, 1975).
Exclusion criteria were history of known allergies to
constituents found in conventional mouthrinses, patients
with untreated/grossly carious teeth, those who had
undergone non-surgical or surgical periodontal therapy
and antibiotic and/or anti-inflammatory therapy within
the past 6 months. Systemically compromised patients
and pregnant and lactating women were also excluded.
The study was carried out in accordance with ‘The
Code of Ethics of the World Medical Association’
(Declaration of Helsinki, 64th WMA General Assembly,
Fortaleza, Brazil, October 2013) for experiments involving humans and the protocol was approved by the Institutional Ethics Review Committee of Mahatma Gandhi
Mission’s Dental College and hospital, Navi Mumbai and
a detailed informed consent was obtained from the 40
empirically selected patients.
Two commercially available solutions of 0.05% of
CPC mouthwash (Colgate Plax® Colgate Palmolive Ltd,
Mumbai, India) and 0.2% CHX mouthwash (Hexidine®
ICPA Health Products Ltd, Ankleshwar, India) were
procured from manufacturers and were transferred into
identical opaque white bottles labelled as A1, B1, A2 and
B2 for the purpose of blinding, by an investigator not
involved in the study. The identity of the samples was
revealed at the completion of the study. To calculate the
sample size at 80% power, the level of significance was
set at 0.05, to detect a standard deviation (SD) in CFU
counts of 18.80 (Konig, 2002) with a ratio of sample sizes
in both groups equal to 1. These data, when analyzed by
MedCalc Statistical Software version 13.3.1 (MedCalc
Software bvba, Ostend, Belgium; http://www.medcalc.
org; 2015) yielded a sample size of 10 per group. Computer-generated random numbers were used for randomization, and 40 patients were divided into four groups of 10
each as follows: Group A1 - warm 0.05% CPC (47ºC);
Group B1 - warm 0.2% CHX (47ºC); Group A2 - cold
0.05% CPC (18ºC); Group B2 - cold 0.2% CHX (18ºC).
For tempering the rinses to 47ºC, 10 mL of specified mouthwash was heated in a calibrated beaker in
a thermostatically regulated water bath. Similarly, the
solutions were cooled to 18ºC using a portable cooler
(Lab Hosp Corporation, Kalbadevi, Mumbai, India). All
patients were asked to rinse with 10 mL of the assigned
rinse for 60 seconds, 10 minutes prior to the ultrasonic
scaling procedure.
Prior to each appointment, a closed operatory was
fumigated using 34% formaldehyde and all surfaces were
disinfected with 70% isopropyl alcohol (A.B Enterprises,
Mumbai). Only one patient was treated per day by the
same right handed operator for the entire span of the
study, so as to allow the room to be free of aerosols. At
the beginning and end of the treatment, the ultrasonic
scaler unit (SUPRASSON® P5 Booster, France) was
flushed with 0.5% sodium hypochlorite (Clorox disinfectant cleaner) for 10 minutes to ensure disinfection
of water lines. Oral prophylaxis for all of the study participants was carried out in a standardized dental chair
using distilled water with controlled frequency (30 KHz)
and water pressure (0.3 MPa). No person other than
the patient, the operator and the assistant was allowed
in the vicinity of the operatory within the diameter of
four feet to avoid contamination of the operating field.
Blood agar plates, used to collect airborne microorganisms were prepared as instructed by the manufacturer (Micro Master Labs Pvt Ltd, Thane, Maharashtra,
India). Briefly, blood agar base was sterilized at 121ºC
for 15 minutes and then cooled to 50ºC in a water bath
to which 5% sterile sheep blood was added aseptically.
Journal of the International Academy of Periodontology (2017) 19/4
This was dispensed in sterile petri plates and stored at
2 - 8ºC until further use. The blood agar plates mounted
firmly on a board were suspended with strings around
the necks of the patient, the assistant and the operator, such that it rested on their chests at a standardized
distance of 12 inches from the patient’s mouth with the
philtrum of the upper lip serving as a fixed reference
point, as shown in Figure 1. These plates were labelled
as P, A and O respectively.
The labelled plates were exposed at the start of the
scaling procedure performed for a duration of 30 minutes, and were left uncovered on the operator’s stool,
assistant’s stool and the back rest of the dental chair
for additional 30 minutes to collect samples of any
aerosolized bacteria. These were then closed with a lid
and placed upside down to prevent contamination of
blood agar with the moisture on the under surface of
the lid. The plates were incubated at 37º C for 48 hours
to facilitate the growth of micro-organisms, following
which they were analyzed for bacterial colony forming
units (CFUs) using a colony counter (Lab Line Stock
Centre, Mumbai, India).
Statistical analysis
Data analysis was done using ‘MedCalc Statistical Software’ version 13.3.1 (MedCalc Software Bvba, Ostend,
Belgium). Scores were averaged for age, number of
teeth present, MGI and GBI across all groups. All
variables were expressed as mean with standard error.
The averaged values were tested for normality using the
Kolmogorov Smirnov test. The data presented normality and the parametric test of analysis of co-variance
(ANCOVA) was used to find which pairs differed
significantly with respect to temperature difference.
Intergroup comparisons were done using unpaired ttests at p < 0.05.
The non-compliance rate for our study was zero and there
were no dropouts. Table 1 represents demographic characteristics of the sampled population. Figure 2 demonstrates
mean ± standard error of the mean (SEM) CFU counts for
all groups at three different locations, namely the chest areas
of patient (P), assistant (A) and operator (O). Among all the
groups, the patients’ chest area of group A2 demonstrated
maximum mean CFU counts (103.60 ± 4.08) while the assistant’s chest area of group B1 showed the lowest counts (38.50
± 2.20). On applying an unpaired t-test to compare groups
A2 and B2, a statistically significant difference was found
for the assistant’s chest area location (p = 0.02). However,
no statistical difference (p > 0.05) was found on intergroup
comparison for all other locations, thereby proving that
both mouthrinses were equally effective irrespective of the
temperature difference (Table 2). Table 3 represents multiple
comparisons using ANCOVA performed to find out which
pairs differed significantly at the 5% level of significance
with respect to temperature. A highly statistically significant
difference was found on comparing group A1 with A2 and
B1 with B2 (p < 0.001).
Table 1: Baseline demographic and clinical characteristics of the four groups.
Group Group Group Group p value
Males/females 7/3
Number of 26.52
32.7 32.64 33.0 NS
27.24 27.11 26.83 NS
NS, not statistically significantly different; MGI, modified gingival index; GBI, gingival bleeding index.
Figure 1. The position of the blood agar plates at three different locations, namely the chest areas of the
patient (P), operator (O) and assistant (A).
Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling
Figure 2. Comparison of mean ± standard error of the mean (SEM) colony forming units (CFU) counts at three
different locations in all four groups.
Table 2: Results of unpaired t-tests comparing mean colony forming unit (CFU) counts between groups.
Location and rinse
Group A1 (Warm CPC)
Group B1 (Warm CHX)
Group A2 (Cold CPC)
Group B2 (Cold CHX)
Group A1 (Warm CPC)
Group B1 (Warm CHX)
Group A2 (Cold CPC)
Group B2 (Cold CHX)
Group A1 (Warm CPC)
Group B1 (Warm CHX)
Group A2 (Cold CPC)
Group B2 (Cold CHX)
p value
*p value < 0.05; NS, non-significant; S, significant; CPC, 0.05% cetylpyridinium chloride; CHX, 0.2% chlorhexidine;
Journal of the International Academy of Periodontology (2017) 19/4
Table 3: Results of ANCOVA test comparing mean CFU counts within groups with respect to temperature difference
Location and rinse
Group A1 (Warm CPC)
Group A2 (Cold CPC)
Group B1 (Warm CHX)
Group B2 (Cold CHX)
Group A1 (Warm CPC)
Group A2 (Cold CPC)
Group B1 (Warm CHX)
Group B2 (Cold CHX)
Group A1 (Warm CPC)
Group A2 (Cold CPC)
Group B1 (Warm CHX)
Group B2 (Cold CHX)
F value
p value
< 0.001*
< 0.001*
< 0.001*
< 0.001*
< 0.001*
< 0.001*
*p value < 0.05; HS, highly significant; CPC, 0.05% cetylpyridinium chloride; CHX, 0.2% chlorhexidine
This analysis revealed that the groups A1 and B1 (47ºC)
showed the maximum reduction in bacterial counts in all
three areas as compared to their cold counterparts A2 and
B2 at 18ºC.
Under the traditional paradigm, a dental health care
worker would be considered to be at high risk for droplet transmission. Legnani et al. (1994) and Bennett et al.
(2000) showed that use of ultrasonic scaling procedures
resulted in peak concentrations of microbial aerosols
in dental treatment rooms. Harrel and Molinari (2004)
enumerated three levels of defence in the reduction of
aerosols as personal protective barriers, routine use of
pre-procedural rinses and high volume evacuation devices. Personal protective equipment, such as a surgical
mask, face shield, or eyewear prevents the projection of
microorganism-laden particles onto the mucosal membranes. However, they do not counter an ever present
hazard of inhaling particulate aerosols when near an
infectious patient.
CHX has already proven its efficacy as a pre-procedural rinse in reducing bacterial aerosol contamination
with the use of an air polisher, as studied by Logothetis
and Martinez-Welles (1995) and ultrasonic scaler as demonstrated by Sawhney et al. (2015). Konig (2002) inferred
that increasing the temperature of 0.2% CHX to 47ºC
was effective in reducing vital plaque content, as assessed
by the vital fluorescence technique. A 25% increase in
bacteria kill rate was observed following irrigation with
the tempered CHX solution. This effect was not solely
due to the physical parameter “temperature,” as the combination of heat and water resulted in unchanged vitality
rate of micro-organisms cultured from plaque samples.
The biologic basis and rationale for this anti-plaque effect
could be attributed to enhanced bactericidal activity at an
elevated temperature. Also, CHX has been found to have
an increase in sporicidal effect when the temperature was
increased to 60 - 70ºC (Shaker, 1986).
However, use of CHX is not free of undesired side
effects. The potential of CPC as an anti-plaque agent
is well documented in the literature (Silva et al., 2009;
Garcia et al., 2011). But there exists lacunae regarding
the role of tempered 0.05% CPC as a pre-procedural
rinse to control aerosol contamination. The Scientific
Committee on Consumer Safety (2015) reported that
degradation of pure CPC occurs at 130ºC, with complete thermal decomposition at 234ºC. Konig (2002) also
reported that tempering rinses to 47ºC exhibits neither
painful sensations nor permanent pulpal damage. To the
best of our knowledge, this study is the first to simultaneously assess the efficacy of two commercially available
mouthrinses, 0.05% CPC and 0.2% CHX, when used
as pre-procedural rinses at two different temperatures,
in reducing aerosol contamination.
Various investigators (Bentley, 1994; Chiramana,
2013) have studied the spread of aerosols within the
range of 2 to 6 feet. In the present study, the perimeter
of the operatory was limited to a diameter of 4 feet
and the aerosols were collected on blood agar plates,
which are considered as a valid non-selective medium
for culturing airborne bacteria (Johnston et al., 1978).
These plates were positioned at three different locations
at a distance of 12 inches from the patient’s philtrum
as performed previously by Gupta et al. (2014). Further
exposure of these plates for an additional 30 min after
ultrasonic scaling was done so as to allow gravitational
settling of airborne bacteria (Larato et al., 1966).
Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling
The results of our study suggest that the pre-procedural rinse containing 0.05% CPC is as effective as
0.2% CHX in reducing aerosol contamination, which
is in agreement with the study conducted by Feres et al.
(2010). Our results are also in accordance with the study
conducted by Reddy et al. (2012), where the tempered
solutions heated to 47ºC were found to be significantly
more effective in reducing aerosol contamination at the
chest areas of patient, operator, and assistant as compared to the cold solutions used at 18ºC. Nevertheless,
on comparing mean bacterial counts at the assistant
chest area after rinsing with only cold CPC and CHX
solutions, a statistically significant difference was noted,
indicating CPC was more effective than CHX at 18ºC.
The CFU estimation in our study includes only
aerobic bacteria capable of growth on blood agar plates;
anaerobic bacteria and viruses that require specialized
media were not isolated, which needs to be addressed
in further investigations. No attempt was made to differentiate these bacteria based on cultural characteristics.
Within the limits of our study, the emerging evidence
beckons towards the role of tempered CPC in effectively
reducing aerosol contamination. Nonetheless, these data
need to be interpreted with caution, as sample size was
small. Hence, there is a need for longitudinal studies of
a single cohort with a larger sample size demonstrating efficiency of tempered CPC in mitigating aerosol
bacteria and viruses.
Overall, the results of our investigation clearly indicate
that a pre-procedural rinse containing 0.05% cetylpyridinium chloride can be considered as a promising
alternative in reducing aerosol contamination during
ultrasonic scaling procedures when compared to the
gold standard 0.2% chlorhexidine, with tempering the
rinse showing the definite edge. Also, it can be concluded
that the amount of viable bacteria in aerosol is maximum
at the patient’s chest area followed by the operator and
assistant in a descending manner, thus reinforcing the
use of personal protective barriers to minimize the risk
to dental professionals.
Department of Microbiology, MGM Medical College
and Hospital, Navi Mumbai, India.
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