Original Article

Efficacy of chlorhexidine/herbal
formulation for microbial reduction in
aerosol generated following ultrasonic
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scaling - A double-blinded randomized

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Department of controlled trial

Periodontology, Faculty
of Dental Sciences, Sri
Ramachandra Institute
of Higher Education and
Research, 1Department
of Microbiology, Sri
Ramachandra Institute
of Higher Education and
Research, Chennai,
Tamil Nadu, India
The work belongs
to Department of
Periodontology, Faculty
of Dental Sciences, Sri
Ramachandra Institute
of Higher Education and
Research, Chennai,
Tamil Nadu, India
Access this article online
Website:
www.jisponline.com
Lekha Ashokkumar, Vamsi Lavu, Kennedy Kumar Palraj,1 Suresh Ranga Rao,
Subbusamy Kanakasabapathy Balaji
Abstract:
Background: Ultrasonic scaling is a potential source of aerosol contamination in dental clinics. The two primary
sources of microbial load in aerosols are from the oral cavity and dental unit water line. Literature evidence suggest
that the use of preprocedural mouth rinse reduce the bacterial load in aerosol generated during ultrasonic scaling.
Aim: The aim of the study is to assess the comparative efficacy of reduction in viable bacteria in the aerosol at
patient’s chest area, doctor’s mask area and two feet beside the patient following use of chlorhexidine/herbal
formulation diluted in the water source by a randomized controlled clinical trial. Materials and Methods: Forty‑five
subjects (with chronic gingivitis) were matched for age, gender, and gingival index score. The subjects
were randomized and received ultrasonic scaling with distilled water (control)/chlorhexidine (tTest)/herbal
formulation (test). Aerosol produced during scaling was collected at patient’s chest area, doctor’s mask area,
two feet beside the patient on blood agar plates, which were incubated at 37°C for 48 h and total colony forming
units (CFUs) were counted. Results: A significant reduction in the total CFUs’ counts was observed at all the three
sites sampled in test groups (chlorhexidine group and herbal formulation group) as compared to control (P < 0.01).
Conclusion: The addition of antiseptic agents to the water source contributed to a significant reduction of the
cultivable microbial counts in the aerosol and hence can be used to reduce the risk of cross‑infection during
ultrasonic scaling.
Key words:
Aerosols, chlorhexidine, colony‑forming units assay, cross infection, periodontal debridement

DOI:
10.4103/jisp.jisp_478_21
Quick Response Code: INTRODUCTION Association of these aerosols with respiratory,
ophthalmic and skin infections, tuberculosis, and

A erosol is a suspension of solid or liquid

particles containing bacteria or viruses.
Particle size may vary from 0.001 to >100 μm.[1]
hepatitis B has been established.[5‑7]

The current literature suggests that having

Aerosol with smaller particle size has been found patients use an antimicrobial rinse before
to have greatest potential to penetrate and lodge treatment will decrease microbial content in
in smaller passages of lungs and thereby transmit aerosols. Chlorhexidine, a broad‑spectrum
Address for infection.[2] Aerosols and splatter pose a main
correspondence: concern in dental community due to possible This is an open access journal, and articles are
Dr. Lekha Ashokkumar, spread of infectious and potentially harmful distributed under the terms of the Creative Commons
Department of Attribution‑NonCommercial‑ShareAlike 4.0 License, which
agents among patient and dental personnel.[2] allows others to remix, tweak, and build upon the work
Periodontology, Faculty
non‑commercially, as long as appropriate credit is given and
of Dental Sciences, Sri
Dental plaque, oral fluids, and respiratory tract the new creations are licensed under the identical terms.
Ramachandra University,
No. 1 Ramachandra Nagar, are the source of numerous bacteria and viruses For reprints contact: WKHLRPMedknow_reprints@
Porur, Chennai ‑ 600 116, in oral cavity. Over six million microorganisms wolterskluwer.com
Tamil Nadu, India. have been reported to be present in one ml of
E‑mail: drlekha@ saliva.[3] How to cite this article: Ashokkumar L,
sriramachandra.edu.in Lavu V, Palraj KK, Rao SR, Balaji SK. Efficacy
The use of ultrasonic scaler is a potential source of chlorhexidine/herbal formulation for microbial
Submitted: 22‑Jul‑2021 reduction in aerosol generated following ultrasonic
Revised: 24‑Jan‑2022 of aerosol production in a dental clinic.[4] Aerosols
scaling – A double‑blinded randomized controlled
Accepted: 05‑Feb‑2022 generated from patient’s mouth contain up
trial. J Indian Soc Periodontol 2023;27:82‑6.
Published: 03-Jan-2023 to a million bacteria per cubic foot of air.[5]

82 © 2023 Indian Society of Periodontology | Published by Wolters Kluwer - Medknow

 Ashokkumar, et al.: Herbal formulation to reduce microbial load
antimicrobial agent, is considered the gold standard and has
substantivity of eight to 12 h.[8] Preprocedural rinsing with
the chlorhexidine mouth wash produced a 94.1% reduction
in recoverable colony forming units (CFUs) compared to the
control (no prerinsing).[9] Chlorhexidine gluconate was found to
be effective even at a dilution of 1/320, which was suggested as
the maximum dilution at which inhibition can be achieved.[10]
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Aerosol‑generated during ultrasonic scaling contains complex
of microbes originating from patient’s oral cavity and from
dental unit water line (DUWL). Preprocedural mouth rinsing
has been proved to be effective in reducing the microbial
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contamination in aerosol primarily originating from oral
cavity. With this background, the objective of this study was to
assess the reduction in viable bacteria in the aerosol at patient
chest area, doctor’s mask area, and two feet beside the patient
following use of chlorhexidine/herbal formulation dilutions
in the water source.
MATERIALS AND METHODS
This study was designed as a randomized controlled
double‑blinded clinical trial. One hundred and fifty patients
attending the out‑patient department were screened, and 45
subjects were selected for the study based on certain specific
inclusion and exclusion criteria. Ethical approval for the
study was obtained from Institutional Ethics Committee
and the trial was registered in the Clinical Trials Registry of
India (CTRI/2017/06/008926).
Written informed consent was obtained from study participants
before inclusion in the study. Systemically healthy subjects above
18 years of age with moderate/severe gingival inflammation
which was assessed based on gingival index score (1.1–3.0),[11]
subjects with fair or poor oral hygiene based on oral hygiene
index–simplified score (1.3–6.0)[12] and subjects with ≥20 teeth
were included in the study. Current smokers, pregnant women,
patients on antibiotics/anti‑inflammatory/any other drugs
in the past 6 months, subjects with healthy gingiva (with no
clinical signs of gingival inflammation) were excluded from
the study. Patient enrollment and elicitation of detailed history
and clinical examination were performed using a prepared
proforma by a single examiner (L. A). Intra‑examiner variability
was assessed and the kappa value was >90%.
Sample size calculation was done based on the study by Gupta
et al.,[13] the power of the study being β = 80% and Type I
error α = 5%. The sample size required to show the difference
between the control and test group was 15 per group. Patients
were divided into three groups (Group I and Group II and
Group III) with 15 patients each. Group I subjects received
ultrasonic scaling with distilled water (placebo) to which food
color (green) was added to match the test group. Group II
subjects received ultrasonic scaling with chlorhexidine
diluted water. Two ml of chlorhexidine gluconate solution
was diluted in one liter of water to obtain a dilution of one
in 320 dilution (maximum inhibitory dilution) as per the
study by Nascimento et al.[10] (with an effective concentration
of 0.0625% of chlorhexidine). Group III subjects received
ultrasonic scaling with herbal formulation diluted water. Ten
ml of herbal formulation (HiOra®) was diluted in one liter
of water (1:100 dilution). HiOra®, a proprietary Ayurvedic
Journal of Indian Society of Periodontology - Volume 27, Issue 1, January-February 2023
medicine solution consisted of a mixture of herbal extracts,
powders, and oils.
Randomization was performed using a computer generated
random table. Randomization list was received by A. L and
sealed envelopes containing the allotted code numbers were
placed in a closed container in the operatory. Before the
scaling procedure, the dilutions of chlorhexidine and herbal
formulation were prepared and added to the water source as
per the code available in the envelope, opened on the day of
the procedure. The operator (L. V) who performed scaling was
blinded regarding use of antiseptic in the water source and the
microbiologist who performed the microbial colony counting
remained blinded throughout the study.
An operatory containing single dental unit was selected to
perform the study. Before the commencement of the trial, a
new DUWL was installed. Only one patient was treated per day
to avoid cross contamination. The operatory was fumigated,
8 h before the procedure to minimize air contamination. The
baseline sample of the microbial count in the room was collected
by exposing the blood agar plates 30 min before procedure.
Blood agar is an enriched media which allows growth of all
fastidious organisms when compared to nutrient agar. During
ultrasonic scaling, aerosol was collected on blood agar plates
kept at three different areas: patient’s chest area, operator mask
level, and two feet to the left side of the patient. The blood
agar plates were exposed for 20 min during the treatment. The
clinical procedure of ultrasonic scaling was performed by the
same operator (L. V) for all the study participants. The plates
were sealed immediately and incubated at 37°C for 48 h in a
sterile incubator. After 48 h, total number of CFUs of aerobic
bacteria were counted on each agar plates under magnification.
ANOVA test was used for determining the significance of
difference in reduction of CFU counts between the test and
control group. Mean, frequency, and standard deviation
were determined for all continuous variables. P < 0.05 was
considered statistically significant.
RESULTS
A total of 45 individuals with an age range of 20–52 years of
age were included in this study. The demographic data of the
study population are summarized in Table 1. The demographic
variables and clinical parameters were similar among the
participants randomized into control and test groups [Table 1].
The total CFUs count at patient’s chest area, doctor’s mask area,
and two feet beside the patient are summarized in Table 2.
Table 1: The mean value of the demographic data and
baseline clinical characteristics of Group I (placebo
control), Group II (chlorhexidine), and Group III (herbal
formulation)
Group I Group II Group III (herbal
(placebo) (CHX) formulation)
Age (years) 34.33±12.90 34.8±10.44 32.46±8.84
Gender (male/female) 7/8 8/7 7/8
OHI‑S score 3.56±0.54 3.64±0.51 3.68±0.49
GI score 2.32±0.23 2.35±0.21 2.33±0.17
CHX - Chlorhexidine; OHI‑S - Oral Hygiene Index‑Simplified; GI - Gingival
index
83
 Ashokkumar, et al.: Herbal formulation to reduce microbial load

Intergroup comparison was made by one‑way ANOVA.
A significant difference in microbial CFUs (P < 0.001) was
observed at the three sites evaluated in comparing the test
groups – chlorhexidine (Group II)/herbal formulation (Group III)
with placebo control [Table 3]. A substantial decrease in CFU was
observed among both the test groups, however, the intergroup
difference between the test groups (chlorhexidine and herbal
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in a significant reduction of the microbial counts in the aerosol
generated during ultrasonic scaling. The herbal formulation
was found to be equally effective as chlorhexidine in reduction
of microbial counts in the aerosol at the sites evaluated.
Pioneering work on aerobiology by Micik et al. used the
terms “aerosol” and “splatter” in the dental environment.

formulation) was not statistically significant at patient’s chest
area (P = 0.607), doctor’s mask area (P = 0.501), and two feet
beside the patient (P = 0.130) [Table 3 and Figures 1‑3]. The
images of blood agar plates for the two interventions and one
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Particles <50 micrometers in diameter were defined as
aerosols. The microbial content in aerosol poses a major threat
for disease transmission through airborne route. The control
and minimization of microbial content in aerosol are of great
importance to the health of both the dentist and the patient.

control group at three different sampling sites are provided in

Supplementary Figures 1-3. Aerosolized bacteria/viruses may remain suspended in the
air for long periods of time and inhaled into the lungs of a
susceptible person.[14‑16]
DISCUSSION
Aerosols generated during dental procedure have gained
The primary observations in this study were addition of
importance in recent times, due to the risk of transmission
chlorhexidine/herbal formulation to the water source resulted
of severe acute respiratory syndrome coronavirus 2 virus. In
light of the COVID‑19 pandemic, a recent review reported
Table 2: The mean and standard deviation of microbial that respiratory bacterial pathogens and influenza viruses are
counts (colony forming units) in three groups at present in saliva and viral shedding occurs in saliva during
different locations acute phases of all respiratory diseases. The microbial load in
Groups Mean±SD aerosol correlates with severity of respiratory diseases. Apart
Patient’s chest Doctor’s mask Two feet beside from the saliva being the primary source of microbial content
area (CFU/cm2) area (CFU) the patient (CFU) in aerosol, the DUWL is also considered as the environmental
Group I 102.13±73.37 77.86±49.61 41.40±18.87 source for aerosol microorganism.[17]
- placebo
Group II 11.60±5.11 12.33±7.45 11.13±7.15 Acharya et al. in 2010 stated that dental handpieces, ultrasonic
- CHX
scalers, air polishing devices, and air abrasion units produce
Group III 19.66±5.49 19.60±7.51 18.13±7.41
- herbal airborne particles by combined action of water sprays,
formulation compressed air, organic particles such as tissue, tooth dust,
CHX - Chlorhexidine; CFU - Colony‑forming unit; SD - Standard deviation and organic fluids such as blood and saliva from the site
where these instruments are used.[2] DUWL is another major
source of microbes in aerosol. The microflora from the
Table 3: Comparison of mean microbial
DUWL and the patient’s oral cavity in the form of aerosol
counts colony‑forming unit between the three
mixes with the surrounding air, thus leading to change in the
interventions (Group I - placebo control, Group
II - chlorhexidine, Group III - herbal formulation) at original composition of the environment. Eventually, it acts
patient’s chest area, doctor’s mask area, and two feet as a source of infection for both the dentist and the patients.
beside the patient area using one‑way ANOVA analysis Regular cleaning, disinfection, and sterilization of the unit
Groups Mean 95% CI P
difference Lower Upper
bound bound
Patient’s chest area
Placebo versus CHX 90.53 +59.15 +121.91 <0.001**
Placebo versus herbal 82.47 +51.08 +113.84 <0.001**
formulation
Herbal formulation versus 8.07 −23.31 +39.44 0.607
CHX
Doctor’s mask area
Placebo versus CHX 65.53 +43.94 +87.12 <0.001**
Placebo versus herbal 58.27 +36.68 +79.85 <0.001**
formulation
Herbal formulation versus 7.27 −14.31 +28.85 0.501
CHX
2 feet beside the patient area
Placebo versus CHX 30.27 +21.11 +39.41 <0.001**
Placebo versus herbal 23.27 +14.11 +32.41 <0.001**
formulation
Herbal formulation versus 7.00 −2.14 +16.14 0.130
CHX
**P value of <0.001 is considered statistically highly significant. Figure 1: Box plot illustrating the colony‑forming units at patient’s chest area with
CHX - Chlorhexidine; CI - Confidence interval; P - P value (Probability value) median and standard deviation

84 Journal of Indian Society of Periodontology - Volume 27, Issue 1, January-February 2023

 Ashokkumar, et al.: Herbal formulation to reduce microbial load
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Figure 2: Box plot illustrating the total microbial count colony‑forming units at
doctor’s mask area showing median and standard deviation
water reservoir, filling it with distilled water, and application
of chemicals to monitor the microbiological quality of DUWL
water, assures effective microbiological control of water and
safety of the unit users.[18]
Several infection control treatment strategies have been tried
and implemented before and during the dental procedures,
especially during ultrasonic scaling to minimize the microbial
load in aerosol generated. Standard protective measures
including gloves, goggles, shields, and masks are used
universally during dental surgeries as an effective barrier
against splatter thereby eliminating inherent danger in
operative site.[19,20]
Literature evidence on studies to reduce the microbial
content of aerosol from oral cavity have focused on use of
preprocedural rinses and prevention/management of biofilm
formation in DUWL. Commonly used agent to reduce the
microbial content in aerosol includes chlorhexidine, povidone
iodine, hydrogen peroxide. Among this chlorhexidine and
povidone iodine has been used as preprocedural rinse to reduce
the microbial contamination in aerosol and all the three has
been used to disinfect DUWL.[21‑24]
Chlorhexidine is considered an effective antiseptic against
free‑floating oral bacteria. Preprocedural mouth rinsing with
antiseptic solutions has been found to be effective in reducing
bacterial count in the air of operatory. Several studies have
evaluated the efficacy of this protocol. Preoperative rinsing
with 0.12% chlorhexidine gluconate diminished the quantity
of aerobic and facultative flora of oral cavity.[25] The use of
0.2% chlorhexidine gluconate or essential oil containing
mouthwashes as preprocedural mouth rinse can cause
substantial reduction in bacterial counts in aerosol.[13]
Preprocedural rinses will be the primary target for reducing
the microbiota in aerosol from oral cavity and does not affect
the water source and water line. By adding antimicrobial agent
in water source, the microbiota from water source, water line,
and oral cavity will be reduced. Addition of 0.2% chlorhexidine
Journal of Indian Society of Periodontology - Volume 27, Issue 1, January-February 2023
Figure 3: Box plot illustrating the total microbial counts colony‑forming units at two
feet beside the patient area showing the median and standard deviation
into dental unit reservoir has shown to significantly reduce the
bacterial and fungal contamination of the DUWL.[22]
Chlorhexidine, a broad‑spectrum antibacterial agent, is
considered the gold standard irrespective of its common side
effects such as temporary loss of taste sensation, staining
of teeth, restoration and mucosa, dryness and soreness of
mucosa, and bitter taste.[8] Chlorhexidine gluconate was found
effective even at a dilution of 1/320, which was suggested as
its maximum inhibitory dilution.[10]
Herbal formulation (HiOra®) comprises components such
as Miswak, nagavalli, gandhapurataila, ela, peppermint
satva, yavani satva which have antimicrobial/antiplaque
property.[26] The antigingivitis and antiplaque efficacy of the
herbal formulation (HiOra®) have been found to be comparable
to chlorhexidine as reported by Deshmukh et al.[27] The herbal
formulation (HiOra®) had a minimum inhibitory concentration
of 1/100 against commercially available strains of aerobic
bacteria ([Streptococcus and Staphylococcus spp.] unpublished
data from in vitro research performed by our team). Hence, in
our present study, the same dilution of commercially available
herbal formulation was prepared and utilized in the test group.
Dental unit water reservoir is the only source of water to the
ultrasonic scaler unit and hence adding a antimicrobial agent
to it will eliminate the DUWL biofilm formation and also
minimize the microbial load from patient mouth when used
during ultrasonic scaling.
To the best of our knowledge, this is the first study to evaluate
the comparative efficacy in reducing microbial levels in aerosols
produced during ultrasonic scaling following the addition of
chlorhexidine and herbal formulation to the dental unit water
reservoir.
In this study, chlorhexidine and herbal mouth wash were
diluted in Dental Unit Water reservoir, and their efficacy
in reducing the microbial level in aerosol produced during
ultrasonic scaling was assessed using culture‑based technique
85
 Ashokkumar, et al.: Herbal formulation to reduce microbial load
with blood agar plates. The results of this study show
significant reduction in microbial levels in aerosol produced
during ultrasonic scaling on addition of chlorhexidine and
herbal formulation into Dental Unit Water reservoir.
The data in this study support the beneficial use of chlorhexidine
or herbal formulation as disinfection agents in the booster water
for scaling devices. This will reduce bacterial contamination
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in the aerosol generated and therefore enhance safety for the
patient and the dental operator.
CONCLUSION
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 10/10/2023
The addition of antiseptic agents to the water source
contributed to a significant reduction of the cultivable microbial
counts in the aerosol and hence can be used to reduce the risk
of cross‑infection during ultrasonic scaling.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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a b c
Supplementary Figure 1: Microbial colonies formed on blood agar plates exposed
at patient’s chest area in (a) Group I- Water/Placebo; (b) Group II- Chlorhexidine
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mouth wash; (c) Group III- Herbal mouth wash

a b c
Supplementary Figure 2: Microbial colonies formed on blood agar plates exposed
at doctor’s mask area in (a) Group I- Water/Placebo; (b) Group II- Chlorhexidine
mouth wash; (c) Group III- Herbal mouth wash

a b c
Supplementary Figure 3: Microbial colonies formed on blood agar plates exposed
at 2 feet beside patient in (a) Group I- Water/Placebo; (b) Group II- Chlorhexidine
mouth wash; (c) Group III- Herbal mouth wash