Effect of chlorhexidine, honey and Propolis on Streptococcus mutans Counts: in vitro 
Study 

 
 

Vol 9 No 1 (2021)    DOI 10.5195/d3000.2021.166 

 http://dentistry3000.pitt.edu 

Effect of chlorhexidine, honey and Propolis on Streptococcus 
mutans Counts: in vitro Study 
 
Abdulrhman Alkhaled1, Laith Alsabek2, Maher Al-assaf3, Faek Badr1 

¹ Department of Pediatric Dentistry, Faculty of Dentistry, Tishreen University, Syria. 

2 Faculty of Dentistry, Damascus University, Damascus, Syria. 

3 Department of Oral Histology and Pathology, Faculty of Dentistry, Damascus University, Damascus, Syria. 

Abstract 

Objective: Streptococcus mutans is considered one of the bacteria that causes necrosis, as it metabolism 
sugar to obtain energy, producing an acidic environment and causing depression in the enamel surface, 
which causes the dissolution of the calcium molecule, a problem of cavited in the tooth; so the aim of 
the research is to evaluate the effect of mouthwashes chlorhexidine 0.12% - honey 50% - propolis 5% on 
Streptococcus mutans in children. 

Study design: A laboratory study to assess the efficiency of chlorhexidine, honey and Propolis on 
Streptococcus mutans counts. The sample included 60 children divided into: G1 (Chlorhexidine 0.12% = 
20 children), G2 (Propolis 5% = 20 children), G3 (Honey 100% = 20 children). The data was analyzed using 
the statistical analysis program SPSS, version 13.00, at a confidence level of 95% (P <0.05(. Paired sample 
T test was used to compare the variable means of the values of the studied variables. 

Results: There is significant difference between three groups. The reduction ratio was 89.97% in G1, 
85.49% in G2 and 76.29% in G3. 

Conclusion: This study demonstrated the 
effectiveness of mouthwashes of 
chlorhexidine, propolis and honey on the 
bacteria of Streptococcus mutans.  

Keywords: Streptococcus mutans; 
chlorhexidine; propolis; honey 

Citation: Alkhaled A, et al. (2021) Effect of chlorhexidine, 
honey and Propolis on Streptococcus mutans Counts: in 
vitro Study. Dentistry 3000. 1:a001 
doi:10.5195/d3000.2021.166 
Received: April 03, 2021 
Accepted: April 21, 2021 
Published: November 19, 2021 
Copyright: ©2021 Alkhaled A, et al. This is an open 
access article licensed under a Creative Commons 
Attribution Work 4.0 United States License. 

Email: dr.maher.assaf1990@gmail.com

 

 

Introduction 

Streptococcus mutans are gram-

positive bacteria of oval or 

aciduric shapes that are lined up in 

the form of acidogenic and 

aciduric nodules or swabs. They 

survive at low pHs and also 

metabolize carbohydrates in an 

acid medium [1]. Streptococcus 

mutans grows at temperatures 

ranging between 18-40 degrees 

Celsius, and it is considered one of 

the bacteria that causes necrosis, 

as it metabolism sugar to obtain 

energy, producing an acidic 

environment and causing 

depression in the enamel surface, 

which causes the dissolution of 

the calcium molecule, a problem 

of cavited in the tooth [2].  It 

found that Streptococcus mutans 

is more prevalent in pit and 

groove and there are a few of 

them on the buccal surfaces, 

where the mutant streptococcus 

forms 39% of the total 

streptococci within the oral cavity 

[3]. A study by Köhler et al.1948 

indicated a relationship between 

early colonization of the 

Streptococcus mutans in the 

mouths of children and a high rate 

of caries [4], Whereas Aas et al. 

2008 study showed that 

Streptococcus mutans is mainly 

responsible for the initiation of 

dental caries despite the 

involvement of other oral bacteria 

in the development of dental 

caries such as Lactobacillus and 

Veillonella [5]. There are two 

types of oral washes (chemical and 

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herbal). Chlorhexidine is chemical. 

While many plant extracts have 

antibacterial properties, they are 

used effectively in oral hygiene. 

Natural herbs such as miswak, 

honey, coconut oil, ginger, clove 

oil, and many other types [6]. 

For a long time, chlorhexidine has 

been the most widely used oral 

washes for reducing plaque and 

gingivitis, and there is no 

difference between an alcohol-

based or a water-based lotion, and 

it is considered the gold standard 

among oral washes [7] , 

Chlorhexidine is considered a 

broad-spectrum antibacterial 

against Gram-positive and Gram-

negative bacteria, fungi, and some 

viruses, and it has an active effect 

against Streptococcus mutans [8]. 

Propolis or what is known as bee 

gum or glue is a natural resinous 

substance that bees collect from 

the buds and parts of the plant, 

mixed with bee enzymes, pollen 

and wax. It has a complex formula 

with a wide range of antibacterial, 

antiviral, antifungal, and 

antioxidant effects [9]. 

Propolis has important uses in 

dentistry and preventive oral 

medicine, as studies in this field 

have shown the protective ability 

of propolis extracts when used to 

reduce the amount of bacterial 

plaque, thus reducing  dental 

caries [10], Barrientos et al 2013 

confirmed the promising efficacy 

of propolis in the prevention of 

dental caries and some diseases of 

the oral cavity, when they 

demonstrated the bio-efficacy of 

the ethanoli extract and metanoli 

of the Chilean propolis samples 

against the bacteria that cause 

caries , Streptococcus sobrinus 

and Streptococcus mutans [11] , 

honey is a natural, viscous, sweet-

tasting food compound, ranging in 

color from light brown to dark, 

resulting from the collection of 

nectar flowers or sugar secretions 

from some trees by bees and the 

addition of compounds to it that 

these bees secrete [12]. 

Honey has antibacterial activity 

which is proven as honey is a 

broad-spectrum inhibitor of many 

types of bacteria , including 

aerobic and anaerobic bacteria , 

the pros and cons of bacteria , as 

one laboratory study showed that 

methanol, ethanol, and ethyl 

acetate extracted from honey 

showed antibacterial activity 

against  Gram-positive 

(Staphylococcus aureus, Bacillus 

subtilis, Bacillus cereus, 

Enterococcus faecalis) and against 

Gram-negative bacteria 

(Escherichia coli, Pseudomonas 

aeruginosa, Salmonella typhi) [13]. 

The aim of this study is to evaluate 

the effect of mouthwashes 

chlorhexidine 0.12% - honey 50% - 

propolis 5% on Streptococcus 

mutans in children. 

Material and Methods 

The sample included 60 children 

(30 males - 30 females) aged 

between 6-9. 

The Tishreen University Research 

and Ethics Committee at the 

Faculty of Medicine and Dentistry 

approved the study protocol, and 

subjects gave their informed 

consent to participate. The study 

was conducted in accordance with 

the CONSORT statement. 

Inclusion criteria: Cooperative 

Children aged from 6 to 9 years 

old. Healthy patients with no 

history of previous systematic 

diseases that can affect their oral 

health.  

Exclusion criteria: Patients with 

gingivitis, having fixed or movable 

braces, and children who is taking 

any antibiotic before enrollment.  

Inform consent was taken from 

the volunteers before starting the 

study according to a special form 

designed for the research that 

includes details of the research 

and the materials used in the 

research. 

We used local products that are 

widespread in pharmacies. The 

children were divided into 3 

groups, each group containing 20 

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children (20 chlorhexidine - 20 

honey - 20 propolis).   

Saliva collection mechanism: [14] 

The child was brought to the clinic 

and the guardian requested the 

following: 

Avoiding food with a high content 

of acids and sugars 60 minutes 

before work in order to cause it to 

reduce the pH of saliva, which 

leads to an increase in bacterial 

growth. And, avoiding foods and 

drinks containing caffeine for 12 

hours before work. That the 

sample be collected between 9 - 

12 p.m. Emphasis on not brushing 

the teeth on the same day in order 

to preserve the oral flora and not 

to have bleeding that affects the 

accuracy of reading the results. 

Patients did not use any oral rinse 

or paste containing chlorhexidine 

7 days before the start of the 

clinical procedures. 

A first saliva sample was taken 

before using rinsing by using a 

sterile salivary swab for this 

procedure. The swab included 

passing the swab head over the 

vestibular surfaces of the teeth, 

the palate, the floor of the mouth, 

and the vestibule of the cheek. 

Then, the child was asked to rinse 

his mouth with solution using 10 

ml of solution for a period of time 

(30) seconds, after that, we took a 

second swab for the child, similar 

to its procedures for the first one, 

and the child's data were recorded 

on it (name - age - gender - swab 

before / after) on each cotton 

swab and sent to the bacterial 

culture laboratory at Hama 

National Hospital - Hama city - 

Syria, to start the laboratory work 

procedures. 

Chlorhexidine sample (20 children) 

The product used is a 0.12% 

chlorhexidine solution ready for 

use, produced by a national 

company. 

Propolis sample (20 children) The 

product used is a 5% propolis 

solution ready for use, produced 

by a national company. 

Honey sample (20 children) The 

honey was 100% concentrated 

from the packing of a national 

company. We drew 5 ml of honey 

and added it to 5 ml of distilled 

water in a sterile sample collection 

package, and we mixed it and then 

gave it to the child to rinse with it. 

Procedures for bacterial culture in 

the laboratory: 

Sampling extension: 

We dilate the saliva samples in 

two stages to reduce the bacterial 

load for ease of counting, 

provided that the real 

concentration of germs is 

calculated later as follows: 

Stage one: Use of 10000 

microliters (10 ml) of salin by 

disposable one use syringe and 

put it in the glass tube. Then, we 

remove 100 microns. Thus, we get 

9900 microliters in the tube. Then 

we add 100 microns of the saliva 

sample by micropipette. Finally, 

we get dilate of saliva sample in 

1/100 radio; 10-2. Then we mixed 

the homogenous dilate saliva 

sample on viberator for 30 

seconds. 

Stage two: Repeating the previous 

stages but by adding 100 

microliters of dilate solution to 

9900 microliters from the saline in 

the other glass tube and the ratio 

became 1/ 10000 and then 

repeated this homogenous 

process by vibrator. 

Bacterial culture: 

Culture medium: Preparation of 

the culture medium for 

Streptococcus mutans. We add 90 

g of Mitis Salivarius Agar (MSA) 

powder to 1 liter (1000 ml) of 

distilled water, then sterilize it in 

Autoclave at 121 C for 20 minutes 

under 15 lbs pressure, and when 

cooled to a temperature of 45-50 ° 

C, 1 ml of 1% Potassium tellurite 

solution is added to it, mixing the 

components well and pouring the 

mixture into sterile Petri dishes in 

the airflow chamber, according to 

the instructions of the 

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manufacturer of the culture 

medium. 

Culture method: we take 10 

microliters of dilate solution by 

micropipette. And then we spread 

it on the surface of culture media 

in Petri dish by sterilize platin loop 

tool in way that we get a distinct 

bacterial colony. And placed 

upside down within the incubator 

at 37 ° C for 48 hours in the 

presence of 5-10% of carbon 

dioxide. 

Bacterial appearance: The MSA 

culture medium performs 

selective isolation of Streptococcus 

mitis, Streptococcus salivirus, 

Streptococcus mutans, 

Streptococcus sanguis, 

Enterococci. 

Streptococcus mutans appear 

convex, crested, blue, pale, with a 

grainy appearance, As shown in 

figure 1 and figure 2. 

Colonies counting: we count the 

colonies by using An Electron 

microscope, After the colonies 

counting is complete. This number 

presents the number of colonies in 

10 dilate microliter. Thus, we 

multiply this number with 10000 

which is the dilation ratio then we 

divide the number by 1000 to get 

the final number of bacterial 

colonies in ml (CFU/ml). 

 

 

Statistical analysis: The data were 

analyzed using the statistical 

analysis program SPSS, version 

13.00, at a confidence level of 95% 

(P <0.05(. 

We used Paired sample T test to 

compare the variable means of 

the values of the studied variables. 

 

 

 

Results 

Table No. 1 shows descriptive 

statistical measures of the number 

of Streptococcus mutans among 

the 60 patients (20 patients in 

each group), while Fig. No. (3) 

shows the arithmetic averages of 

the number of Streptococcus 

mutans in the research patients. 

 

Figure 1 Indicates the number of 
Streptococcus mutans before 
applying honey. 

Figure 2 Indicates the number of 
Streptococcus mutans after 
applying honey. 

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Table No. 2 shows the results of 

using Paired Samples T Test when 

comparing the arithmetic means 

of the number of Streptococcus 

mutans in each patient group 

before and after the experiment. 

 

 

Table No. 3 shows the percentages 

of the amount of decrease in 

number of Streptococcus mutans 

between the two times (before 

using the substance and after 

using the substance) between the 

three experimental groups. 

 

 

As for Figure No. 4, it shows the 

percentage values of the amount 

of decrease in number of 

Streptococcus mutans between 

the two times (before using the 

substance and after using the 

substance) between the three 

experimental groups. 

 

max 

value 

minimal 

value 
SD mean Studied variables Sample Material 

222 96 43.11 161.30 
number of Streptococcus mutans before using 

honey 

20 Honey 

53 23 10.23 38.25 
number of Streptococcus mutans after using 

honey 

222 101 40.24 158.45 
number of Streptococcus mutans before using 

propolis 
20 propolis 

25 9 4.48 15.90 
number of Streptococcus mutans after using 

propolis 

250 117 33.87 173.70 
number of Streptococcus mutans before using 

chlorhexidine 
20 chlorhexidine 

37 17 4.94 25.20 
number of Streptococcus mutans after using 

chlorhexidine 

Table No 1. Statistical measures of the number of Streptococcus mutans in the research patients 

Figure 3 Mean of the number of Streptococcus mutans in 
the research patients. 

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sig: significance. 

*: statistically significant. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SIG P-value df 
T 

Test value 

difference 

between 

arithmetic means 

Comparisons Materials 

* 0.000 19 16.731 123.05 

number of 

Streptococcus mutans 

before using honey - 

number of 

Streptococcus mutans 

after using honey 

Pair 1 

 

 

Honey 

* 0.000 19 22.876 148.50 

number of 

Streptococcus mutans 

before using propolis - 

number of 

Streptococcus mutans 

after using propolis 

Pair 1 propolis 

* 0.000 19 17.428 142.55 

number of 

Streptococcus mutans 

before using 

chlorhexidine - number 

of Streptococcus 

mutans after using 

chlorhexidine 

Pair 1 chlorhexidine 

Table No. 2. Results of using Paired Samples T Test when comparing the arithmetic means of the number of 

Streptococcus mutans before and after the experiment. 

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Discussion  

The saliva count is a reasonable 

indicator of a complete bacterial 

load in the oral cavity, and the 

count of Streptococcus mutans or 

Lactobacillus in total dental plaque 

samples does not explain the 

variation in the incidence of caries  

 

 

 

as well as it does when counted in 

induced saliva [15]. 

In this study we studied the count 

of Streptococcus mutans in saliva 

because there is an association 

between the count of 

Streptococcus mutans in saliva and 

the occurrence of dental caries in 

children, and often there  

 

 

is a correlation between the levels 

of the Streptococcus mutans and 

caries activity. Identification 

persons with a high risk of caries 

[16]. 

bacteria have developed 

antibiotic-resistant strains, and  

 

Reduction ratio 

% 

The amount 

of decrease 

Streptococcus mutans 

variable value after using the 

material 

Streptococcus mutans 

variable value before using 

the material 

Number of 

patients 
Materials 

76.29% 123 38.25 161.30 20 Honey 

85.49% 149 25.20 173.70 20 Propolis 

89.97% 143 15.90 158.45 20 Chlorhexidine 

76.19% 125 39.167 164.48 60 Total 

Table No. 3. The percentages of the amount of decrease in number of Streptococcus mutans between the two times 

(before using the substance and after using the substance) between the three experimental groups. 

Figure 4 The percentages of the amount of 
decrease in number of Streptococcus 
mutans between the two times (before 
using the substance and after using the 
substance) between the three experimental 
groups. 

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pharmaceutical factories found it 

difficult to develop new 

antibacterial agents to meet this 

new challenge due to the high 

costs of drug research, which 

prompted researchers to search 

for an alternative to low-cost 

natural materials [17]. 

Reports have shown the efficacy 

of honey against a wide spectrum 

of clinically resistant multi-

bacteria, so its use has emerged as 

a promising alternative to 

industrial pharmaceutical products 

[17]. 

The rate of decrease in the count 

of Streptococcus mutans was 

statistically significant after one 

minute of using chlorhexidine, as 

it reached 89.97%, and in propolis 

it was 85.49%, and in honey it was 

76.29%. 

The results of this study showed 

that the three oral rinse had an 

immediate and direct effect on the 

bacteria of the Streptococcus 

mutans, as chlorhexidine had a 

higher direct efficacy on the count 

of Streptococcus mutans , then 

propolis and then honey. 

The results of this study regarding 

the antibacterial effect of honey 

oral washes are in agreement with 

the results of Rupesh et al. 2014, 

which confirmed the antibacterial 

properties of New Zealand honey 

oral washes [18]. 

The results of our study also 

agreed with the study of Nassar 

and colleagues 2012, where they 

compared natural honey with 

synthetic honey in its effect on the 

Streptococcus mutans found in 

plaque, and found the effect of 

natural honey much higher than 

synthetic [19], This negates the 

effect of the high sugar content in 

honey on the oral bacteria.  

We agreed with the results of 

Hegde and colleagues' 2013 study 

in which they indicated that 

propolis has anti-Streptococcus 

mutans activity in the oral cavity 

and can be used as a measure to 

prevent dental caries [20]. 

We also agreed with the results of 

the study by Elbaz and colleagues 

2012, where the study compared 

New Zealand honey and Egyptian 

propolis extract on Streptococcus 

mutans and Lactobacillu, and the 

study Draw a Conclusion that 

Egyptian propolis was superior to 

New Zealand honey in affecting 

the count of Streptococcus mutans 

and Lactobacillus [21]. 

The results of our study on the 

effect of chlorhexidine on 

Streptococcus mutans are in 

agreement with the results of the 

Fredri Lundström and Krasse 1987 

study where the use of 

chlorhexidine reduced the count 

of Streptococcus mutans in fixed 

orthodontic patients [22]. 

Conclusion 

This study demonstrated the 

effectiveness of mouthwashes of 

chlorhexidine, propolis and honey 

on the bacteria of Streptococcus 

mutans. The results of the present 

study showed the effectiveness of 

chlorhexidine mouthwashes 0.12% 

- honey 50% - propolis 5% in 

reducing oral bacterial count 

rates. Chlorhexidine showed the 

maximum effectiveness, followed 

by propolis, then honey. 

Acknowledgment 

None. 

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