Vol 51 No 1 Jan-Mrt 2018.indd


4747

An in-vitro antimicrobial effect of 405 nm laser diode combined 
with chlorophylls of Alfalfa (Medicago sativa L.) on Enterococcus 
faecalis

Suryani Dyah Astuti 1,2
1 Biomedical Engineering Magister Program, Post Graduate School, Universitas Airlangga
2 Department of Physics, Faculty of Science and Technology, Universitas Airlangga
Surabaya - Indonesia

ABSTRACT

Background: Enterococcus faecalis (E. faecalis) is a bacterium commonly detected in the root canals of teeth with 
post-treatment apical periodontitis or advanced marginal periodontitis. It has the ability to live in an extreme environment 
and survive as an organism with its virulence factor possibly contributing to the pathogenesis of post-treatment apical 
and marginal periodontitis. Photodynamic therapy (PDT) is an urgently required alternative method of improving therapy 
effectiveness. Photodynamic therapy combined with conventional endodontic treatment decreases the number of antibiotic-
resistant bacteria and biofilms. Chlorophyll is one of the photosensitizers added to enhance the absorption of light in 
photodynamic therapy. Purpose: The purpose of this study was to determine the antimicrobial effect of the combination 
of photodynamic laser therapy and Alfalfa chlorophyll in E. faecalis. Methods: In vitro study using E. faecalis distributed 
between negative control (C-) and positive control (C+), treatment groups using various energy doses of a 405 nm diode 
laser (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 J/cm2) with (G1) and without alfalfa chlorophyll as organic photosensitizer (G2). 
The suspension was inoculated on Tryptocase Soy Agar (TSA) and incubated at 37° C for 24 hours. The number of colony-
forming units per milliliter (CFU/ml) was determined. The results were analyzed by ANOVA with p value ≤0.05. Results: 
A 405 nm irradiating laser with or without a photosensitizer can decrease E. faecalis viability percentage through the 
administering of various energy doses. The highest decrease (42%) was obtained in the group without a photosensitizer 
using 20 J/cm2, while 10 J/cm2 in the group with a photosensitizer proved the most effective dose (25%). Conclusion: The 
results of this study showed a decrease in the viability of E. faecalis exposed to a 405 nm (40 mW) laser. An irradiating 
process using a 405 nm laser without a photosensitizer (Alfalfa chlorophyll) resulted in the highest percentage decrease 
(42%) in E. faecalis bacterial viability.

Keywords: antimicrobial Photodynamic therapy; Enterococcus faecalis; diode laser 405 nm; Alfafa chlorophyll

Correspondence: Suryani Dyah Astuti, Department of Physics, Faculty of Science and Technology, Universitas Airlangga. Jl. Mulyorejo, 
Mulyorejo, Surabaya 60115, Indonesia. E-mail: suryanidyah@fst.unair.ac.id 

Research Report

INTRODUCTION

Enterococcus faecalis (E. faecalis), a Gram-positive 
bacterium commonly found in the root canal, is ovoid 
in shape with a diameter of between 0.5 and 1 μm. This 
bacterium is a facultative anaerobe and possesses the ability 
to survive in an extreme environment such as in a highly 
alkaline pH and high salt concentrated condition.1

E. faecalis is resistant to calcium hydroxide and 
antibiotics.2 Tetracycline produce a poor antimicrobial 
effect on periodontal E. faecalis, with more than 50% of 
the E. faecalis periodontal isolates showing resistance. 
Furthermore, E. faecalis isolates from root canals 
demonstrate a high prevalence of the genetic determinant of 
tetracycline resistance (tetM). Recent studies have indicated 
that tetM genes were detected in approximately 50% of 
isolates from the root canal.1 

Dental Journal
(Majalah Kedokteran Gigi)

2018 March; 51(1): 47–51

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
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48 Astuti/Dent. J. (Majalah Kedokteran Gigi) 2018 March; 51(1): 47–51

E. faecalis demonstrate high frequency in cases of poor 
response to either endodontic or periodontal treatment.3 
Therefore, alternative methods are urgently needed to 
improve the therapy effectiveness, one such method being 
photodynamic therapy which is a medical treatment that 
utilizes light to activate photosensitizer agents. Exposure of 
the photosensitizer to light produces a wide range of oxygen 
species and free radicals that cause localized damage and 
the death of bacteria.3 Photodynamic therapy is also known 
as an antimicrobial photodynamic therapy (aPDT) and 
photodynamic inactivation (PDI).4 

Photodynamic therapy uses light sources and light-
sensitive photosensitizer agents. The suitability of the 
light spectrum and photosensitizer agent to photodynamic 
therapy will produce ROS. Light sources such as Light 
Emitting Diodes (LED) and a variety of lasers can be 
used to activate the photosensitizer agent.3 These lasers 
generally have complicated systems resulting in a high 
cost. Currently, diode lasers are widely used because they 
offer the advantages of simple systems, portability and 
low cost.5 Research results showed that photodynamic 
therapy combined with conventional endodontic treatment 
decreases the prevalence of bacteria and biofilms.6,7

Certain photosensitizer agents have been tested for their 
ability to efficiently produce reactive oxygen species (ROS) 
formed through photochemical type I or II that inactivate 
microbial cells.8 Antimicrobial photodynamic therapy 
success relies heavily on the type of photosensitizer, the 
light source wavelength and output power and the irradiation 
time used.9 The use of low power laser photodynamic 
therapy with different exogenous photosensitizers, toluidine 
blue (TBO),10,11 methylene Blue (MB),12–14 and organic 
photosensitizer curcumin15 has been investigated. For these 
reasons, chlorophyll is more appropriate for development as 
a photodynamic therapy in cases of tumors and cancer.16

Chlorophyll is a substantial bioorganic molecule 
with the core function of absorbing light and transferring 
excitation energy to the reaction center in photosynthetic 
devices. High absorption energy during the photosynthesis 
process is caused by a relatively long excitation process 
of chlorophyll (≤ 10-8 seconds). The longer the process of 
singlet excitation of chlorophyll, the greater the electronic 
energy converted. The conversion process may occur 
from a basic level to ones of triplet excitation. The excess 
energy produced by chlorophyll at the level of triplet 
excitation provides an opportunity to transfer energy to 
oxygen molecules, a process producing reactive singlet 
oxygen.17

In this study, chlorophyll was extracted from the 
leaves of Alfalfa (Medicago sativa L), a type of perennial 
leguminous plant with high chlorophyll content.18 Other 
research showed that chlorophyll a and b, in addition to 
pheophytin are all powerful antioxidants that can reduce 
ROS and prevent lipid peroxidation and DNA damage.19 
The purpose of this study was to determine the antimicrobial 
photodynamic effect of the photodynamic therapy laser 

diode in combination with alfalfa chlorophyll in E. faecalis 
bacteria.

MATERIALS AND METHODS

The sample strains used in this research consisted of 
purely cultured bacteria from E. faecalis ATCC 29212. 
The bacteria strains were grown in Tryptic soy agar (Oxoid, 
England, UK), subsequently inoculated in Triptic Soy Broth 
(TSB) solution (Merk, Darmstadt, Germany), incubated for 
24 hours and, finally, diluted to a value of optical density of 
OD600nm = 1.6/0.142, which is equal to ~ 10

3 CFU/mL.
Alfalfa chlorophyll (K-Link liquid chlorophyll, Jakarta 

Selatan, Indonesia) at a concentration of 1.6 mg/ml was 
diluted in normal saline. The absorption spectrum of 
chlorophyll was measured using a Shimadzu UV-VIS 1800 
spectrometer (Shimadzu, Tokyo, Japan).

Laser irradiation was carried out using Sony diode lasers 
with output wavelengths of 405 nm. The power outputs 
were 40 mW with a focal spot ®xed: 0.3 cm2 

This research was conducted using post-test only control 
group design. The samples were distributed into four groups: 
(1) E. faecalis exposed to neither a 405 nm diode laser nor 
Alfalfa chlorophyll as the Negative Control (C-); (2) E. 
faecalis exposed only to 1.6 mg/ml Alfalfa chlorophyll as 
the Positive Control (C+); (3) E. faecalis exposed to various 
energy doses of 405 nm diode laser (2.5; 5.0; 7.5; 10.0; 
12.5; 15.0; 17.5; 20.0) and 1.6 mg/ml Alfalfa chlorophyll 
as Group 1 (G1); (4) E. faecalis exposed to various energy 
doses of diode laser 405 nm (2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 
17.5; 20.0 J/cm2) without Alfalfa chlorophyll as Group 2 
(G2). After treatment, the suspension was inoculated on 
TSA and incubated at 37o C for 24 hours. The number of 
colony-forming units per milliliter was counted manually 
to determine the antimicrobial effect on E. faecalis. The 
percentage of decrease bacterial viability was defined as: 

(Ʃ control colony – Ʃ treatment colony) ×100%
Ʃ control colony

A broth microdilution method was applied to determine 
the minimum inhibitory concentration (MIC) of Alfafa 
chlorophyll against E. faecalis. Testing was conducted 
using a 96-well flat-bottomed microplate (Nunc, Sjælland, 
Denmark). Each well contained 90 μl TSB, 90 μl Alfafa 
chlorophyll at various concentrations (0.8, 1.0, 1.4, 1.6 
mg/ml), and 20 μl of E. faecalis culture at a concentration 
of 10 CFU/ml1. The microplates were incubated for two 
hours at 37° C under microaerophilic conditions. The MIC 
was defined after two hours of incubation. All tests were 
repeated at least four times. The number of colony-forming 
units per milliliter (CFU/ml) was calculated manually to 
determine the MIC test results which were log-transformed 
and analyzed with SPSS version 10.05 (SPSS Inc., Chicago, 
Illinois, USA) using ANOVA (p=0.05).

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v51.i1.p47–51

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49Astuti/Dent. J. (Majalah Kedokteran Gigi) 2018 March; 51(1): 47–51

RESULTS

The photosensitizer used in this study was Alfalfa 
chlorophyll, the absorption spectrum of which20 is shown 
in Figure 1.The MIC test for Alfalfa chlorophyll confirmed 
that the dye had no toxic effects on E. faecalis. Statistical 
results showed that the chlorophyll concentrations (0.8, 
1.0, 1.4, 1.6 mg/ml) did not significantly differ from each 
other at p=0.12 (p>0.05). 

Figure 2 shows the viability of E. faecalis exposed to 405 
nm diode laser at the same concentration of photosensitizer 
(chlorophylls). The 405 nm diode laser treatment group 
resulted in statistically significant increases and decreases  
of CFU p=0.00 (p<0.05) compared to the control group. 
The statistical test results showed the largest percentage of 
reduction in bacterial viability to be in the treatment of 405 
nm 20.0 J/cm2 laser exposure to be 41.75%.

Figure 1. The absorption spectrum of chlorophyll Alfalfa (Medicago sativa L.)

Figure 2. The viability of E. faecalis exposed to diode laser 405 nm.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v51.i1.p47–51

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50 Astuti/Dent. J. (Majalah Kedokteran Gigi) 2018 March; 51(1): 47–51

Figure 3 shows the percentage (%) of decreasing 
bacterial viability at various irradiating laser energy doses 
with chlorophyll as a photosensitizer. The results in figure 
3 indicate that the irradiating process using a 405 nm laser 
with and without chlorophyll can reduce the viability of 
E. faecalis bacteria. A 405 nm laser without a photosensitizer 
(Alfalfa chlorophyll) produced the highest decrease in the 
E. faecalis bacterial viability percentage (42%) and that of 
the photosensitizer (25%).

DISCUSSION

Antimicrobial photodynamic therapy requires a light 
source at a specific wavelength that activates the PS. This 
study used a laser diode at an output power wavelength of 
405 nm with a range of 40 mW. For medical applications, 
the mechanism of photochemical interaction plays a 
significant role in photodynamic therapy. Similarly with 
biostimulation, photochemical interactions occur at a very 
low power density (1 W/cm2) and an exposure time of one 
second.21 The results of temperature measurements in figure 
3 showed that during irradiation the temperature stablized 
below 45o C, which remained in the optimum growth range 
of the E. faecalis bacterium. This suggested that bacterial 
death was not caused by an increase in temperature but, 
rather, was due to irradiation.

The photosensitizer used in this study was Alfalfa 
chlorophyll at a concentration of 1.6 mg/ml. The 
absorption spectrum of exogenous photosensitizer in 
figure 4 indicated effective absorption at a blue and red 
wavelength. Based on the data, the quantum yield of laser 
diode with a wavelength of 405 nm could be calculated. The 
chlorophylls absorption stood at 92.97%.22 The absorption 
percentage of photosensitizer affects the production of 
ROS. The concentration of photosensitizer and conformity 
of the light wavelength with the absorption spectrum 

of the photosensitizer leads to successful antimicrobial 
photodynamic therapy.23 

This study used a diode laser with a 405 nm wavelength 
and the same focal spot. Table 1 shows the energy doses 
and time duration of laser irradiation applied in this study. 
Irradiation at different wavelengths produces a variety of 
quantum yields. The similarity of the wavelength of the 
light source with the photosensitizer absorption spectrum 
will produce a photophysical mechanism, i.e. the absorption 
of light energy. Absorption of light energy will excite 
photosensitizer molecules that trigger the occurrence 
of photochemical reactions resulting in radical oxygen 
species. Another deciding factor was the radiation energy 
dosage. The appropriate dose of energy activates a chemical 
reaction producing a wide range of reactive oxygen species 
that causes photoinactivation in bacteria. The energy dose 
of laser irradiation per total area of irradiation (power 
density, unit J/cm2) is the magnitude of radiation energy 
(power multiplied by the longer exposure time) divided 
by the total area of irradiation. This determines the time 
duration of the laser irradiation adjusted to the energy dose 
and quantum yield.

The results of this study showed a decrease in the 
viability of E. faecalis that were exposed to a 405 nm 
laser with a power output of 40 mW. The irradiating 
process using a 405 nm laser at an energy dose of 20 J/cm2 
without a photosensitizer (Alfalfa chlorophyll) resulted in 
the highest decrease in the E. faecalis bacterial viability 
percentage (42%). An irradiating 405 nm 10 J/cm2 laser 
with a photosensitizer resulted in the highest percentage 
decrease (25%) in the bacterial viability of E. faecalis. 

Singlet Dioxygen is capable of causing permanent 
damage to various parts of the cells, including: the plasma, 
mitochondria, lysosomes, nuclear membranes, in addition 
to the modification of proteins. Photodynamic therapy 
within various photosensitizer molecules is more effective 
in immobilizing Gram-positive than Gram-negative 

Figure 3. The percentage of the E. faecalis decrease exposed to laser 405 nm.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v51.i1.p47–51

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51Astuti/Dent. J. (Majalah Kedokteran Gigi) 2018 March; 51(1): 47–51

bacteria.24,25 Differential susceptibility to photodynamic 
therapy arises because of contrasting cell wall structure 
of the respective groups. The outer wall (15–80 nm 
thick) of Gram-positive bacteria consists of more than 
100 layers of peptidoglycan-related lipotechoic and 
teichuronic acids. This layer is relatively porous allowing 
macromolecules with a molecular weight of 30,000–60,000 
Da to diffuse across the plasma membrane. Therefore, a 
photosensitizer with a molecular weight of 1500-1800 Da 
can be diffused. Gram-negative bacterial cells consist of 
the cytoplasmic membrane and outer membrane separated 
by peptidoglycan-containing periplasm. Moreover, 
the latter is of heterogeneous composition, including: 
proteins that function as porin, lipopolysaccharide and 
lipoprotein. The outer membrane forms an effective barrier 
to permeability. Only hydrophilic compounds with a low 
molecular weight of 600-700 Da can diffuse and limit 
the penetration of photosensitizer. Type 1 photochemical 
reactions occur in Gram (+), while type II occur in Gram (-). 
Photoinactivation in bacteria consists of several processes: 
(a) photosensitizer translocation to the plasma inner 
membrane, (b) photoinactivation of the photosensitizer, (c) 
the generating of ROS, (d) oxidative modification of the 
target, (e) a decrease in cell function and metabolism and 
(f) inhibition of cell growth and cell death.25  

The results of this study showed a decrease in the 
viability of E. faecalis that had been exposed to a 405 nm 
(40 mW) laser. An irradiating process using a 405 nm laser 
without a photosensitizer (Alfalfa chlorophyll) resulted 
in the highest percentage decrease (42%) in E. faecalis 
bacterial viability. The use of photosensitizer (Alfalfa 
chlorophyll) decreases bacterial viability to 25%. 

ACKNOWLEDGEMENT

The research reported here was funded by a 2016 grant 
from the Ministry of Research, Technology, and Higher 
Education.

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Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v51.i1.p47–51

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51