In situ substantivity of the essential oils in the oral cavity

I. Prada-López1, V. Quintas1, and I. Tomás.2

1
PhD Student. School of Medicine and Dentistry. Santiago de Compostela University. Spain.
2
Special Needs Unit. School of Medicine and Dentistry. Santiago de Compostela University. Spain.

The proved antimicrobial and anti-inflammatory activities of the Essential Oils (EO) led, in 1879, to their combination
with a phenolic based formula to reach a higher potential. This new product was called Listerine ®. After that, it was
gradually acquiring more importance as an oral antiseptic until nowadays, when it is considered as the most popular
phenolic compound used in the oral cavity. Due to this, the number of studies about its mechanism of action has been
increasing, especially in the last decade. The antiseptic activity of the EO comes from their hydrophobicity, which
produces a disturbance in the bacterial membrane that affects to diverse cellular compounds causing a cascade effect. The
purpose of this chapter is to review the former published studies about the in situ antibacterial activity of the EO in
different oral micro-niches, as well as presenting our own results in this field.
One of the ideal characteristic that antiseptics must have is the ability to adhere to the substrate and persist at effective
concentrations, which is called substantivity. Thus, there are some published papers about the substantivity of the EO in
the saliva, which concluded that these last ones kept lower bacterial vitality levels than the negative control between 1-5
hours after their application; nevertheless, there are few papers which study the in situ antibacterial activity of the EO on
structured biofilm and, in those cases, their application was done ex vivo. Up to now, there has not been published any
paper comparing the EO antibacterial activity with Chlorhexidine’s (CHX) –the Gold-standard- after a single application,
and there are only few cases in which the immediate antibacterial activity of the EO was evaluated. What is common to
most of the papers is the using of the Confocal Laser Scanning Microscope (CLSM) combined with a staining solution.
This fact is due to its consideration as an effective technique when analysing both the structure and the bacterial vitality in
an oral biofilm.
Consequently, it seems to be interesting and necessary to continue studying with CLSM techniques the EO antibacterial
activity. The methodology should be directed to in situ antiseptic application, and the results obtained should be compared
with other antiseptics. Thereby, a more reliable approach about the ability of the EO as oral antiseptics could be obtained.

Keywords: essential oils; Listerine; substantivity, oral biofilm; bacterial vitality; confocal laser scanning microscopy.

1. Introduction

Since ancient times, the essential oils (EO) have been used for the treatment of a large variety of
diseases all over the world, from the Egyptians to the Mayas or Aztecs [1]. In 1879, the Dr. Josep Lawrence and the
pharmaceutic Jordan Wheat Lambert developed a phenolic compound [2] whose antimicrobial activity was enhanced by
the combination with some EO (Thyme, Eucaliptus, Baptisia, Gahulteria and Mentha Arvensis) [3]. This formula was
called Listerine® in honor of Sir Joseph Lister, father of the antisepsis in medicin [4]. Although it had been designed as
an antiseptic for surgeries and it also had demonstrated its antimicrobial abilities, it had a poor aceptation among the
surgical medicin field. It soon adquired other indications such as: treatment of gonorrhea, floor cleaner, anti-dandruff
solution, aftershave lotion or remedy for baldness. After that, it was observed that it was especially effective against
germens commonly found in the oral cavity. As a result, in 1895, Lambert extended the sale and promotion of his
product to the dental profession [2]. It was his son, Gerald, who introduced the product to Americans as a palliative for
halitosis, becoming so popular that in 1914 was one of the first prescription oral products and technically reached the
category of mouthwash. By this time, the oils included in the original formula had been already replaced by eucalyptol,
thymol, menthol and methyl salicylate - the latter two are replaced by synthetic derivates today- [5]. In the decades of
70s-80s, it started to be considered as a mouthwash against not only the halitosis but other mouth diseases [6]. This
condition became true in 1986 when the American Dental Association approved them for the control of the dental
plaque and gingivitis [7] based on some existing studies that sastified their criteria [8-11].

Nowadays, Listerine® is widely used all over the world and has been used for millions of costumers,
particularly in the United States [12]. Furthermore, it has been considered as effective and safe by the Expert Comitee in
Oral Health of the Food and Drug Administration (FDA) [13].
One of the most important characteristic that the EO have
is their ability to adhere to the substrate and persist at effective concentrations, which is called substantivity . The
purpose of this chapter is to review the findings published in the literature on the in situ substantivity of EO in the oral
cavity, and to report our own results in this field.
 2. General characteristics of the essential oils
The EO are natural products that plants produce for their own needs. Different types of techniques are used to
extract them which include: the microwaves, the liquid carbon dyoxide or the steam distillation [1, 14]. At present, the
pharmaceutical industry replaces the plant extraction by the chemical synthesis in case its natural production is difficult
[5].

2.1 Chemical structure

They can be classified in two groups: terpenes (and terpenoids) or aromatic derivates (Fig. 1). The first are the
most common group and they are characterized by a extensive variety in their structural groups [15]. In this group there
are also included the terpenoids which are terpenes with an oxygen. The second group is based on an aromatic
compound that, being less common than terpenes, comes from phenylpropane by-products.

Fig. 1 Chemical formulas

exemplifying the different types
of EO.

2.2 Physical properties

They are volatile and water-insoluble which makes very difficult the determination of their antibacterial effect [16].
Moreover, their high hydrophobicity and viscosity cause an irregular distribution when testing this antiseptic activity.
Due to this, it is always necessary a solvent agent to prevent the unequal dilution in the culture medium [17].

2.3 Mechanism of antibacterial action

The EO have a complex mechanism of antibacterial action [18], in fact, it was unknown or even perceived until 1985
[10]. Even so, the antimicrobial actions of the EO are intimately attached to their major characteristic, its
hydrophobicity, which produces an increase in the bacterial membrane permeability and the consequent loss of their
main cellular elements [19-22]. It is important to remark that a perturbation in the bacterial covers (membrane and
wall) can affect to other cellular compounds and produce a cascade of events [23] that are explained in the figs. 2 and 3.

Fig. 2
Graphic of the effects of the EO on the bacterial cells.
 1. Cell wall and membrane disturbance. Increase in

the cell
permeability.
2. Loss of the cell membrane potential.
3. Disturbed trans-membrane transport, accumulation of toxins inside the cell.
4. Intracellular ATP decrease. The ATPs go out of the cell due to the membrane increased permeability.
5. Intracellular pH falls down because of the inability of the membrane to block the extracellular protons.
6. Appearance of coagulated material in the intracytoplasmatic cell space.
7. Inhibition of the “Quorum Sensing”. Stopping of the intra- and inter-cellular communications.
8. Appearance of mutations in the DNA.

 Perturbación de la membrana y la pared celular

Los aceites esenciales producen una pérdida del potencial de membrana, lo que afecta seriamente a los mecanismos de transporte [24] (Bouhdid 2009). Estas modificaciones estructurales suceden en la bicapa lípidica, condicionando el transporte trans-membrana, produciendo una limitación en la liberación de toxinas al medio. Por lo que existe una acción indirecta inhibitoria sobre la producción de toxinas[25] (Somoza 2010).

 Producción de ATP
La producción de ATP en procariotas ocurre tanto en la membrane como en el cytosol. Por tanto, se espera que el balance de ATP intra y extracelular esté alterado debido a la desestructuración de membrana producida por los EO. Y así lo demuestran los estudios que revelan una pérdida de ATP a través de la membrana alterada por la acción de los EO[21] (Turgis 2009).

 Sintesis de proteinas
El studio de [26] Burt en 2007, inducen la formación de HSPs (Heat Shock Proteins) que inhibieron la síntesis de flagenlina, dando como resultado bacterias no-motrices.

 Perturbación del Ph intracellular

. Probablemente debido a la pérdida de la capacidad de la membrana para bloquear protones[21] (Turgis 2009).

 Cambios intraplasmáticos

En el estudio de [27] Becerril 2007 se identificaron cambios tales como material coagulado, espacio periplásmico más grande e irregular, además de ausencia de fimbrias en el mismo.

 Alteraciones en el ADN

Las mutaciones que producen los EO confieren a las células bacterianas una apariencia de pronunciada rugosidad y que las vuelve permeables y completamente inofensivas [28, 29] (Mezzoug, 2007 y De Martino, 2009).

 “Quorum Sensing”

El QS está involucrado en la producción de biofilm, motilidad, resistencia al estrés y virulencia (Klelleberg y Molin 2002). [30]

Fig. 3. Scheme of the mechanism of antibacterial action of the EO.

2.4 Chemi cal composition of a mouthwash.

The exact composition of Listerine® (Menthol) is an aqueous-ethanolic solution (21.6% - 26.9%) which serves as a
vehicle of a combination of four EO: eucalyptol, thymol, methyl salicilate and menthol (see Table 1) [5, 18, 31, 32].

Table 1. Listerine containing EO with its chemical structure, natural origin, chemical synthesis, pharmaceutic properties and its
concentration.

Chemical Pharmaceutic Concentration

Name Natural Origin Chemical Synthesis
Structure Properties in Listerine
C10H18O

Eucalyptol Eucalyptus globulus Ø 0.092%

Anesthesic
& antiseptic

Bactericid &
Thymol C10H14O Thymus vulgans Phenolic Alquiderivate 0.064%
fungicid
 C8H8O3
Esterification of
Gaultheria and/or
Methyl Salicilate salicylic acid with Antiseptic 0.06%
Betülla
methanol

C10H20O

Hydrogenation of
Menthol Mentha arvensis Antiseptic 0.042%
thymol

3. In situ antimicrobial activity of EO in different oral ecosystems

Study of the antibacterial activity of an antiseptic involves an analysis of its immediate effect and substantivity. This
term is defined as the prolonged adherence of the antiseptic to the oral surfaces (teeth and mucosas) and its slow release
in effective doses, that guarantee the persistence of its antimicrobial activity [33]. This property is essential for an
antiseptic to be clinically effective .
Although in vitro studies do not have to be predictive of clinical activity, they may elucidate its subsequent
mechanism and guide the objectives of the in situ studies. Thereby, in vitro studies, whose objective is to determine the
antimicrobial activity of the EO, can be found in the literature. In this same way, Fine et al [34] concluded that a single
application of EO could achieve up to 97% of decrease in the bacterial charge, being for Streptoccocus a 99.7%. In
other studies, their results are compared with the clorhexidine’s (CHX) with diverse conclusions depending on the
consulted paper. That is the case of the study of Pan et al [35] who found that after 60 seconds-single application of EO
and CHX, the first were a 57.5% more effective than CHX. In the contrary, Sliepen et al [36] concluded that the CHX
reduced the bacterial vitality in a higher proportion than the EO. However, the scientific community recognizes that in
vitro oral biofilms are not comparable to those formed in situ [37-39]. For this reason, some authors consider that the
results of in vitro studies should be taken with caution [37, 40, 41] and highlight the necessity of the development of in
situ biofilm models that permit their posterior analysis without being disturbed [37, 41-43].
To a better understanding of the clinical effects that the antiseptic agents produce on the oral biofilm is necessary to
apply a methodology in which this biofilm can directly grow inside the oral cavity. Moreover, its three-dimensional
structure should not be distorted with manipulation [44, 45].

3.1 In situ antimicrobial activity of the EO on salivary flora

The analysis of the salivary flora provides information on antiseptic agent antimicrobial activity and is considered to be
predictive of its substantivity [46, 47]. This is due to the ability of the saliva to be a microorganism dissemination
medium inside the oral cavity, since it wets the entire inner mouth surface. That is why Weiger [48] suggested that the
first phenomena of microbial colonisation after contact with a clean tooth surface occur mainly due to the adhesion of
salivary bacteria. However, other authors argued that a single determination of the reduction in the bacterial load in
saliva does not demonstrate any correlation with effects on plaque inhibition [49].

 Methodological approach

In the majority of published studies, the quantification of the antimicrobial activity of EO in saliva was performed using
plate culture microbiological techniques [34, 50-53]. However, some authors have questioned the reliability of this
methodology [54], referring numerous difficulties that could underestimate the bacterial vitality [55] or, in some cases,
overestimate specific bacterial genera. As a consequence, the epifluorescence microscopy technique (Fig. 4) with
specific fluorochromes, such as LIVE/DEAD® BacLight™ [55, 56] has been posed as an alternative for the plate culture
techniques. This fluorescence solution detects bacterial vitality based on the integrity of the cytoplasmic membrane. The
combination of fluorochromes offers the possibility of staining live and dead bacteria in a selective manner. That is why
they have been widely used to analyse bacterial vitality before and after the application of oral antiseptics such as CHX
on saliva [57, 58] or EO on oral biofilm [59, 60] (no data has been found over its use on saliva after the application of
EO). The main advantages of this technique include: the rapidity and simplicity of the technique, which quantifies
bacterial vitality in real time [55]; the SYTO-9/propidium iodide dual staining allows vital and non-vital bacteria to be
counted simultaneously [55]; and the possibility of detecting bacteria that cannot be cultured using plate culture
techniques [54, 61].
 Fig. 4. Images from
epifluorescence
microscope, bacterial
vitality shown before and
after a mouthwash. Note
the predominance of green
in the image before the
mouthwash (live bacteria)
and after rinsing when
there is a predominance of
red (dead bacteria).

 Immediate effect and substantivity of EO in saliva

As previouly said, published data about the EO substantivity has been obtained through culture plate techniques,
measuring the CFU/mL after a certain period (24 - 48 h). Jenkins et al [50] studied the antibacterial activity and its
permanence on salivary microbiota in 14 volunteers using 5 different mouthwashes. They concluded that the effect of
the EO was already patent 7 hours after a single application, being this effect lower than 0.2% CHX. Later, in 1996,
DePaola et al [51] stated that the EO might have clinical utility as a pre-procedural rinse to decrease the level of viable
microorganisms in aerosols generated during dental procedures. They studied its activity on saliva after 2, 5, 15, 30 and
60 minutes, concluding that a single EO mouthwash decreased up to 60-65% the bacterial counts with regard to the
baseline, keeping its effects, at least until 1 hour after the a single application in comparison to the basal sample. Other
results partially related to this are those obtained by the group of Fine [34] in which they show a decrease of 50.8% in
Streptococcus in comparison to the negative control, after 11 days rinsing twice a day with a containing EO mouthwash.
Botelho et al [53] added some clinical indexes to the plaque culture techniques to compare the effect of rinsing with EO
or CHX after 7 days. There was a decrease in all analysed indexes and Streptoccocus mutans in saliva with regard to the
basal in both mouthwashes, being their effects quite similar.
The EO not only appear to be effective against oral bacteria but also against virus. In this sense, there is an
interesting study conducted by Meiller [62] who concluded that after a single EO mouthwash there was a significant
decrease in the presence of the HSV in saliva. In this case, they use direct immunofluorescence of cytological smears of
the oral fluids.

3.2 In situ antimicrobial activity of EO on plaque-like biofilm

In the oral cavity biofilms are developed spontaneously on the teeth surface, prosthesis, dental implants and on the oral
epithelium [63].
The scientific community has demonstrated that the growth of structured bacterial populations over a surface differs
phenotypically from their counterpart in planktonic phase [42, 64, 65]. As a result, it has been suggested that the
bacteria present in the biofilms behaves like cells in a multicellular organism, collaborating and communicating like a
basic circulatory system [66]. This affirmation can explain the reason why the bacteria in biofilms may be from 10 to
1000 times more resistant to an antimicrobial treatment than those grown in planktonic phase [67, 68]. This finding
could be related to the slower growth rate of biofilm, to problems of antimicrobial agent penetration into biofilm, or to
inactivation of the agent in the biofilm [44].
It is a fact that for the success of an antiseptic, the bacteria present in the biofilm must be exposed to an appropriate
concentration of the antimicrobial agent for a certain time [69]. Therefore, it is important the antiseptic penetration rate,
which is related not only with its physicochemical characteristics, but also with other biofilm-related factors such as its
structure and composition [39, 44], although its thickness [70] and physicochemical characteristics are probably more
important [39, 44].

 Methodological approach

The in vitro studies highlight the necessity to develop in situ biofilm models to provide samples which can be analysed
ex vivo without any disturbance. In the first in situ studies over de antimicrobial activity of the EO in oral biofilm,
dental plaque was recollected from the teeth surfaces using paper points [71] or with a dental scaler [59, 72]. These
techniques produced an interruption in the delicate three-dimensional existing relationship between cells, the
extracellular matrix and the substrate [44, 73]. Another disadvantage is that the penetration rate of the antimicrobial
agent cannot be evaluated due to the dental plaque samples do not conserve the original in situ architecture and
organization.
To a better understanding of the clinical effects of the antimicrobial agents inside the biofilm is necessary to apply a
specific methodology to permit the biofilm growing directly in the oral cavity. This will permit that its three-
dimensional structure is not distorted with manipulation [44, 45]. As a consequence, the most recent studies about in
situ antimicrobial activity of the EO analyse a biofilm which is growing on the surface of small disks made by different
materials. These disks are introduced in the mouth for a certain period of time during which they are exposed to the oral
cavity conditions [60, 74]. At this point, it should be remarked that they are not analysing dental plaque itself, but a
biofilm which is presumably very similar, which is generated in the same conditions and it is set on an artificial
substrate, which is called plaque like-biofilm (PL-Biofilm).
In 1998, following the objective of studying the “non-destructured” biofilm, the confocal laser scanning microscope
(CLSM) was incorporated. This technique combined with a fluorescence staining solution permits the analysis of the
biofilm structure and the distribution of the vital and no vital bacteria in the biofilm [75, 76]. With CLSM, biofilms can
be studied in their natural hydrated state, with no need for dehydration, fixation, or staining [37, 38, 77].
In addition, the optical sectioning properties of CLSM mean that very thin optical sections in the horizontal plane
(X-Y axes) can be taken at 0.5 to 2 μm intervals, at increasing depths through the biofilm (from the surface of the
biofilm to its base), and they will be free from out-of-focus blurring [40, 73, 77-79]. Consequently, at present, the
scientific community considers that the methodological design based on the use of special removable appliances
(including disks) to obtain biofilm samples and its analysis by CLSM (in combination with other microscopic and
microbiological techniques) is the most suitable approach for studying the in situ architecture and physiology of
undisturbed PL-biofilm formed on surfaces, as well as the antibacterial effect of antiseptics on this microbial structure
[41, 44, 80]. The CLSM associated with a fluorescence staining solution are an essential part of the methodology
followed by some of the studies existing in the literature about the in situ antimicrobial activity of the EO [59, 60].
Other used techniques are the spectophotometric method [71, 74], that quantifies the bacterial mass existing in a
dissolution by the diffraction of the light when passing through it. The biggest limitation of this technique is the fact that
it is impossible to know if the bacteria found in the solution are alive or dead. Fine et al [72] used the culture in plaque
technique which presents considerable limitations already explained. Furthermore, these latter two techniques do not
permit the study of the biofilm structure.
Apart from the methodological limitations of the named studies, it lacks the comparison of the effect of the EO not
only with a negative control or with an antiseptic with lower efficacy, but also with a positive control, the Gold-
standard, the CHX. Thus, a more accurate view of the EO activity as an antiseptic could be obtained.

 Immediate effect and substantivity of EO

To the best of the author’s knowledge, there are only 5 studies in the literature which analyse the in situ antimicrobial
activity of the EO in the PL-Biofilm [59, 60, 71, 72, 74]. In only 2 of them the authors studied a “non-destructured”
biofilm [60, 74] and just Gosau et al [60] did it using CLSM.
Charles et al [71] determinate the interproximal antibacterial activity of the EO. For that, they count on 34 volunteers
who did a mouthwash (EO or saline negative control) after the dental brushing. Five minutes after that, they collected
interproximal plaque samples using paper points. After a single EO mouthwash, they obtained a decrease in the
bacterial vitality of 44% in the interproximal spaces with regard to the negative control, 5 minutes post-application.
After their in vitro test [81], the research group of Pan [59] examined in 17 subjects the one day-formation PL-
biofilm. For that purpose, using a dental scaler, they collect the dental plaque from the vestibular surfaces of an upper
quadrant and its lower contralateral before the EO mouthwash. The plaque was deposed on a glass disk and stained with
the fluorescence solution LIVE/DEAD® BacLightTM to differ from live and dead bacteria, obtaining a vitality of 22.3%
at 30 minutes, while with the control was 72.1% at that specific moment.
Fine et al [72] carried out a study in which they determinated the antimicrobial effect of a single EO mouthwash 12
hours after its application and 14 days after a continuous daily use. A curette was used as the recollection method for
the dental plaque. They obtained a decrease in the vitality that went from 56.3% to 87.7% compared to the negative
control (5% hydro-alcoholic solution) 12 hours after the mouthwash application. After the daily use (twice a day / 2
weeks) the decrease rate for the bacterial vitality was 72.5-93.8% with regard to the negative control.
 Dong et al [74] tried to establish a new in situ model to recollect intact PL-bioiflm for the evaluation of its structure,
the immediate penetration tax and the antibacterial effect of a single EO mouthwash. For this, they used hydroxyapatite
disks in which they made 500 µm deep grooves. A total of six volunteers wore these disks for 6, 24 and 48 hours. Disks
were subsequently divided in 2 halves. One of them was summerged in an EO solution for 1 minute and the plaque was
visualized 5, 15 and 30 minutes after. They obtained a significant reduction in the bacterial vitality in comparisson to
the control group.
Gosau et al [60] evaluated the antimicrobial activity of the EO in the 12 hour PL-biofilm formed on the surface of
titan disks hold in an upper splint in a total of 4 volunteers. The application of the mouthwash was done extraoraly, for a
minute. After the using of the fluorescence staining solution LIVE/DEAD ® BacLightTM and the CLSM for the
visualization of the PL-biofilm in situ, the dead bacteria rate was 76.8% (range = 65.09 - 95.87%).
One interesting point that should be remarked about these latter two studies is the fact that having developed a
model which permits the growth of the biofilm in the oral cavity, it seems, at least, surprising that they had applied the
EO mouthwash ex vivo; what they did was just take the disks from the mouth and immerse them in the EO solution for a
certain period of time (60 seconds). By doing this, they are assuming that this immersion is the same as doing an active
mouthwash, obviating the intrinsic factors of the rinse itself such as the washing effect and the strength applied by the
muscle movement.

3.3 In situ substantivity of EO vs 0.2 CHX on PL-biofilm

According to the theory of the investigation in the evidence based science new techniques, treatments, drugs or
substances should be always compared with their respective considered Gold-standard. Up to now, to the author’s
knowledge, there is not any published study which compares the in situ antimicrobial effect on the biofilm of the
application of a single EO mouthwash with the CHX.
Quintas et al (unpublished data), studied the in situ antimicrobial activity of a single EO mouthwash on a 2 days
“non-destructured” PL-biofilm formed on glass disks in comparison to a single water mouthwash (negative control) and
a single 0.2% CHX mouthwash (positive control). In the field of the methodology, they used the Intraoral Device of
Overlaid Disk-holding Splints (IDODS) (Fig. 5).

Fig. 5. Clinical views

of the Intraoral
Device of Overlaid
Disk-holding Splints.
Note in the second
photograph the places
where the glass disks
are positioned.

The apparatus carried 6 glass disks which permitted the formation of the PL-biofilm in similar conditions to the
dental plaque itself from the volunteers. Each of the six disks were analysed at different moments; at baseline, 30
seconds after the mouthwash, 1, 3, 5 and 7 hours after the application. The samples were analysed using the CLSM after
the LIVE/DEAD® BacLightTM fluorescence staining solution. They studied the bacterial vitality and the thickness of all
samples and the penetration rate of each mouthwash.
After the application of the EO mouthwash, the bacterial vitality was 1.18%, while after the 0.2% CHX the bacterial
vitality was 5.08%. The antimicrobial activity of the EO was detected until 7 hours after the mouthwash application
when the bacterial vitality reduction was still the 61% with regard to the basal (Fig. 6). The effects of the water
mouthwash were almost imperceptible (Fig. 6).
Fig. 6. Images of different samples at different moments before and after the three mouthwashes; they are obtained with the CLSM
after the fluorescence staining solution LIVE/DEAD ® BacLightTM (SYTO 9 / propidium idodide – green = live bacteria / red = dead
bacteria-). Note that 7 hours after the EO and 0.2 CHX mouthwashes the effects are clearly visible, while after the water mouthwash
the changes in the PL-biofilm are almost imperceptible.

An interesting aspect that this study shows is the higher penetration ability of the EO in comparison to the 0.2%
CHX. There were significant differences between both antiseptics in the bacterial vitality reduction in the deepest layers
of the biofilm (Fig. 7), the differences increased as time passed by (difference in terms of vitality at 7 hour mouthwash
post-application EO vs 0.2% CHX = -36.88%; p<0.001). This may indicate that the penetration ability of a single EO
mouthwash is higher than 0.2% CHX’s; besides, its effect lasts longer in the deepest layers (those which are nearest to
what in theory is the tooth surface).

Fig. 7. Sectional images

from basal and 7 hours
samples for EO and 0.2%
CHX. Note that starting
from a similar bacterial
vitality at basal; this
vitality keeps lower in the
deepest layers of the PL-
biofilm 7 hours after a
single EO mouthwash.

In terms of thickness reduction, the EO mouthwash did not show any significant effects (the same as the water),
while the 0.2% CHX showed significant differences between basal and post-application measures (difference in terms
of thickness between basal and 1 hour sample for 0.2 CHX = 9.97 µm; p < 0.05). This result could suggest a possible
antiplaque effect caused by 0.2% CHX after a single antiseptic application.
 4. Conclusions
Although the effects of the EO have been already tested both in saliva and in biofilm, results have not been uniform.
Furthermore, the plaque culture techniques do not seem to be the most efficient manner to prove the EO in situ
antibacterial effect due to their limitations. In the other side, there are not many studies in which the CLSM or the
epifluorescence techniques associated with bacterial vitality techniques had been used to determine the in situ effects of
the EO in the oral cavity.
The CLSM asscociated with fluorochromes seem to be the best option for the in situ study of the antimicrobial effect
of the EO in the oral biofilm. This last one must grow inside the oral cavity and should be analysed without being
destructured. In this case, it will be possible to study the architecture, the vitality distribution inside the biofilm and its
thickness. Moreover, the immediate effect, substantivity and penetration rate of an antiseptic could be analysed.
Finally, the obtained results should be compared with a positive and negative control and with the antiseptic effects
observed in other oral ecosystems such as the saliva. Thereby, a more reliable approach about the ability of the EO as
oral antiseptics could be obtained.

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