TUGAS REFRAT
SALIVA DAN KELENJAR SALIVA
Disusun Oleh :
1. M. Iqbal J5201100222. Yusifa Edo R J5201100233. Kurnia Indah P J520110027
FAKULTAS KEDOKTERANPROGRAM STUDI KEDOKTERAN GIGI
UNIVERSITAS MUHAMMADIYAH SURAKARTA 2012
GMS Krankenhhyg Interdiszip. 2012;7(1):Doc06. Epub 2012 Apr 4.
XTT assay of ex vivo saliva biofilms to test antimicrobial influences.
Koban I, Matthes R, Hübner NO, Welk A, Sietmann R, Lademann J, Kramer A, Kocher T.
Source
Unit of Periodontology, Dental School, University of Greifswald, Greifswald, Germany.
Abstract
Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial
substances, in particular, requires a high sample throughput and a realistic model, the evaluation
must be as quick and as simple as possible. For this purpose, a colorimetric assay of the
tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-
nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of
XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored
formazan product which is measured photometrically.Materials and method: The suitability of
the XTT assay for detecting the vitality of ex vivo saliva biofilms was tested to determine the
efficacy of chlorhexidine and ozone versus saliva biofilms grown on titanium discs.Results: The
XTT method lends itself to testing the vitality of microorganisms in saliva biofilms. The
sensitivity of the arrays requires a specific minimum number of pathogens, this number being
different for planktonic bacteria and those occurring in biofilms. The antibacterial effect after
treatment with chlorhexidine or ozone was measured by XTT conversion that was significantly
reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment was equal
and comparable with 60 s ozone treatment. Conclusion: The XTT assay is a suitable method to
determine the vitality in saliva biofilms, permitting assessment of the efficacy of antimicrobial
substances. Its quick and easy applicability renders it especially suitable for screening.
GMS Krankenhhyg Interdiszip. 2012; 7(1): Doc06.
Published online 2012 April 4. doi: 10.3205/dgkh000190
PMCID: PMC3334957
XTT assay of ex vivo saliva biofilms to test antimicrobial influences
Ina Koban,*,1 Rutger Matthes,2 Nils-Olaf Hübner,2 Alexander Welk,1 Rabea Sietmann,3 Jürgen
Lademann,4 Axel Kramer,2 and Thomas Kocher1
Author information ► Copyright and License information ►
Abstract
Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial
substances, in particular, requires a high sample throughput and a realistic model, the evaluation
must be as quick and as simple as possible. For this purpose, a colorimetric assay of the
tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-
nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of
XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored
formazan product which is measured photometrically.
Materials and method: The suitability of the XTT assay for detecting the vitality of ex
vivo saliva biofilms was tested to determine the efficacy of chlorhexidine and ozone versus saliva
biofilms grown on titanium discs.
Results: The XTT method lends itself to testing the vitality of microorganisms in saliva
biofilms. The sensitivity of the arrays requires a specific minimum number of pathogens, this
number being different for planktonic bacteria and those occurring in biofilms. The antibacterial
effect after treatment with chlorhexidine or ozone was measured by XTT conversion that was
significantly reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment
was equal and comparable with 60 s ozone treatment.
Conclusion: The XTT assay is a suitable method to determine the vitality in saliva biofilms,
permitting assessment of the efficacy of antimicrobial substances. Its quick and easy
applicability renders it especially suitable for screening.
Keywords: biofilm model, saliva, S. mutans, ozone, chlorhexidine, XTT assay
Introduction
Bacterial infections play a specific role in dentistry. After the supragingival tooth surfaces and
mucous membranes have been wetted with saliva, microbes settle there and form a biofilm [1].
This biofilm accommodates dental pathogens and protects them against environmental stress
factors, such as chemotherapeutics, the immune system, acids, hunger periods, and reactive
oxygen products [2], [3].
Antimicrobially effective substances and techniques should be tested on a suitable biofilm
model, as the efficacy against planktonic pathogens has little predictive value for the efficacy
against biofilms.
For a number of years, several mono-species biofilm models have been available which
accommodate typical oral microbes [4], [5]. Streptococci are frequently used as a caries model,
although other scientists found out that they do not represent the etiological pathogens of the
disease [6]. For the treatment of periodontal diseases, anaerobic periodontal marker pathogens
are important [7]. But in vivo plaque microbiota is highly diverse and complex [8]. The oral
cavity harbors more than 1,000 different microorganisms, which join to form multispecies
biofilms [9]. Guggenheim et al. used biofilms consisting of a maximum of six different
pathogens [10]. The oral fluid, too, is an essential component in the formation of dental biofilms.
The proteins in the saliva are a significant source of food for microbes. Pellicle proteins settle on
the dental surfaces forming the so-called conditioning film. This conditioning film forms the
basis for the development of biofilms, as the adhesins of the bacteria directly bind to these
oligosaccharide-containing glycoproteins [11], [12]. In addition salivary antimicrobial factors are
important stressors that can enhance biofilm formation [13], [14]. Many artificial saliva
formulations have been designed that attempt to imitate this process in order to ensure realistic
biofilm formation. In most cases, however, artificial saliva fails to provide all the organic and
inorganic components that exist in natural saliva. Moreover, no evidence has been supplied that
artificial saliva promotes biofilm formation [15].
For their biofilm models, other researchers used volunteers’ saliva which was filtered under
sterile conditions, diluted and centrifuged [16].
A simple, more realistic multispecies biofilm model can be obtained by culturing the saliva of
volunteers, without prior filtration under sterile conditions [17]. Of course, this is only a model
because the circadian rhythm of salivation and the correspondingly variable composition of
saliva cannot be imitated, nor can regular food intake [18], [19]. Furthermore, the oral conditions
are different in each patient [20]. A subsequent detection of biofilm formation is difficult. Even
when using a non-specific agar, such as Columbia blood agar, it is not possible to definitely
detect every species in the culture. Alternatively, colorimetric methods, e.g., the XTT assay, are
appropriate to measure metabolic activity and vitality. XTT is a yellow salt that is reduced by
dehydrogenases of metabolically active cells to a colored formazan product. Colorimetric
methods are attractive because they have the potential to generate clear-cut endpoints based on a
visible color change.
The objective of this study, therefore, was the development of a method suitable for testing
saliva biofilms using XTT.
Materials and method
Cultivation of biofilms
Biofilms were cultured on titanium discs 5 mm in diameter and 1 mm thick (Straumann, Basel,
Switzerland).
The sterile titanium discs were positioned in 96-well microtitre plates (Techno Plastic Products
AG, Trasadingen, Switzerland), covered with 100 µl fresh, unstimulated saliva of healthy
volunteers (aged between 20 and 30 years, non-smokers), and incubated aerobically at 37°C. The
donors did not take any medication three months prior the study and did not have active carious
lesions or periodontal disease. After 24 h, the saliva was drawn off and replaced by sterile brain-
heart infusion broth (BD, BBL™, Heidelberg, Germany), as a highly nutritious general-purpose
growth medium. After 48 h, the medium was drawn off, and the discs were washed with 0.9%
NaCl solution and transferred onto a new, sterile microtitre plate.
Antiseptic treatment with chlorhexidine
Chlorhexidine digluconate was used as a 0.1% and 0.05% aqueous solution. The discs were
covered with 100 µl of the antiseptic and incubated for 1 min.
After this exposure, the chlorhexidine was drawn off, and the antiseptic effect was stopped by
adding 1 ml of inactivator (Lipofundin MCT 20%, B.Braun, Melsungen, Germany). The
inactivation of chlorhexidine by the inactivator was validated by the quantitative suspension test
according to EN 1040. Physiological saline was used for control.
Application of ozone
The test objects were direct treated for 20 s, 30 s, 40 s, or 60 s with gaseous ozone provided by a
HealOzone device (KAVO, Biberach, Germany). The ozone is delivered via a hose into a
disposable sterile cup at a concentration of 2,100 ppm ± 10%. The ozone gas is refreshed in this
disposable cup at a rate of 615 cc/minute changing the volume of gas inside the cup over 300
times every second [21].
Inactivation was unnecessary as the device suctions off any residual ozone after application.
Vitality measurement by XTT assay
Bioreduction of XTT could be potentiated by addition of electron coupling agents such as
phenazine methosulfate (PMS) or menadione (Men) [22].
To optimize the staining solution, 200 µl of the XTT solution was added to each disc bearing the
grown biofilm after 24 h and 48 h, respectively. The added XTT solution was composed of the
following:
1. XTT (180 mg/l) (AppliChem, Darmstadt, Germany) and menadione (0.688 mg/l) (Sigma-
Aldrich, Munich, Germany) (hereafter called „XTT+Men“)
2. XTT (180 mg/l) and phenazine methosulfate (20 mg/l) (PMS, AppliChem, Darmstadt,
Germany) (hereafter called „XTT+PMS“)
3. XTT (180 mg/l), menadione (0,688 mg/l) and phenazine methosulfate (20 mg/l)
(hereafter called „XTT+Men+PMS”)
To determine the measuring range, saliva was diluted with physiological saline and incubated
with the XTT staining solution specified in test 1 (XTT+PMS+menadione).
To test the antimicrobial efficacy of chlorhexidine and gaseous ozone, the treated discs were also
incubated with 200 µl of the staining solution (XTT+PMS+menadione).
After 3 h of incubation while shaking (Titramax, Heidolph Instruments, Schwabach, Germany)
at 37°C, 100 µl of all solutions were transferred onto a new sterile microtitre plate and analyzed
at 450 nm (reference wavelength 620 nm) using a photometer (anthos Mikrosysteme, Krefeld,
Germany) [23].
Determination of CFU
After treatment, the titanium discs were placed into wells with 200 µl 0.9% NaCl solution and
the biofilm was removed by ultrasonic scaling (Branson 2510, 130 W, 42 kHz, Dietzenbach,
Germany). Serial dilutions were made by transferring 0.1 ml of the resultant suspension to 0.9 ml
of fresh 0.9% NaCl solution. After that, an aliquot portion of 0.1 ml from each dilution was
plated on Columbia sheep blood agar (BBL™, BD, Heidelberg, Germany) and incubated aerobe
at 37°C for 48 h. The colonies were counted and expressed as colony forming units (CFU). The
CFU values were log transformed.
Confocal laser scanning microscopy
Live/Dead-Staining (BacLight, Invitrogen, Darmstadt, Germany) was used for microscopical
analysis of the bacterial vitality. The biofilms were incubated immediately with the dye
according to manufacturer’s instructions. After incubation the discs were rinsed with 0.9% NaCl
solution to remove dye residues from the biofilm. The samples were evaluated in the confocal
laser scanning microscope (CLSM510 Exciter, Zeiss, Jena, Germany).
Scanning electron microscopy
For electron microscopy, the biofilms were prepared as follows: after a fixation step (1 h in 1%
glutaraldehyde, 2% paraformaldehyde, 0.2% picric acid, 5 mM HEPES (pH 7.4), and 50 mM
NaN3), the samples were treated with 2% tannic acid for 1 h, 1% osmium tetroxide for 1 h, 1%
thiocarbohydrazide for 30 min, 1% osmium tetroxide at 4°C overnight, and with 2% uranyl
acetate for 2 h with washing steps in between. The samples were dehydrated in a graded series of
acetone solutions (10 to 100%) and then critical-point dried. Finally, samples were mounted on
aluminum stubs, sputtered with gold-palladium and examined in an EVO LS10 (Zeiss,
Oberkochen, Germany) (Figure 1 (Fig. 1)).
Figure 1
Schematic view of the experiment
Analysis
For all experiments, at least eight test objects each were used. The chlorhexidine treatment
required 23 discs. In addition, eight test objects each were available for control tests. Continuous
data are presented as mean ± standard deviation. Statistical analyses were performed with
STATVIEW® 5.0 (SAS, Cary, NC) software. Graphs were also created using STATVIEW.
Medians are given with their standard errors. Nonparametric correlations (Mann-Whitney U-test)
were estimated for comparison of absorptions. P-values below 0.05 were considered statistically
significant.
Results
Cultivation of biofilms
The cultivation procedure was constantly checked for cultures by determining the CFU and for
reproducibility by microscopy. Cell densities of 108 CFU/ml were regularly be removed from the
test objects by ultrasound scaling.
Optimization of the staining solution
The absorption of the colored formazan derivate of XTT converted by the microbes is a measure
of the cellular vitality.
A high absorption value indicates high metabolic activity. Figure 2 (Fig. 2) shows the absorption
values (at 450 nm) of the different staining solutions (XTT+Men, XTT+PMS and
XTT+Men+PMS) on 24-h- and 48-h-old biofilms. For all staining solutions, significant (p<0.01)
differences were visible between the 24-h and 48-h biofilms.
Figure 2
Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT metabolized
by saliva biofilm (XTT+ menadione, XTT+ phenazine methosulfate, XTT+ menadione+ N-
methyl-phenazinium methylsulfate).
In the case of XTT+Men, the absorption at 450 nm increased to a value an average of 30 times
higher (from 0.01 to 0.30) after 48 h than after 24 h. Within these 24 h, the absorption value of
XTT+PMS increased to five times (from 0.10 to 0.50) the original value, while the absorption of
XTT+Men+PMS was increased three times from 0.20 to 0.65.
The absorption values of XTT+Men+PMS were always significantly (p<0.01) higher than those
of the other straining solutions.
Determination of the measuring range
Figure 3 (Fig. 3) shows the absorption values at 450 nm determined by dilution of the saliva and
subsequent staining with XTT+Men+PMS. There are significant differences (p<0.01) between
the absorption values of the biofilms up to increments of 4.5 log10. Absorption was no longer
measurable in concentrations from 3.5 log10 (CFU/ml), except for negative controls.
Figure 3
Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT + menadione
+ phenazine methosulfate metabolized by various saliva dilutions.
Antimicrobial treatment
A reduced XTT conversion was observed in saliva biofilms which had been subjected to
antimicrobial treatment with gaseous ozone and chlorhexidine. This reduction was significant
after a 60-s or 120-s ozone treatment (p<0.02) and a one-minute chlorhexidine treatment
(p<0.01) (see Figure 4 (Fig. 4)). There was no difference in the conversion of XTT using a
0.05% or a 0.1% chlorhexidine solution. No difference could be seen in the scanning electron
micrographs between the untreated biofilm and the biofilm treated with ozone. In both samples,
the cells appear plump and the biofilm has a loosely bound structure. The bacteria in the biofilm
treated with chlorhexidine are damaged and the overall structure appears to be tighter (Figure
5 (Fig. 5)). The CLSM images confirmed these observations (Figure 6 (Fig. 6)). After treatment
with ozone, parts of the biofilm were dyed red (cells with damaged membranes). After CHX
treatment, no green colored areas (cells with intact membranes) were identified.
Figure 4
Diagram of the absorption at 450 nm (reference wavelength 620 nm) of XTT+ menadione+
phenazine methosulfate, metabolized by saliva biofilms for 48 h after treatment with 0.9%
NaCl solution (control), gaseous ozone and chlorhexidine.
Figure 5
SEM micrograph: 48-h mature saliva biofilm: (A) untreated, (B) after ozone treatment, (C)
after chlorhexidine treatment. Magnification 10,000 x
Figure 6
CLSM micrographs: 48-h mature saliva biofilm: (A) untreated, (B) after ozone treatment,
(C) after chlorhexidine treatment. Magnification 100 x
Discussion
Causing typical dental diseases, such as caries and periodontitis, biofilms complicate the
elimination of microbes responsible for forming the biofilms by antimicrobial substances. The
objective of this study was to develop a biofilm model suitable for testing the efficacy of
antimicrobial substances with non-culture-based detection of the vitality of saliva biofilms using
XTT, and to prepare a suitable XTT assay.
Our study had several limitations. We used saliva of volunteers to create a practically relevant
biofilm model. For better understanding of the interactions between bacteria and XTT, we
performed our experiments on machined titanium discs to exclude material hydrophobicity,
retention niches such as cavities and porosities into which the biofilm could adhere. Titanium
implants have been successfully used in dentistry and biofilms on titanium are the central
problem in peri-implantitis. Peri-implantitis of osseointegrated oral implants is not a
monoinfection by single pathogens; rather, they show the characteristics of mixed infections.
Whereas the single species of a mixture of bacteria could not induce experimental abscesses, the
combination of these species could do it [24]. Plaque biofilms containing multiple species of
appropriate bacteria should be more relevant for studying dental diseases and antimicrobial
efficacies [25]. The first step was the use of only aerobe cultivation methods. Most XTT assays
are carried out aerobically. In the next step we will use a subgingival plaque biofilm under
anaerobic conditions for XTT assays. But studies also showed that the same microbiota that can
be found around implants (under anaerobic conditions) also can be found around teeth (under
aerobic conditions) [26], [27].
XTT is a colorless tetrazolium salt, which is converted into a colored, water-soluble formazan
derivate by dehydrogenases, with succinate dehydrogenase being particularly important [23] as it
plays a major role in the energy supply of each individual living cell [28]. Unlike other
tetrazolium salts (e.g., CTC and TTC), XTT does not require any insoluble formazans to be
extracted. Since the color change in the solution can be directly determined by photometry, the
XTT test permits a large number of test objects to be tested for their vitality very quickly. The
incubation of bacteria with XTT for 3 h at 37°C has already been the subject of several
publications [23]. What is new is the composition of the XTT staining solution applied. The
saliva biofilm contains a large variety of different microbes (Gram-positive, Gram-negative
bacteria and fungi). To convert XTT, these microbes require various additives which function as
electron carriers. The standard additive for Candida spp. is menadione [29], [30], [31], [32], but
PMS can be applied as well [33]. For Gram-positive cocci bacteria, PMS is used in most
instances [34], but sometimes menadione is also used [35]. In the case of Gram-negative rod-
shaped bacteria, mainly menadione is applied [35], [36]. Our own preliminary (unpublished)
investigations confirm these results on the suitability of menadione for Candida albicans and
PMS for Streptococcus mutans andStreptococcus sanguinis as representatives in dentistry. As
for Pseudomonas aeruginosa, the combined application of menadione and PMS turned out to be
suitable [37]. By means of a culture-based analysis of the saliva, Gram-positive cocci bacteria, i.
a., could be isolated. Therefore, the addition of PMS seemed to be indispensable for the
colorimetric detection using XTT. McCluskey et al. also exclusively used PMS for the
colorimetric detection of microbes occurring in activated sludge, and were able to prove that
there is a direct correlation between formazan production and oxygen consumption [23].
In a direct comparison, the combined PMS-menadione electron mediators showed the highest
XTT conversion in saliva biofilms (see Figure 2 (Fig. 2)). It turned out, however, that a high cell
count of at least 4.5 log10 (CFU/ml) is required to detect the XTT reduction (see Figure 4 (Fig.
4)). At 5.5 log10(CFU/ml), a very high absorption (1.91) was detected at 450 nm. The higher the
number of metabolically active cells, the higher the colorimetric signal. In addition, the higher
the metabolism of cells, the higher the signal. Obviously, there is no linear relation between the
number of cells and the colorimetric signal [38]. When other dyes (FDA und Syto9) were used,
the adsorption even remained constant [39]. After 48 h of biofilm formation, cell densities of ca.
8 log10 (CFU/ml) were reached, but following the XTT test, the absorption in the biofilm was
0.65, i.e., less than in planktonic bacteria although the cell density was higher. The amount of
retained product may vary between planktonic bacteria and biofilms [38]. Moreover, biofilms are
subjected to other conditions than are fresh bacteria suspensions. Many pathogens are persistent
and, therefore, exhibit lower metabolic activity [40]. For this reason, a minimum number of
pathogens cannot be determined from suspensions and biofilms in exactly the same manner. In
addition, planktonic cells can invest more energy in routine metabolism [30]. In our experiments,
the absorption was 30 times higher after 48 h than after 24 h, i.e., the conversion of XTT
increased due to the growth of the biomass. However, the different metabolic levels do not lead
to a logarithmic increase of the XTT reduction. This XTT-related observation was also made by
other research groups [39].
Reduced formazan formation was observed due to the antimicrobial treatment of the saliva
biofilm. Consequently, the XTT test is suitable for determining the efficacy of antimicrobial
substances, especially for screening. The low chlorhexidine concentration used has already been
investigated by other researchers, who noted insufficient antimicrobial efficacy in the biofilms
[41]. On the other hand, chlorhexidine proved to be slightly superior to ozone. This has also been
published by other research groups who applied alternative methods [42]. However, in
vivo Hauser-Gerspach et al. could not find significant antimicrobial effects of CHX and ozone
[43]. The scanning electron micrographs confirmed this result. Following the ozone treatment,
the morphology of the cells showed no differences. Although the biofilm treated with
chlorhexidine appeared to be damaged compared to the control, only a few cells were
morphologically deformed.
In spite of some disadvantages, XTT with the addition of menadione and PMS is a suitable
method for determining the vitality in bacterial saliva biofilms and permits assessment of the
efficacy of antimicrobial substances.
The assay is easy to perform, and allows a large number of test objects to be tested. It is
particularly suited to screening various factors influencing the biofilm, such as antiseptics or
other physical or chemical treatments, for instance, ozone, photodynamic therapy or atmospheric
pressure plasma.
Notes
Acknowledgements
This work was realized within the framework of the multi-disciplinary research cooperation
“Campus PlasmaMed”, particularly within the project “PlasmaDent”. The authors acknowledge
that this work was supported by a grant funded by the German Ministry of Education and
Research (BMBF, grant no, 13N9779).
We thank Tina Dornquast and Hartmut Fischer for their excellent technical assistance. We also
thank PD Dr. Lutz Netuschil for the critical discussions.
Competing interests
The authors declare that they have no conflict of interest.
References
1. Bortolaia C, Sbordone L. I biofilm del cavo orale. Formazione, sviluppo e implicazioni
nell'insorgenza delle malattie correlate all'accumulo di placca batterica. [Biofilms of the oral
cavity. Formation, development and involvement in the onset of diseases related to bacterial
plaque increase]. Minerva Stomatol. 2002 May;51(5):187–192. (Ger). [PubMed]
2. Carlsson J. Bacterial metabolism in dental biofilms. Adv Dent Res. 1997 Apr;11(1):75–80.
doi: 10.1177/08959374970110012001. Available
from: http://dx.doi.org/10.1177/08959374970110012001.[PubMed] [Cross Ref]
3. Gilbert P, Das J, Foley I. Biofilm susceptibility to antimicrobials. Adv Dent Res. 1997
Apr;11(1):160–167. doi: 10.1177/08959374970110010701. Available
from:http://dx.doi.org/10.1177/08959374970110010701. [PubMed] [Cross Ref]
4. Noorda WD, van Montfort AM, Purdell-Lewis DJ, Weerkamp AH. Developmental and
metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human
enamel slabs in an artificial mouth model. I. Plaque Data. Caries Res. 1986;20(4):300–307. doi:
10.1159/000260949.Available from: http://dx.doi.org/10.1159/000260949. [PubMed] [Cross
Ref]
5. Noorda WD, van Montfort AM, Purdell-Lewis DJ, Weerkamp AH. Developmental and
metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1 grown on human
enamel slabs in an artificial mouth model. II. Enamel Data. Caries Res. 1986;20(4):308–314. doi:
10.1159/000260950.Available from: http://dx.doi.org/10.1159/000260950. [PubMed] [Cross
Ref]
6. Beighton D. The complex oral microflora of high-risk individuals and groups and its role in
the caries process. Community Dent Oral Epidemiol. 2005 Aug;33(4):248–255. doi:
10.1111/j.1600-0528.2005.00232.x. Available from: http://dx.doi.org/10.1111/j.1600-
0528.2005.00232.x. [PubMed][Cross Ref]
7. Zafiropoulos GG, Kasaj A, Beaumont C, Willershausen B, Frohberg U. Der Einsatz von
Antibiotika in der Paro-Behandlung. Die spezifische medikamentöse Plaque-
Kontrolle. Stomatologie. 2006;103(2):39–47.
8. Moore WE, Moore LV. The bacteria of periodontal diseases. Periodontol 2000. 1994
Jun;5:66–77. doi: 10.1111/j.1600-0757.1994.tb00019.x. Available
from: http://dx.doi.org/10.1111/j.1600-0757.1994.tb00019.x. [PubMed] [Cross Ref]
9. Netuschil L. Die dentale Plaque – ein Paradebiofilm. Plaquencare. 2006;2:6–8.
10. Guggenheim B, Giertsen E, Schüpbach P, Shapiro S. Validation of an in vitro biofilm model
of supragingival plaque. J Dent Res. 2001 Jan;80(1):363–370. doi:
10.1177/00220345010800011201.Available
from: http://dx.doi.org/10.1177/00220345010800011201. [PubMed] [Cross Ref]
11. Scheie AA. Mechanisms of dental plaque formation. Adv Dent Res. 1994 Jul;8(2):246–253.
[PubMed]
12. Castro P, Tovar JA, Jaramillo L. Adhesion of Streptococcus mutans to salivary proteins in
caries-free and caries-susceptible individuals. Acta Odontol Latinoam. 2006;19(2):59–
66. [PubMed]
13. Rosin M, Hanschke M, Splieth C, Kramer A. Activities of lysozyme and salivary peroxidase
in unstimulated whole saliva in relation to plaque and gingivitis scores in healthy young
males. Clin Oral Investig. 1999 Sep;3(3):133–137. doi: 10.1007/s007840050091. Available
from:http://dx.doi.org/10.1007/s007840050091. [PubMed] [Cross Ref]
14. Arweiler NB, Auschill TM, Donos N, Sculean A. Antibacterial effect of an enamel matrix
protein derivative on in vivo dental biofilm vitality. Clin Oral Investig. 2002 Dec;6(4):205–209.
doi: 10.1007/s00784-002-0185-0. Available from: http://dx.doi.org/10.1007/s00784-002-0185-0.
[PubMed] [Cross Ref]
15. Honraet K. In vitro studie van Candida albicans en Streptococcus mutans biofilms
[dissertation][dissertation]. Ghent: Ghent University; 2005. (Ger). Available
from:https://biblio.ugent.be/publication/471547.
16. Sedlacek MJ, Walker C. Antibiotic resistance in an in vitro subgingival biofilm model. Oral
Microbiol Immunol. 2007 Oct;22(5):333–339. doi: 10.1111/j.1399-
302X.2007.00366.x. Available from:http://dx.doi.org/10.1111/j.1399-302X.2007.00366.x. [PMC
free article] [PubMed] [Cross Ref]
17. Sissons CH, Wong L, Cutress TW. Patterns and rates of growth of microcosm dental plaque
biofilms.Oral Microbiol Immunol. 1995 Jun;10(3):160–167. doi: 10.1111/j.1399-
302X.1995.tb00137.x. Available from: http://dx.doi.org/10.1111/j.1399-
302X.1995.tb00137.x. [PubMed] [Cross Ref]
18. Albegger KW, Müller O. Zur Circadianstruktur der Glandula submandibularis. [Proceedings:
Circadian structure of the submandibular gland]. Arch Klin Exp Ohren Nasen
Kehlkopfheilkd. 1973 Dec 17;205(2):122–125. doi: 10.1007/BF02412518. (Ger). Available
from:http://dx.doi.org/10.1007/BF02412518. [PubMed] [Cross Ref]
19. Arweiler NB, Lenz R, Sculean A, Al-Ahmad A, Hellwig E, Auschill TM. Effect of food
preservatives on in situ biofilm formation. Clin Oral Investig. 2008 Sep;12(3):203–208. doi:
10.1007/s00784-008-0188-6. Available from: http://dx.doi.org/10.1007/s00784-008-0188-
6. [PubMed] [Cross Ref]
20. Scheie AA, Fejerskov O, Lingström P, Birkhed D, Manji F. Use of palladium touch
microelectrodes under field conditions for in vivo assessment of dental plaque pH in
children. Caries Res. 1992;26(1):44–51. doi: 10.1159/000261426. Available
from: http://dx.doi.org/10.1159/000261426. [PubMed][Cross Ref]
21. Holmes J. Clinical reversal of root caries using ozone, double-blind, randomised, controlled
18-month trial. Gerodontology. 2003 Dec;20(2):106–114. doi: 10.1111/j.1741-
2358.2003.00106.x. Available from: http://dx.doi.org/10.1111/j.1741-
2358.2003.00106.x. [PubMed] [Cross Ref]
22. Roehm NW, Rodgers GH, Hatfield SM, Glasebrook AL. An improved colorimetric assay for
cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods. 1991 Sep
13;142(2):257–265. doi: 10.1016/0022-1759(91)90114-U. Available
from:http://dx.doi.org/10.1016/0022-1759(91)90114-U. [PubMed] [Cross Ref]
23. McCluskey C, Quinn JP, McGrath JW. An evaluation of three new-generation tetrazolium
salts for the measurement of respiratory activity in activated sludge microorganisms. Microb
Ecol. 2005 Apr;49(3):379–387. doi: 10.1007/s00248-004-0012-z. Available
from:http://dx.doi.org/10.1007/s00248-004-0012-z. [PubMed] [Cross Ref]
24. Mombelli A. In vitro models of biological responses to implant microbiological models. Adv
Dent Res. 1999 Jun;13:67–72. doi: 10.1177/08959374990130011701. Available
from:http://dx.doi.org/10.1177/08959374990130011701. [PubMed] [Cross Ref]
25. Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of
oral bacteria as an enamel and root caries model system. Arch Oral Biol. 2000 Jan;45(1):27–40.
doi: 10.1016/S0003-9969(99)00111-9. Available from: http://dx.doi.org/10.1016/S0003-
9969(99)00111-9.[PubMed] [Cross Ref]
26. Leonhardt A, Renvert S, Dahlén G. Microbial findings at failing implants. Clin Oral Implants
Res.1999 Oct;10(5):339–345. doi: 10.1034/j.1600-0501.1999.100501.x. Available
from:http://dx.doi.org/10.1034/j.1600-0501.1999.100501.x. [PubMed] [Cross Ref]
27. Leonhardt A, Bergström C, Lekholm U. Microbiologic diagnostics at titanium implants. Clin
Implant Dent Relat Res. 2003;5(4):226–232. doi: 10.1111/j.1708-
8208.2003.tb00205.x. Available from:http://dx.doi.org/10.1111/j.1708-
8208.2003.tb00205.x. [PubMed] [Cross Ref]
28. Samokhvalov V, Ignatov V, Kondrashova M. Inhibition of Krebs cycle and activation of
glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae.
Compensatory role of succinate oxidation. Biochimie. 2004 Jan;86(1):39–46. doi:
10.1016/j.biochi.2003.10.019. Available
from:http://dx.doi.org/10.1016/j.biochi.2003.10.019. [PubMed] [Cross Ref]
29. Tsang CS, Ng H, McMillan AS. Antifungal susceptibility of Candida albicans biofilms on
titanium discs with different surface roughness. Clin Oral Investig. 2007 Dec;11(4):361–368.
doi: 10.1007/s00784-007-0122-3. Available from: http://dx.doi.org/10.1007/s00784-007-0122-3.
[PubMed] [Cross Ref]
30. Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. Antifungal susceptibility
of Candida biofilms: unique efficacy of amphotericin B lipid formulations and
echinocandins. Antimicrob Agents Chemother. 2002 Jun;46(6):1773–1780. doi:
10.1128/AAC.46.6.1773-1780.2002. Available from: http://dx.doi.org/10.1128/AAC.46.6.1773-
1780.2002. [PMC free article] [PubMed] [Cross Ref]
31. Thein ZM, Samaranayake YH, Samaranayake LP. In vitro biofilm formation of Candida
albicans and non-albicans Candida species under dynamic and anaerobic conditions. Arch Oral
Biol. 2007 Aug;52(8):761–767. doi: 10.1016/j.archoralbio.2007.01.009. Available
from:http://dx.doi.org/10.1016/j.archoralbio.2007.01.009. [PubMed] [Cross Ref]
32. Jin Y, Yip HK, Samaranayake YH, Yau JY, Samaranayake LP. Biofilm-forming ability of
Candida albicans is unlikely to contribute to high levels of oral yeast carriage in cases of human
immunodeficiency virus infection. J Clin Microbiol. 2003 Jul;41(7):2961–2967. doi:
10.1128/JCM.41.7.2961-2967.2003. Available from: http://dx.doi.org/10.1128/JCM.41.7.2961-
2967.2003. [PMC free article] [PubMed] [Cross Ref]
33. Hawser SP, Norris H, Jessup CJ, Ghannoum MA. Comparison of a 2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric
method with the standardized National Committee for Clinical Laboratory Standards method of
testing clinical yeast isolates for susceptibility to antifungal agents. J Clin Microbiol. 1998
May;36(5):1450–1452.[PMC free article] [PubMed]
34. Cerca N, Martins S, Cerca F, Jefferson KK, Pier GB, Oliveira R, Azeredo J. Comparative
assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus
planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. J Antimicrob
Chemother. 2005 Aug;56(2):331–336. doi: 10.1093/jac/dki217. Available
from: http://dx.doi.org/10.1093/jac/dki217.[PMC free article] [PubMed] [Cross Ref]
35. Al-Bakri AG, Afifi FU. Evaluation of antimicrobial activity of selected plant extracts by
rapid XTT colorimetry and bacterial enumeration. J Microbiol Methods. 2007 Jan;68(1):19–25.
doi: 10.1016/j.mimet.2006.05.013. Available
from: http://dx.doi.org/10.1016/j.mimet.2006.05.013.[PubMed] [Cross Ref]
36. Tunney MM, Ramage G, Field TR, Moriarty TF, Storey DG. Rapid colorimetric assay for
antimicrobial susceptibility testing of Pseudomonas aeruginosa. Antimicrob Agents
Chemother. 2004 May;48(5):1879–1881. doi: 10.1128/AAC.48.5.1879-1881.2004. Available
from:http://dx.doi.org/10.1128/AAC.48.5.1879-1881.2004. [PMC free article] [PubMed] [Cross
Ref]
37. Hübner NO, Matthes R, Koban I, Rändler C, Müller G, Bender C, Kindel E, Kocher T,
Kramer A. Efficacy of chlorhexidine, polihexanide and tissue-tolerable plasma against
Pseudomonas aeruginosa biofilms grown on polystyrene and silicone materials. Skin Pharmacol
Physiol. 2010;23 Suppl:28–34. doi: 10.1159/000318265. Available
from: http://dx.doi.org/10.1159/000318265. [PubMed] [Cross Ref]
38. Kuhn DM, Balkis M, Chandra J, Mukherjee PK, Ghannoum MA. Uses and limitations of the
XTT assay in studies of Candida growth and metabolism. J Clin Microbiol. 2003 Jan;41(1):506–
508. doi: 10.1128/JCM.41.1.506-508.2003. Available
from: http://dx.doi.org/10.1128/JCM.41.1.506-508.2003.[PMC free article] [PubMed] [Cross
Ref]
39. Honraet K, Goetghebeur E, Nelis HJ. Comparison of three assays for the quantification of
Candida biomass in suspension and CDC reactor grown biofilms. J Microbiol Methods. 2005
Dec;63(3):287–295. doi: 10.1016/j.mimet.2005.03.014. Available
from: http://dx.doi.org/10.1016/j.mimet.2005.03.014.[PubMed] [Cross Ref]
40. Lewis K. Persister cells and the riddle of biofilm survival. Biochemistry (Mosc) 2005
Feb;70(2):267–274. doi: 10.1007/s10541-005-0111-6. Available
from: http://dx.doi.org/10.1007/s10541-005-0111-6.[PubMed] [Cross Ref]
41. Seidler V, Linetskiy I, Hubálková H, Stanková H, Smucler R, Mazánek J. Ozone and its
usage in general medicine and dentistry. A review article. Prague Med Rep. 2008;109(1):5–
13. [PubMed]
42. Botha FS, van der Vyver PJ. Evaluation of Antibacterial Effects of Different Root Canal
Disinfection Methods. The IADR/AADR/CADR 83rd General Session; 2005 March 9-12;
Baltimore, MD. 2005. Available
from: http://iadr.confex.com/iadr/2005Balt/techprogram/abstract_61353.htm.
43. Hauser-Gerspach I, Pfäffli-Savtchenko V, Dähnhardt JE, Meyer J, Lussi A. Comparison of
the immediate effects of gaseous ozone and chlorhexidine gel on bacteria in cavitated carious
lesions in children in vivo. Clin Oral Investig. 2009 Sep;13(3):287–291. doi: 10.1007/s00784-
008-0234-4.Available from: http://dx.doi.org/10.1007/s00784-008-0234-4. [PubMed] [Cross
Ref]
PEMBAHASAN JURNAL
Banyak penyakit gigi yang disebabkan biofilm. Pemutaran zat antimikroba, khususnya,
memerlukan throughput sampel tinggi dan model yang realistis, evaluasi harus secepat dan
sesederhana mungkin. Untuk tujuan ini, alat tes kolorimetri dari tetrazolium garam XTT (natrium
3'-[1 - [(phenylamino)-carbony] -3,4-tetrazolium]-bis (4-metoksi-6-nitro) benzena-sulfonat hidrat
asam ) dikonversi oleh biofilm air liur dianjurkan. Pembelahan oleh enzim dehidrogenase XTT
sel yang aktif secara metabolik dalam biofilm menghasilkan produk formazan sangat berwarna
yang diukur photometrically.
Kesesuaian uji XTT untuk mendeteksi vitalitas biofilm air liur ex vivo diuji untuk
menentukan kemanjuran dari klorheksidin dan ozon terhadap air liur biofilm tumbuh pada
cakram titanium. Metode XTT cocok untuk menguji vitalitas mikroorganisme dalam biofilm air
liur. Sensitivitas dari array membutuhkan jumlah minimum tertentu patogen, jumlah ini menjadi
berbeda bagi bakteri plankton dan yang terjadi dalam biofilm. Efek antibakteri setelah
pengobatan dengan klorheksidin atau ozon diukur dengan konversi XTT yang berkurang secara
signifikan. Efektivitas antimikroba dari 60 0,5% s dan pengobatan klorheksidin 0,1% adalah
sama dan sebanding dengan pengobatan ozon 60 detik. Uji XTT adalah metode yang cocok
untuk mencari vitalitas dalam biofilm air liur, yang memungkinkan penilaian efektivitas zat
antimikroba. Penerapannya cepat dan mudah menjadikan itu sangat cocok untuk penyaringan.
Infeksi bakteri memainkan peran khusus dalam kedokteran gigi.Setelah permukaan gigi
supragingiva dan selaput lendir telah dibasahi dengan air liur, mikroba menetap di sana dan
membentuk biofilm. Biofilm ini mengakomodasi patogen gigi dan melindungi mereka terhadap
faktor stres lingkungan, seperti kemoterapi, sistem kekebalan tubuh, asam, periode kelaparan,
dan produk oksigen reaktif .
Zat Antimicrobially efektif dan teknik harus diuji pada model biofilm yang cocok, sebagai
efektivitas terhadap patogen planktonik memiliki sedikit nilai prediktif untuk efektivitas terhadap
biofilm.
Selama beberapa tahun, beberapa mono-spesies model biofilm telah tersedia yang
mengakomodasi mikroba mulut khas. Streptococcus sering digunakan sebagai model karies,
meskipun para ilmuwan lainnya menemukan bahwa mereka tidak mewakili patogen etiologi
penyakit .Untuk pengobatan penyakit periodontal, anaerob patogen periodontal penanda penting.
Namun dalam mikrobiota plak vivo sangat beragam dan kompleks. Rongga mulut pelabuhan
lebih dari 1.000 mikroorganisme yang berbeda, yang bergabung untuk membentuk biofilm
spesies lain. Guggenheim dkk. biofilm digunakan terdiri dari maksimal enam patogen yang
berbeda. Cairan oral, juga merupakan komponen penting dalam pembentukan biofilm
gigi. Protein dalam air liur adalah sumber penting makanan untuk mikroba. Protein pelikel
menetap pada permukaan gigi membentuk film penyejuk disebut. Film pengkondisian
membentuk dasar untuk pengembangan biofilm, sebagai adhesins bakteri langsung mengikat ini
oligosakarida yang mengandung glikoprotein. Selain faktor antimikroba ludah adalah stressor
penting yang bisa meningkatkan pembentukan biofilm. Banyak formulasi saliva buatan telah
dirancang yang mencoba untuk meniru proses ini untuk memastikan pembentukan biofilm
realistis. Dalam kebanyakan kasus, bagaimanapun, saliva buatan gagal untuk menyediakan
semua komponen organik dan anorganik yang ada dalam air liur alam. Selain itu, tidak ada bukti
telah diberikan bahwa air liur buatan mempromosikan pembentukan biofilm.
Untuk model biofilm mereka, peneliti lainnya menggunakan air liur relawan yang
disaring dalam kondisi steril, dan disentrifugasi diencerkan.
Secara sederhana lebih realistis spesies lain Model biofilm dapat diperoleh dengan kultur
air liur relawan, tanpa filtrasi sebelumnya dalam kondisi steril. Tentu saja, ini hanya model
karena irama sirkadian dari air liur dan komposisi Sejalan variabel air liur tidak dapat ditiru, juga
tidak dapat asupan makanan biasa. Selanjutnya, kondisi lisan berbeda dalam setiap pasien.
Sebuah deteksi selanjutnya pembentukan biofilm sulit. Bahkan ketika menggunakan agar-agar
yang tidak spesifik, seperti agar darah Columbia, tidak mungkin untuk pasti mendeteksi setiap
spesies dalam budaya. Atau, metode kolorimetri, misalnya, uji XTT, sesuai untuk mengukur
aktivitas metabolisme dan vitalitas. XTT adalah garam kuning yang dikurangi dengan
dehydrogenases sel yang aktif secara metabolik untuk produk formazan berwarna.Metode
kolorimetri yang menarik karena mereka memiliki potensi untuk menghasilkan yang jelas titik
akhir berdasarkan perubahan warna terlihat.
Tujuan dari penelitian ini, oleh karena itu, adalah pengembangan metode yang cocok
untuk menguji air liur menggunakan biofilm XTT.
Budidaya biofilm
Biofilm dikultur pada cakram titanium diameter 5 mm dan 1 mm tebal (Straumann, Basel,
Swiss). Cakram titanium steril diposisikan dalam 96-well piring microtitre (Techno Produk
Plastik AG, Trasadingen, Swiss), ditutupi dengan 100 ml air liur segar, tidak distimulasi
sukarelawan sehat (berusia antara 20 dan 30 tahun, bukan perokok), dan diinkubasi aerobikpada
37 ° C. Para donor tidak mengambil obat tiga bulan sebelum studi dan tidak memiliki lesi karies
aktif atau penyakit periodontal.Setelah 24 jam, air liur tersebut diambil off dan digantikan oleh
steril otak-jantung kaldu infus (BD, BBL ™, Heidelberg, Jerman), sebagai media untuk tujuan
umum pertumbuhan yang sangat bergizi. Setelah 48 jam, medium ditarik off, dan cakram dicuci
dengan larutan NaCl 0,9% dan dipindahkan ke piring, microtitre baru steril.
Antiseptik pengobatan dengan klorheksidin
Chlorhexidine digluconate digunakan sebagai% 0,1 dan larutan 0,05%. Cakram ditutupi
dengan 100 ml antiseptik dan diinkubasi selama 1 menit. Setelah paparan ini, klorheksidin
tersebut diambil off, dan efek antiseptik dihentikan dengan menambahkan 1 ml inactivator
(Lipofundin MCT 20%, B.Braun, Melsungen, Jerman). Inaktivasi klorheksidin oleh inactivator
divalidasi dengan uji suspensi kuantitatif sesuai dengan EN 1040. Garam fisiologis digunakan
untuk kontrol.
Penerapan ozon
Benda uji langsung dirawat selama 20 s, 30 detik, 40 detik, atau 60 dengan ozon gas yang
disediakan oleh perangkat HealOzone (KAVO, Biberach, Jerman). Ozon ini disampaikan
melalui selang ke dalam cangkir steril sekali pakai pada konsentrasi 2.100 ppm ± 10%. Gas ozon
di-refresh dalam cangkir sekali pakai dengan kecepatan 615 cc / menit perubahan volume gas di
dalam cangkir lebih dari 300 kali setiap detik.
Inaktivasi itu tidak perlu sebagai perangkat suctions dari setiap ozon sisa setelah aplikasi.
Vitalitas pengukuran dengan alat tes XTT
Bioreduction dari XTT bisa diperkuat dengan penambahan agen kopling elektron seperti
methosulfate phenazine (PMS) atau menadione (Pria). Untuk mengoptimalkan solusi pewarnaan,
200 ml larutan XTT ditambahkan ke setiap disk menyandang biofilm tumbuh setelah 24 jam dan
48 jam, masing-masing. Solusi XTT menambahkan terdiri dari:
o XTT (180 mg / l) (AppliChem, Darmstadt, Jerman) dan menadione (0,688 mg / l)
(Sigma-Aldrich, Munich, Jerman) (selanjutnya disebut "XTT + Pria")
o XTT (180 mg / l) dan methosulfate phenazine (20 mg / l) (PMS, AppliChem, Darmstadt,
Jerman) (selanjutnya disebut "XTT + PMS")
o XTT (180 mg / l), menadione (0,688 mg / l) dan methosulfate phenazine (20 mg / l)
(selanjutnya disebut "XTT + Pria + PMS")
Untuk menentukan rentang pengukuran, air liur diencerkan dengan garam fisiologis dan
diinkubasi dengan larutan pewarnaan XTT ditentukan dalam tes 1 (XTT + PMS + menadione).
Untuk menguji efektivitas antimikroba dari klorheksidin dan ozon gas, cakram diobati juga
diinkubasi dengan 200 ml larutan pewarnaan (XTT + PMS + menadione).
Setelah 3 jam inkubasi sambil geleng-geleng (Titramax, Heidolph Instrumen, Schwabach,
Jerman) pada 37 ° C, 100 ml dari semua solusi dipindahkan ke piring microtitre baru steril dan
dianalisis pada 450 nm (referensi panjang gelombang 620 nm) dengan menggunakan fotometer
sebuah ( anthos Mikrosysteme, Krefeld, Jerman)
Penentuan CFU
Setelah pengobatan, cakram titanium ditempatkan ke dalam sumur dengan 200 ml larutan
NaCl 0,9% dan biofilm telah dihapus dengan skala ultrasonik (Branson 2510, 130 W, 42 kHz,
Dietzenbach, Jerman). Pengenceran Serial dibuat dengan mentransfer 0,1 ml suspensi yang
dihasilkan menjadi 0,9 ml larutan NaCl 0,9% segar.Setelah itu, porsi alikuot dari 0,1 ml dari
pengenceran masing-masing disebar pada agar darah domba Columbia (BBL ™, BD,
Heidelberg, Jerman) dan aerob diinkubasi pada 37 ° C selama 48 jam. Koloni dihitung dan
dinyatakan sebagai unit pembentuk koloni (CFU). Nilai CFU transformasi log.
Laser confocal pemindaian mikroskop
Live / Mati-Pewarnaan (BacLight, Invitrogen, Darmstadt, Jerman) digunakan untuk
analisis mikroskopis dari vitalitas bakteri. Biofilm diinkubasi segera dengan pewarna sesuai
dengan instruksi produsen. Setelah inkubasi cakram dibilas dengan larutan NaCl 0,9% untuk
menghilangkan residu zat warna dari biofilm. Sampel dievaluasi dalam mikroskop laser scanning
confocal (CLSM510 Exciter, Zeiss, Jena, Jerman).
Scanning elektron mikroskop
Untuk mikroskop elektron, biofilm disusun sebagai berikut: setelah langkah fiksasi (1
jam dalam 1% glutaraldehid, paraformaldehyde 2%, asam picric 0,2%, HEPES mM 5 (pH 7,4),
dan 50 mM NaN3), sampel diperlakukan dengan asam tanat 2% selama 1 jam, 1% osmium ferri
selama 1 jam, thiocarbohydrazide 1% selama 30 menit, 1% osmium ferri pada 4 ° C semalam,
dan dengan uranil asetat 2% selama 2 jam dengan langkah-langkah mencuci di
antaranya. Sampel penelitian adalah dehidrasi dalam serangkaian bergradasi solusi aseton (10
sampai 100%) dan kemudian kritis-point kering. Akhirnya, sampel yang dipasang di bertopik
aluminium, tergagap dengan emas-paladium dan diperiksa dalam LS10 EVO (Zeiss,
Oberkochen, Jerman)
gambar 1.
Skema pandangan percobaan
Analisa
Untuk semua percobaan, sedikitnya delapan objek setiap tes yang digunakan. Perlakuan
klorheksidin diperlukan 23 disc. Selain itu, benda uji masing-masing delapan yang tersedia untuk
tes kontrol.Data kontinu disajikan sebagai rata-rata ± standar deviasi. Analisis statistik dilakukan
dengan STATVIEW ® 5.0 (SAS, Cary, NC) perangkat lunak. Grafik juga dibuat menggunakan
STATVIEW.Median diberikan dengan kesalahan standar mereka. Korelasi nonparametrik
(Mann-Whitney U-test) diperkirakan untuk perbandingan serapan. P-nilai di bawah 0,05
dianggap signifikan secara statistik.
Hasil
Budidaya biofilm
Prosedur budidaya terus-menerus diperiksa budaya dengan menentukan CFU dan untuk
reproduktifitas dengan mikroskop. Sel kepadatan dari 108 CFU / ml secara teratur dihapus dari
benda uji dengan skala USG.
Optimasi solusi pewarnaan
Penyerapan turunan formazan berwarna XTT dikonversi oleh mikroba adalah ukuran vitalitas
sel. Nilai penyerapan yang tinggi menunjukkan aktivitas metabolisme tinggi. Gambar 2 (Gambar
2) menunjukkan nilai penyerapan (pada 450 nm) dari solusi pewarnaan yang berbeda (XTT +
Pria, XTT + PMS dan XTT + Pria + PMS) pada 24-h-dan 48-jam berusia biofilm. Untuk semua
solusi pewarnaan, bermakna (p <0,01) terlihat perbedaan antara 24-jam dan 48-jam biofilm.
Gambar 2
Diagram absorpsi pada 450 nm (referensi panjang gelombang 620 nm) dari XTT
dimetabolisme oleh biofilm air liur (XTT + menadione, XTT + phenazine methosulfate,
XTT + menadione + N-metil-phenazinium methylsulfate).
Dalam kasus XTT + Pria, penyerapan pada 450 nm meningkat menjadi nilai rata-rata 30 kali
lebih tinggi (0,01-0,30) setelah 48 jam dari setelah 24 jam. Di dalam 24 jam, nilai penyerapan
XTT + PMS meningkat menjadi lima kali (0,10-0,50) nilai asli, sementara penyerapan XTT Pria
+ + PMS meningkat tiga kali 0,20-0,65.
Nilai penyerapan dari XTT + Pria + PMS selalu nyata (p <0,01) lebih tinggi dibandingkan
dengan solusi yang tegang lainnya.
Penentuan rentang pengukuran
Gambar 3 (Gambar 3) menunjukkan nilai serapan pada 450 nm ditentukan oleh cairan air liur
dan pewarnaan selanjutnya dengan XTT + Pria + PMS. Ada perbedaan yang signifikan (p <0,01)
antara nilai penyerapan biofilm hingga penambahan sebesar 4,5 log10.Penyerapan tidak lagi
diukur dalam konsentrasi dari 3,5 log10 (CFU / ml), kecuali untuk kontrol negatif.
Gambar 3
Diagram penyerapan pada 450 nm (referensi panjang gelombang 620 nm) dari XTT + +
methosulfate menadione phenazine dimetabolisme oleh pengenceran air liur berbagai.
Antimikroba pengobatan
Sebuah konversi XTT berkurang diamati pada biofilm air liur yang telah mengalami
pengobatan antimikroba dengan ozon gas dan chlorhexidine. Penurunan ini signifikan setelah
pengobatan ozon 60-s atau 120-s (p <0,02) dan satu menit klorheksidin pengobatan (p <0,01)
(lihat Gambar 4 (Gbr. 4)). Tidak ada perbedaan dalam konversi XTT menggunakan 0,05% atau
larutan klorheksidin 0,1%.Tidak ada perbedaan terlihat di mikrograf elektron scanning antara
biofilm yang tidak dirawat dan biofilm diobati dengan ozon. Dalam kedua contoh, sel-sel muncul
montok dan biofilm memiliki struktur longgar terikat. Bakteri dalam biofilm diobati dengan
klorheksidin yang rusak dan struktur keseluruhan tampaknya lebih ketat (Gambar 5 (Gbr.
5)). Gambar CLSM dikonfirmasi pengamatan ini (Gambar 6 (Gbr. 6)). Setelah pengobatan
dengan ozon, bagian dari biofilm dicelup merah (sel dengan membran yang rusak). Setelah
pengobatan CHX, tidak ada bagian berwarna hijau (sel dengan membran utuh) diidentifikasi.
Gambar 4
Diagram penyerapan pada 450 nm (referensi panjang gelombang 620 nm) dari XTT + +
methosulfate menadione phenazine, dimetabolisme oleh biofilm air liur selama 48 jam
setelah pengobatan dengan larutan NaCl 0,9% (kontrol), ozon gas dan chlorhexidine.
Gambar 5
SEM mikrograf: 48-h biofilm air liur matang: (A) yang tidak diobati, (B) setelah
pengobatan ozon, (C) setelah pengobatan klorheksidin.Perbesaran 10.000 x
Gambar 6
CLSM mikrograf: 48-h air liur biofilm matang: (A) yang tidak diobati, (B) setelah
pengobatan ozon, (C) setelah pengobatan klorheksidin.Pembesaran 100 x
Diskusi
Menyebabkan penyakit gigi yang khas, seperti karies dan periodontitis, biofilm
menyulitkan penghapusan mikroba bertanggung jawab untuk pembentukan biofilm oleh zat
antimikroba. Tujuan dari penelitian ini adalah untuk mengembangkan model biofilm cocok
untuk menguji kemanjuran zat antimikroba dengan non-berbasis budaya deteksi vitalitas biofilm
air liur menggunakan XTT, dan untuk mempersiapkan alat tes XTT cocok.
Penelitian kami memiliki beberapa keterbatasan. Kami menggunakan air liur dari
sukarelawan untuk membuat model biofilm praktis yang relevan. Untuk lebih memahami
interaksi antara bakteri dan XTT, kami melakukan percobaan kami pada cakram titanium mesin
untuk mengecualikan bahan relung hidrofobik retensi, seperti gigi berlubang dan porositas di
mana biofilm bisa menempel. Implan titanium telah berhasil digunakan dalam kedokteran gigi
dan biofilm pada titanium adalah masalah sentral dalam peri-implantitis. Peri-implantitis implan
osseointegrasi lisan bukan monoinfeksi oleh patogen tunggal, melainkan, mereka menunjukkan
karakteristik infeksi campuran. Sedangkan spesies tunggal dari campuran bakteri tidak dapat
menyebabkan abses eksperimental, kombinasi dari spesies ini bisa melakukannya. Plak biofilm
yang mengandung beberapa spesies bakteri yang sesuai harus lebih relevan untuk mempelajari
penyakit gigi dan khasiat antimikroba. Langkah pertama adalah penggunaan metode budidaya
aerob saja. Tes paling XTT dilakukan aerobik.Pada langkah berikutnya kita akan menggunakan
biofilm plak subgingiva dalam kondisi anaerob untuk tes XTT. Tetapi penelitian juga
menunjukkan bahwa mikrobiota yang sama yang dapat ditemukan di sekitar implan (di bawah
kondisi anaerobik) juga dapat ditemukan di sekitar gigi (dalam kondisi aerobik).
XTT adalah garam tetrazolium tidak berwarna, yang diubah menjadi turunan, berwarna
larut air formazan oleh dehydrogenases, dengan dehidrogenase suksinat menjadi sangat penting
karena memainkan peran utama dalam penyediaan energi setiap sel hidup individu. Tidak seperti
garam tetrazolium lain (misalnya, CTC dan TTC), XTT tidak memerlukan formazans larut harus
diekstrak.Karena perubahan warna dalam larutan dapat langsung ditentukan oleh fotometri, tes
XTT memungkinkan sejumlah besar objek pengujian yang akan diuji untuk vitalitas mereka
sangat cepat.Inkubasi bakteri dengan XTT selama 3 jam pada 37 ° C telah menjadi subyek dari
beberapa publikasi. Apa yang baru adalah komposisi dari solusi pewarnaan XTT
diterapkan. Biofilm air liur mengandung berbagai macam mikroba yang berbeda (Gram positif,
bakteri Gram-negatif dan jamur). Untuk mengkonversi XTT, mikroba ini memerlukan berbagai
aditif yang berfungsi sebagai pembawa elektron. Aditif standar untuk Candida spp.adalah
menadione, tetapi PMS dapat diterapkan juga. Untuk bakteri Gram-positif cocci, PMS digunakan
dalam kebanyakan, tapi kadang-kadang menadione juga digunakan. Dalam kasus Gram-negatif
berbentuk batang bakteri, terutama menadione diterapkan. Awal kami sendiri (tidak diterbitkan)
investigasi mengkonfirmasi hasil ini pada kesesuaian menadione untuk Candida albicans dan
PMS untuk Streptococcus mutans dan Streptococcus sanguinis sebagai wakil dalam kedokteran
gigi. Adapun Pseudomonas aeruginosa, aplikasi gabungan dari menadione dan PMS ternyata
cocok. Dengan menggunakan analisis berbasis budaya dari air liur, Gram-positif cocci bakteri,
i. a., dapat diisolasi. Oleh karena itu, penambahan PMS tampaknya sangat diperlukan untuk
mendeteksi kolorimetri menggunakan XTT. McCluskey dkk. juga digunakan secara eksklusif
PMS untuk deteksi kolorimetri mikroba terjadi di lumpur aktif, dan mampu membuktikan bahwa
ada korelasi langsung antara produksi dan konsumsi oksigen formazan.
Dalam perbandingan langsung, PMS-menadione gabungan elektron mediator
menunjukkan konversi XTT tertinggi dalam biofilm air liur (lihat Gambar 2 (Gambar
2)). Ternyata, bagaimanapun, bahwa jumlah sel tinggi minimal 4,5 log10 (CFU / ml) diperlukan
untuk mendeteksi penurunan XTT (lihat Gambar 4 (Gbr. 4)). Sebesar 5,5 log10 (CFU / ml),
penyerapan sangat tinggi (1,91) dideteksi pada 450 nm. Semakin tinggi jumlah sel yang aktif
secara metabolik, sinyal semakin tinggi kolorimetri. Selain itu, semakin tinggi metabolisme sel,
semakin tinggi sinyal. Jelas, tidak ada hubungan linier antara jumlah sel-sel dan sinyal
kolorimetri.Ketika pewarna lainnya (FDA und Syto9) digunakan, adsorpsi bahkan tetap
konstan. Setelah 48 jam biofilm, kepadatan sel pembentukan ca. 8 log10 (CFU / ml) tercapai,
tetapi mengikuti tes XTT, penyerapan dalam biofilm adalah 0,65, yaitu, kurang dari pada bakteri
planktonik meskipun kepadatan sel lebih tinggi.Jumlah produk yang ditahan dapat bervariasi
antara bakteri planktonik dan biofilm. Selain itu, biofilm dikenakan kondisi selain segar bakteri
suspensi. Banyak patogen gigih dan, karenanya, menunjukkan aktivitas metabolik yang lebih
rendah.Untuk alasan ini, jumlah minimum patogen tidak dapat ditentukan dari suspensi dan
biofilm secara persis sama. Selain itu, sel planktonik dapat berinvestasi lebih banyak energi
dalam metabolisme rutin. Dalam percobaan kami, penyerapan adalah 30 kali lebih tinggi setelah
48 jam dari setelah 24 jam, yaitu konversi XTT meningkat karena pertumbuhan
biomassa. Namun, tingkat metabolisme yang berbeda tidak menyebabkan peningkatan
logaritmik dari pengurangan XTT. Pengamatan XTT terkait juga dibuat oleh kelompok
penelitian lain.
Mengurangi formazan pembentukan diamati karena pengobatan antimikroba dari biofilm
air liur. Akibatnya, tes XTT cocok untuk menentukan kemanjuran zat antimikroba, terutama
untuk penyaringan. Konsentrasi klorheksidin rendah digunakan telah diselidiki oleh peneliti lain,
yang mencatat khasiat antimikroba tidak cukup dalam biofilm. Di sisi lain, klorheksidin terbukti
menjadi sedikit lebih unggul ozon. Ini juga telah diterbitkan oleh kelompok penelitian lain yang
menerapkan metode alternatif. Namun, in vivo Hauser-Gerspach dkk. tidak menemukan efek
antimikroba yang signifikan dari CHX dan ozon. Para mikrograf elektron scanning
mengkonfirmasi hasil ini. Setelah pengobatan ozon, morfologi sel-sel menunjukkan tidak ada
perbedaan. Walaupun biofilm diobati dengan klorheksidin tampaknya rusak dibandingkan
dengan kontrol, hanya beberapa sel yang secara morfologis cacat.
Terlepas dari beberapa kelemahan, XTT dengan penambahan menadione dan PMS
adalah metode yang cocok untuk menentukan vitalitas dalam biofilm bakteri air liur dan
penilaian izin dari efektivitas zat antimikroba.
Pengujian adalah mudah dilakukan, dan memungkinkan sejumlah besar benda uji yang
akan diuji. Hal ini sangat cocok untuk skrining berbagai faktor yang mempengaruhi biofilm,
seperti antiseptik atau perawatan fisik atau kimia lainnya, misalnya, terapi ozon, photodynamic
atau plasma tekanan atmosfer.
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