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NR 1-3/2009
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In vitro
photodynamic properties of methylene blue in a combination with
laser illumination at 630 nm concerning Candida albicans
Oddziaływanie na Candida
albicans fotodynamicznych własności błękitu metylenowego w
połączeniu z promieniowaniem laserowym o długości fali 630 nm –
badanie in vitro
Professor Pasyechnikova Nataliya, MD, PhD
Zborovskaya Olexandra, MD, PhD,
Kustrin Taras, MD
State Institution „The Filatov`s Institute of Eye Diseases and
Tissue Therapy of AMS of Ukraine”
Director: Professor Pasyechnikova Nataliya, MD, PhD
Department: Laboratory of Microbiology and Department of Uveitis |
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| Summary: |
The study had to define
influence of combined applications of laser radiation
and methylene blue (MB) on pathogenic culture of Candida
albicans in vitro. The experimental study was done at
standard techniques of method of cultivations in a broth.
The laser irradiation of cultures was done at once after
addition of MB in concentration 0.05%, 0.1% and 0.2%.
During studying action of MB in dark, influence of MB to
the growth of test-shtam without laser radiation, were
prepared fluid Gissa’s broth with glucose without
Andrede’s indicator. Activation of MB was done by laser
with wave length 630 nm during 3 or 5 min. All
experiments were passed in 4 parallels and 3 repeats.
Maximal suppression of growth of microorganisms was
noted in group with using 0.1% MB with laser radiation 3
minutes without centrifugation after 24 hours. Maximal
suppression of growth was noted in group after
centrifugation with 0.05% MB with exposure of laser 3
min. after 48 hours. Sensitivity of pathogenic culture
of Candida albicans to application of МB and laser
raises accordingly to increase of concentration of MB. |
| Słowa kluczowe: |
methylene blue,
photosensitizer, Candida albicans. |
| Key words: |
błękit metylenu,
substancja uczulająca na światło, Candida albicans. |
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Currently, new antibacterial
preparations are developed. But occurrence of new generations of
antibiotics is accompanied by occurrence of new cultures of
microorganisms with stability to these medicines (1). It is
continuing active development of photodynamic antimicrobic
chemical therapy (PACT) (2). At the some time usage of PACT is
at the initial stage of development, despite of the knowledge
since 19 century about abilities of some chemical substances as
photosensitizers, in particular aniline dyes (3,4). Absence of
effective antimycotic agents for the local use, makes mycotic
keratitis an important problem in ophthalmology (5,6,7).
The purpose
Of the study was to define influence of combined applications of
laser radiation and methylene blue (MB) on pathogenic culture of
Candida albicans in vitro.
Design
Pilot, openlabel, comparative laboratory study.
Methods
Study was conducted with a culture of pathogenic test-shtam of
Candida albicans. Test-shtam were kept and conducted to the
surface of meat-pepton oblique agar at temperature 4°C. For
experiment were used daily cultures which were grown up in
test-tubes on meat-pepton oblique agar at 37°C. Initial solution
of MB in distilled water was done.
For studying of action of MB in dark, influence of MB to the
growth of test-shtam without laser radiation, were prepared
fluid Gissa’s broth with glucose without Andrede’s indicator.
Broth was spilled into test-tubes for 1 ml. Concentrations of MB
were 0.05%, 0.1% and 0.2%. A number of parallels for each
variant were 4. Test-tubes with the broth were sterilized in
autoclave at 0.5 atm. Culture of test-microorganisms was swabbed
by sterile physiological solution. Suspension were diluted by
sterile physiological solution until concentration 2·104 cells/
ml. From this inoculum were selected 50 mcl and were added into
the each test-tube. Terminal concentration of cells in 1 ml of
the broth were 1·103 cells/ ml.
Culture with MB was incubated in a thermostat at temperature
37°C during 24 and 48 hours. Intensity of growth of test-shtam
carried out by optic density of culture by spectrophotometer
“Spekol-10” at wave length 540 nm. For control were used same
cultures of microorganisms without adding of MB. Estimation of
results – intensity of the growth of culture by optical density
of solution, was done after 24 and 48 hours after added of MB in
test-tubes with culture.
Defining of photoinducted influence of methylene blue to the
microorganisms
Culture of test-microorganisms was swabbed by sterile
physiological solution. Into the test-tubes with 1 ml of sterile
physiological solution, which contained 0.05%, 0.1% and 0.2% MB,
was added 50 mcl of this suspension with final concentration
1·107 cells/ ml in test-tubes. Suspension with MB was incubated
30 min. at room temperature, for binding substance with the
cells. Laser with wave length 630 nm during 3 or 5 min was used
for activation of MB. Radiated suspensions were passed for 1
hour at room temperature, and diluted by sterile physiological
solution until concentration 1·103 cells/ ml. From the last
dilution selected 50 mcl and added into the sterile Gissa’s
broth with glucose without indicator (variant without
centrifugation). In parallel the initial suspension were
centrifuged (1200 circles/ min, 20 min), after this supernatant
liquide was poured with added sterile physiological solution,
and than selected 50 mcl into sterile broth (variant with
centrifugation), with final concentration 1·103 cells/ ml. The
culture after radiation without MB was used as control. A number
of repeats, conditions of incubating and estimation of results
were done in the same way with the previous technique. All
experiments were passed in 3 repeats.
Results
Maximal suppression of growth of microorganisms was noted in
group with 0.05% solution of MB with exposure of laser 3 min
with centrifugation after 24 hours (Tab. I). In groups with 0.1%
and 0.2% solution of MB were noted stimulation of growth of
microorganisms with exposure of laser radiation 3 min. at 1.2
and 5 times accordingly. In groups with exposure of laser 5 min.
increasing of growth of microorganisms were: MB 0.1% – in 1.5
times, 0.2% – in 3 times.
After 24 hours without centrifugation maximal suppression was
noted with 0.1% solution of MB with exposure of laser 3 min
(Tab. II). In group with concentration of MB 0.05% was noted
stimulation of growth, 0.2% – suppression of growth in 1.3 times.
In all groups with radiation 5 min. were noted stimulation of
growth of cells. After 48 hours suppression of growth was
significant in groups with 0.2% solution of MB with duration of
radiation 3 and 5 min, and also in groups with 0.1%
concentration of MB with duration of radiation 5 min. After 48 hours
maximal suppression of growth was noted in group after
centrifugation with 0.05% MB and exposure of laser 3 min.
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Significant suppression of growth
of fungoid flora was not observed after centrifugation after 24
and 48 hours with action of MB in a dark (Tab. III, IV).
Obtained results prove significant suppression of Candida
albicans by using methylene blue as photosensitizer.
Discussion
After 24 hours the maximal suppression was noted in group after
centrifugation with 0.05% MB and exposition of laser 3 min.
However, in group without centrifugation the maximal suppression
was noted in test-tubes with 0.1% MB and laser 3 min. After 48
hours the maximal suppression of microorganisms was noted in
group after centrifugation with 0.05% MB and exposition of laser
3min. It is possible to explain these results, most likely, by
the expressed thermal effects at increase of duration of laser.
This condition could stimulate growth of Candida albicans. MB is
distributed in tissues of eye with gradual full removing from
structures of an eye after 24 hours (8). So it is necessary to
take into account the results of group without centrifugation
after 24 hours. In our next study we shall research efficiency
of MB as photosensitizer in the treatment of fungoid keratitis.
Conclusions
1. Growth of Candida albicans may be suppressed by methylene
blue as photosensitizer in a combination with laser radiation
with wave length 630 nm.
2. After 24 hours maximal suppression of growth of
microorganisms was noted in group without centrifugation with
using of 0.1% MB and duration of laser radiation 3 minutes.
3. After 48 hours maximal suppression of growth of
microorganisms was noted in group after centrifugation with
using of 0.05% MB and exposure of laser radiation 3 minutes.
References:
1. Finegold SM: Intestinal microbial changes and disease as a
result of antimicrobial use. Pediatric Infectious Disease 1986,
5, 88-90.
2. Foote CS: Future directions and applications in photodynamic
theory. SPIE Institute Series 1S6 1990, 115-126.
3. Wainwright M: Photodynamic antimicrobial chemotherapy (PACT).
Journal of Antimicrobial Chemotherapy 1998, 42, 13-28.
4. Wainwright M: Non-porphyrin photosensitisers in biomedicine.
Chemical Society Reviews 1996, 25, 351-259.
5. Tzu GW, Kirk RW, Bradley MM: Experimental Keratomycosis in a
Mouse Model. Invest Ophthalm & Vis Sci 2003, 1, 210-216.
6. Gopinathan U, Garg P, Fernandes M, Sharma S, Athmanathan S,
Rao GN: The epidemiological features and laboratory results of
fungal keratitis: A 10-year review at a referral eye care
center in south India. Cornea 2002, 21, 555-559.
7. Prajna NV, Nirmalan PK, Mahalakshmi R, Lalitha P, Srinivasan M:
Concurrent use of 5% natamycin and 2% econazole for the
management of fungal keratitis. Cornea 2004, 23(8), 793-796.
8. Аль-Асталь МС: Фотодинамическая терапия неоваскуляризации
роговицы с применением метиленового синего и лазерного излучения
длины волны 578нм: Автореф.дис… к-та мед.наук: 14.00.08 /
Респуб.гос.казённое предпр. «Казахский ордена «Знак почёта»
научно-исследовательский институт глазных болезней»: Алматы,
2006, 24с.
The study was originally received/ 13.12.2008 (1093)
Praca wpłynęła do redakcji 13.12.2008 r. (1093)
Accepted for publication 21.01.2009/
Zakwalifikowano do druku 21.01.2009 r.Adres do
korespondencji (Reprint requests to):
Author-correspondent Zborovskaya O. MD, PhD
State Institution „The Filatov`s Institute of Eye Diseases and
Tissue Therapy of AMS of Ukraine”
49/51, Frantsuzki blvd., Odessa, Ukraine
e-mail: filatoveye@mail.ru
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Concentration
of methylene
blue, %
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Time of influence by the laser, min
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3
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5
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M ±m
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d |
M ±m
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d
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24 hours
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0.05
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0.075 ± 0.005
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0.015
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0.334 ± 0.004
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0.0120
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0.1
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0.232 ± 0.008
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0.023
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0.306 ± 0.008
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0.0024
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0.2
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0.586 ± 0.008
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0.024
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0.300 ± 0.011
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0.0330
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48 hours
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0.05
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0.275 ± 0.021
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0.061
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0.304 ± 0.019
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0.0540
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0.1
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0.433 ± 0.026
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0.074
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0.329 ± 0.018
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0.0520
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0.2
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0.388 ± 0.039
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0.111
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0.305 ± 0.019
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0.0550
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Tab. I. Growth of Candida albicans
with presence of methylene blue after laser influence after
centrifugation.
Tab. I. Wzrost Candida albicans w obecności błękitu metylenowego
po laserowaniu i wirowaniu.
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Concentration
of methylene
blue, % |
Time of influence by the laser, min |
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3 |
5 |
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M ±m |
d |
M ±m |
d |
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24 hours |
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0.05 |
0.377 ± 0.005 |
0.015 |
0.300 ± 0.009 |
0.024 |
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0.1 |
0.161 ± 0.003 |
0.008 |
0.366 ± 0.004 |
0.011 |
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0.2 |
0.282 ± 0.005 |
0.014 |
0.434 ± 0.005 |
0.013 |
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48 hours |
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0.05 |
0.470 ± 0.055 |
0.155 |
0.333 ± 0.018 |
0.051 |
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0.1 |
0.633 ± 0.021 |
0.062 |
0.321 ± 0.022 |
0.063 |
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0.2 |
0.308 ± 0.007 |
0.021 |
0.329 ± 0.008 |
0.023 |
Tab. II. Growth of Candida albicans with
presence of methylene blue after laser influence without
centrifugation.
Tab. II. Wzrost Candida albicans w obecności błękitu
metylenowego po laserowaniu bez wirowania.
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Concentration of
methylene blue, % |
M ±m |
d |
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24 hours |
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0.05 |
0.160 ± 0.016 |
0.031 |
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0.1 |
0.189 ± 0.016 |
0.046 |
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0.2 |
0.102 ± 0.005 |
0.015 |
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48 hours |
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0.05 |
0.314 ± 0.008 |
0.016 |
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0.1 |
0.389 ± 0.014 |
0.041 |
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0.2 |
0.328 ± 0.011 |
0.032 |
Tab. III. Growth of Candida albicans with
presence of methylene blue after centrifugation.
Tab. III. Wzrost Candida albicans w obecności błękitu
metylenowego po wirowaniu.
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Concentration of
methylene blue, % |
M ±m |
d |
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24 hours |
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0.05 |
0.170
± 0.021 |
0.042 |
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0.1 |
0.231
± 0.006 |
0.016 |
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0.2 |
0.394
± 0.007 |
0.019 |
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48 hours |
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0.05 |
0.257
± 0.021 |
0.042 |
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0.1 |
0.353
± 0.012 |
0.035 |
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0.2 |
0.396
± 0.023 |
0.067 |
Tab. IV. Growth of Candida albicans with
presence of methylene blue without centrifugation.
Tab. IV. Wzrost Candida albicans w obecności błękitu
metylenowego bez wirowania.
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