Lasers in Surgery and Medicine 12450458 (1992)
Dye-Mediated Bactericidal Effect of He-Ne Laser Irradiation on Oral Microorganisms Hayato Okamoto, DDS, Tatsuo Iwase, DDSC, and Toshio Morioka, DMSC Department of Preventive Dentistry, Faculty of Dentistry, Kyushu University, Fukuoka 8 12, Japan
Little attention has been given to the bactericidal effect of laser irradiation, particularly using low-power energy lasers. It has been demonstrated that He-Ne laser light has an inhibitory action on dental plaque. The purpose of this study was to investigate the bactericidal effect of He-Ne laser irradiation on cariogenic microorganisms. The bactericidal effect was determined by the formation of a growth-inhibitory zone or by the counting of viable bacterial colonies. Streptococcus sobrinus AHT that is a Gram-positive microorganism was sensitive to He-Ne laser light, but Escherichia coli, a Gram-negative microorganism, was resistant. The effect of several dyes necessary to instigate a bactericidal action was also examined. A growth-inhibitory zone was observed using 10 kinds of blue, purple, or green dyes, which were mainly phenylmethane dyes. The leakage of potassium from S. sobrinus AHT following laser irradiation was determined using an atomic absorption spectrophotometer. The leakage began to increase following irradiation for 2 min, and reached a plateau following irradiation for 30-60 min. Moreover, to examine some changes in the dye itself following laser irradiation in the absence of bacteria, ultraviolet-visible absorption spectra and 'H NMR spectra were recorded. In this study, it was indicated that the bactericidal effect on cariogenic bacteria by He-Ne laser irradiation was efficient only in the presence of specific dyes. It is suggested that this laser may be suitable for clinical applications in preventive dentistry. 0 1992 Wiley-Liss, Inc.
Key words: growth inhibition, low power laser, potassium leakage, proton NMR, Streptococcus mutans, ultraviolet-visibleabsorption spectrum
enhanced in the presence of a dark colored dye. Klein et al. [61 observed the death of microorganRecently, laser application has been ex- isms or the partial inhibition of their growth as a panded in the field of dentistry. Basic and clinical result of 60-250 J of ruby laser irradiation. In research on the biostimulative effects by laser irstudies by Zakariasen et al. [7], bactericidal acradiation (wound healing, pain relief, etc.) has tion in the experimental dental root canal was been published [l-41.There have also been many achieved by carbon dioxide laser irradiation. In reports about the effects of laser irradiation on microorganisms [5-151. Schultz et al. [51 indicated that Pseudomonas aeruginosa, Staphylococ- Accepted for publication January 16, 1992. cus aureus, and Escherichia coli were killed by Address reprint requests to Hayato Okamoto, DDS, Departneodymium-YAG laser irradiation, where the en- ment of Preventive Dentistry, Faculty of Dentistry, Kyushu ergy density was greater than 1,667 J/cm2, and University 61, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Jathe sensitivity of P. aeruginosa t o laser light was pan. INTRODUCTION
0 1992 Wiley-Liss, Inc.
Bactericidal Effect of He-NeLaser Irradiation 451 other cases, laser irradiation was used for the diameter and 1.8 m in length, while the latter sterilization of instruments [8,91. The mechanism reflected the laser beam on the mirror without behind the bactericidal action reported above re- any fiber. Both these lasers were used with a garding high-power laser irradiation is assumed wavelength of 632.8 nm and a CW mode. to be the destruction of bacterial cells by heat proPreparation of Cell Suspension duced from high energy. All strains were cultured overnight in BHI Studies on low-power energy laser irradiation are extremely scarce. Iwase et al. [13] re- broth. Aliquots were inoculated onto the new meported that the accumulation of dental plaque in dium and preincubated at 37°C anaerobically. hamsters was prevented by He-Ne laser irradia- The optical density (530 nm) was 0.7 and 0.6 on S. tion. McGuff and Bell [141 examined the effect of sobrinus AHT and E . coli, respectively. The cells He-Ne gas laser irradiation (continuous wave) were harvested by centrifugation at 3,000 rpm for (CW) (0.5 mW) on P. aeruginosa, Proteus vulgaris, 10 min, washed twice with sterile physiological S. aureus, and Bacillus subtilis, but did not obtain saline, and then recentrifuged. The pellets were any bactericidal effect. Macmillan et al. [151 re- resuspended in sterile physiological saline. ported that microorganisms of seven species were rapidly killed when they were irradiated in tolu- Preparation of Dyes idine blue solution by a 21-30 mW CW gas laser The dyes were dissolved in distilled water or 100% ethyl alcohol and then diluted by distilled with a wavelength of 632.8 nm. As regards some of the studies of these pho- water to obtain various concentrations. The final toreactions, it is known that the existence of a concentration of ethyl alcohol was 1%,a concenphotosensitizer, such as dye, can result in various tration which could not affect the survival of bacteria. phenomena [5,6,14,151. The purpose of this investigation was to study the bactericidal action of low-power He-Ne Experimental Design Assay for the bactericidal effect of Helaser irradiation together with the influence of Ne laser irradiation by means of the formadyes acting as a photosensitizer. tion of a growth-inhibitoryzone. The effect on the serological group of S. mutans. Two milliliMATERIALS AND METHODS ters of 0.5% agar plus 0.5 ml of cell suspension Cultures were poured on a Mitis-Salivarius (MS) agar Representative strains of the each serotype (Difco, USA) plate and the laser was perpendicuof mutans streptococci were used for this study. larly irradiated at 6 mW. The distance from the These included Streptococcus mutans GS5 (sero- tip of the fiber to the surface of the agar plate was type c), LM7 (el, and OMZ175 (f); S. rattus FA1 0.5 cm. On this condition, the area of an irradi(b);S. cricetus HS-1 (a); and S. sobrinus OMZ176 ated spot was 0.126 cm2.The irradiation time was (d) and AHT (g). In addition, E . coli W3350 was 2, 5 , and 10 min; therefore, the energy density also used as a Gram-negative [G(->] bacterium. was 5.7, 14.3, and 28.6 J/cm2, respectively. After Within these strains, S. sobrinus AHT was irradiated plates were anaerobically cultured at mainly used for the experiments because this 37°C for 48 hr, it was visually estimated whether strain was often isolated from human caries le- or not a growth-inhibitory zone had formed. Moresions. All strains were maintained in brain-heart over, the ratio of the inhibitory zone area was calculated for 2,5, and 10 min of irradiation using infusion (BHI) medium (BBL, USA). the image analysis processor (Nexus Qube, Nexus Laser Systems Inc., Tokyo, Japan). The effect on S. sobrinus A H T using various Two He-Ne gas lasers each with a different energy level were used. One (SOFT LASER 632, dyes. In the same manner as mentioned above, Worldwide Lasers Industry, Switzerland) was em- the effect of various dyes was estimated by means ployed at the output power of 6 mW, while the of the formation of a growth-inhibitory zone on an other (KHL-50, NEC, Japan) was employed agar plate. In this experiment, crystal violet (CV) within a range from 30 t o 40 mW. The output and trypan blue (TB), dyes which are originally power was measured by a power meter (SCIEN- contained in MS agar, were removed from that TECH 361, Boulder, CO, USA). The former device agar, and then the effects were estimated on the was used coupled to a glass optical fiber, 2 mm in agar plates containing each experimental dye
Okamoto et al.
452
TABLE 1. Ratios of Growth-Inhibitory Zones Following Laser Irradiation in the Case of the Serological Group of S. mutuns Genotwe I
SDecies S. mutans
I1 I11
S. ruttus S. sobrinus
IV
S. cricetus
Strain GS5 LM7 OMZ175 FA1 OMZ176 AHT HS-1
SerotvDe C
e
f b d g a
Irradiation time (min) 2 5 10 1 1.70 1.91 1 1.86 2.23 1 2.14 2.76 1 1.36 1.66 1 1.73 2.02 1 1.44 1.65 1
1.68
2.23
only, or else no dye at all. The final concentra- was irradiated at an output power of 40 mW for 2, tions of CV and TB were 0.8 pg/ml and 75 pg/ml, 5,10,20,30, and 60 min. The mixtures were then respectively. Moreover, a total of 17 dyes were filtered (pore size: 0.2 pm). The potassium concenexamined whose concentrations were 75 pg/ml. In tration in the filtrates was determined, using an addition, to examine the influence of a different atomic absorption spectrophotometer (180-60 poconcentration of dye, the effects of CV at final larized Zeeman, Hitachi, Tokyo, Japan). Prior to concentrations of 8x1O3, 80, 8, 0.8, 8 ~ 1 0 - ~ the , filtration, aliquots were taken from both the 8 x lop3, and 8 x lop5 pg/ml were examined. irradiated and unirradiated cell suspensions in Assay for the bactericidal effect of He- order to count the viable colonies. Ne laser irradiation by means of the counting Ultraviolet-visible absorption spectroof viable colonies. In this assay, S. sobrinus photometric analysis (UV analysis). CV and AHT and E . coli W3350 were used. A total of 0.5 TB solutions at a final concentration of 7.5 pg/ml ml of cell suspension (-lo7 CFU/ml) containing were irradiated at an output power of 40 mW for CV, whose final concentration was 8 pg/ml, were 30 min. UV absorption spectra were recorded on placed in a glass test tube. The laser beam was irradiated and unirradiated dye solution with an perpendicularly led into each cell suspension. automatic spectrophotometer (U-3210, Hitachi, During irradiation, the glass tube was covered Japan). The absorption peak value between 400 with aluminum foil to prevent other various in- and 800 nm was measured for each spectrum. fluences. The output power of the laser was 30 Nuclear magnetic resonance analysis mW and the irradiation time was 2,5,10,20, and (NMR analysis). CV was dissolved with deute30 min. Tenfold serial dilutions of irradiated and rium oxide (D,O) as the solvent for NMR analysis unirradiated cell suspensions were plated on BHI at a final concentration of 7.5 x l o 2 pg/ml. Irradiagar. Viable colonies were counted after anaero- ation was carried out at an output power of 40 bic incubation of the plates at 37°C for 48 hr. mW for 30 min and 5 hr. The 'H NMR spectra The influence of irradiated dye. This ex- were recorded for both the irradiated and unirraperiment was undertaken to examine whether or diated CV solution with a FT NMR spectrometer not irradiated dye had any bactericidal ability. A (JNM-GX400, JEOL, Japan). total of 0.5 ml of the CV solution containing 8 pg/ml were irradiated at an output power of 40 mW for 30 min. A cell suspension of S. sobrinus RESULTS AHT (-lo4 CFU/ml) was added to both the irraA growth-inhibitory zone was observed in all diated and unirradiated dye solutions. The mix- the strains of mutans streptococci. It was suptures were continuously shaken for 30 min at posed that the area of a growth-inhibitory zone room temperature. Viable colonies were counted observed following irradiation for 2 min was 1; by the usual method. In addition, the mixture of then the ratios of those for 2, 5, and 10 min were cell suspension and unirradiated dye solution of indicated (Table 1). The longer the irradiation CV was irradiated and this was also evaluated as time, the larger the inhibitory area became. Figures 1, and 2a-d and Table 2 show the a reference. Assay for the leakage of potassium from influence of CV and TB on the bactericidal effect bacterial cells. A cell suspension of S. sobrinus on S. sobrinus AHT. If CV was not included, an AHT (-lo9 CFU/ml) containing 8 pg/ml of CV inhibitory zone was not observed. TB was ineffec-
Bactericidal Effect of He-Ne Laser Irradiation
453
TABLE 2. Ratios of Growth-InhibitoryZones Following Laser Irradiation With Each of the Two Dyes Contained in MS Agar in the Case of S. sobrinus AHT* Irradiation time (min) Dye CV + TB CV TB No dve
2 1 1
-
5 1.70 1.27 -
10 2.73 1.72 -
*MS, Mitis-Salivarius; CV, crystal violet 0.8 pg/ml; TB, trypan blue 75 pg/ml; -, there was no detectable inhibitory zone.
tive for the expression of the bactericidal effect of laser irradiation. Table 3 shows the bactericidal effect of various concentrations of CV on S. sobrinus AHT. When the concentration of CV was greater than 80 p.g/ml, bacteria did not grow, even without laser irradiation. At concentrations of 8, 0.8, and 8X p.g/ml, growth-inhibitory zones were pg/ml, manifested. At a concentration of 8 x an unclear growth-inhibitory zone was observed following irradiation for 10 min. Table 4a,b shows the bactericidal effects of laser irradiation on S. sobrinus AHT using various dyes. As for the structural characteristics of chromophore, by which most dyes were usually classified, thiazine, oxazine, anthraquinone, and phenylmethane dyes-except for brilliant blue and thymol blue-were all effective. All the azo dyes which were used, proved ineffective. As for the characteristics of color tone, most blue or purple dyes proved effective, except for TB and brilliant blue. All red and yellow dyes which were tested proved ineffective. This is probably because these colors reflect the red light of the laser, and so the bactericidal effect of the laser may be lost. As shown in Figure 3a, a decrease in the number of viable colonies on S. sobrinus AHT was not found in the control without CV, but when CV was added to the bacterial suspension, viable colonies began to decrease following 2 min of irradiation, with the bactericidal ratio becoming greater than 99% following 20 min of irradiation. A decrease in the number of viable colonies on E. Fig. 1. Photograph of the growth-inhibitory zone of S . sobricoli was not found, even when the irradiation nus AHT on MS agar following laser irradiation (6 mW, 2, 5, time was 30 min (Fig. 3b). and 10 rnin). As shown in Figure 4, when irradiated and Fig. 2. A higher magnification of the photographic views of unirradiated CV solutions were mixed with the the growth-inhibitory zone. a = both crystal violet and trypan blue; the same plate as that in Figure 1; b=crystal violet bacterial suspension of s-sobrinus AHT, respeconly; c = trypan blue only; d = no dye. tively, a bactericidal effect was not observed at
Okamoto et al.
454
TABLE 3. Bactericidal Effects of Laser Irradiation on S . sobrinus AHT Using Various Concentrations of Crystal Violet* Concentration of crvstal violet (I*g/ml) 8 x lo3
80 8 0.8 8 x lop2 8 x 10-3
Irradiation time ( m i d 0 2 5 No growth No growth 2+ 2+ 2+ 2+ + f -
-
~ ~ i n - 5
-
10
2+ 24-
+
+ -
- 31 a
" 0
;i
1
5
10
20
30
Irradiation Time ( m i d
*,
*2 + , very clear inhibitory zone;
+ , clear inhibitory zone; unclear inhibitory zone; -, there was no detectable inhibitory zone. TABLE 4. Effective and Ineffective Dyes for Instigating Bactericidal Action Following Laser Irradiation Color tone a: Effective Blue
Purple
Green b: Ineffective Blue
Yellow
Red
Dvea
Structural classification
Toluidine blue 0 Brilliant cresyl blue Victoria blue B Bromocresol green Alizarin blue S Crystal violet Bromocresol purple Bromophenol blue Fast green Bromothymol blue
Thiazine Oxazine Phenylmethane Phenylmethane Anthraquinone Phenylmethane Phenylmethane Phenylmethane Phenylmethane Phenylme thane
Trypan blue Brilliant blue Thymol blue Bismarck brown Evans blue Pontamine sky blue Eosin Y
Azo Phenylmethane Phenylmethane Azo Azo Azo Xanthine
-$ 31 b
" 0
I
5
:2
1'0
20 Irradiation Time (min)
30
Fig. 3. Bactericidal effect of laser irradiation (30 mW) on S. sobrinus AHT (a)and E . coli W3350 (b) according to the number of viable colonies. A = condition in the absence of crystal violet; =in the presence of crystal violet.
-
"The concentration of all dyes was 75 pg/ml.
all. Only when bacteria, dye, and laser light coexisted did a bactericidal effect appear. Figure 5 shows potassium leakage and the survival ratio of S. sobrinus AHT following laser irradiation. With 2 min of irradiation, potassium ions had already begun to leak from the cells. The leakage of potassium ions reached a plateau at 30-60 min, but most bacteria had already been killed after 10 min of irradiation. Among the dyes used for UV analysis, CV proved to be an effective dye for the bactericidal effect of laser irradiation. TB proved t o be an in-
Fig. 4. Bactericidal effect of irradiated (40 mW, 30 min) and unirradiated dye. I = viable colonies when the mixture of irradiated crystal violet and S. sobrinus AHT (-lo4 CFUlml) was continuously shaken for 30 min at room temperature; UI =those of unirradiated; R = viable colonies when laser light was led to the mixture of unirradiated crystal violet and S. sobrinus AHT for 30 min as a reference.
effective dye. When comparing the absorption spectrum of CV prior to irradiation, the absorbance intensity following irradiation decreased, but the wavelength at the absorbance peak was
Bactericidal Effect of He-Ne Laser Irradiation 455 0 the expression of a bactericidal effect of laser irradiation. Generally, laser light of the visible region is mostly absorbed by complementary colors [171, d2 0 and it is thought that in the absorbed area, selec.^ c Q tive biostimulation may occur. Since the He-Ne CT 50 laser used in the present study has red light, the .-> laser may efficiently absorbed by the blue color 5 contained be (I) in MS agar. Therefore, the growth-inhibitory zone which we observed is probably due to the absorption of the red light from the laser by the blue dyes of the MS agar. Klein et al. [61 sugJ JlOO gested that the presence of dye, such as methyl5 10 20 30 60 ene blue, in the broth increased the sensitivity of Irradiation Time ( m i d microorganisms to the effects of laser irradiation Fig. 5. Potassium leakage and survival ratio of S. sobrinus using a ruby laser with red light and a waveAHT (-lo9 CFU/ml) containing crystal violet following laser irradiation (40 mW).*=potassium leakage from the cells length of 694.3 nm. McGuff and Bell [14] hypothwith laser irradiation; o = those of non-irradiated control; esized that the bacterial preparation might abA = survival ratio of S. sobrinus AHT with laser irradiation. sorb an increased amount of laser energy by the incorporation of dyes. The formation of a growthinhibitory zone observed on MS agar in our exthe same both before and after irradiation (Table periment may be supported by their suggestions. 5 , Figs. 6a,b). As for the absorption spectrum of However, because of the ineffectiveness of TB, in TB, no changes were seen following irradiation spite of its blue tone, it was assumed that both the (Table 5 , Figs. 6c,d). color tone and the chemical structural characterFigure 7a-c shows the 'H NMR spectra with istics of a dye are probably related to any bacteunirradiated CV, with CV irradiated for 30 min ricidal effect. and with CV irradiated for 5 hr, respectively. The In this study, 10 dyes were found to be effecpeak at 6 4.7 ppm is due to the proton of DHO in tive while the others proved ineffective for prothe solvent, D20. The two peaks near 6 6.5 and 6.9 ducing a bactericidal effect of laser irradiation ppm are due to the hydrogen atoms of the aro- (Table 4a,b). Yamamoto [181 attempted to clarify matic ring. The singlet near 6 2.95 ppm is due to the conditions needed for the photodynamic inacthe methyl hydrogens. There were no differences tivation of bacteriophages. They reported the pho(e.g., chemical shift, intensity of each peak, etc.) todynamic activity of dyes which belong to thiazamong the three 'H NMR spectra. ine, oxazine and acridine. These dyes were all basic and hetero-tricyclic compounds. From the basis of such common properties and chemical DISCUSSION structures of dyes, the ionic bonding between dye The bactericidal effect of laser irradiation and bacteriophage was suggested. In addition, could be noted for all strains of mutans strepto- they proposed that these dyes might associate cocci with different serotypes (Table 1).According with the nucleic acid of bacteriophages. Herczegh to Blum [16], it was discovered the phenomenon et al. [19]reported that the photon of a ruby laser that paramecia swimming in solution containing light was absorbed by the dye molecule attached a dye, were rapidly killed when exposed t o the to the protein coat of the phage, and the dissipasun's rays, whereas they survived for long periods tion of the absorbed energy took place in the proin sunlight when no dye was present. We there- tein coat, with the release of energy causing damfore investigated the influence of various dyes on age to the protein. Mutans streptococci are very the bactericidal effect of laser irradiation. Firstly, different microorganisms from bacteriophages. CV and TB, dyes which are contained in MS agar, However, there is the possibility that a dye may were examined. CV was found to be effective, combine with a certain site on the bacterial cell whereas TB was not. When no dyes were con- and that this binding may be concerned with the tained into the medium, it was not effective at all chemical structure of the dye. Since there has been a report on the inhibi(Figs. 1 and 2a-d, Table 2). It was therefore confirmed that the presence of dye was essential for tory effect of dye on bacteria without any laser h Y
42
Okamoto et al.
456
b
- -. 0" - AO" 200 300 400 500 600 700 800 900200 300 400 500 600 700 800 900 I
C
Q,
1-
d
0
C
e0 v)
D
a Wavelength (nm)
Wavelength (nm)
Fig. 6. Absorption spectra of dye both before and after laser irradiation (40 mW, 30 min). Each spectra represent crystal violet before (a) and after (b) irradiation, and trypan blue before (c) and after (d) irradiation.
TABLE 5. Wavelength and Absorbance at the Peak of the Spectrum Between 400 and 800 nm for CV and TB* Dve"
cv TB
Before irradiation WL(nm) Abs' 590.0 0.95 577.2 0.35
After irradiation WL(nm) Abs2 589.6 0.72 573.2 0.35
Absl -Abs2 0.23 0
*CV, crystal violet; TB, trypan blue; WL, wavelength; Abs, absorbance. "The concentration of all dyes was 7.5 pg/ml.
light [20,21], it would suggest that an appropriate concentration of dye is necessary for the expression of a bactericidal effect of laser irradiation. Since the inhibitory zone was most clearly observed at 8 pg/ml of CV, we used this concentration to count the viable colonies. A decrease in the number of colonies of S. sobrinus AHT with CV could be seen, but not without the use of dye (Fig. 3a). There was no decrease in the case of E . coli, in spite of the existence of dye (Fig. 3b). There have been a few reports of photoinactivation using both Gram-positive [G( + )] and G(-) microorganisms [14,22-241. In a study on the bactericidal effect induced by laser irradiation and Haematoporphyrin (Hpr) against G( + ) and G( - ) microorganisms, Martinetto et al. [22] reported that a higher Hpr concentration and greater laser energy than those used with G( +>microorganisms had to be applied to achieve the same results for G(-) microorganisms. The suggestions in their report support our findings. It was also speculated that our results might have been due t o the differences in chemi-
cal or structural properties between G ( + ) and G(-) microorganisms. Leakage of potassium ions by laser irradiation was observed (Fig. 5). In the review of Belkin et al. 1251, it was reported that 5 min of 2 mW/cm2 He-Ne laser irradiation of erythrocytes in vitro decreased the intracellular level of K + . There have also been reports discussing membrane damage following near-ultraviolet radiation [26,271. Bertoloni et al. [231 reported Hpr almost exclusively bound with the cytoplasmic membrane. In this study, it was not clear how much the dye bound with the cytoplasmic membrane. The membrane component might have been degraded by laser irradiation, which would lead t o a leakage of potassium ions from the bacterial cells. An alteration of the electrolytic balance may thus lead to osmotic shock [231. We measured the UV absorption spectrum in order to examine whether or not any changes were formed on irradiated dye compared with unirradiated dye. In the absorption spectrum, the wavelength of the absorption peak and molar ex-
Bactericidal Effect of He-Ne Laser Irradiation
1
4""B
' " * '
"6"
a
I
5
K " ' 3 '"1
'
i ' ' " b -PPM -
Fig. 7. 'H NMR spectra of crystal violet. a=non-irradiated control; b = irradiated for 30 min; c = irradiated for 5 hr.
tinction coefficient (E) are important for elucidating the molecular structure. As for the absorption spectrum of CV, which is an effective dye for the bactericidal effect, it is conjectured that there would be some kind of twist around the single bond connecting the chromophores due to a decrease in absorbance intensity following irradiation (Table 5, Fig. 6a,b), and that these twists
457
may influence the electronic spectrum, thus resulting in a decrease of &. However, there would not be any large structural changes, such as a degradation of the chromophores or a replacement of the substituent. This is because if such large structural changes took place, the shifts in wavelength of the absorption peak would be pronounced [28]. As for the absorption spectrum of TB, which is an ineffective dye, no changes were seen following irradiation (Table 5, Fig. 6c,d). Regarding UV analysis, it was suggested that any changes in the electronic conditions of CV were caused by laser irradiation. Moreover, there were no changes in the spectra of CV among unirradiated, 30-min irradiated and 5-hr irradiated CV according t o NMR analysis (Fig. 7a-c). Consequently, we can speculate that structural changes, such as degradation, rearrangement, and conformational changes as detected by some chemical shift or change in intensity of signal, may not come about. We could find some change in the irradiated dye by UV analysis, but we could not relate this change with the bactericidal effect. It was argued that there were no differences in the number of viable colonies between the mixture of unirradiated dye with bacteria and that of irradiated dye with bacteria (Fig. 4). In addition, it was understood that the bacteria, dye, and laser light were needed at the same time for the expression of a bactericidal effect. Karu [29,30] reported two effects of irradiation of cells with visible light of the same wavelength and absorption of this light by the same molecules. One of these effects was the acceleration of the electron transfer in the redox pairs in some sections of the respiratory chain, and the other was the transfer of the excitation energy t o oxygen to form '0,. The latter seems to cause damage to intracellular systems and the death of cells. Moan [311 stated that photodynamic treatment led to lipid peroxidation and that this type of damage played a major part in cell inactivation. It was concluded that cariogenic bacteria were briefly killed by He-Ne laser irradiation in the presence of specific dyes. A He-Ne laser beam can be safely led into human mouth. We have already confirmed that dental plaque bacteria were killed by direct He-Ne laser irradiation for 2 min once a day for 3 days on human tooth surface. Therefore the present study seemed to support the possibility that, in clinical practice, dental plaque control might be successfully achieved by some application of laser irradiation.
458
Okamoto et al.
ACKNOWLEDGMENTS
We wish to express sincere appreciation t o the late Dr. Yoshio Nara (Associate Professor of Our department) for his continuous guidance and valuable discussion. We also thank Prof. T. Inazu of the Department of Chemistry, Faculty of Science, Kyushu University, Japan, for the analysis of the ‘H NMR spectrum of dye and his helpful suggestions. REFERENCES 1. Marin VTW, Corti L, Velussi c. An experimental study of the healing effect of the HeNe laser and the infrared laser. Lasers Med Sci 1988; 3:151-163. 2, Mester E, spiry T, Szende B, Tota JG. Effect of laser rays on wound healing. Am J Surg 1971; 122532-535. 3. Mester E, Szende B, Spiry T, Scher A. Stimulation of wound healing by laser rays. Acta Chir Acad Sci Hung 1972; 13(3):315-324. 4. Iwase T, Hori N, Morioka T. Possible mechanisms of the He-Ne laser effects on the cell membrane characteristics. Laser Med Surg 1988; 4:166-171. 5. Schultz FkJ, Harvey GP, Fernandez-Beros ME, Krishnamurthy S, Rodriguez JE, Cabello F. Bactericidal effects of the neodymium: YAG laser: vitro study, L~~~~~surg Med 1986; 6:445-448. 6. Klein E, Fine S, Ambrus J , Cohen E, Neter E, Ambrus C, Bardos T, Lyman R. Interaction of laser radiation with biologic systems. 111. Studies on biologic systems in vitro. Fed Proc 1965; 24:104-110. 7. Zakariasen KL, Dederich DN, Tulip J , DeCoste S, Jensen SE, Pickard MA. Bactericidal action of carbon dioxide laser radiation in experimental dental root canals. Can J Microbiol 1986; 32:942-946. 8. Adrian JC, Gross A. A new method of sterilization: The carbon dioxide laser. J Oral Pathol 1979; 8:60-61. 9. Hooks TW, Adrian JC, Gross A, Bernier WE. Use of the carbon dioxide laser in sterilization of endodontic reamers. Oral Surg 1980; 49:263-265. 10. Keates RH, Drago PC, Rothchild EJ. Effect of excimer laser on microbiological organisms. Ophthalmic Surg 1988; 19(10):715-718. 11. Korn MY, Chel’nyi AA. Effect of laser microirradiation on filamentous forms of Escherichia coli. Microbiol (USSR) 1970; 39:944-949. 12. Saks NM, Roth CA. Ruby laser as a microsurgical instrument. Science 1963; 141:46-47.
13. Iwase T, Saito T, Nara Y, Morioka T. Inhibitory effect of He-Ne laser on dental plaque deposition in hamsters. J Periodont Res 1989; 24:282-283. 14. McGuff PE, Bell EJ. The effect of laser energy radiation on bacteria. Med Biol I11 1966; 16:191-194. 15. Macmillan JD, Maxwell WA, Chichester CO. Lethal photosensitization of microorganisms with light from a continuous-wave gas laser. Photochem Photobiol 1966; 5: 555-565. 16. Blum HF. “Photodynamic Action and Diseases Caused by Light.” New York: Reinhold Publishing Corp., 1941:3-5. 17. Morioka T, ed. “Laser in Dental Medicine.” Tokyo: Ishiyaku Publishers, Inc., 1986:50. 18. Yamamoto N. Photodynamic inactivation of bacteriophage and its inhibition. J Bacteriol 1958; 75443-448. 19. Herczegh M, Mester E, Ronto Gy. Examination of laserinactivation on “7 phages. Acta Biochim Biophys Acad Sci Hung 1971; 6(1):41-44. 20. Churchman JW. The selective bactericidal action of gentian violet. J Exp Med 1912; 16:221-247. 21. Fung DYC, Miller RD. Effect of dyes on bacterial growth. Appl Microbiol 1973; 25:793-799. 22. Martinetto P, Gariglio M, Lombard GF, Fiscella B, Boggio F. Bactericidal effects induced by laser irradiation and haematoporphyrin against Gram-positive and Gramnegative microorganisms. Drugs Exp Clin Res 1986; 12(4):335-342. 23. Bertoloni G, Salvato B, Dall’Acqua M, Vazzoler M, Jori G. Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis. Photochem Photobiol 1984; 39(6): 811-816. 24. Baugh CL, Clark JB. Photodynamic response in bacteria. J Gen Physiol 1959; 42(5):917-922. 25. Belkin M, Zaturunsky B, Schwartz M. A critical review of low energy laser bioeffects. Lasers Light Ophthalmol 1988; 2(1):63-71. 26. Nakata H, Kamikawa K. Effects of near UV laser irradiation on 3T3 cells and SV3T3 cells. J Jpn SOCLaser Med 1982; 3(1):241-246. 27. Ito A, Ito T. Cell membrane damage by near-UV radiation in yeast cells. Photomed Photobiol 1981; 3(2):65-66. 28. Okamoto H, Iwase T, Nara Y, Morioka T. Studies on mechanism of bactericidal action by low energy laser. J Dent Health 1990; 40(4):536-537. 29. Karu TI. Molecular mechanism of the therapeutic effect of low-intensity laser radiation. Lasers Life Sci 1988; 2(1):53-74. 30. Karu TI. Photochemical effects upon the cornea, skin and other tissues. Health Phys 1989; 56(5):691-704. 31. Moan J. Porphyrin-sensitized photodynamic inactivation of cells: a review. Lasers Med Sci 1986; 15-12.
Dye-Mediated Bactericidal Effect of He-Ne Laser Irradiation on Oral Microorganisms Hayato Okamoto, DDS, Tatsuo Iwase, DDSC, and Toshio Morioka, DMSC Department of Preventive Dentistry, Faculty of Dentistry, Kyushu University, Fukuoka 8 12, Japan
Little attention has been given to the bactericidal effect of laser irradiation, particularly using low-power energy lasers. It has been demonstrated that He-Ne laser light has an inhibitory action on dental plaque. The purpose of this study was to investigate the bactericidal effect of He-Ne laser irradiation on cariogenic microorganisms. The bactericidal effect was determined by the formation of a growth-inhibitory zone or by the counting of viable bacterial colonies. Streptococcus sobrinus AHT that is a Gram-positive microorganism was sensitive to He-Ne laser light, but Escherichia coli, a Gram-negative microorganism, was resistant. The effect of several dyes necessary to instigate a bactericidal action was also examined. A growth-inhibitory zone was observed using 10 kinds of blue, purple, or green dyes, which were mainly phenylmethane dyes. The leakage of potassium from S. sobrinus AHT following laser irradiation was determined using an atomic absorption spectrophotometer. The leakage began to increase following irradiation for 2 min, and reached a plateau following irradiation for 30-60 min. Moreover, to examine some changes in the dye itself following laser irradiation in the absence of bacteria, ultraviolet-visible absorption spectra and 'H NMR spectra were recorded. In this study, it was indicated that the bactericidal effect on cariogenic bacteria by He-Ne laser irradiation was efficient only in the presence of specific dyes. It is suggested that this laser may be suitable for clinical applications in preventive dentistry. 0 1992 Wiley-Liss, Inc.
Key words: growth inhibition, low power laser, potassium leakage, proton NMR, Streptococcus mutans, ultraviolet-visibleabsorption spectrum
enhanced in the presence of a dark colored dye. Klein et al. [61 observed the death of microorganRecently, laser application has been ex- isms or the partial inhibition of their growth as a panded in the field of dentistry. Basic and clinical result of 60-250 J of ruby laser irradiation. In research on the biostimulative effects by laser irstudies by Zakariasen et al. [7], bactericidal acradiation (wound healing, pain relief, etc.) has tion in the experimental dental root canal was been published [l-41.There have also been many achieved by carbon dioxide laser irradiation. In reports about the effects of laser irradiation on microorganisms [5-151. Schultz et al. [51 indicated that Pseudomonas aeruginosa, Staphylococ- Accepted for publication January 16, 1992. cus aureus, and Escherichia coli were killed by Address reprint requests to Hayato Okamoto, DDS, Departneodymium-YAG laser irradiation, where the en- ment of Preventive Dentistry, Faculty of Dentistry, Kyushu ergy density was greater than 1,667 J/cm2, and University 61, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Jathe sensitivity of P. aeruginosa t o laser light was pan. INTRODUCTION
0 1992 Wiley-Liss, Inc.
Bactericidal Effect of He-NeLaser Irradiation 451 other cases, laser irradiation was used for the diameter and 1.8 m in length, while the latter sterilization of instruments [8,91. The mechanism reflected the laser beam on the mirror without behind the bactericidal action reported above re- any fiber. Both these lasers were used with a garding high-power laser irradiation is assumed wavelength of 632.8 nm and a CW mode. to be the destruction of bacterial cells by heat proPreparation of Cell Suspension duced from high energy. All strains were cultured overnight in BHI Studies on low-power energy laser irradiation are extremely scarce. Iwase et al. [13] re- broth. Aliquots were inoculated onto the new meported that the accumulation of dental plaque in dium and preincubated at 37°C anaerobically. hamsters was prevented by He-Ne laser irradia- The optical density (530 nm) was 0.7 and 0.6 on S. tion. McGuff and Bell [141 examined the effect of sobrinus AHT and E . coli, respectively. The cells He-Ne gas laser irradiation (continuous wave) were harvested by centrifugation at 3,000 rpm for (CW) (0.5 mW) on P. aeruginosa, Proteus vulgaris, 10 min, washed twice with sterile physiological S. aureus, and Bacillus subtilis, but did not obtain saline, and then recentrifuged. The pellets were any bactericidal effect. Macmillan et al. [151 re- resuspended in sterile physiological saline. ported that microorganisms of seven species were rapidly killed when they were irradiated in tolu- Preparation of Dyes idine blue solution by a 21-30 mW CW gas laser The dyes were dissolved in distilled water or 100% ethyl alcohol and then diluted by distilled with a wavelength of 632.8 nm. As regards some of the studies of these pho- water to obtain various concentrations. The final toreactions, it is known that the existence of a concentration of ethyl alcohol was 1%,a concenphotosensitizer, such as dye, can result in various tration which could not affect the survival of bacteria. phenomena [5,6,14,151. The purpose of this investigation was to study the bactericidal action of low-power He-Ne Experimental Design Assay for the bactericidal effect of Helaser irradiation together with the influence of Ne laser irradiation by means of the formadyes acting as a photosensitizer. tion of a growth-inhibitoryzone. The effect on the serological group of S. mutans. Two milliliMATERIALS AND METHODS ters of 0.5% agar plus 0.5 ml of cell suspension Cultures were poured on a Mitis-Salivarius (MS) agar Representative strains of the each serotype (Difco, USA) plate and the laser was perpendicuof mutans streptococci were used for this study. larly irradiated at 6 mW. The distance from the These included Streptococcus mutans GS5 (sero- tip of the fiber to the surface of the agar plate was type c), LM7 (el, and OMZ175 (f); S. rattus FA1 0.5 cm. On this condition, the area of an irradi(b);S. cricetus HS-1 (a); and S. sobrinus OMZ176 ated spot was 0.126 cm2.The irradiation time was (d) and AHT (g). In addition, E . coli W3350 was 2, 5 , and 10 min; therefore, the energy density also used as a Gram-negative [G(->] bacterium. was 5.7, 14.3, and 28.6 J/cm2, respectively. After Within these strains, S. sobrinus AHT was irradiated plates were anaerobically cultured at mainly used for the experiments because this 37°C for 48 hr, it was visually estimated whether strain was often isolated from human caries le- or not a growth-inhibitory zone had formed. Moresions. All strains were maintained in brain-heart over, the ratio of the inhibitory zone area was calculated for 2,5, and 10 min of irradiation using infusion (BHI) medium (BBL, USA). the image analysis processor (Nexus Qube, Nexus Laser Systems Inc., Tokyo, Japan). The effect on S. sobrinus A H T using various Two He-Ne gas lasers each with a different energy level were used. One (SOFT LASER 632, dyes. In the same manner as mentioned above, Worldwide Lasers Industry, Switzerland) was em- the effect of various dyes was estimated by means ployed at the output power of 6 mW, while the of the formation of a growth-inhibitory zone on an other (KHL-50, NEC, Japan) was employed agar plate. In this experiment, crystal violet (CV) within a range from 30 t o 40 mW. The output and trypan blue (TB), dyes which are originally power was measured by a power meter (SCIEN- contained in MS agar, were removed from that TECH 361, Boulder, CO, USA). The former device agar, and then the effects were estimated on the was used coupled to a glass optical fiber, 2 mm in agar plates containing each experimental dye
Okamoto et al.
452
TABLE 1. Ratios of Growth-Inhibitory Zones Following Laser Irradiation in the Case of the Serological Group of S. mutuns Genotwe I
SDecies S. mutans
I1 I11
S. ruttus S. sobrinus
IV
S. cricetus
Strain GS5 LM7 OMZ175 FA1 OMZ176 AHT HS-1
SerotvDe C
e
f b d g a
Irradiation time (min) 2 5 10 1 1.70 1.91 1 1.86 2.23 1 2.14 2.76 1 1.36 1.66 1 1.73 2.02 1 1.44 1.65 1
1.68
2.23
only, or else no dye at all. The final concentra- was irradiated at an output power of 40 mW for 2, tions of CV and TB were 0.8 pg/ml and 75 pg/ml, 5,10,20,30, and 60 min. The mixtures were then respectively. Moreover, a total of 17 dyes were filtered (pore size: 0.2 pm). The potassium concenexamined whose concentrations were 75 pg/ml. In tration in the filtrates was determined, using an addition, to examine the influence of a different atomic absorption spectrophotometer (180-60 poconcentration of dye, the effects of CV at final larized Zeeman, Hitachi, Tokyo, Japan). Prior to concentrations of 8x1O3, 80, 8, 0.8, 8 ~ 1 0 - ~ the , filtration, aliquots were taken from both the 8 x lop3, and 8 x lop5 pg/ml were examined. irradiated and unirradiated cell suspensions in Assay for the bactericidal effect of He- order to count the viable colonies. Ne laser irradiation by means of the counting Ultraviolet-visible absorption spectroof viable colonies. In this assay, S. sobrinus photometric analysis (UV analysis). CV and AHT and E . coli W3350 were used. A total of 0.5 TB solutions at a final concentration of 7.5 pg/ml ml of cell suspension (-lo7 CFU/ml) containing were irradiated at an output power of 40 mW for CV, whose final concentration was 8 pg/ml, were 30 min. UV absorption spectra were recorded on placed in a glass test tube. The laser beam was irradiated and unirradiated dye solution with an perpendicularly led into each cell suspension. automatic spectrophotometer (U-3210, Hitachi, During irradiation, the glass tube was covered Japan). The absorption peak value between 400 with aluminum foil to prevent other various in- and 800 nm was measured for each spectrum. fluences. The output power of the laser was 30 Nuclear magnetic resonance analysis mW and the irradiation time was 2,5,10,20, and (NMR analysis). CV was dissolved with deute30 min. Tenfold serial dilutions of irradiated and rium oxide (D,O) as the solvent for NMR analysis unirradiated cell suspensions were plated on BHI at a final concentration of 7.5 x l o 2 pg/ml. Irradiagar. Viable colonies were counted after anaero- ation was carried out at an output power of 40 bic incubation of the plates at 37°C for 48 hr. mW for 30 min and 5 hr. The 'H NMR spectra The influence of irradiated dye. This ex- were recorded for both the irradiated and unirraperiment was undertaken to examine whether or diated CV solution with a FT NMR spectrometer not irradiated dye had any bactericidal ability. A (JNM-GX400, JEOL, Japan). total of 0.5 ml of the CV solution containing 8 pg/ml were irradiated at an output power of 40 mW for 30 min. A cell suspension of S. sobrinus RESULTS AHT (-lo4 CFU/ml) was added to both the irraA growth-inhibitory zone was observed in all diated and unirradiated dye solutions. The mix- the strains of mutans streptococci. It was suptures were continuously shaken for 30 min at posed that the area of a growth-inhibitory zone room temperature. Viable colonies were counted observed following irradiation for 2 min was 1; by the usual method. In addition, the mixture of then the ratios of those for 2, 5, and 10 min were cell suspension and unirradiated dye solution of indicated (Table 1). The longer the irradiation CV was irradiated and this was also evaluated as time, the larger the inhibitory area became. Figures 1, and 2a-d and Table 2 show the a reference. Assay for the leakage of potassium from influence of CV and TB on the bactericidal effect bacterial cells. A cell suspension of S. sobrinus on S. sobrinus AHT. If CV was not included, an AHT (-lo9 CFU/ml) containing 8 pg/ml of CV inhibitory zone was not observed. TB was ineffec-
Bactericidal Effect of He-Ne Laser Irradiation
453
TABLE 2. Ratios of Growth-InhibitoryZones Following Laser Irradiation With Each of the Two Dyes Contained in MS Agar in the Case of S. sobrinus AHT* Irradiation time (min) Dye CV + TB CV TB No dve
2 1 1
-
5 1.70 1.27 -
10 2.73 1.72 -
*MS, Mitis-Salivarius; CV, crystal violet 0.8 pg/ml; TB, trypan blue 75 pg/ml; -, there was no detectable inhibitory zone.
tive for the expression of the bactericidal effect of laser irradiation. Table 3 shows the bactericidal effect of various concentrations of CV on S. sobrinus AHT. When the concentration of CV was greater than 80 p.g/ml, bacteria did not grow, even without laser irradiation. At concentrations of 8, 0.8, and 8X p.g/ml, growth-inhibitory zones were pg/ml, manifested. At a concentration of 8 x an unclear growth-inhibitory zone was observed following irradiation for 10 min. Table 4a,b shows the bactericidal effects of laser irradiation on S. sobrinus AHT using various dyes. As for the structural characteristics of chromophore, by which most dyes were usually classified, thiazine, oxazine, anthraquinone, and phenylmethane dyes-except for brilliant blue and thymol blue-were all effective. All the azo dyes which were used, proved ineffective. As for the characteristics of color tone, most blue or purple dyes proved effective, except for TB and brilliant blue. All red and yellow dyes which were tested proved ineffective. This is probably because these colors reflect the red light of the laser, and so the bactericidal effect of the laser may be lost. As shown in Figure 3a, a decrease in the number of viable colonies on S. sobrinus AHT was not found in the control without CV, but when CV was added to the bacterial suspension, viable colonies began to decrease following 2 min of irradiation, with the bactericidal ratio becoming greater than 99% following 20 min of irradiation. A decrease in the number of viable colonies on E. Fig. 1. Photograph of the growth-inhibitory zone of S . sobricoli was not found, even when the irradiation nus AHT on MS agar following laser irradiation (6 mW, 2, 5, time was 30 min (Fig. 3b). and 10 rnin). As shown in Figure 4, when irradiated and Fig. 2. A higher magnification of the photographic views of unirradiated CV solutions were mixed with the the growth-inhibitory zone. a = both crystal violet and trypan blue; the same plate as that in Figure 1; b=crystal violet bacterial suspension of s-sobrinus AHT, respeconly; c = trypan blue only; d = no dye. tively, a bactericidal effect was not observed at
Okamoto et al.
454
TABLE 3. Bactericidal Effects of Laser Irradiation on S . sobrinus AHT Using Various Concentrations of Crystal Violet* Concentration of crvstal violet (I*g/ml) 8 x lo3
80 8 0.8 8 x lop2 8 x 10-3
Irradiation time ( m i d 0 2 5 No growth No growth 2+ 2+ 2+ 2+ + f -
-
~ ~ i n - 5
-
10
2+ 24-
+
+ -
- 31 a
" 0
;i
1
5
10
20
30
Irradiation Time ( m i d
*,
*2 + , very clear inhibitory zone;
+ , clear inhibitory zone; unclear inhibitory zone; -, there was no detectable inhibitory zone. TABLE 4. Effective and Ineffective Dyes for Instigating Bactericidal Action Following Laser Irradiation Color tone a: Effective Blue
Purple
Green b: Ineffective Blue
Yellow
Red
Dvea
Structural classification
Toluidine blue 0 Brilliant cresyl blue Victoria blue B Bromocresol green Alizarin blue S Crystal violet Bromocresol purple Bromophenol blue Fast green Bromothymol blue
Thiazine Oxazine Phenylmethane Phenylmethane Anthraquinone Phenylmethane Phenylmethane Phenylmethane Phenylmethane Phenylme thane
Trypan blue Brilliant blue Thymol blue Bismarck brown Evans blue Pontamine sky blue Eosin Y
Azo Phenylmethane Phenylmethane Azo Azo Azo Xanthine
-$ 31 b
" 0
I
5
:2
1'0
20 Irradiation Time (min)
30
Fig. 3. Bactericidal effect of laser irradiation (30 mW) on S. sobrinus AHT (a)and E . coli W3350 (b) according to the number of viable colonies. A = condition in the absence of crystal violet; =in the presence of crystal violet.
-
"The concentration of all dyes was 75 pg/ml.
all. Only when bacteria, dye, and laser light coexisted did a bactericidal effect appear. Figure 5 shows potassium leakage and the survival ratio of S. sobrinus AHT following laser irradiation. With 2 min of irradiation, potassium ions had already begun to leak from the cells. The leakage of potassium ions reached a plateau at 30-60 min, but most bacteria had already been killed after 10 min of irradiation. Among the dyes used for UV analysis, CV proved to be an effective dye for the bactericidal effect of laser irradiation. TB proved t o be an in-
Fig. 4. Bactericidal effect of irradiated (40 mW, 30 min) and unirradiated dye. I = viable colonies when the mixture of irradiated crystal violet and S. sobrinus AHT (-lo4 CFUlml) was continuously shaken for 30 min at room temperature; UI =those of unirradiated; R = viable colonies when laser light was led to the mixture of unirradiated crystal violet and S. sobrinus AHT for 30 min as a reference.
effective dye. When comparing the absorption spectrum of CV prior to irradiation, the absorbance intensity following irradiation decreased, but the wavelength at the absorbance peak was
Bactericidal Effect of He-Ne Laser Irradiation 455 0 the expression of a bactericidal effect of laser irradiation. Generally, laser light of the visible region is mostly absorbed by complementary colors [171, d2 0 and it is thought that in the absorbed area, selec.^ c Q tive biostimulation may occur. Since the He-Ne CT 50 laser used in the present study has red light, the .-> laser may efficiently absorbed by the blue color 5 contained be (I) in MS agar. Therefore, the growth-inhibitory zone which we observed is probably due to the absorption of the red light from the laser by the blue dyes of the MS agar. Klein et al. [61 sugJ JlOO gested that the presence of dye, such as methyl5 10 20 30 60 ene blue, in the broth increased the sensitivity of Irradiation Time ( m i d microorganisms to the effects of laser irradiation Fig. 5. Potassium leakage and survival ratio of S. sobrinus using a ruby laser with red light and a waveAHT (-lo9 CFU/ml) containing crystal violet following laser irradiation (40 mW).*=potassium leakage from the cells length of 694.3 nm. McGuff and Bell [14] hypothwith laser irradiation; o = those of non-irradiated control; esized that the bacterial preparation might abA = survival ratio of S. sobrinus AHT with laser irradiation. sorb an increased amount of laser energy by the incorporation of dyes. The formation of a growthinhibitory zone observed on MS agar in our exthe same both before and after irradiation (Table periment may be supported by their suggestions. 5 , Figs. 6a,b). As for the absorption spectrum of However, because of the ineffectiveness of TB, in TB, no changes were seen following irradiation spite of its blue tone, it was assumed that both the (Table 5 , Figs. 6c,d). color tone and the chemical structural characterFigure 7a-c shows the 'H NMR spectra with istics of a dye are probably related to any bacteunirradiated CV, with CV irradiated for 30 min ricidal effect. and with CV irradiated for 5 hr, respectively. The In this study, 10 dyes were found to be effecpeak at 6 4.7 ppm is due to the proton of DHO in tive while the others proved ineffective for prothe solvent, D20. The two peaks near 6 6.5 and 6.9 ducing a bactericidal effect of laser irradiation ppm are due to the hydrogen atoms of the aro- (Table 4a,b). Yamamoto [181 attempted to clarify matic ring. The singlet near 6 2.95 ppm is due to the conditions needed for the photodynamic inacthe methyl hydrogens. There were no differences tivation of bacteriophages. They reported the pho(e.g., chemical shift, intensity of each peak, etc.) todynamic activity of dyes which belong to thiazamong the three 'H NMR spectra. ine, oxazine and acridine. These dyes were all basic and hetero-tricyclic compounds. From the basis of such common properties and chemical DISCUSSION structures of dyes, the ionic bonding between dye The bactericidal effect of laser irradiation and bacteriophage was suggested. In addition, could be noted for all strains of mutans strepto- they proposed that these dyes might associate cocci with different serotypes (Table 1).According with the nucleic acid of bacteriophages. Herczegh to Blum [16], it was discovered the phenomenon et al. [19]reported that the photon of a ruby laser that paramecia swimming in solution containing light was absorbed by the dye molecule attached a dye, were rapidly killed when exposed t o the to the protein coat of the phage, and the dissipasun's rays, whereas they survived for long periods tion of the absorbed energy took place in the proin sunlight when no dye was present. We there- tein coat, with the release of energy causing damfore investigated the influence of various dyes on age to the protein. Mutans streptococci are very the bactericidal effect of laser irradiation. Firstly, different microorganisms from bacteriophages. CV and TB, dyes which are contained in MS agar, However, there is the possibility that a dye may were examined. CV was found to be effective, combine with a certain site on the bacterial cell whereas TB was not. When no dyes were con- and that this binding may be concerned with the tained into the medium, it was not effective at all chemical structure of the dye. Since there has been a report on the inhibi(Figs. 1 and 2a-d, Table 2). It was therefore confirmed that the presence of dye was essential for tory effect of dye on bacteria without any laser h Y
42
Okamoto et al.
456
b
- -. 0" - AO" 200 300 400 500 600 700 800 900200 300 400 500 600 700 800 900 I
C
Q,
1-
d
0
C
e0 v)
D
a Wavelength (nm)
Wavelength (nm)
Fig. 6. Absorption spectra of dye both before and after laser irradiation (40 mW, 30 min). Each spectra represent crystal violet before (a) and after (b) irradiation, and trypan blue before (c) and after (d) irradiation.
TABLE 5. Wavelength and Absorbance at the Peak of the Spectrum Between 400 and 800 nm for CV and TB* Dve"
cv TB
Before irradiation WL(nm) Abs' 590.0 0.95 577.2 0.35
After irradiation WL(nm) Abs2 589.6 0.72 573.2 0.35
Absl -Abs2 0.23 0
*CV, crystal violet; TB, trypan blue; WL, wavelength; Abs, absorbance. "The concentration of all dyes was 7.5 pg/ml.
light [20,21], it would suggest that an appropriate concentration of dye is necessary for the expression of a bactericidal effect of laser irradiation. Since the inhibitory zone was most clearly observed at 8 pg/ml of CV, we used this concentration to count the viable colonies. A decrease in the number of colonies of S. sobrinus AHT with CV could be seen, but not without the use of dye (Fig. 3a). There was no decrease in the case of E . coli, in spite of the existence of dye (Fig. 3b). There have been a few reports of photoinactivation using both Gram-positive [G( + )] and G(-) microorganisms [14,22-241. In a study on the bactericidal effect induced by laser irradiation and Haematoporphyrin (Hpr) against G( + ) and G( - ) microorganisms, Martinetto et al. [22] reported that a higher Hpr concentration and greater laser energy than those used with G( +>microorganisms had to be applied to achieve the same results for G(-) microorganisms. The suggestions in their report support our findings. It was also speculated that our results might have been due t o the differences in chemi-
cal or structural properties between G ( + ) and G(-) microorganisms. Leakage of potassium ions by laser irradiation was observed (Fig. 5). In the review of Belkin et al. 1251, it was reported that 5 min of 2 mW/cm2 He-Ne laser irradiation of erythrocytes in vitro decreased the intracellular level of K + . There have also been reports discussing membrane damage following near-ultraviolet radiation [26,271. Bertoloni et al. [231 reported Hpr almost exclusively bound with the cytoplasmic membrane. In this study, it was not clear how much the dye bound with the cytoplasmic membrane. The membrane component might have been degraded by laser irradiation, which would lead t o a leakage of potassium ions from the bacterial cells. An alteration of the electrolytic balance may thus lead to osmotic shock [231. We measured the UV absorption spectrum in order to examine whether or not any changes were formed on irradiated dye compared with unirradiated dye. In the absorption spectrum, the wavelength of the absorption peak and molar ex-
Bactericidal Effect of He-Ne Laser Irradiation
1
4""B
' " * '
"6"
a
I
5
K " ' 3 '"1
'
i ' ' " b -PPM -
Fig. 7. 'H NMR spectra of crystal violet. a=non-irradiated control; b = irradiated for 30 min; c = irradiated for 5 hr.
tinction coefficient (E) are important for elucidating the molecular structure. As for the absorption spectrum of CV, which is an effective dye for the bactericidal effect, it is conjectured that there would be some kind of twist around the single bond connecting the chromophores due to a decrease in absorbance intensity following irradiation (Table 5, Fig. 6a,b), and that these twists
457
may influence the electronic spectrum, thus resulting in a decrease of &. However, there would not be any large structural changes, such as a degradation of the chromophores or a replacement of the substituent. This is because if such large structural changes took place, the shifts in wavelength of the absorption peak would be pronounced [28]. As for the absorption spectrum of TB, which is an ineffective dye, no changes were seen following irradiation (Table 5, Fig. 6c,d). Regarding UV analysis, it was suggested that any changes in the electronic conditions of CV were caused by laser irradiation. Moreover, there were no changes in the spectra of CV among unirradiated, 30-min irradiated and 5-hr irradiated CV according t o NMR analysis (Fig. 7a-c). Consequently, we can speculate that structural changes, such as degradation, rearrangement, and conformational changes as detected by some chemical shift or change in intensity of signal, may not come about. We could find some change in the irradiated dye by UV analysis, but we could not relate this change with the bactericidal effect. It was argued that there were no differences in the number of viable colonies between the mixture of unirradiated dye with bacteria and that of irradiated dye with bacteria (Fig. 4). In addition, it was understood that the bacteria, dye, and laser light were needed at the same time for the expression of a bactericidal effect. Karu [29,30] reported two effects of irradiation of cells with visible light of the same wavelength and absorption of this light by the same molecules. One of these effects was the acceleration of the electron transfer in the redox pairs in some sections of the respiratory chain, and the other was the transfer of the excitation energy t o oxygen to form '0,. The latter seems to cause damage to intracellular systems and the death of cells. Moan [311 stated that photodynamic treatment led to lipid peroxidation and that this type of damage played a major part in cell inactivation. It was concluded that cariogenic bacteria were briefly killed by He-Ne laser irradiation in the presence of specific dyes. A He-Ne laser beam can be safely led into human mouth. We have already confirmed that dental plaque bacteria were killed by direct He-Ne laser irradiation for 2 min once a day for 3 days on human tooth surface. Therefore the present study seemed to support the possibility that, in clinical practice, dental plaque control might be successfully achieved by some application of laser irradiation.
458
Okamoto et al.
ACKNOWLEDGMENTS
We wish to express sincere appreciation t o the late Dr. Yoshio Nara (Associate Professor of Our department) for his continuous guidance and valuable discussion. We also thank Prof. T. Inazu of the Department of Chemistry, Faculty of Science, Kyushu University, Japan, for the analysis of the ‘H NMR spectrum of dye and his helpful suggestions. REFERENCES 1. Marin VTW, Corti L, Velussi c. An experimental study of the healing effect of the HeNe laser and the infrared laser. Lasers Med Sci 1988; 3:151-163. 2, Mester E, spiry T, Szende B, Tota JG. Effect of laser rays on wound healing. Am J Surg 1971; 122532-535. 3. Mester E, Szende B, Spiry T, Scher A. Stimulation of wound healing by laser rays. Acta Chir Acad Sci Hung 1972; 13(3):315-324. 4. Iwase T, Hori N, Morioka T. Possible mechanisms of the He-Ne laser effects on the cell membrane characteristics. Laser Med Surg 1988; 4:166-171. 5. Schultz FkJ, Harvey GP, Fernandez-Beros ME, Krishnamurthy S, Rodriguez JE, Cabello F. Bactericidal effects of the neodymium: YAG laser: vitro study, L~~~~~surg Med 1986; 6:445-448. 6. Klein E, Fine S, Ambrus J , Cohen E, Neter E, Ambrus C, Bardos T, Lyman R. Interaction of laser radiation with biologic systems. 111. Studies on biologic systems in vitro. Fed Proc 1965; 24:104-110. 7. Zakariasen KL, Dederich DN, Tulip J , DeCoste S, Jensen SE, Pickard MA. Bactericidal action of carbon dioxide laser radiation in experimental dental root canals. Can J Microbiol 1986; 32:942-946. 8. Adrian JC, Gross A. A new method of sterilization: The carbon dioxide laser. J Oral Pathol 1979; 8:60-61. 9. Hooks TW, Adrian JC, Gross A, Bernier WE. Use of the carbon dioxide laser in sterilization of endodontic reamers. Oral Surg 1980; 49:263-265. 10. Keates RH, Drago PC, Rothchild EJ. Effect of excimer laser on microbiological organisms. Ophthalmic Surg 1988; 19(10):715-718. 11. Korn MY, Chel’nyi AA. Effect of laser microirradiation on filamentous forms of Escherichia coli. Microbiol (USSR) 1970; 39:944-949. 12. Saks NM, Roth CA. Ruby laser as a microsurgical instrument. Science 1963; 141:46-47.
13. Iwase T, Saito T, Nara Y, Morioka T. Inhibitory effect of He-Ne laser on dental plaque deposition in hamsters. J Periodont Res 1989; 24:282-283. 14. McGuff PE, Bell EJ. The effect of laser energy radiation on bacteria. Med Biol I11 1966; 16:191-194. 15. Macmillan JD, Maxwell WA, Chichester CO. Lethal photosensitization of microorganisms with light from a continuous-wave gas laser. Photochem Photobiol 1966; 5: 555-565. 16. Blum HF. “Photodynamic Action and Diseases Caused by Light.” New York: Reinhold Publishing Corp., 1941:3-5. 17. Morioka T, ed. “Laser in Dental Medicine.” Tokyo: Ishiyaku Publishers, Inc., 1986:50. 18. Yamamoto N. Photodynamic inactivation of bacteriophage and its inhibition. J Bacteriol 1958; 75443-448. 19. Herczegh M, Mester E, Ronto Gy. Examination of laserinactivation on “7 phages. Acta Biochim Biophys Acad Sci Hung 1971; 6(1):41-44. 20. Churchman JW. The selective bactericidal action of gentian violet. J Exp Med 1912; 16:221-247. 21. Fung DYC, Miller RD. Effect of dyes on bacterial growth. Appl Microbiol 1973; 25:793-799. 22. Martinetto P, Gariglio M, Lombard GF, Fiscella B, Boggio F. Bactericidal effects induced by laser irradiation and haematoporphyrin against Gram-positive and Gramnegative microorganisms. Drugs Exp Clin Res 1986; 12(4):335-342. 23. Bertoloni G, Salvato B, Dall’Acqua M, Vazzoler M, Jori G. Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis. Photochem Photobiol 1984; 39(6): 811-816. 24. Baugh CL, Clark JB. Photodynamic response in bacteria. J Gen Physiol 1959; 42(5):917-922. 25. Belkin M, Zaturunsky B, Schwartz M. A critical review of low energy laser bioeffects. Lasers Light Ophthalmol 1988; 2(1):63-71. 26. Nakata H, Kamikawa K. Effects of near UV laser irradiation on 3T3 cells and SV3T3 cells. J Jpn SOCLaser Med 1982; 3(1):241-246. 27. Ito A, Ito T. Cell membrane damage by near-UV radiation in yeast cells. Photomed Photobiol 1981; 3(2):65-66. 28. Okamoto H, Iwase T, Nara Y, Morioka T. Studies on mechanism of bactericidal action by low energy laser. J Dent Health 1990; 40(4):536-537. 29. Karu TI. Molecular mechanism of the therapeutic effect of low-intensity laser radiation. Lasers Life Sci 1988; 2(1):53-74. 30. Karu TI. Photochemical effects upon the cornea, skin and other tissues. Health Phys 1989; 56(5):691-704. 31. Moan J. Porphyrin-sensitized photodynamic inactivation of cells: a review. Lasers Med Sci 1986; 15-12.
