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Comparative biocidal activities of lasers operating at seven different wavelengths

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Seven laser instruments, delivering radiation at a selection of wavelengths in the range of 0.355 to 118 μm, were investigated for their ability to kill Escherichia coli as a lawn of the bacteria on nutrient agar culture plates. Easily the most effective was a 600-W CO2 laser operating at 10.6 μm, which produced 1.2-cm2 circular zones of sterilization at energy densities of around 8 J cm-2 in a 30-msec exposure. Circular zones with an area of 0.7 cm2 were achieved with 200 W from a Nd:YAG laser delivering 8-ms, 10-J pulses of 1.06 μm radiation at 20 Hz. The exposure time, however, was 16 s and the energy density (1940 J cm-2) was more than 240 times higher than with the CO2 laser. This difference is believed to be partly due to the much higher absorption of radiation at 10.6 μm than at 1.06 μm, by water in the bacterial cells and the surrounding medium (nutrient agar). Sterilization was observed after exposure to frequency-tripled Nd:YAG laser radiation at 355 nm (3.5 J cm-2). Lasers that were totally ineffective in killing Escherichia coli (with their wavelength and maximum energy densities tested) were the far infrared laser (118 μm; 7.96 J cm-2), the laser diode array (0.81 μm; 13,750 J cm-2), and the argon ion laser (0.488 μm; 2210 J cm-2). The speed at which laser sterilization can be achieved is particularly attractive to the medical and food industries.
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COMPARATIVE BACTERICIDAL ACTIVITIES OF
LASERS OPERATING AT SEVEN
DIFFERENT WAVELENGTHS
Ian A. Watson,Glenn D. Ward,Ruikang K. Wang,James H. Sharp,
David M. Budgett,Duncan E. Stewart-Tull,Alastair C. Wardlaw,
and Chris R. Chatwin*
University of Glasgow, Lasers & Optical Systems Engineering Centre, Department of Mechanical
Engineering, Glasgow G12 8QQ, United Kingdom; University of Glasgow, Division of Molecular and
Cellular Biology, Institute of Biomedical and Life Sciences, Laboratory of Microbiology, Joseph
Black Building, Glasgow G12 8QQ, United Kingdom; *University of Sussex, School of Engineering,
Research Centre, Industrial Informatics & Manufacturing Systems, Falmer, Brighton, East
Sussex, United Kingdom
(Paper JBO-075 received Feb. 15, 1996; revised manuscript received Aug. 5, 1996; accepted for publication Aug. 29, 1996)
ABSTRACT
Seven laser instruments, delivering radiation at a selection of wavelengths in the range of 0.355 to 118
m
m,
were investigated for their ability to kill Escherichia coli as a lawn of the bacteria on nutrient agar culture
plates. Easily the most effective was a 600-W CO2laser operating at 10.6
m
m, which produced 1.2-
cm2circular zones of sterilization at energy densities of around 8 J cm22in a 30-msec exposure. Circular
zones with an area of 0.7 cm2were achieved with 200 W from a Nd:YAG laser delivering 8-ms, 10-J pulses of
1.06
m
m radiation at 20 Hz. The exposure time, however, was 16 s and the energy density (1940 J cm22) was
more than 240 times higher than with the CO2laser. This difference is believed to be partly due to the much
higher absorption of radiation at 10.6
m
m than at 1.06
m
m, by water in the bacterial cells and the surrounding
medium (nutrient agar). Sterilization was observed after exposure to frequency-tripled Nd:YAG laser radia-
tion at 355 nm (3.5 J cm22). Lasers that were totally ineffective in killing Escherichia coli (with their wavelength
and maximum energy densities tested) were the far infrared laser (118
m
m; 7.96 J cm22), the laser diode array
(0.81
m
m; 13,750 J cm22), and the argon ion laser (0.488
m
m; 2210 J cm22). The speed at which laser steriliza-
tion can be achieved is particularly attractive to the medical and food industries. ©1996 Society of Photo-Optical
Instrumentation Engineers.
Keywords Ar ion; CO2;Escherichia coli; far infrared; frequency doubled; frequency tripled; inactivation;
laser; laser diode array; Nd:YAG; Q-switched; sterilization.
1INTRODUCTION
In 1963 Saks and Roth1demonstrated that ruby la-
sers had significant biocidal capacity against Spiro-
gyra and Amoeba. Since then, a number of laser
sources have been used to sterilize a range of bac-
teria and yeasts, with most applications occurring
in dentistry and medicine. For example, Adrian and
Gross2demonstrated that within 1.5 min, a 10 W
CO2laser could sterilize metal scalpel blades con-
taminated with spores of Bacillus subtilis and
Clostridium sporogenes. A comparison of the steril-
ization efficacy of CO2, Nd:YAG, and argon ion la-
sers was made by Powell et al.,3who concluded
that the argon ion laser provided the best steriliza-
tion efficiency for dental instruments in that it re-
quired an exposure of 120 s at 1 W. The beam area,
however, was not given and therefore the energy
density required for sterilization was not deter-
mined. Schultz et al.4found that Pseudomonas
aeruginosa was more sensitive than Escherichia coli
and Staphylococcus aureus to exposure from an
Nd:YAG laser; moreover, the addition of methylene
blue reduced the sterilization threshold energy den-
sity.
Medical applications of laser sterilization have
centered on reducing wound infections during sur-
gery. Clinical trials comparing laser sterilization
and iodine for controlling infection in amputee
cases indicated that the former proved most
effective.5Mullarky, Norris, and Goldberg6used a
CO2laser to sterilize skin seeded with bacteria, and
showed that contamination by the laser plume was
only a small risk. Ruby, Nd:YAG, and He-Ne lasers
were found to have no effect on S. aureus and P.
aeruginosa by McGuff and Bell.7However, low-
Address all correspondence to Ian A. Watson. E-mail:
i.watson@mech.gla.ac.uk 1083-3668/96/$6.00 © 1996 SPIE
JOURNAL OF BIOMEDICAL OPTICS 1(4), 466472 (OCTOBER 1996)
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power lasers have proved effective in sterilization if
they are used in conjunction with photosensitizers.
For example, Wilson et al.8evaluated 27 com-
pounds to sensitize Streptococcus sanguis,Porphy-
romonas gingivalis,Actinobacillus actinomycetemcomi-
tans, and Fusobacterium nucleatum to exposure from
a 7.3-mW He-Ne laser, and found that these bacte-
ria were killed after 30 s exposures with toludine
blue O, azure B chloride, and methylene blue.
Black-pigmented bacteria, for example P. gingivalis,
were more sensitive to light than nonpigmented
strains.
It is clear from these reports that lasers have the
capacity to achieve sterilization in remarkably short
periods of time compared with some conventional
methods, such as autoclaves, which typically re-
quire a 15-min exposure at 121 °C. From the results
reported here, sterilization by laser was achieved
with exposure times as short as tens of millisec-
onds. The present study investigated the perfor-
mance of seven different laser wavelengths to find
the rate of sterilization for the different laser de-
vices and the optimum laser sterilization or inacti-
vation source.
2MATERIALS AND METHODS
The wavelengths ranged from 118
m
mforaCW
far-infrared (FIR) laser to 355 nm with a
Q-switched, frequency-tripled Nd:YAG operating
with 5-ns pulses. A standardized assay was devel-
oped to assess each laser’s bactericidal capacity.
Plates that had been lawned with E. coli were ex-
posed to various energy densities of laser irradia-
tion, incubated for 24 h, and examined for growth.
Any cleared zones on the surface indicated that the
laser sterilization had been successful. Because of
the nonuniform spatial distribution of energy
within the laser beam, the energy density delivered
to the sample had a spatial variation. Consequently,
for a given exposure, the area of clearing was in-
dicative of how effective the laser was at steriliza-
tion. The laser’s effect was quantified by measuring
the area of the cleared zones as a function of the
energy density.
2.1 LASER SOURCES
Table 1 shows the laser characteristics and their
manufacturers, namely; wavelength and mean
power and where applicable the pulse energy,
pulse duration, peak power, and frequency. The
mean power of the lasers ranged from 0.04 to 600
W, the peak power from 150 to 108 MW, and the
minimum pulse duration was about 4 ns.
2.2 PREPARATION OF LAWNED PLATES
Escherichia coli B10537 was obtained from the cul-
ture collection at the University of Glasgow, where
it is maintained on nutrient agar (Difco) slopes at 4
°C and subcultured once monthly. The culture was
grown in nutrient broth (Difco) and incubated over-
night at 37 °C. Aliquots (1.5 ml) of the culture (ap-
proximately 108/ml) were pipetted onto nutrient
agar plates and allowed to flood the surface; the
excess culture was decanted. The plates were dried
for 30 min in a Petric Class III microbiological
safety cabinet. The approximate concentration of E.
coli on the surface of the lawned plates was 8.5
3105cm22.
2.3 EXPOSURE OF BACTERIA TO LASER
RADIATION
Plates lawned with E. coli were irradiated with vari-
ous laser beams and exposures; typically four or
five separate exposures were made on each plate,
and two exposures were made for each condition.
The laser beam and petri dish were stationary dur-
ing each exposure. A detailed statistical analysis
has been done on Nd:YAG laser sterilization for a
range of bacteria and yeasts by Ward et al.;9these
results indicate the high degree of repeatability of
this process. After exposure, the plates were incu-
bated for 24 h at 37 °C and analyzed for growth.
Unlawned plates were kept for control for up to 14
days, and because only a small part of each plate
was exposed, the nonexposed area served as an ad-
ditional control. As further control, unlawned
plates were exposed to the range of energy densi-
ties, then lawned and incubated in the usual man-
ner. This control was designed to see if the laser
exposure affected the nutrients in the agar, leading
to subsequent death of the bacteria. If the laser had
a significant effect on the E. coli, then after the incu-
bation period an area free from bacterial growth
was observed and the average area was calculated.
If the plate had no such areas after incubation, the
laser was deduced to have had no significant effect.
The zones of sterilization were measured as a func-
tion of the applied energy density. An imaging sys-
tem consisting of a Sun workstation (ARS, Edin-
burgh, UK) and an ITEX frame grabber (Bedford,
MA) was used to reduce errors in the measure-
ments of the areas.
To compare the bactericidal capacity of the seven
lasers, the energy density at which bacteria were
killed over an area greater than 15% of the beam
area, over an area less than 15% of the beam area,
and where no killing was observed was plotted. Of
the lasers where sterilization/inactivation was ob-
served, the average zones of clearing were plotted
as a function of energy density. To compare the
relative performance and time over which steriliza-
tion could be achieved for these lasers, these data
were normalized to the laser beam area and applied
energy density, and plotted as a function of time.
2RESULTS
Table 2 shows the beam diameters, mean power,
exposure times, and energy densities that were ap-
plied to E. coli for the different lasers under inves-
tigation. The maximum applied energy density was
COMPARATIVE BACTERICIDAL ACTIVITIES OF LASERS
467JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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13,754 J cm22from Opto Power Corporation’s laser
diode array. The minimum and maximum beam di-
ameters were 1.5 and 40 mm respectively; both of
these diameters were for the Ar ion laser. A number
of exposures were made on each lawned plate, ex-
cept for the 40-mm Ar ion beam, where only one
exposure per plate was made. In all other cases the
beam area was kept constant for each laser.
Sterile areas were observed after exposing plates
to radiation from the CO2, Lumonic’s Nd:YAG
MS830, and the frequency-tripled output from Con-
tinuum’s Nd:YAG Minilite 10 and Surelite II-10, in-
dicating that these lasers had significant biocidal ef-
fect at these energy densities. For Lumonic’s
Nd:YAG, the energy density had to be above a
minimum value, about 1200 J cm22,to ensure
growth inhibition. It was found that the zones of
inhibition after exposure from the CO2and Lumon-
ic’s Nd:YAG lasers were strongly dependent on the
applied energy density. The laser-exposed plates
were incubated for up to 14 days, but in no case
was delayed growth observed. Similarly, no further
growth occurred when sections of the laser-exposed
areas were removed and imprinted on fresh agar.
In every case growth was observed on the
controls—unexposed lawned plates—indicating
that the laser exposure was the cause of the ob-
served zones of clearing. Growth was observed af-
ter exposure from the following lasers: FIR, both
Q-switched Nd:YAG lasers operating at 1.06
m
m
and 532 nm, the laser diode array, and the Ar ion
laser, indicating that these lasers had no effect at
these applied energy densities.
Figure 1 compares the performance of each laser
investigated by plotting the energy density against
the observed response, i.e., no killing; bacteria
killed, but only over an area less than 15% of the
beam area; and bacteria killed over an area greater
than 15% of the beam area. It is seen that the
frequency-tripled Minilight laser only produced
sterilization areas below 15% of its beam area,
whereas the tripled Surelite sterilized areas were all
greater than 15% of its beam area. The Nd:YAG
MS830 produced areas above 15% of its beam area
Table 1 Laser characteristics.
Laser/
model Manufacturer
Wavelength
(
m
m)
Pulse
energy
(J)
Pulse
duration
(s)
Frequency
(Hz)/CW
Mean
power
(W)
Peak
power
(W)
FIR/
FIRL 100
Edinburgh
Instruments,
Edinburgh,
UK
118 NA NA CW 0.150 0.150
CO2/MFKP Laser Ecosse,
Dundee, UK
10.6 NA NA CW 600 600
Nd:YAG
MS830
Lumonics,
Rugby, UK
1.06 10 8310−3 20 200 1.33103
Nd:YAG/
Minilite
Continuum,
Santa Clara,
CA,
1.06 2.5310−2 5310−9 10 0.25 5.03106
Doubled 0.532 1310−2 4310−9 10 0.10 23106
Tripled 0.355 4310−3 4310−9 10 0.04 13106
Nd:YAG/
Surelite II-10
1.06 0.65 6310910 6.5 1.083108
Doubled 0.532 0.3 5310−9 10 3 6.03107
Tripled 0.355 0.1 5310−9 10 1 2.03107
Laser Diode
Array OPC-
AO15
Opto Power
Corporation,
CA
0.810 NA NA CW 15 15
Ar ion/
Beamlok
2060
Spectra
Physics,
Hemel
Hempstead,
UK
0.488 NA NA CW 2 2
Note: NA=not applicable; CW=continuous wave.
WATSON ET AL.
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Table 2 Bactericidal effect of exposure of
E. coli
colonies to various lasers.
Laser/
model
Beam area
(cm2)
Mean
power
(W)
Exposure
time
(s)
Energy
density
(J cm−2)
Area of
zone of
bacterial
killing
(cm2)
FIR/
FIRL 100
0.385 0.15 60a7.96a0
CO2/MFKP 2.30 600 0.005 1.31 0.160
0.010 2.63 0.660
0.020 5.26 1.04
0.030 7.88 1.21
Nd:YAG/
MS830
1.65 200 9 1090 0
10 1210 0.0380
12 1460 0.310
14 1700 0.540
16 1940 0.715
Nd:YAG/
Minilite 10
0.283 0.25 10a8.84a0c
Doubled
Nd:YAG/Minilite 10
0.283 0.1 15a5.31a0c
Tripled 0.283 0.04 5 0.710 0.0107
Nd:YAG/ 15 2.12 0.0178
Minilite 10 20 2.82 0.0277
30 4.22 0.0362
60 8.49 0.0365
Nd:YAG/
Surelite II-10
0.283 6.5 3a69a0d
Doubled
Nd:YAG/
Surelite II-10
0.283 3.0 3b31.8b0c
Tripled 0.283 1.0 1 3.54 0.121
Nd:YAG/ 2 7.07 0.112
Surelite II-10 3 10.6 0.123
Laser diode
Array OPC-
AO15
0.196 15 120a13750a0
Ar ion/
Beamlok
2060
12.56e0.65 60a2210a0
aShorter times and smaller energy densities were also nonbactericidal.
bA single-shot exposure produced a visible but unquantifiable effect.
cThe plastic of the petri dish was burnt.
dThe plastic of the petri dish was melted.
eMinimum beam diameter tested was 0.15 cm.
COMPARATIVE BACTERICIDAL ACTIVITIES OF LASERS
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for energy densities above 1460 J cm22, and the
CO2laser produced areas greater than 15% for en-
ergy densities above 2.63 J cm22.
Figure 2 shows a graph of the zones of clearing as
a function of the applied energy density for each
laser that demonstrated a biocidal capacity. To
achieve similar areas of no growth, the energy den-
sities were over two orders of magnitude greater
for Lumonic’s Nd:YAG laser than the CO2laser.
For example, with the CO2laser and an energy den-
sity of 2.63 J cm22, a clear area of 0.66 cm2was
measured; this increased to an area of 1.215 cm2for
an applied energy density of 7.88 J cm22. Areas ap-
proximately 18% lower were produced with Lu-
monic’s Nd:YAG laser after applying energy densi-
ties about 650 times larger than those used for the
CO2laser. For the frequency-tripled lasers, the ar-
eas of sterilization did not vary much as the energy
density was increased. The areas, however, were
greater for the Surelite II-10, which had the same
beam diameter as the Minilite (as quoted by the
manufacturer) but a greater pulse energy. Areas of
partially inhibited growth were observed after ex-
posure to a single pulse from the frequency-tripled
Nd:YAG laser (Surelite II-10), but these areas were
not quantifiable.
Figure 3 shows the zones of inhibition normal-
ized to the average applied energy density and the
beam area as a function of exposure time. The
greatest fractional value was observed for the Sure-
lite II-10 laser operating at 355 nm, closely followed
by the CO2laser (600 W), the minilite (355 nm), and
Lumonic’s Nd:YAG (200 W). The CO2laser pro-
vided the most rapid sterilization, achieving zones
of inhibition of 1.2 cm2in 30 msec (7.88 J cm22),
whereas a 16 s exposure from the Nd:YAG laser
produced a zone of inhibition of about 0.7 cm2
(1940 J cm22). It is interesting to note that even
though the peak powers were over three orders of
magnitude greater for the Q-switched lasers, oper-
ating at 1.06 and 0.532
m
m, than Lumonic’s
Nd:YAG, no effect on the E. coli was observed for
these devices. The Q-switched irradiances were suf-
ficiently high for the petri dish to melt or burn at
the junction between the agar and the dish and on
the rear side of the dish. No such effect was ob-
served on the dishes exposed to any of the other
lasers tested.
3DISCUSSION
Although there is now a substantial literature on
the bactericidal activity of laser radiation, there
have been few studies in which different laser
wavelengths were compared for activity in relation
to energy density with a standardized bacterial tar-
Fig. 1 Comparison of the bactericidal capacity of the seven lasers investigated. Energy densities are
shown at which there was no killing observed, killing at less than 15% of the total beam area, and killing
over 15% of the total beam area.
WATSON ET AL.
470 JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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get. In the present study, where seven wavelengths
of laser radiation were explored, by far the most
effective was the 10.6-
m
m radiation delivered by a
600-W CO2laser. This instrument, operating with a
beam area of 2.3 cm2,was able to sterilize a circular
1.21-cm2patch on the E. coli colony in 30 msec. The
energy delivered to the patch under these condi-
tions was about 18 J.
The sterilization mechanism will differ for lasers
operating in the UV and IR. Interestingly, steriliza-
tion was not observed for the Q-switched lasers op-
erating at 1.06 and 0.532
m
m, where the intensities
were above the damage threshold of the plastic pe-
tri dishes but below that required to kill the bacte-
ria. Because high peak power radiation at 1.06
m
m
had no bactericidal effect, whereas high mean
power at this wavelength did, it is apparent that the
sterilization mechanism at this wavelength is prob-
ably thermally dominated, i.e., it is not dependent
on rapid photochemical or ablative processes occur-
ring over short pulse durations, as was observed
for exposure at 355 nm, where even a high-power
single pulse of 4 ns duration affected the growth of
the bacteria. As far as the authors are aware, there
are no reports on laser sterilization at 355 nm; how-
Fig. 2 Zones of clearing as a function of the applied energy den-
sity for each laser that demonstrated a bactericidal capacity
against
Escherichia coli
grown on nutrient agar culture plates.
Fig. 3 Exposure times to generate zones of clearing normalized to the laser beam area and applied
energy density, for each laser that demonstrated a bactericidal capacity against
Escherichia coli
grown
on nutrient agar culture plates.
COMPARATIVE BACTERICIDAL ACTIVITIES OF LASERS
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ever, the observed inactivation may be due to fluo-
rescence occurring at wavelengths normally associ-
ated with UV sterilization, i.e., between 254 and 260
nm, which may be a result of a biphotonic process;
this hypothesis was not examined further.
A plausible explanation for the sterilization from
exposure to the IR laser is that the rapid transient
temperature rise of the bacteria and the underlying
agar is sufficient to kill by thermal effects. Since wa-
ter absorbs strongly at 10.6
m
m and the bacterial
cell is about 80% H2O, it can be calculated that 18 J
absorbed by the top 0.6
m
m of thickness of agar in a
1.21-cm circular patch on the culture plate would
raise its temperature from 20 to 80°, which would
kill the bacteria very rapidly. At energy densities
500 to 1000 times greater than those used with the
CO2laser (i.e., around 2000 J cm22), the Nd:YAG
laser at 1.06
m
m was fully effective in sterilizing
circular areas on the bacterial colony, and such
doses were delivered in about 16 s with the laser
operating at 200 W.
If the values of the fractional beam area that
cause sterilization per unit energy density were
known for bacterial vegetative cells and spores, en-
vironmental conditions, lasers, and laser param-
eters, then such information would allow develop-
ment of laser sterilization systems across a number
of industrial sectors. At their present stage of devel-
opment, however, it is convenient to standardize
the experiment on seeded agar surfaces because of
the simplicity of these experiments and the ease
with which the biocidal capacity of different lasers
can be compared.
It is clear that lasers offer a novel way to achieve
inactivation or sterilization, with a capacity to ster-
ilize much faster than conventional methods. The
rate of exposure from the frequency-tripled YAGs
was limited because the highest pulse repetition
frequency was only 10 Hz. In practice, the 3 s expo-
sure was only 30 pulses, each of 5 ns, totaling an
exposure of 150 ns. This represents an extremely
fast and efficient sterilization system which may
have useful implications for sterilization and hy-
giene practices in general. High-power excimer la-
sers, operating in the UV and at high pulse repeti-
tion frequency, are being developed10 and could be
used for extremely rapid laser sterilization systems
that may have applications across a number of in-
dustrial sectors. Studies are now under way to re-
fine and extend knowledge of the bactericidal capa-
bilities of the CO2and Nd:YAG high-power lasers
for a range of bacterial and ambient conditions.
4CONCLUSIONS
Of the lasers tested, significant ability to kill Escheri-
chia coli was observed with the CO2(600 W),
frequency-tripled Nd:YAGs (1 and 0.04 W) and
Nd:YAG (200 W) lasers. The CO2laser provided the
most rapid sterilization: a zone of inhibition of 1.2
cm2was achieved in 30 msec, whereas 16-s expo-
sure of Nd:YAG (200 W) irradiation produced a
zone of inhibition of 0.7 cm2.Growth inhibition was
observed after exposure to laser radiation at 355
nm. No bactericidal capacity was observed with the
FIR device, Q-switched lasers operating at 1.06
m
m and 532 nm, the Ar ion laser, or the laser diode
array.
Acknowledgments
This work was funded by the Ministry of Agricul-
ture, Fisheries and Food. Craig Henry from Spectra
Physics and John Macleod at Edinburgh Instru-
ments kindly made available a number of lasers for
this investigation.
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472 JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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... However, a direct comparison of the present results with most of the work done earlier is not possible because of the differences in the wavelength of the laser light used and the intensities and/or energy doses employed. Most of the earlier studies employed laser light in the IR region with very high power density (Ward et al., 2000;Watson et al., 1996). However, Watson et al. (1996) studied the bactericidal abilities of lasers operating at various wavelengths ranging from 355 to 1180 nm and found no bactericidal activities at 532 nm for the energy densities tested (between 1 and 100 J/cm 2 ). ...
... Most of the earlier studies employed laser light in the IR region with very high power density (Ward et al., 2000;Watson et al., 1996). However, Watson et al. (1996) studied the bactericidal abilities of lasers operating at various wavelengths ranging from 355 to 1180 nm and found no bactericidal activities at 532 nm for the energy densities tested (between 1 and 100 J/cm 2 ). ...
Article
The role of stationary phase sigma factor gene (rpoS) in the stress response of Moraxella strain when exposed to radiation was determined by comparing the stress responses of the wild-type (WT) and its rpoS knockout (KO) mutant. The rpoS was turned on by starving the WT cultures for 24 h in minimal salt medium. Under non-starved condition, both WT and KO planktonic Moraxella cells showed an increase in mortality with the increase in duration of irradiation. In the planktonic non-starved Moraxella, for the power intensity tested, UV radiation caused a substantially higher mortality rate than did by the visible laser light (the mortality rate observed for 15-min laser radiation was 53.4 +/- 10.5 and 48.7 +/- 8.9 for WT and KO, respectively, and 97.6 +/- 0 and 98.5 +/- 0 for 25 s of UV irradiation in WT and KO, respectively). However, the mortality rate decreased significantly in the starved WT when exposed to these two radiations. In comparison, rpoS protected the WT against the visible laser light more effectively than it did for the UV radiation. The WT and KO strains of Moraxella formed distinctly different types of biofilms on stainless steel coupons. The KO strain formed a denser biofilm than did the WT. Visible laser light removed biofilms from the surfaces more effectively than did the UV. This was true when comparing the mortality of bacteria in the biofilms as well. The inability of UV radiation to penetrate biofilms due to greater rates of surface absorption is considered to be the major reason for the weaker removal of biofilms in comparison to that of the visible laser light. This result suggests that high power visible laser light might be an effective tool for the removal of biofilms.
... However, the fact that there are a large numbers of different lasers, operating over a range of wavelengths makes the selection of an ideal device for specific applications a difficult process. Watson et al. (1996) investigated the effect of a range of laser wavelengths against Escherichia coli, ranging from 118 lm to 355 nm and noted varying bactericidal efficacies, with practical systems dependent on laser design and costs. In addition to the wavelength, there are a number of other laser characteristics which affect laser inactivation efficacy. ...
Article
The effect of laser (pulse repetition frequency, pulse energy and exposure time) and environmental parameters (pH, NaCl concentration and wet or dry samples) of Nd:YAG laser decontamination of stainless steel inoculated with Escherichia coli, Staphylococcus aureus and Listeria monocytogenes was investigated. Stainless steel discs were inoculated with the bacterial samples and exposed to laser energy densities to about 900 J cm(-2). These inactivation curves allowed selection of laser parameters for two-level multifactorial designed experiments, the results of which allowed comparisons to be made between effects of individual and combined parameters on the laser inactivation efficiency. Escherichia coli was inactivated most effectively as a wet film with L. monocytogenes and S. aureus showing similar response. For the multifactorial experiments all laser parameters were significant and were smallest for S. aureus as a wet film. pH and NaCl concentration had little effect on the efficacy of laser inactivation but dry or wet states and all laser parameters were significant. Such systems may prove to be applicable in industrial processes where stainless steel may be contaminated with acidic solutions or salt, e.g. in the food industry with laser inactivation seeming to be independent of these parameters. Parameters have been identified that allow optimization of the treatment process.
... Whilst most, if not all, of these studies used incoherent light, Warriner et al. (2000) used a UV KrFl excimer laser (248 nm) to demonstrate sporicidal inactivation. Laser inactivation of microorganisms has mainly focused on bacterial organism rather than spores as the target, where bactericidal action has been demonstrated across a range of wavelengths (Watson et al., 1996) and with high power IR lasers (Ward, Watson, Stewart-Tull, Wardlaw, & Chatwin, 1996; Yeo, Watson, Armstrong, Stewart-Tull, & Wardlaw, 1998). Few studies, however, have focussed on the effect of high power infra-red (IR) laser treatment on spores. ...
Article
Nd:YAG laser irradiation at 1.06 μm and germicidal ultraviolet irradiation (UV) were combined to treat B. cereus spores lawned on agar surfaces, stainless steel discs or distilled water. On the agar surface the applied laser energy density was 3000 J cm−2 (applied as 100 or 200 W) and the UV (2×8 W at 24 cm) irradiance was 190 μW cm−2 over 2 to 10 min. Laser treatment alone was insufficient to produce zones of clearing and the UV was only effective for exposures >6 min, where confluent growth was slightly reduced. However, the combined treatment produced cleared zones that increased with UV exposure. The spores proved relatively resistant to laser treatment in distilled water with ∼0.5 log reduction in viability after exposure to 2400 J cm−2 which raised the sample temperature to 90 °C. A multifactorial experiment was devised to investigate the interaction between the laser pre-treatment, the delay between treatments and the subsequent UV exposure. There was a significant decrease in spore viability for the interactions between the laser and UV treatment and the laser treatment and the delay time.From the results of this study it was deduced that the biocidal action of combined Nd:YAG laser and UV irradiation was additive and it provided an increased rate of inactivation of B. cereus spores. The ability to combine such biocidal inactivation treatments leads to possibilities to attack spores at different sites simultaneously, such as the spore's DNA, cytoplasm or spore coat.Such a multilevel approach could lead to a combination of treatments that are complementary and also provide more effective treatments against a wider range of microorganisms.
... Various electromagnetic radiations such as laser in the IR region (Vasilenko V., http://www.mbaa.com/TechQuarterly/ Abstracts/2001/tq01ab26.htm), laser irradiation at various wavelengths (Dobson and Wilson 1992;Watson et al. 1996;Yeo et al. 1998;Kawamoto et al. 2000;Ward et al. 2000;Charvalos and Karoutis 2001;Nandakumar et al. 2002a), UV irradiation (Jeffrey et al. http:// www.nbiap.vt.edu/brarg/brasym94/jeffrey.htm), c-radiations (Rossmoore and Hoffman 1971) and microwave (Yeo et al. 1999) -were reported to cause damages in bacteria. ...
Article
To study the molecular level damages in a marine bacterium, Pseudoalteromonas carrageenovora, exposed to low power pulsed laser radiation from an Nd:YAG laser. The laser damages in bacterial DNA were monitored by studying the formation of apurinic/apyrimidinic (AP) sites. Molecular probe kits were used for this purpose. Occurrence of lesions in the cell walls was monitored under a transmission electron microscope (TEM). The results showed that laser radiation significantly increased the number of AP sites in the bacterial DNA. This increase corresponded to the laser fluence (J cm(-2)) and to the duration of laser irradiation. TEM observation showed the occurrence of lesions in bacterial cell walls upon laser irradiation. It is concluded that bacteria exposed to laser irradiation suffers DNA damages and resulted in broken cell walls. These events led to bacterial mortality. These are in addition to the mechanisms reported earlier such as the photochemical reactions occurring inside the cells upon exposure to low power laser. These results help us to understand the mechanisms of bacterial mortality on exposure to low power pulsed laser irradiation and are useful in formulating a laser treatment strategy to kill bacteria.
... Pulsed UV light has been used on liquid and agar plates and achieved rates of decontamination of 7-8 log 10 CFU ml )1 over 30 s (Krishnamurthy et al. 2004). Previous work in our laboratories revealed that a wide variety of high-power lasers had the capacity for the rapid inactivation of micro-organisms on different substrates with a significant reduction in CFU Watson et al. 1996;Yeo et al. 1998). ...
Article
The performance of three scanning CO(2) laser inactivation systems was assessed and included: a gantry system, a rapidly rotating mirror and a low-power hybrid system combining an oscillating mirror and rotary motion of the sample. Escherichia coli and Staphylococcus aureus were the target organisms on stainless steel, nutrient agar or moist collagen film and the laser power was varied from 2 to 1060 W (two laser sources). In general, a threshold energy density was identified, above which no inactivation was observed because the scanning velocity was too high (10 cm s(-1) for stainless steel, 660 W). Reducing the velocity increased the inactivation process until complete inactivation was observed at 1.3 cm s(-1) (E. coli, approximately 10(6) CFU per sample) and 0.82 cm s(-1) (S. aureus, approximately 10(8) CFU per sample); consequently, S. aureus organisms showed a greater resistance to laser irradiation. For the nutrient agar and collagen samples, the averages of the width of clearing were measured as a function of the translation velocity and the rates of inactivation (I(R), cm(2) s(-1)) were found; an optimum velocity was observed that produced the maximum rate of inactivation. At a laser power of 1060 W, the maximum value of I(R) was 140 cm(2) s(-1) ( approximately 10(7) CFU cm(-2)) for S. aureus on collagen and slightly less on nutrient agar (114 cm(2) s(-1), estimated from a best-fit polynomial, r(2) = 0.98). A comparison of the low- and high-power lasers produced values of 0.09 cm(2) s(-1) W(-1) (i.e. I(R) per Watt delivered) for S. aureus on nutrient agar with the low-power laser at 13 W and on collagen 0.13 cm(2) s(-1) W(-1) for 1060 W. The rate of inactivation was found to be a function of the laser power, translation velocity and properties of the substrate media. The three laser inactivation systems successfully demonstrated the potential speed, efficiency and application of such systems. Laser scanning systems offer the potential for rapid and efficient inactivation of surfaces, eliminating the need for chemical treatment.
Conference Paper
Decontamination of micro-organisms plays an important role in society. Applications range, for example, from the medical to food industries. Implementation of industrial sterilization systems reduces the likelihood of infection and cross-contamination. In the food industry, there has been an increasing incidence of microbial food poisoning over the last decade in Europe, Asia and the USA, with the result of significant loss of life. Medical applications are broad, and include for example sterilization of prosthesis, sterile packaging and clean room systems. There are, however, many approaches to achieve decontamination, and each process seems to have its niche application. Novel approaches have included: microwave, high pressure, pulsed electric fields and pulsed incoherent light. One of the more recent methods is the use of laser light. Lasers can be used in two ways to achieve decontamination, either by indirect use, where a photosensitize is applied to the surface, or directly, where the energy density delivered by the laser is sufficiently high to achieve killing. In the present case, the direct approach was used. Seven laser instruments, delivering radiation at a selection of wavelengths in the range of 0.355-118μm, were investigated for their ability to kill Escherichia coli as a lawned suspension of the bacteria on nutrient agar culture plates. One of the most effective was a 600 W CO2 laser operating at 10.6 μm, which produced 1.2 cm² circular zones of sterilization at energy densities of around 8 Jcm⁻² in a 30 ms exposure. The Nd:YAG laser was used to sterilize liquids inoculated with concentrations of E. coli ranging from 10⁵ to 10⁸ cells ml⁻¹. The time-dependent heat diffusion equation was solved to calculate the three dimensional temperature distribution in the liquid and the death kinetics of laser inactivation were analysed.
Article
46 children with acute intestinal infections were studied. The development of pathological process was associated with the activation of lipid peroxidation, the decrease of superoxide dismutase and catalase activities in erythrocytes as well as with the fall of vitamin E content in blood plasma. Vitamin E and IR laser irradiation use in complex treatment showed the best therapeutic effect.
Article
A time-dependent, heat diffusion equation was used to predict the three-dimensional temperature distribution of Escherichia coli in liquid-suspension during irradiation by a high-power Nd:YAG laser. The model may be used to calculate the temperature rise and the transient 3-D temperature profile in the liquid suspension under arbitrary combinations of laser wavelength, pulse shape, pulse width, repetition rate, energy density, and for different concentrations of bacteria. The temperature profiles in the liquid, for a range of energy densities, were measured to validate the theoretical model. The experimental results were in good agreement with the theoretical ones. A temperature gradient was found in the sample in the radial and axial directions during laser irradiation. The model enables the parameters that affect the temperature distribution of the liquid suspension to be identified and optimized when designing laser sterilization systems.
Article
The effect of laser radiation on Staphylococcus aureus 6571 (Oxford strain) was studied with high-power Nd:YAG laser radiation between 50 and 300 W. A range of laser pulse repetition frequencies (PRF) from 5 to 30 Hz, with a combination of pulse energies from 2 to 30 J were applied; this covered a range of energy densities from 800 to . The area of inactivation of S. aureus, lawned on nutrient agar plates, was quantified as a function of energy density and exposure time. The shortest exposure time which produced an area of inactivation equal to 50% of the beam area was achieved at a PRF of 30 Hz, pulse energy of 10 J, and with an exposure time of 10.75 s; this was equivalent to an applied energy density of . No bacterial inactivation was observed at relatively low-power settings for PRF, pulse energies and exposure time of: 20 Hz, 3 J and 34 s; 25 Hz, 2 J and 45 s and 30 Hz, 2 J and 35 s, respectively. These results shows that pulse energy, PRF and exposure time are important criteria when considering inactivation of micro-organisms by laser radiation.
Article
Eric Bornstein, DMD, presents a protocol for using the laser and other technologies to minimize certain potential complications of implant surgery.
Article
The effects of laser energy on three bacterial strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were studied utilizing the neodymium: YAG laser. Cell suspensions of each strain were divided into four groups. In group I, suspensions from each strain were exposed to laser energy densities of 555–3,333 J/cm2. In groups II and III, two artificial dyes, congo red or methylene blue, were added to the suspensions prior to lasing. In group IVa, no laser energy was used, and group IVb was used to measure the bactericidal thermal effects of the laser. It was concluded that: 1) Low dosages of laser energy exceeding 1,667 J/cm2 resulted in a 2 to 8 log decline in the number of viable bacterial colonies in vitro. 2) Compared to the other two bacterial strains, P aeruginosa was the most senstive to YAG laser irradiation. 3) Addition of methylene blue, a dark-colored dye, enhanced the bactericidal effects of the YAG laser as indicated by the significantly reduced viability of P aeruginosa after irradiation with 2,222 J/cm2.
Article
The use of the carbon dioxide (CO2) laser for sterilization of metal instruments was investigated. Scalpal blades contaminated with bacterial spores were exposed to the laser and subsequently cultured. Our results demonstrate that the CO2 laser effectively sterilizes metal instruments.
Article
Twenty-seven compounds were screened for their ability to sensitize Streptococcus sanguis to killing by light from a 7.3-mW Helium/Neon (HeNe) laser. Bacteria were mixed with various concentrations of the test compounds, spread over the surfaces of agar plates, and then exposed to light from the HeNe laser for various time periods. The plates were then incubated and examined for zones of inhibition. Those compounds found to be effective photosensitizers were then tested against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum. Toluidine blue O, azure B chloride, and methylene blue at concentrations of 0.005% (wt/vol) were effective photosensitizers of all four species, enabling killing of bacteria following exposure to laser light for only 30 s.
Article
The sterilization of dental instruments is an area of great interest and recent concern in the field of dentistry. The purpose of this study was to compare the ability of three lasers (argon, CO2, and NdYAG) to sterilize dental instruments. Endodontic reamers were contaminated with microorganisms, lased at various levels of energy, placed in Trypticase soy broth, incubated, and read for growth or no growth to determine sterility. Results indicated that the argon laser is capable of sterilizing selected dental instruments at the lowest energy level (1 watt for 120 seconds) of the three lasers tested. The other two lasers were able to sterilize the instruments also, but at higher energy levels. Results indicated all three lasers capable of sterilizing selected dental instruments; however, the argon laser was able to do so consistently at the lowest energy level of 1 watt for 120 seconds.
Article
Control of infection in a surgical wound remains a challenge, especially if further surgery in the area is needed. This study was designed to compare the effectiveness of sterilization of a standard experimental infected wound by surgical skin preparation (Betadine) as compared to treatment with the CO2 laser. Standard wounds (5 x 6 cm) were created superficial to the panniculus carnosus on each flank of 37 adult male New Zealand rabbits. Each wound was infected with a standard dose of Pseudomonas aeruginosa. All wounds became grossly infected. On the third day one flank wound was treated with the CO2 laser, the other with the Betadine solution, and a punch biopsy (4 mm) was taken from each wound for quantitative bacterial counts. Less than 10% of the laser-treated wounds grew Pseudomonas, whereas nearly 40% of the iodine-treated wounds remained infected (P less than 0.005). Our early clinical experience using the CO2 laser for the sterilization of infected wounds is also reported.
Article
A quantitative study on the survival of bacteria following exposure to the CO2 laser was determined at the skin surface and in the plume. Known quantities of bacteria were inoculated onto the surface of fresh pig skin and exposed to timed bursts of the radiation. Results indicate that the bacterial population at the skin surface was reduced by several orders of magnitude while the potential for spread of bacteria by the plume of smoke was negligible.
Article
Near infrared light from a high-powered, 1064 nm, Neodymium:Yttrium Aluminium Garnet (Nd:YAG) laser killed a variety of Gram-positive and Gram-negative bacteria and two yeasts, lawned on nutrient agar plates. A beam (cross-sectional area, 1.65 cm2) of laser light was delivered in 10 J, 8 ms pulses at 10 Hz, in a series of exposure times. For each microbial species, a dose/response curve was obtained of area of inactivation vs energy density (J cm-2). The energy density that gave an inactivation area (IA) equal to 50% of the beam area was designated the IA50-value and was plotted together with its 95% confidence limits. Average IA50-values were all within a threefold range and varied from 1768 J cm-2 for Serratia marcescens to 4489 J cm-2 for vegetative cells of Bacillus stearothermophilus. There were no systematic differences in sensitivity attributable to cell shape, size, pigmentation or Gram reaction. At the lowest energy densities where inactivation was achieved for the majority of organisms (around 2000 J cm-2), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localized melting of the agar.
Article
Morphological changes at the cell level have been produced experimentally by a pulsed ruby laser (optical maser). A pulse duration of approximately 500 microseconds caused discrete damage to structures in the cell without irreversible damage to the surrounding area.