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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), 466–472 (OCTOBER 1996)
466 JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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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
469JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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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
471JOURNAL OF BIOMEDICAL OPTICS dOCTOBER 1996 dVOL.1NO.4
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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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