STUDY OF OXYGEN PLASMA FOR APPLICATION IN
STERILIZATION PROCESSES
A. Moreira , T. Pinto
College of Pharmaceutical Sciences, Sao Paulo University, São Paulo, Brazil
R. Mansano, N. Ordonez; L. Vilhegas
University of São Paulo Laboratory of Integrated Systems Polytechnic School, University of São Paulo, Brazil
Keywords: Sterilization, Plasma, Microorganisms, contamination.
Abstract: The main objective of this work was to propose a technique of sterilization for medical devices with less
exposition time than current plasma techniques, and also determine if this technique can be applied to
temperature sensitive materials. Therefore, it was used as biological sensor Bacillus subtilis spores var.
niger ATCC 9372 and Bacillus stearothermophilus. For Bacillus subtilis indicators were used two
substrates: glass with 2,0 x 107 CFU/substrate of microbial load initial, and paper strips with 3,8 x 10
6
CFU/
substrate of microbial load initial. The efficacy of process was evaluated with the count of survivors and it
respective value of decimal reduction (D value). In this work it was used RIE (Reactive Ion Etching). For all
processes were used Petri dishes with the sample in triplicates for both microorganisms types. The process
parameters was varied as follow: gas flow - 100, 200 and 500 sccm, pressure – 100 and 330 mTorr, RF
power – 50, 100 and 150 Watts and the time were of 2 minutes up to 60 minutes. After these processes, we
made the count of survivors, in order to evaluate the plasma efficiency as sterilizer agent.
Espectrophotometric analysis was made to evaluate the oxygen consumption during all process, and was
used a scanning electronic microscope to visualize the plasma effect over microorganisms. With these
results it was possible to adapt the process parameters for each type of substrate.
1 INTRODUCTION
Sterilization processes aim at full elimination or
destruction of microorganisms (viruses, bacteria,
fungi in sporulated or vegetative state) in a certain
material to attain an acceptable security level for
both patients and operators. Sterilizing methods can
be divided into physical, chemical or physical-
chemical. Physical processes are e.g. saturated
steam/autoclaves, dry heat/ovens, and
radiation/gamma rays.
These methods’ main problem is the high
temperature that can degrade polymeric materials
used in catheters and other devices. This limitation
affects materials used in endoscopy and a wide
variety of plastic and elastomeric materials used in a
wide range of clinical, therapeutic and surgical
techniques (Holy, 2001).
Plasma sterilization techniques provide a wide range
of advantages compared with other methods used,
because they can be effective in microbial load,
besides working at room temperature and not using
toxic gases. They expose materials with
microorganisms to reactive species generated by a
gas ionization (generally oxygen), using
electromagnetic fields (Lerouge, 2000; Oshma,
1997; Hermelin, 1998; Rutala, 1999).
Ideal characteristics for a low temperature
sterilization process include the following: high
efficiency, fast action, great penetration power,
compatibility with the largest number of materials
possible, non use or generation of toxic products,
efficiency even with organic material, easy
adaptation in each environment (hospital and
industry), possibility to be monitored and easy
operation (Hoefel, 2002).
Considering those items, the best method
approaching the ideal is the plasma process;
however an effective method using plasma as the
main sterilizing element has not been developed.
134
Moreira A., Pinto T., Mansano R., Ordonez N. and Vilhegas L. (2008).
STUDY OF OXYGEN PLASMA FOR APPLICATION IN STERILIZATION PROCESSES.
In Proceedings of the First International Conference on Biomedical Electronics and Devices, pages 134-137
DOI: 10.5220/0001047901340137
Copyright
c
SciTePress
Current equipment use the plasma step to eliminate
toxic residues generated by hydrogen peroxide or
peracetic acid sterilization methods, as this plasma
step is only performed at the end of the sterilization
cycle (Hayakawa, 1998). Hence, there does not exist
a specific method to use plasma for an effective
sterilization, neither does there exist commercial
equipment that allows using this sterilization method
(Moisan, 2001).
Furthermore, possible limits regarding aspects of
validation are known, be it on the dimensional limits
of the sterilization chamber, be it on the
configuration of the product. The reasons for these
concerns are the homogeneous distribution of the
formed plasma and the average lifetime of its
constituents, which are known to be unstable.
2 EXPERIMENTAL
Reactive Ion Etching (RIE) system consists of a
stainless steel chamber with 330 mm of diameter
and 114 mm high. Inside is a 6-inch diameter
electrode, which creates the plasma. The electrode is
cooled at 20 °C, maintaining a constant temperature
during the processes. The Radio Frequency for the
generation of the plasma is 13.56 MHz; at this
frequency, strong ion bombardment and high electric
fields are the typical plasma characteristics.
In order to compare the process results, other
sterilization tests were performed in an autoclave,
Lutz Ironing, Model 39.211, with 6 kg/h of vapour
production, 5 KW of power, and an X-ray
Diffraction equipment, model URD6, with a
molybdenum tube and a power capacity of up to
1000 W.
The indicators of B. stearothermophilus,
inoculated on paper, with an initial load of 1 million
UFC/ml, were submitted to plasma exposures at the
following process conditions: 500 sccm of oxygen
flow, 330 mTorr pressure, 100 W power and process
times of 5 up to 60 minutes. For the indicators of B.
subtilis, inoculated on glass plates, the gas flow was
200 sccm, for a pressure of 100 mTorr, and process
times of 2 up to 120 minutes. Power levels of 100
W and 150 W were applied for each flow and
pressure parameter. For all the processes, oxygen
was injected into the chamber for 10 minutes before
igniting the plasma, to guarantee that the chamber
was full filled with this gas.
The sterilization process in the autoclave was
performed for 15 minutes at a temperature of 121
°C.
The sterilization processes using X rays were
done during 30 minutes, with power levels of 200,
400, 600, 800 and 1000 W. Finally, the UV
processes were performed during 1, 5, 10, 30, 45 and
60 minutes, with an effective lamp power density of
14 mW/cm
2
; hence the effective dose, being the
effective power multiplied by the exposure time,
was varied between 0,84 J/cm² and 50.4 J/cm²
The indicators of B. stearothermophilus used in
the plasma sterilization processes, were produced on
cellulosic strips of 50 mm by 5 mm with loads of 1.0
x 10
6
UFC/strips, and fabricated by the Steris
Corporation. The celulosic strips, which support the
B. subtilis, have dimensions of 40 mm by 3 mm and
an initial load of 3.8 x 10
6
UFC/ml, fabricated by
3M. The indicators deposited on the glass plates,
with dimensions of 18 mm by 18 mm, have an initial
load of 2.0 x 10
7
UFC/glass and were fabricated by
the college of Pharmaceutical Sciences of the
University of São Paulo
.
The procedure of counting the surviving spores
was made like follows: first the spores had to be
removed through mechanical agitation in test tubes
with sterile water; then they were stirred in a bath of
water at 70 °C for B. Suitilis and 82 °C for B.
Stearothermophilus during 15 minutes. These
samples were then diluted and 1 ml of these
dilutions were placed on Petri dishes together with a
culture of agar casein soy and placed in an oven.
Incubation for the B. Stearothermophilus indicators
was done at 45 °C during 72 hours and for the B.
Subtilis indicators at 37 °C during 24 hours.
3 RESULTS AND DISCUSSIONS
Figure 1 shows the results of the RIE sterilization
processes, using B. stearothermophilus indicators,
inoculated on paper. It’s possible to observe that
after only 7 minutes, there already occurred
significant reduction of microbial load. The process
was performed in the following way: Petri glass
dishes with strips containing B. stearothermophilus
were placed, without a cover, inside the reactor. The
pressure was lowered down to 10
-3
Torr and the
process gas was injected. The process duration
varied from 2 to 60 minutes, with power level of 100
W, oxygen pressure of 330mTorr and oxygen flow
of 500 sccm.
Figure 2 shows the relation of B.
stearothermophilus samples before and after
suffering an RIE type plasma sterilization process
and a comparison with the autoclave sterilization
STUDY OF OXYGEN PLASMA FOR APPLICATION IN STERILIZATION PROCESSES
135
process. It can be observed that the plasma process
is much more aggressive than the autoclave process.
Each sample was analysed by scanning electron
microscopy. After two minutes of plasma
processing, only a few microorganisms could still be
found on the paper, and many of those had their
morphology modified. After 5 minutes of
processing, the concentration of modified spores was
higher. After 7 minutes, a lot of spores had been
destroyed and the few that remained had a
completely changed morphology. After 10 minutes
of sterilization, it was very difficult to find any
spore; many spores were split into several
fragments. These results show that the process is
efficient to sterilize these biological indicators.
However, it is necessary to be careful with the time
of exposure to the plasma, because for times higher
than 15 minutes, the cellulose material starts to
suffer degradation and it disintegrates completely
after 30 minutes of plasma (Moreira, 2003).
Figure 1: B. stearothermophilus exposed to RIE plasma at
330 mTorr pressure, 100 W power and 500 sccm O
2
flow.
(a) (b) (c)
Figure 2: (a)- Spore before exposure to a plasma process;
(b)- Spore after a 10-minute exposure to the plasma
process; (c)- Spore after exposure to autoclave
sterilization.
Figure 3 shows the number of survivors of B.
subtilis microorganisms (inoculated on glass plates)
when exposed to the oxygen plasma process. It can
be observed that, after 20 minutes of processing,
there is a reasonable decrease in the number of
survivors at a power of 100 W, with this number
being reduced to zero after around 60 minutes.
When 150 W is applied, the microbial load is rapidly
reduced, being zeroed out after 20 minutes of
processing.
Figure 3.: Graphic of the number of survivors of the
indicators B. subtilis, inoculated on glass plates and
exposed to the plasma process for 2 to 120 minutes, under
O
2
flow of 200 sccm and pressure of 100 mTorr.
Figure 4 shows a comparison of B. subtilis indicators
before and after exposure to the plasma, X-ray and
UV processes. When exposed to the X-ray and UV
processes, the form of the microbes does not change
when compared to it before the application of the
processes. These micrographs corroborate the results
of the survivor counts in which it was possible to
verify the non-reduction of the microbial load. When
it is used the oxygen plasma processes, deformation
of the microorganisms is observed as well as spaces
between them, something that does not happen in the
other processes.
(a) (b)
(c) (d) (e)
Figure 4.: (a) Sample before the processes; (b) Sample
after the UV process; (c) Sample after the X-ray process;
(d) Samples after the plasma process with 100 W of
power; (e) Sample after the plasma process with 150 W.
4 CONCLUSIONS
The system proves to be efficient for sterilization;
the process is fast and does not attack the materials
being sterilized. As the sterilization time is quite
short, the oxygen plasma can be applied to test its
0 102030405060
0
1
2
3
4
5
6
log of the number of survivors
Time (min)
0 20406080100120
-1
0
1
2
3
4
5
6
7
8
100 W
150 W
llog of the number survivors
Time min)
BIODEVICES 2008 - International Conference on Biomedical Electronics and Devices
136
potential to sterilize cellulose materials inoculated
with B. subtilis and B. stearothermophillus. The tests
with materials on small glass plates suggest the
utilization of this technique for the sterilization of
several medical products, because of the fact that
with shorter exposure times, the microbial load is
reduced to zero and the sterilized material is not
attacked during the process. Besides, this process is
safe for the operator and the environment. Once the
process is fast and conducted at room temperature, it
can also be used for sterilization of polymeric
materials since tests are performed before to adjust
the parameters of the process to the characteristics of
the material to be sterilized.
The microbial load is reduced to zero in few
minutes when 150 W of power, 100 mTorr of
pressure and 200 sccm of oxygen flow is applied,
requiring at the most a 20 minute process for B.
subtilis. This duration must be increased to 60
minutes when 100 W of power is applied.
For the samples of B. stearothermophillus, the
processing time to zero out the microbial load is
around 15 minutes, using in this case 100 Watts of
power, 330 mTorr of pressure and 500 sccm of flow.
For the X-ray sterilization tests, 200 to 1000 W
were utilized in 30 minute processes, despite the
high power levels applied (when compared with the
plasma processes), the logarithmic reduction of the
viability of the biological indicators remains very
low to indicate a sterilization effect, what
discourages the application of this technique for
sterilization purposes. The UV sterilization tests at
14 W/cm² and 2 to 60 minute periods did not present
satisfactory results, because the microbial load was
only slightly reduced, showing that the UV radiation
does not contribute to the sterilization effect.
ACKNOWLEDGEMENTS
The authors thank CNPQ for financial support, and
Alexandre Marques Camponucci, José Antônio
Rodrigues Porto, Rubens Pereira de Alcântara,
Elisio José de Lima and Msc. Peter L. Polak for
technical support.
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