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

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

 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

; 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

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

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

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

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

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)

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

log of the number of survivors

Time (min)

0 20406080100120

-1

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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