COMPERSION OF STERILIZED OPERATION THEATER

EQUIPMENTS THROUG LOW PRESSURE PLASMA AND

AUTOCLAVE

BY
Ziaud Din
M.phi Scholar

DEPARTMENT OF PHYSICS
UNIVERSITY OF PESHAWAR
Session 2018-2020
Literature Revive
INTRODUCTION

Sterilization is an act or process, physical or chemical that destroys or eliminates all forms of
life, especially micro organisms. Conventional sterilization techniques, such as those using
autoclaves, ovens, and chemicals like ethylene oxide (EtO), rely on irreversible metabolic
inactivation or on breakdown of vital Structural components of the microorganism. Plasma
sterilization operates differently because of its Specific active agents, which are ultraviolet (UV)
photons and radicals (atoms or assembly of atoms with unpaired electrons, therefore chemically
reactive, e.g., O and OH, respectively). An advantage of the plasma method is the possibility,
under appropriate conditions, of achieving such a process at relatively low temperatures (≤50
C), preserving the integrity of polymer based instruments, which cannot be subjected to
autoclaves and ovens Furthermore, plasma sterilization is safe, both for the operator and the
patient, in contrast to EtO. The UV photons are expected to be more strongly (if not totally)
reabsorbed by the ambient gas at atmospheric pressure than at reduced pressure (≤10 tor). This
led us to consider two types of sterilizers: Atmospheric and reduced pressure systems The
respective roles of UV photons and radicals at reduced pressure are already well understood
which is not the case yet at atmospheric pressure [5,6]. Our presentation focuses on reduced-
pressure operation and is based on the analysis of the specific characteristics of the
microorganism survival curves resulting from plasma sterilization[1].
NON-THERMAL [1] gas discharge plasma sterilization is being investigated as a possible
alternative to conventional sterilization methods for heat sensitive medical devices [1]–[3]. Non-
thermal plasmas possess many attractive features for both aseptic and terminal sterilization
applications such as enhanced efficacy without the need for elevated temperatures, low
penetration depths that achieve surface inactivation without changing the bulk properties of
materials, cost efficiency (both capital and operational), the absence of residues or toxic
emissions (including x-ray), and the utility of installation as either standalone or inline
application. Plasma technology has been evaluated extensively in a number of biomedical fields
besides microbial inactivation, including biomaterial surface modification (living tissue
treatment and cell behavior manipulation [4]), material modification (surface activation and
functionalization [5] and deposition of films and coatings [6]). Some of the main features lending
to the utility of modern plasma technologies are their high energy density, chemical selectivity,
and high energy efficiency.[K] Sterilization and disinfection methods should be implemented
meticulously and their effectiveness carries a crucial importance for the physician and the
patient’s health. Assurance of sterility of instruments and devices can be obtained through the
use of one of several tests and these tests must be performed regularly to ensure that the sterilizer
is sterilizing all instruments and devices and that these are safe for use on patients.

Why plasma sterilization offers unique benefits?

A plasma sterilization platform offers a number of unique benefits distinguishing this technology
from alternatives including the following: (1) potential application for heat labile/sensitive
materials;(2) potential for aseptic and terminal sterilization applications;( 3) possible installation
of equipment as stand alone or for inline applications; (4) reduced space and shielding
requirements in comparison with low energy electron beam applications; (5) faster treatment and
product turnout time in comparison with low temperature gas sterilization applications(e.g., EO);
(6) safe operations due to the absence of residuals and toxic emissions; (7) safety to both the
operator and the product due to the absence of radiation; (8) reduced energy consumption due to
the low power of non-thermal plasmas;(9) simultaneous layer processing possibilities to improve
gas barrier properties, hydrophilic property improvement, reduced friction, etc., while processing
for microbial inactivation;(10) deep penetration capabilities due to enhanced diffusion properties
of plasma for mated surfaces, cracks, crevices, etc.;(11) flexibility in physical configurations as
plasma-based or plasma-assisted (accompanying cidal gas compositions) systems to obtain
varying degrees of benefits; and (12) cost effectiveness, both capital and operational [2].

[T] Table 5. Typical classification of plasma [10,17].

Classification Temperature [K] Electron Density Discharge Type Examples

[ 𝑚 −3 ]
High-temperature Laser fusion Fusion plasma for
𝑇𝑒 ≈ 𝑇𝑖𝑜𝑛 ≈ 𝑇𝑔𝑎𝑠 20
Plasma 𝑛𝑒 ≥ 10 Tokamak energy
(Equilibrium =
plasma) 106 –108
Thermal plasma Arc plasma, Radiation, welding
(Quasi-equilibrium 𝑇𝑒 ≈ 𝑇𝑖𝑜𝑛 ≈ 𝑇𝑛 𝑛𝑒 ≥ 1020 Plasma torch, and cutting,Waste
plasma) ≈ 𝑇𝑔𝑎𝑠 ≥ 2 × 104 Radio frequency treatment, Material
(RF) processing, etc.
Plasma,
Microwave
plasma etc.
Non-thermal Glow discharge, Ozonizer, Plasma
plasma 𝑇𝑒 ≥ 𝑇𝑖𝑜𝑛 ≥ 𝑇𝑛 ≈ Corona discharge, medicine, Volatile
(Non-equilibrium 𝑇𝑔𝑎𝑠 =300 – 1000 𝑛𝑒 ≈ 1010 atmospheric organic compound
plasma) pressure plasma jet (VOC) treatment,
(APPJ), Plasma agriculture,
dielectric barrier Surface
discharge (DBD), modifications
micro-hollow (coating, etching,
cathode discharge activation,
(MHCD), Plasma cleaning, nitration,
needle, Low- etc.),
pressure plasma Illumination
etc. (plasma
screen, fluorescent
lamps, etc.)
𝑻𝒆 = electron temperature, 𝑻𝒊𝒐𝒏 = ion temperature, 𝑻𝒈𝒂𝒔 = gas temperature, 𝒏𝒆 = electron density.

[4] Comparison of Oxygen, Nitrogen & Argon Gas plasma

 For sterilization purposes
1. Oxygen Gas Plasma:
 The most popular gas investigated for plasma sterilization is oxygen (𝑂2) this gas
produces O, OH, OOH, and a mixture of other radicals that contribute to the sterilization
effects. Among these OH, radical is most efficient for microbial inactivation b/c of
extremely short life time. grater the amount of 𝑂2 plasma exposure the more shrinkage of
the spore is observed so there is significant relationship b/w the degree of shrinkage and
spore death. The dissociation energy of oxygen is (5.21eV)
 The penetration depth of oxygen reactive species (ions, and radicals) are predicted to be
<1000 nm this suggests that the penetration depth of oxygen is ten time s deeper then
nitrogen reactive species (ions, and radicals)
 Oxygen containing plasmas increase the surface energy of materials by introducing
oxygen-containing polar groups onto the material surface

2. Nitrogen Gas Plasma:

 To be clarified. 𝑁2 Gas plasma had no or insufficient ability to shrink bacterial
endospores. There is no specific theory as to why this is the case. But it may be due to the
difficulty of ionizing of 𝑁2 b/c Nitrogen gas plasma does not present any shrinkage. So
the difference b/w 𝑂2 & 𝑁2 gas plasma yet chemically 𝑁2 is a molicule with a triple bond
so the dissociation energy is higher upto (9.91eV)
 Penetration depth of nitrogen reactive species (ions, and radicals) is predicted to be
approximately 10-40 nm from the surface of the spores, as observed by atomic force
microscopy.

3. Argon Gas Plasma: [3]

 This study confirmed that the sterilization effects of microwave-induced argon plasma at
atmospheric pressure on the microorganisms caused by the generation of free radicals,
and UV light, as well as the etching process. All the bacteria and fungi tested showed
time-dependent effects in the sterilization test using microwave-induced argon plasma at
atmospheric pressure, with the fungi requiring much less exposure time than the bacteria.
 Furthermore, the microwave plasma system required much less exposure time than what
has been reported from most published systems, because of the high plasma density, the
large number of free radicals and the strong intensity of generated UV light.
 These results suggest that this sterilization method is easy to use, requires significantly
less exposure time than other methods, such as traditional methods and different
 established plasma sterilization methods. In addition, it is nontoxic. Certainly, more tests
and in-depth studies of this process are needed. Nevertheless, this study represents the
first step in showing that microwave-induced argon plasma at atmospheric pressure is a
powerful sterilization tool for materials contaminated with microorganisms[3].

[7] Table 1. List of various reactive oxygen, nitrogen species [10, 22, 23].

Radical Non-radical Radical Non-radical

Reactive oxygen species (ROS) Reactive nitrogen species (RNS)

Superoxide, O−2 H2O2 Nitric oxide, NO Nitrous acid, HNO2

Hydroxyl, OH Ozone, O3 Nitrogen dioxide, Nitrosyl cation, NO+
Hydroperoxyl, HO2 Singlet oxygen (O2 1 Nitrate radical, NO3 Nitroxyl anion, NO−
Carbonate, CO−3 Hypobromous acid, HOBr Dinitrogen trioxide, N2O3
Peroxyl, RO2 Hypochlorous acid, HOC Dinitrogen tetroxide, N2O4
Alkoxyl, RO Dinitrogen pentoxide, N2O5
Carbon dioxide radical CO−2 Hypoiodous acid, HOI Alkyl peroxynitrites, ROONO
Singlet (1O2) Organic peroxides, ROOH Alkyl peroxynitrates, RO2ONO
Peroxynitrite, ONOO− Nitryl chloride, NO2Cl
Peroxynitrate, O2NOO− Peroxyacetyl nitrate, CH3C(O)OONO2
Carbon monoxide, CO
Peroxomonocarbonate, HOOCO−2
Carbon monoxide, CO

Reactive chlorine/bromine species Reactive sulfur species

Atomic chlorine, Cl Chloramines
Atomic Bromine, Br Chlorine gas, Cl 2
Bromine gas, Br2
Bromine chloride, BrCl
Chlorine dioxide, ClO2
Thiyl radical S. Hydrogen sulfide, H2S
Disulfide, RSSR
Disulfide-S-monoxide, RS(O)SR
Disulfide-S-dixide, RS(O)2SR
Sulfenic acid, RSOH
Thiol/sulfide, RSR_

[8] Beneficial Effects of ROS and RNS.

 The deleterious role of ROS and RNS in promoting demyelinating pathology, but it should be
appreciated that these species also serve many beneficial, physiological functions (e.g. NO
functions as a messenger by binding to guanylate cyclase) which are beyond the scope of this
review ( NO, in particular, may also serve beneficial functions under pathological circumstances.
It can, for example, react with organic radicals and it can stop chain radical reactions. Perhaps as
a consequence of such reactions, several experiments have found that blocking NO production in
EAE is sometimes detrimental (see “Antioxidant Therapy” below). Indeed, NO is known to
exhibit some anti-inflammatory properties, such as decreasing the proliferation of lymphocytes
in vitro (65), inhibiting the adherence of leukocytes to the vasculature and infiltration into the
tissue (131), and decreasing IFN𝛾 production by Th1 T lymphocytes (228). Other protective
effects of NO include protection from superoxide- or hydrogen peroxide-mediated damage to
cells in vitro, perhaps achieved by blocking the formation of hydroxyl radicals (247).

[6] Table 2. Methods for disinfection and sterilization of patient-care

items and environmental surfaces.

Process, method Level of microbial inactivation Example(s) (processing time) Health care application (example)

Sterilization

High temperature Destroys all Steam (∼40 min) and dry heat Heat-tolerant critical
microorganisms, (1–6h,depending on (surgical instruments) and
Including bacterial temperature) semicritical patient-care
spores items

Low temperature Destroys all ETO gas (∼15 h) and Heat-sensitive critical and
microorganisms, hydrogen peroxide gas plasma semicritical patient-care
including bacterial (∼50min) items
spores
Liquid immersion Destroys all Chemical sterilants:a ≥2.4% Heat-sensitive critical and
microorganism glut (∼10h), 1.12% glut and semicritical patient-care
including bacterial 1.93% phenol(12 h), 7.35% items that can be
spores HP and 0.23% PA (3h), 7.5% immersed
HP (6 h), 1.0% HP and0.08%
PA (8h) and ≥ 0.2% PA(∼50
min at 50 ℃ –56 ℃ )

High-level Destroys all Pasteurization (∼50 min) Heat-sensitive semicritical

disinfection Heat microorganisms patientcare items
automated except high (respiratory-therapy
numbers of equipment)
bacterial spores

Liquid immersion Destroys all Chemical sterilants or high- Heat-sensitive semicritical

microorganisms level disinfectants:a >2% glut patientcare items (GI
except high (20–45 min),0.55% OPA (12 endoscopes and
numbers of min), 1.12% glutand 1.93% bronchoscopes)
bacterial spores phenol (20 min), 7.35%HP
and 0.23% PA (15 min),
7.5%HP (30 min), 1.0% HP
and 0.08%PA (25 min), and
650–675 pm chlorine (10
min)

Intermediate-level Destroys EPA-registered hospital Noncritical patient-care

disinfection, vegetative disinfectants with label items (blood pressure
liquid contact bacteria,my claiming tuberculocidal cuff) or surfaces (bed side
cobacteria, most activity, such as chlorine- table), with visible blood
viruses, and most based products and phenolics
fungi but not (at least 60s)
bacterial spores

Low-level Destroys EPA-registered hospital Noncritical patient-care

disinfection, vegetative bacteria disinfectants with no items (bloodpressure
liquid contact and some fungi tuberculocidal claim, such cuff) or surfaces (bedside
and viruses but not as chlorine-based products, table), with no visible
mycobacteria or phenolics,and quaternary blood
spores ammonium compounds (at
least 60 s), or 70%–90%
alcohol
 [5] Table 3. Summary of advantages and disadvantages of
commonly used sterilization technologies
Sterilization method Advantages
Steam Nontoxic to patient, staff, and
environment Cycle is easy to
control and monitor Rapidly
microbicidal Least affected by
organic/inorganic soils, among
sterilization processes listed
Rapid cycle time Penetrates
medical packing and device
lumens
Hydrogen peroxide gas plasma Safe for the environment
Leaves no toxic residuals Cycle
time is 45–73 min, and no
Disadvantages
Deleterious for heat-sensitive
instruments Microsurgical
instruments damaged by repeated
exposure May leave instruments
wet, causing them to rust
Potential for burns
Cellulose (paper), linens, and
liquids cannot be processed
Sterilization chamber is small
 aeration is necessary Used for
heat- and moisture-sensitive
items, because process
temperature is !50 ℃ Equipment
is simple to operate, install (208
V outlet), and monitor
Compatible with most medical
devices Equipment requires
electrical outlet only
100% ETO Penetrates packaging materials
and device lumens Single-dose
cartridge and negative-pressure
chamber minimizes the potential
for gas leak and ETO exposure
Equipment is simple to operate
and monitor Compatible with
most medical materials
ETO mixture Penetrates medical packaging
and many plastics Compatible
with most medical materials
Cycle is easy to control and
monitor
Peracetic acid Rapid cycle time (30–45 min)
Low-temperature (50 ℃–55 ℃
) liquid-immersion sterilization
Environmentally friendly by-
products Sterilant flows through
endoscope, which facilitates salt,
protein, and microbe removal
(∼3.5–7.3 ft3) Endoscope or
medical-device restrictions based
on lumen internal diameter and
length (see manufacturer’
recommendations)
Requires synthetic packaging
(polypropylene wraps or
polyolefin pouches) or special
container tray Hydrogen peroxide
may be toxic at levels 11 pm
TWA
Requires aeration time to remove
ETO residue Sterilization
chamber is small (∼4–8.8 ft3)
ETO is toxic, a carcinogen, and
flammable ETO emission
regulated by states, but catalytic
cell removes 99.9% of ETO and
converts it to CO2 and H2O ETO
cartridges should be stored in
flammable liquid– storage
cabinet Lengthy cycle and
aeration time
Requires aeration time to remove
ETO residue Sterilization
chamber is small (∼4–8.8 ft3)
ETO is toxic, a carcinogen, and
flammable ETO emission
regulated by states, but catalytic
cell removes 99.9% of ETO and
converts it to CO2 and H2O ETO
cartridges should be stored in
flammable liquid–storage cabinet
Lengthy cycle and aeration time
Some states (e.g., CA, NY, and
MI) require ETO-emission
reduction of 90%–99.9% CFC
(inert gas that eliminates
explosion hazard) banned
in 1995 Potential hazards to staff
and patients Lengthy cycle and
aeration time ETO is toxic, a
carcinogen, and flammable
 TABLE (A)
[B] VARIOUS STERILIZATION METHODS AND THEIR
CHARACTERISTICS

Methods Heat Pressure Vacuum Exposure Sterilization Damage to Environmentally

Time Effectiveness the Medium Friendly

Incineration Yes No No > 20min Good Yes No

Autoclaving Yes Yes No > 20min Good Yes No

UV No No No < 20min Good …….. Yes

irradiation

Electron No No Yes …….. Good …….. Yes

beam
irradiation

Glow No No No < 10min Good No Yes

Discharge at
one
Atmosphere

Filtration No No No No …….. No Yes

[P] Inactivation of Microorganisms by Plasma

Some microorganisms such as bacteria, viruses, and fungi act as pathogens and cause diseases.
There is a resistance hierarchy of microorganisms against disinfection/sterilization that can be
divided into the following five categories: most resistant, highly resistant, intermediately
resistant, less resistant, and very susceptible [43]. The most resistant infectious agents are prions
(proteinaceous infectious particles), which are the causative agents of prion diseases, such as the
Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle,
and chronic wasting disease (CWD) in cervids (deer family). Bacterial spores, protozoan
oocysts, and helminth eggs are categorized as highly resistant microorganisms. Intermediately
resistant microorganisms include mycobacteria, protozoan cysts, small non-enveloped viruses,
and fungal spores. Vegetative bacteria, protozoa, helminthes, fungi, and algae, as well as large
non-enveloped viruses are less resistant. Enveloped viruses such as human immunodeficiency
virus (HIV) are generally highly susceptible to various disinfectants.
However, in reality, the situation is more complex. For example, although the enveloped virus
HIV is very sensitive to disinfection and is rapidly inactivated even at room temperature without
any treatment, the influenza virus (another enveloped virus) can remain infectious for up to 48 h.
Orthopoxviruses (e.g., smallpox, vaccinia) and filoviruses (e.g., Ebola virus, Marburg virus) are
all enveloped viruses that remain infectious for up to several weeks. Furthermore, susceptibility
against biocides depends on the environment where the microorganisms are present (e.g., blood,
serum, spinal fluid, saliva). Certain soils can prevent drying and stabilize the viral structure,
extending the survival time of viruses. For example, viruses normally interact with external
materials including proteins, lipids, salts, and cell debris. In other cases, viruses cause the
aggregation of host cells [44] or make aggregates themselves [45], which could reduce the
virucidal effect of disinfectants. Therefore, the susceptibility of microorganisms to
disinfection/sterilization should be examined under a range of different conditions.
The most impressive results in terms of microbicidal activity have come from studies using
bacteria, including bacterial spores [4–49]. Although some bacteria can form biofilms in certain
environments, this does not prevent their successful inactivation by plasma treatment [50,51].
Furthermore, the recent emergence of drug-resistant pathogens is now a serious public health
concern that has been acknowledged by both the World Health Organization (WHO) [52] and the
US Centers for Disease Control (CDC) [53]. The extensive and indiscriminate use of antibiotics
may have altered the environmental micro biome, contributing to the emergence of drug-resistant
bacteria [54]. Consequently, there is an urgent requirement to devise novel methods to eliminate
these multidrug-resistant bacteria from the food production process. Plasma treatment is an
especially promising method because the mechanism of bactericidal action is unlikely to differ
between multidrug-resistant and normal bacteria. The main mechanisms of bactericidal action in
plasma are thought to involve exposure to reactive chemical species for which multidrug-
resistant bacteria are unlikely to be resistant [55].Furthermore, plasma pre-treatment enhances
the sensitivity of methicillin-resistant Staphylococcus aureus to antibiotics [56].Several reports
suggest that plasma can be effective in inactivating fungi [57–59]. However,our own
investigations have shown that the viable cell number of Aspergillus brasiliensis was not
significantly impacted after 5 min plasma treatment using a nitrogen gas plasma device, BLP-
TES(bi-polar and low-pressure plasma-triple effects sterilization) [57], whereas Salmonella
enterica serovar Abony was completely inactivated after employing the same treatment regime
[55] (Figure 2). Indeed, a 15 min treatment with nitrogen gas plasma was required to reduce the
viability of A. brasiliensis.Thus, by comparison to bacteria, extended treatment time with
nitrogen gas plasma must be used to inactivate fungi.

NON THERMAL PLASMA

Non-thermal plasma is easy to obtain under low-pressure conditions because the collisions
between electrons, ions, and neutral molecules occur infrequently. Low-pressure plasma can be
generated by a low breakdown voltage in a vacuum chamber evacuated with a vacuum pump.
Low-pressure plasma systems are important for the manufacture of semiconductor components.
Furthermore, research into low-pressure plasma systems has also focused on the decontamination
and sterilization of medical devices [23,24]. Although low pressure plasma can generate high
concentrations of active species with a uniform glow plasma, it involves high maintenance costs
because of the requirement for a vacuum system. Atmospheric pressure plasma requires a high
voltage and relatively high temperature due to frequent collisions between electrons and ions
accompanying the high particle density [9]. However, it is possible to generate plasma under
non-thermal conditions by using a pulse discharge and APPJ [25], DBD [26], and floating
electrode barrier discharge (FE-DBD) [27,28], or MHCD [21,29]. These non-thermal conditions
allow applications involving exposure of the plasma with tissues such as skin [30]. Similarly,
non-thermal plasma can be used to disinfect agricultural products and medical devices with
relatively little impact on their structural integrity [31,32]. Alternatively, the plasma could be
transferred to a target site where the object for treatment is located using a plasma afterglow
(Figure 1). Indirect treatment using solutions treated with plasma, known as “plasma-activated
water (PAW)” [33], “plasma-activated medium (PAM)” [34], “plasma-stimulated medium
(PSM)” [35], “plasma-treated water (PTW)” [36,37], “plasma-treated phosphate-buffered saline
(pPBS)” [38], or “non-thermal plasma-conditioned media (NTP media)” [39], is also possible.
The constituents in these solutions react with samples and act as disinfectants [40] or anti-cancer
agents [41]. In cases where samples are in contact with plasma bulk in the discharging area,
plasma components such as UV radiation and reactive chemical species directly interact with the
samples. Thus, short-life reactive chemical species, such as reactive oxygen species (ROS) and
reactive nitrogen species (RNS), efficiently interact with the sample components [42]. By
contrast, in cases where the sample is in contact with plasma bulk away from the discharging
area, the contribution of UV radiation is significantly lower. In addition, there is a greatly
reduced concentration of reactive chemical species in the post discharging area due to their short
half-life at ambient temperature. In the case of plasma-treated solutions, freezing can extend the
storage time and minimize the loss of reactive chemical species [37].

[T] Table 2. Various types of electrical discharge methods

for non-thermal plasma generation.

Discharge Type * Representative Pressure Gas Temperature Application

Conditions (V, A,
Freq, Gas)
Direct current (DC) 5–30 kV direct Room temperature Biomedical
corona discharge current (DC) 1 atm Applications
(positive and
negative);10-250μA;
dry or wet; O2, N2,
Ar, He at 10 L/min
Atmospheric P = 2.5W; 2.45 GHz; 1 atm Max. 50.8 ℃ on a Biomedical
pressure plasma jet He/O2/N2 at dentin surface; Applications
(APPJ) microwave 2.0/1.2/1.5 L/min 20 ℃ on an agar
Surface
Dielectric barrier ±2.5 kV; 5 kHz; air, 1 atm Approximately Biomedical
discharge (DBD) humidity 64.4% 50 ℃ Applications
(Flexible sheet-type)
Micro-hollow 1.5–2.5 kV DC; 20 1 > atm Room temperature Medical
cathode discharge mA; air (0.1–8 (220 mL/min); Applications
(MHCD) jet L/min) >55 ℃ (5 mm from
nozzle,220 mL/min)
Pin-to-hole spark 4 kV DC; average 1 atm 9030 ± 320 K (by Medical
discharge (PHD) ~1.8 J/pulse Boltzmann applications
plasma calculation) (wound healing)

* These types of electrical discharge are for atmospheric pressure plasmas.

 [A] RESISTANCE OF VARIOUS MICROORGANISMS TO PLASMA

A number of studies have compared the resistance of different microorganisms with plasma,
although it is often difficult to compare results of studies using different plasmas and different
types of apparatus. However, when different microbes have been used under the same
conditions, invariably, spores of Bacillus species and its close relatives, Bacillus atrophaeus,
Bacillus cereus, Bacillus subtilis, or Geobacillus stearothermophilus are significantly more
resistant to plasma than are the vegetative forms of these species or other bacteria [13]–[18].
There have, however, been no thorough studies of the effect of growth stage on the killing of
vegetative bacteria by plasma, and given that increases in bacterial resistance are not uncommon
in stationary phase, this might also be the case with plasma resistance. The plasma resistance of
the generally hyper-resistant bacterium Deinococcus radiodurans has also not been compared
with that of spores, although Deinococcus radiodurans is effectively killed by plasma [19]. While
spores of Bacillus atrophaeus, Bacillus cereus, Bacillus subtilis, and Geobacillus
stearothermophilus are much more plasma resistant than the corresponding growing bacteria, few
studies have compared the resistance of spores of different Bacillus species. However, one study
found that Bacillus cereus spores were more plasma sensitive than spores of Geobacillus
stearothermophilus [16], and another found that Bacillus subtilis spores were slightly more
plasma sensitive than Geobacillus stearothermophilus spores [20]. While these observations
suggest that Geobacillus stearothermophilus spores would be the most appropriate BI for plasma
sterilization, this may be an oversimplified view, since the precise plasma composition could
influence the relative resistance of spores of different species. Indeed, other studies found that
Geobacillus stearothermophilus spores were slightly more sensitive to plasma than were Bacillus
subtilis spores [21], and Bacillus atrophaeus spores were more plasma resistant than were spores
of the radiation indicator organism Bacillus pumilus [13]. However, it seems unlikely that
variations in plasma composition would alter the generally much greater plasma sensitivity of
growing cells compared with spores.

Limitations of Plasma Sterilization

Our experience has shown us that the principal limitation is the weak penetrating power of
plasma species: in the presence of organic residues, of packaging material, of complex
geometries, or of a large number of devices to be sterilized in a given chamber, the effectiveness
of plasma is severely limited, particularly in the case of “down-stream” (afterglow) approaches.
Several possible solutions may be envisaged to counter this weakness: one may greatly enhance
the efficacy of plasma by adding UV radiation with ¸ near 254 nm, which has been shown to be
most effective against microorganisms.(5;41) By selecting packaging materials which are
relatively transparent to this radiation, one can at least partly resolve the packaging problem.
This can also be accomplished using atmospheric pressure discharges, which can create plasma
inside packages, and without the need for expensive vacuum systems. Again, this calls for much
further investigation.

THE ROLE OF VARIOUS PLASMA PARAMETERS

To better understand variations among results of studies regarding plasma sterilization, it is
useful to review the numerous experimental parameters which can influence the efficacy of the
plasma process on microorganisms.
4.1. Gas Composition
As already mentioned, gas composition is a determining factor in the effectiveness of plasma. It
dictates the types of active species present, for example, radicals and excited molecules (atomic
and singlet molecular oxygen, hydroxyl radicals, ozone, . . .) and, at the same time, the intensity
and wavelengths of emitted UV and VUV radiation. For example, oxygen-based plasmas are
much more effective than argon plasmas.(16) Among oxygen-containing gases or gas mixtures
we have examined, pure O2, CO2, O2/H2, O2/Ar, O2/CF4, the latter proved to be by far the
most effective.(1;4) Mixtures of O2 and CF4 are well known to enhance plasma efficiency for
etching carbon-based and silicon-based solids.(17;18) In O2-rich plasmas, the addition of some
CF4 leads to the creation of fluorine atoms and to a higher yield of O atoms, responsible for
etching; the F atoms abstract hydrogen from organic surfaces, thereby creating highly reactive
free radical sites which are readily prone to etching reactions with O or O2.(17;19) This effect
peaks at CF4 concentrations which are typically below 20%. (4;17;18) The addition of N2 to O2
plasma also boosts the yield of O and raises the intensity of UV emission, hence the sterilization
effectiveness.(17;20) Hydrogen peroxide, H2O2, on the other hand, a powerful bactericide in its
own right, can act even in the absence of plasma, hence its choice for Sterrad° R .(21) Relatively
minor differences have been observed among pure O2, CO2, H2/O2 and air.(4;22) Our studies
have also shown that plasma sterilization induced significant surface modifications on polymeric
devices.(1;3) Since etching and oxidation are nonspecific process, plasma parameters should be
chosen in such a way as to compromise between plasma efficiency and damage to the sterilized
biomedical instruments. As an example, the afore mentioned O2/CF4 plasma is prone to also
modify the surfaces of polymeric devices.(1;18)
4.2. Gas Flow Rate (F)
The parameter F (in standard cm3/min. sccm) is also important, since it strongly affects the rate
of generating reactive species, for example, the flow of O atoms toward the sample surface. In
our own experiments, doubling F was found to increase by three orders of magnitude the number
of spores killed during a 5 min. plasma exposure.(4) However, one may expect a plateau effect
(when too many active species, N, are present to be used effectively), or even a reduction with
rising F (when their residence time in the treatment chamber, D V=F (V being the volume of the
chamber), becomes too short). In our experiments, F could not be raised above 110 sccm without
increasing the pressure, p, in the chamber, on account of limited pumping capacity.
4.3. Gas Pressure (p)
In our studies, p was not varied, even though pressure is known to influence the etch rate of
polymers.(18) Generally, rising p produces competing effects in plasmas: up to a certain limit, it
increases the residence time of the gas molecules, influencing the volume concentration, N, of
active species.18 Thereafter, the concentration drops on account of increasing recombination
rates, changes in the electron energy distribution function (EEDF, f (E)) and other effects.(18)
Furthermore, the plasma volume may decrease with rising p, and the gas temperature increases.
For example, Chau and coworkers observed incomplete destruction of Escherichia coli bacteria
at p D 400 mtorr, while at 43 and 220 mtorr their inactivation was total.(23) Khomisch et al. on
the other hand, found the effectiveness of their direct current (d.c.) glow discharge plasma to be
practically independent of p in the range 80 · p · 250 mtorr.(24;25)
4.4. Power (P)
Rising P leads to increased electron density, ne, hence to an increased concentration of active
species in the plasma, N. Thus, both Boucher (16) and Khomisch (24) observed increased
Sporicidal activity as they raised P .However, excessive heating of the gas and the materials to
be sterilized set an obvious upper limit to the value of P.
4.5. Excitation Frequency of the Plasma ( f )
In the course of our research, we have demonstrated superior efficacy of MW plasma in
sterilization, compared with its RF counterpart, other parameters being identical.(5) This is
thought to be related to the known greater etch rate of MW plasma,(26;27) as we explained in
Ref. 5: the parameter f determines the EEDF, one of the most fundamental plasma
characteristics; in MW plasma, the EEDF tends to be “Maxwell an”, which raises the population
of high-energy electrons (those responsible for creating reactive atomic, ionic and excited
molecular plasma species). RF plasma is “non-Maxwell an” and has a lower population density
of such “hot” electrons, which explains the lower concentration of O atoms in RF compared with
MW O2 plasma, for example. The role of ions and ion bombardment of exposed surfaces is also
very different between MW and RF plasmas.(24¡26) Suffice it to say here, however, that we
have not found charged particles to play a very important role in plasma sterilization,(5) in
agreement with the findings of Boucher(16) and of Khomisch.(24;25) For more details about the
effects of plasma excitation frequency on the concentration of active species and on the
intensities of UV emissions, the reader may consult several articles from these
laboratories.(27¡30)
4.6. Geometrical Factors
The type of reactor design can also strongly influence the concentrations of active species. For
example, the specimens to be sterilized may be placed in direct contact with the plasma, as we
have done,(1¡5) or in the so-called “afterglow”, downstream from the primary glow
discharge.(13;20) In the former case, the specimens are exposed to the combined effects of all
species (reactive neutrals, ions, photons, and the exciting electromagnetic field). Under suitable
conditions, this can even lead to plasma being generated inside a package containing the material
to be sterilized (described later). Afterglow, on the other hand, does not expose the material to
the effects of ions or to the electromagnetic field, only to long lived excited species; evidently,
the concentration of these decreases rapidly with increasing distance from the primary plasma,
which may result in much slower kinetics of sterilization.(13;20) Furthermore, depending on the
geometry of the system, part or all of the photon flux emitted from the primary discharge does
not reach the sterilization chamber. Even in the case of direct plasma systems, the concentration
of reactive species can decrease with increasing distance of the specimen from the source: thus,
we observed reduced effectiveness in our MW reactor, as the sample distance from the MW
power applicator was increased.(1;4) Finally, the materials making up the reactor chamber
and/or the specimen holder can strongly influence the recombination rates of active species, for
example the flux of O atoms reaching the sample surface.(20¡22)
4.7. Substrate Temperature (Ts )
Hury and coworkers observed that T s influences the effectiveness of plasma sterilization(22);
however, since the effect of T s was not monotonic (C60±C > ¡15±C > C15±C), the authors
concluded that competing effects must exist. Enhanced effectiveness with rising Ts between
C15±C and C60±C is readily understood, in terms of the classical Arrhenius law; it is often
observed to be valid in plasma etching of organic solids,(17;18) in spite of the multiple chemical
pathways which are probably active: R D A exp (¡Ea=kNT ) (1) where R is the etch rate (for
example), Ea the activation energy (in kJ/mole), and k and N are Boltzmann’s constant and
Avogadro’s number, respectively. As to the relatively high effectiveness at ¡15±C, this may be
due to a greater sensitivity of the spores at low temperature: it is known that frozen spores are
hypersensitive to UV radiation,(31) which may then also apply to the case of other sterilization
methods. Considering that the effect of Ts has already be studied by others, we were satisfied
simply to assure that Ts did not exceed 60±C in our experiments. This was accomplished (a) with
the help of a thermocouple in the sample holder; and (b) with the use of “Tempilstick °R ”, a
kind of resin which can be deposited directly on surfaces to be sterilized, and which changes
appearance beyond specific threshold values of Ts .
4.8. Packaging
The presence or absence of packaging material is obviously highly relevant to the effectiveness
of a plasma sterilization process. In our work, a drastic reduction in spore mortality was
observed, as expected, when the spores were inside a polymeric package, due to the small
number of active species passing through the packaging material.(1) Instead, they recombine on
the outer surface of the material or, more likely, even react with it. This fundamental obstacle
can only be overcome by creating plasma directly inside the package, but this entrains another
set of problems. The most logical approach is to place the sterile materials or medical
instruments inside sealed packages, into which microorganisms cannot penetrate; however, this
must be done under sterile conditions, and is quite impractical in a hospital environment.
4.9. Nature and Quantity of Substrates to be sterilized
The nature of the substrate material has been found to influence the “D-value” (where D-value is
the time necessary to decrease the number of living microorganisms by one order of magnitude)
of a given microorganism,(32) for still unknown reasons. Four possible explanations have been
proposed, namely:

1. Moisan, M., et al., Plasma sterilization. Methods and mechanisms. Pure and applied
chemistry, 2002. 74(3): p. 349-358.
2. Yardimci, O. and P. Setlow, Plasma sterilization: opportunities and microbial
assessment strategies in medical device manufacturing. IEEE Transactions on Plasma
Science, 2010. 38(4): p. 973-981.
3. Park, B.J., et al., Sterilization using a microwave-induced argon plasma system at
atmospheric pressure. Physics of Plasmas, 2003. 10(11): p. 4539-4544.