Journal of Ophthalmology Clinics & Research

: 2021  |  Volume : 1  |  Issue : 1  |  Page : 9--15

Resurgence of plasma sterilization: A review

NV Annapurna1, N Rohit Goud2, Swathi Nadella3,  
1 MS, FCAS, Consultant - Cornea, Cataract and Refractive Surgery Services, Sankara Netra Chikitsalaya, Vijayawada, Andhra Pradesh, India
2 MS, FVRS, Consultant - Vitreoretinal Services, Sankara Netra Chikitsalaya, Vijayawada, Andhra Pradesh, India
3 MS, Consultant - Comprehensive Ophthalmology, Sankara Netra Chikitsalaya, Vijayawada, Andhra Pradesh, India

Correspondence Address:
Dr. N V Annapurna
Department of Cornea and Cataract Surgery, Sankara Netra Chikitsalaya, 59-14-1/2, Above Federal Bank, Bharati Square Building, Nirmala Convent Circle, Vijayawada - 520 008, Andhra Pradesh


Sterilization is the backbone of a health-care organization and ensures high-quality patient care. While the horizon of medical and surgical devices has undergone vast expansions, not many discoveries have been made as far as novel sterilization procedures are concerned. Steam sterilization remains the most widely used modality of sterilization of equipment to date in most health-care organizations. Its limitation lies in the fact that there are many medical and surgical devices in the market today which are heat and pressure sensitive and can be damaged by the high temperature and pressure levels of steam sterilization. Novel sterilization techniques which are helpful in the sterilization of such sensitive instruments that are being widely used today include ethylene oxide (ETO) gas sterilization and plasma sterilization. ETO sterilization requires the instruments to undergo aeration after the sterilization process, which takes a significant amount of time. Since the sterilized materials can be used only after the aeration period, stocking up of medical instruments is required which incurs extra cost and entails further investment in this regard. In the last few decades, there have also been concerns over the safety of ETO gas itself. Hence, attention has shifted to plasma sterilization which has spiked the interest of medical professionals due to both its safety and economic running costs. This article reviews the evolution of plasma sterilization along with its working principle, methods of inactivation of microorganisms, advantages, and disadvantages. A literature search using the keywords “plasma sterilization” was carried out in PubMed and Google Scholar platforms. Out of the suggestions available, the search was zeroed down to the most relevant articles and a few landmark articles, with focus on the origin of plasma sterilization as a procedure, methods of generation of gas plasma, phases of plasma sterilization, and antimicrobial properties of plasma. A review of articles comparing the efficacy of steam sterilization, ETO sterilization, and plasma sterilization was performed. Standard textbooks, as cited in the references, were referred to as required. The operation and maintenance instruction manual for low-temperature hydrogen peroxide sterilizer ACTIPLAZR model HP-3041, Hanshin Medical Co. Ltd, South Korea, 2019, was used as the primary reference when describing the working of the aforementioned model (HP-3041) of plasma sterilizer.

How to cite this article:
Annapurna N V, Goud N R, Nadella S. Resurgence of plasma sterilization: A review.J Ophthalmol Clin Res 2021;1:9-15

How to cite this URL:
Annapurna N V, Goud N R, Nadella S. Resurgence of plasma sterilization: A review. J Ophthalmol Clin Res [serial online] 2021 [cited 2022 Aug 11 ];1:9-15
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Sterilization by definition refers to “the process by which all living cells, spores, and acellular entities (e.g., viruses, viroids, and prions) are either destroyed or removed from an object or habitat.”[1] Sterilization is a quintessential part of ensuring safe medical and surgical practice. While keeping pace with the changing times, the medical and surgical device industry has noted rapid growth, whereas the sterilization technologies have remained essentially unchanged.

The most common techniques of sterilization that are in use today in eye care organizations are steam sterilization, ethylene oxide (ETO) sterilization, and plasma sterilization using hydrogen peroxide (H2O2). This article briefly reviews steam and ETO sterilization and provides an in-depth analysis of plasma sterilization.

 Steam Sterilization

Steam sterilization is a conventionally used and inexpensive[2] method of sterilization where moist heat in the form of saturated steam at the required temperature and pressure for a specified time is used for the inactivation of microorganisms.[3] Its lethality is due to the denaturation of microbial enzymes and structural proteins.[3] Most of the medical and surgical instruments are made of heat-stable materials and hence can be safely sterilized using steam sterilization.

With an increase in devices made of materials like plastic which cannot withstand high temperature and pressure, several new low-temperature sterilization systems, such as ETO sterilization and H2O2 gas plasma sterilization, have been developed in the recent past and are currently in use to sterilize medical devices.

 Ethylene Oxide Sterilization

ETO gas is used instead of steam sterilization for equipment that is heat and moisture sensitive. ETO is a colorless and flammable gas.[4] It causes alkylation of protein, DNA, and RNA, thus disrupting the normal cell cycle.[4] The main disadvantages of this technique are the lengthy cycle time, higher running cost,[4] and its potential hazards[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17] to the patients and staff.[18] Apart from the acute and chronic hazards due to ETO gas itself, [Table 1][5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22] its usage as a mixture along with chlorofluorocarbons (CFCs) and hydrochlorofluorocarbons (HCFCs), to reduce its flammable properties,[19] is of additional concern. CFC and HCFC are considered hazardous due to their effect on the depletion of the ozone layer in Earth's atmosphere. Due to the environmental regulations on CFCs, the early versions of ETO sterilizers were discarded[19] and newer models of ETO sterilizers without CFC were developed. Despite the elimination of CFCs, concerns over the carcinogenic properties of the ETO gas itself, which remains adsorbed on the materials after processing, still loom large.[20],[21],[22]{Table 1}

 Hydrogen Peroxide Gas Plasma Sterilization

In 1927, Irving Langmuir coined the term “plasma” to describe an ionized gas.[23] Gas plasmas are the fourth state of matter,[24] the other three being solids, liquids, and gases. For the first time, the sterilization property of plasma was introduced in the year 1968.[25] Further patents were made by Ashman and Menashi, Boucher (Gut), and Bithell.[26],[27],[28] Lerouge et al. have extensively studied plasma sterilization as a technique and eluded on its various properties.[29],[30],[31],[32],[33]

Gas plasmas are produced under a deep vacuum, in an electric field, using radiofrequency or microwave energy to produce charged particles or free radicals. (“A free radical is an atom with an unpaired electron and is a highly reactive species.”)[3] These free radicals (hydroxyl and hydroperoxyl ions) interact with cell components such as enzymes and nucleic acids, thus deranging the metabolism of microorganisms.[3] The antimicrobial property of plasma[34],[35],[36] is primarily due to H2O2 gas. This antimicrobial action of H2O2 is further enhanced by plasma by causing an accelerated breakdown of residual H2O2.[37] The greater the amount of reactive oxygen species, the more is the antimicrobial activity, as has been observed experimentally, with the shrinkage of spores.[38] Studies show that spores are much less resistant to gas plasmas.[39],[40] Plasma is not only effective against spores but also prions, which are resistant to many routinely used chemicals.[41],[42]

The by-products of the cycle (e.g., water vapor and oxygen) are nontoxic. Hence, the sterilized materials are safe, both for immediate use and storage. Studies show that plasma sterilization is compatible with >95% of medical devices tested.[42] Its only drawback is the inability to sterilize liquids, powders, or strong absorbers (e.g., cellulose).

 Phases of Plasma Sterilization

Five phases of plasma sterilization are vacuum, injection, diffusion, plasma, and vent cycles.[43]

Items to be sterilized must be thoroughly dried first since moisture will prolong the evacuation phase and can lead to the cancellation of the cycle. During the vacuum stage, the pressure is decreased to 0.3 mmHg, followed by injection of liquid H2O2 at a temperature of 40°–45°C. The relative humidity is between 6% and 14% throughout the process.[44]

H2O2 acts on all surfaces of the load in the chamber, in the diffusion phase. After this, radio-frequency plasma discharge is initiated, which causes H2O2 vapor to form reactive oxygen species, which in turn causes lethal damage to the cell wall of microorganisms. The final by-products are oxygen and water.[45] In the end, the chamber is vented through a high-efficiency particulate air (HEPA) filter and a catalytic filter to safely decompose all remaining traces of H2O2 into water and oxygen.

 Mechanisms of Inactivation of Microorganisms by Plasma

According to a study that analyzed methods of inactivation of microorganisms during plasma sterilization,[46] there are three basic mechanisms for the same:

Direct destruction by UV irradiation of the genetic material of microorganismsErosion of the microorganisms through intrinsic photodesorption by UV irradiation to form volatile compounds combining atoms intrinsic to the microorganismsErosion of the microorganisms through etching to form volatile compounds as a result of slow combustion using oxygen atoms or radicals emanating from the plasma.

 Advantages of Plasma Sterilization

Rapid and gentle sterilization – Results in longer life span of instrumentsCost-effective technology – No aeration time is required after sterilization. Therefore, a large stock of instruments need not be stored, thus effectively minimizing the costEnvironment-friendly end products of the sterilization processConvenience of installation – Electricity is the only primary requirement for the working of plasma sterilizer.

 Disadvantages of Plasma Sterilization

The only known limitation is that it cannot be used for sterilizing highly absorbent materials [Table 2].{Table 2}

When H2O2 gas plasma sterilization is compared to other methods of sterilization, one area of concern is its efficacy in the sterilization of instruments that have a lumen.[47] Only two comparative studies[48],[49] done on some models of plasma sterilizers have confirmed their efficacy in this regard. Some models performed better than the others in one of these studies.[49]

An important point to note here is that studies[50],[51] have emphasized adequate removal of serum and salt deposits from the lumen of instruments before beginning H2O2 gas sterilization. However, in the presence of salt and serum, steam sterilization is considered the best,[52] whereas both ETO gas sterilization and H2O2 gas plasma are a close second.[52]

Some of the other surgical specialties such as gastroenterology and urology, routinely use instruments containing long and narrow lumens. Few recent studies focusing on the effect of sterilization using H2O2 gas plasma, on instruments with long and narrow lumen like the ones used in endourology, have been published with encouraging results.[52]

Similar studies focusing on the efficacy of plasma sterilization on ophthalmic instruments containing lumen are required before any conclusions can be drawn in this regard.

 Safely Handling the Sterilant

H2O2 is a valid sterilant only for 6 months once inserted into the sterilizer or when stored intact at room temperature or in the refrigerator. It should be discarded according to regulations of the waste control law. H2O2 used as a sterilant in plasma sterilization has a characteristic of high oxidation and low inflammability. But in high temperatures and humidity, it is flammable. Hence, protective equipment such as gloves and goggles should be worn while operating, inspecting, or repairing the sterilizer.

 Models of Plasma Sterilizers

Quite a few models of plasma sterilizers exist in the market, for example, Sterrad from Johnson and Johnson, U.S.A; Steris with V-Pro and Renosem from Korea; Laoken from China; Concalves from Portugal; Tuttnauer from Germany; and ACTIPLAZR HP-3041 from South Korea.

The working methods of each of these plasma sterilizers are quite similar in most aspects. However, for ease of understanding its operation, we have explained the working of ACTIPLAZR HP-3041, from Hanshin Medical Co. Ltd in South Korea, with which we have considerable experience in sterilization.


The details enlisted below are as per the manufacturer of the machine,[53] ACTIPLAZR HP-3041 [Figure 1] and [Figure 2] is a low-temperature, low-pressure sterilizer that works on the principle of plasma sterilization. It is a microprocessor-controlled 74-kg machine with a 1600-W electricity consumption, 40-l capacity, and a working temperature of 40°–70°C.{Figure 1}{Figure 2}

Specifications for installation

Surroundings of sterilizer should have a space of at least 300 mm at the back and both sides for ventilation and 500 mm near the door for smooth door operationElectric power source has to be grounded completely and its fluctuation should be kept within 10% of nominal. The minimum power-supplying capacity must be over 1600 W for covering the power consumption of the sterilizerAmbient temperature within 40°C and relative humidity within 80%Minimum of ten air exchanges per hour should be providedAvoid installing the sterilizer near a water or heat source.

Operation procedure

Only distilled water or RO water (reverse osmosis method) should be used for cleaning the instrumentsThe instruments are packed in standardized TyvekR pouches for sterilization [Figure 3]. A change in color of the indicator present on the top of the pouch from blue to pink indicates adequate sterilizationIdeally, the transparent side of a pouch should face the opaque side of the next pouch, thus H2O2 and plasma can surround them perfectlyVacuum test is run first in an empty chamber before the start of daily sterilizationThe sterilizer has two kinds of built-in sterilization cycles, i.e., standard cycle and the flash cycle, apart from the vacuum leak test cycle. The standard cycle sterilizes the devices perfectly whereas the flash cycle sterilizes the surface of medical devices in a short time [Table 3].{Figure 3}{Table 3}

Steps in sterilization

These are similar to the phases in plasma sterilization as discussed earlier. However, it will be briefly discussed to elaborate on the difference between standard and flash cycles.

Preheat – The cool air from the chamber is sucked out and heated air is let into the chamberFirst vacuum – A vacuum pump is operated to exhaust the air in the chamberFirst sterilization – The vaporized H2O2 is injected into the evacuated chamber for a set duration of time. When the set time has elapsed, air is introduced into the chamber until atmospheric pressure is reached, so that the sterilant penetrates within the lumen of the sterilizing device by the pressure difference.

In the flash cycle, the plasma phase is followed immediately after this process [Figure 4] and [Figure 5].{Figure 4}{Figure 5}

Second vacuum – The vacuum pump is operated again to evacuate the chamberSecond sterilization – Vaporized H2O2 is re-injected into the chamber and maintained for a preset time. When the set time has elapsed, air is again introduced into the chamber until it reaches the atmospheric pressure.Plasma and exhaustion – When the set second sterilization time has elapsed, the plasma generator mounted externally is operated, and the chamber is evacuated.Air-in and completion – Upon completion of the plasma and exhaustion phase, air filtered through the HEPA filter is introduced into the chamber until it reaches the atmospheric pressure, and the cycle is completed. An automated and detailed printout is given [Figure 6].{Figure 6}

Monitoring quality of sterilization

Monitoring by chemical indicator

Chemical indicators (CIs) are designed in the form of strips or tapes, which change color from blue [Figure 7]a to pink [Figure 7]b on adequate sterilization. They provide definite visual evidence of the quality of sterilization. If the color of the CI strip has not changed completely after sterilization, the load in the chamber should not be used until the cause is clarified.{Figure 7}

Monitoring by biological indicator

This is the most definitive method to monitor sterility [Figure 8]. The strain used as a biological indicator (BI) for H2O2 gas plasma sterilization is Geobacillus stearothermophilus. Incubate the processed BIs immediately for 24–48 h at a temperature of 55°–60°C after completing sterilization, along with unprocessed BIs, to estimate the sterility. For adequate sterilization, the processed BI must not change color (remains orange-red) and the nonprocessed BI must change its color (from orange-red to yellow).{Figure 8}

Technical advantages of ACTIPLAZR HP-3041

Cheap sterilization cost – One-bottle (150cc) sterilant can be used for ten cyclesSafe handling of sterilant – H2O2 solution is contained a specially designed bottleHEPA filter for bacteria capture – It is capable of capturing 99.999% of particles of 0.3 μm size, which prevents re-contaminationFully automatic cycle processing and tracking of sterilization on the LCD screenConvenient operation by a one-touch control panel [Figure 9]Minimal consumables: H2O2 solution, TyvekR pouches, chemical, and BI.{Figure 9}

As per the manufacturing company,[53] ACTIPLAZR HP-3041 can sterilize instruments with an internal diameter of 2 mm and a length of 1.5 m.


Since the 19th century, steam sterilization using autoclave has remained the conventional method of sterilization for instruments which are not damaged by steam, heat and pressure. Low-temperature sterilization technologies (e.g., ETO and H2O2 gas plasma) are used for critical patient-care equipment that is heat or moisture sensitive. Serious concerns over the safety profile of ETO are beginning to limit its widespread usage. Plasma sterilization has largely replaced ETO as an effective alternative to sterilize heat- and pressure-sensitive devices since it is quick, safe, and cost-effective. The only limitation of plasma sterilization is its inability to sterilize absorbent materials. Hence, plasma sterilization is likely to remain one of the key methods of sterilization until a novel technique that is quick, safe, and compatible with all kinds of materials is invented.


We would like to thank Hanshin Medical Co. Ltd, South Korea, for technical help.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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