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Development of a Practical Method for Using Ozone Gas as a Virus Decontaminating Agent



Our objective was to develop a practical method of utilizing the known anti-viral properties of ozone in a mobile apparatus that could be used to decontaminate rooms in health care facilities, hotels and other buildings. Maximum anti-viral efficacy required a short period of high humidity (>90% relative humidity) after the attainment of peak ozone gas concentration (20–25 ppm). All 12 viruses tested, on different hard and porous surfaces, and in the presence of biological fluids, could be inactivated by at least 3 log10, in the laboratory and in simulated field trials. The ozone was subsequently removed by a built-in catalytic converter.
Ozone: Science & Engineering-accepted (Jan 09)
Development Of A Practical Method For Using Ozone Gas
As A Virus Decontaminating Agent
Hudson, JB. Sharma, M. Vimalanathan, S.
Viroforce Systems Inc. Laboratory, Vancouver, Canada.
Affiliations: Drs. James B Hudson, Professor; Manju Sharma, Research Associate;
Selvarani Vimalanathan, Research Associate
Address Correspondence to:
Dr. JB. Hudson, Department of Pathology & Laboratory Medicine, University of
British Columbia, C-360 Heather Pavilion, 2733 Heather Street,Vancouver V5Z 1M5,
Canada. I-604-875-4351 Fax no. 1-604-875-4351 e-mail:
Running Head: Ozone Gas: a Practical Antiviral Agent
Our objective was to develop a practical method of utilizing the known anti-viral
properties of ozone in a mobile apparatus that could be used to decontaminate rooms
in health care facilities, hotels and other buildings. Maximum anti-viral efficacy
required a short period of high humidity (> 90% relative humidity) after the attainment
of peak ozone gas concentration (20-25 ppm). All 12 viruses tested, on different hard
and porous surfaces, and in the presence of biological fluids, could be inactivated by at
least 3 log
in the laboratory and in simulated field trials. The ozone was
subsequently removed by a built-in catalytic converter.
The anti-viral and anti-microbial properties of ozone have been well documented,
although the mechanisms of action are not well understood, and several
macromolecular targets could be involved (Carpendale and Freeberg, 1991; Wells et al
1991; Khadre and Yousef, 2002; Shin and Sobsey,2003; Cataldo, 2006; Lin and Wu,
2006; Lin et al 2007). Aqueous solutions of ozone are in use as disinfectants in many
commercial situations, including waste water treatment, laundries, and food
processing (Kim et al, 1999; Shin and Sobsey, 2003; Naitou and Takahara, 2006;
2008; Cardis et al 2007), but the use of the gas on a commercial scale as a
decontamination device has not been exploited. Ozone gas however has a number of
potential advantages over other decontaminating gases and liquid chemical
applications (McDonnell and Russell, 1999; Barker et al 2004). Thus ozone is a
natural compound, is easily generated in situ from oxygen or air, and breaks down to
oxygen with a half life of about 20 minutes (± 10 min depending on the environment).
As a gas it can penetrate all areas within a room, including crevices, fixtures, fabrics,
and the undersurfaces of furniture, much more efficiently than manually applied liquid
sprays and aerosols (Barker et al, 2004; Malik et al, 2006; Hudson et al 2007).
The only significant disadvantages are its ability to corrode certain materials, such as
natural rubber, on prolonged exposure, and its potential toxicity to humans. The
recognition of the risk of pathologic consequences following exposure of people and
experimental animals to ozone gas has led to restrictions in its use in public areas.
However the latter consideration can be offset to some extent by the potential benefits
of ozone therapy in medicine and dentistry (Devlin et al 1996; Bocci, 2004;
Ciencewicki and Jaspers 2007; Huth et al 2007).
The health hazard can be overcome in practice by ensuring that the room to be treated
is temporarily closed to people during the treatment and is sealed to prevent escape of
the gas into the environment. Sensitive materials can be temporarily covered or
removed if necessary. In addition the ozone gas can be removed quickly after
treatment by use of a catalytic converter, which can transform the ozone back into
oxygen within minutes.
We evaluated the feasibility of using ozone gas as an effective means of
decontaminating various hard and porous surfaces containing dry or wet films of
different viruses, in the presence and absence of cell debris and biological fluids.
Following successful laboratory experiments, we then developed an efficient
prototype ozone generator and catalytic converter which could be used in a room
containing viral contaminants. We also examined a role for high humidity in
enhancing the virus inactivation process, and incorporated this feature into the field
Materials and Methods
The laboratory test chamber was a molded polycarbonate box with a transparent
plastic front window that could be lifted to allow access to samples. Within the test
chamber was a small ozone generator (corona discharge system, from Treated Air
Systems, Vancouver) fitted with a control dial that could be pre-set to determine the
approximate ozone dosage in ppm, an ozone sampler tube connected to the exterior
ozone measuring system (for accurately recording ozone concentration, see below),
and the probe of a hygrometer for measuring relative humidity and
temperature. Humidity was provided in the form of a mist of deionized sterile water by
means of a spray bottle, which had been washed out with 70% ethanol
The model 1000 Viroforce ozone generator (Fig 1) was a portable module containing
multiple corona discharge units, a circulating fan, and an efficient catalytic converter
(scrubber) to reconvert ozone to oxygen at the termination of the ozone exposure
period (further details are available in In addition a portable
commercial humidifier (Humidifirst Inc, Florida) was used to provide a burst of water
vapor (at ambient temperature) when required. All the components were controlled
remotely from outside the test room. Ozone concentration was monitored continuously
by means of an Advanced Pollution Instrumentation Inc. model 450 system (from
Teledyne, San Diego), which measured samples of ozonated air passed through a UV
spectrometer. This apparatus was used for all accurate ozone measurements in all test
locations. The input teflon sampling tube was taped in an appropriate location for the
duration of the experiment. Relative humidity and temperature were recorded by a
portable hygrometer (VWR Scientific, Ontario). The probe was taped in a convenient
location inside the test room.
The lids of sterile polystyrene tissue culture trays were used as plastic surfaces. Glass
slides, 75 x 25 mm; stainless steel circular disks, 1.0 cm diameter; and pieces of fabric
and cotton (typical of those used in hospital and hotel rooms) were cleaned in
detergent, washed, dried, and sterilized by autoclaving. Cotton tips (Q-tips) were
heated for 2 min in a microwave oven. Fetal bovine serum and PBS (phosphate
buffered saline) were obtained from Invitrogen (Ontario). Sterile plastic 24 well plates
and other supplies were BD-Falcon brand obtained from VWR Scientific (Ontario).
Cell Lines & Viruses:
All cell lines (Vero monkey kidney cells; MDCK canine kidney cells; H-1 sub clone
of HeLa cells; A549 human lung epithelial cells; feline kidney cells; all acquired
originally from ATCC; mouse DBT cells, from Dr. Pierre Talbot) were passaged
regularly in Dulbecco MEM, in cell culture flasks, supplemented with 5-10% fetal
bovine serum, at 37° C in a 5% CO2 atmosphere, with the exception of the H-1 cells,
which were grown at 35
C. No antibiotics or antimycotic agents were used.
The following 12 viruses were used: influenza, strain H3N2, human isolate (from BC
Centre for Disease Control), propagated in MDCK cells; HSV (herpes simplex virus
type 1, BC-CDC), propagated in Vero cells; rhinovirus types 1A and 14 (RV 1A and
RV 14, from ATCC), propagated in H-1 cells; Adenovirus types 3 and 11 (ATCC), in
A549 cells; mouse coronavirus (MCV, from Dr. Pierre Talbot) in DBT cells. Sindbis
virus (SINV), yellow fever virus (YFV), vesicular stomatitis virus (VSV), poliovirus
(PV,vaccine strain), vaccinia virus (VV), all ATCC strains, were grown in Vero cells.
All the stock viruses were prepared as clarified cell-free supernatants, with titers
ranging from 10
to 10
pfu (plaque-forming units) per mL.
Experimental Protocol
Aliquots of virus, diluted when necessary in PBS, usually 100 uL, were spotted onto
the appropriate sterile surface, spread into a film by means of a sterile tip, and allowed
to dry, within a biosafety cabinet (normally 30-40 min). In some experiments the
spread films were left wet for the ozone treatment. The samples were then transported
in sterile containers to the appropriate chamber or room for ozone treatment. Controls
consisted of equivalent samples transported to the test site but not exposed to ozone,
and others retained in the biosafety cabinet for the entire duration of the experiment.
All control samples were contained were contained within sealed sterile plastic boxes
and kept outside the ozone- exposed room or chamber for the duration of the
Test Rooms
1. The initial field trials were conducted in an unused laboratory, volume 65 m
, in
which we used three small ozone generators (Treated Air Systems) located in different
parts of the room, together with a circulating fan. These tests were carried out at
ambient humidity (40-45% RH).
2. In most of the subsequent field tests we used an office, volume 35 m
, containing
normal office furniture, which was located adjacent to the laboratory. We placed the
prototype ozone generator (Viroforce model 1000) in the centre of the room, together
with the humidifier. Test samples were placed in various locations of the room, and
the probes for the ozone monitor and the hygrometer were taped in convenient
locations. All instruments were controlled remotely from outside the test room. At the
beginning of the test the air vent was covered with plastic and the door was sealed
with duct tape. The standard program adopted for most of the tests involved increasing
the ozone level over a period of 15 min to 25 ppm, maintaining this level for 10 min,
at which point the humidifier was activated to produce a rapid burst of water vapor.
This resulted in the RH increasing to > 95% within 5 min. Following this the
humidifier and generator were switched off and the catalytic converter was switched
on, which resulted in a decrease in ozone to < 1ppm within 15 min. The door was then
opened and the samples retrieved and covered for transport back to the biosafety
cabinet. These samples, and equivalent control samples that had been kept in the
biosafety cabinet for the duration of the test, were then reconstituted in 1.0 mL PBS
and stored at -70
C until assayed by plaque formation (plaque forming units, pfu) in
the appropriate cells. Unless otherwise indicated, results are presented as pfu/mL.
3. A similar protocol was employed for use in the test hotel room, a typical room with
a double bed, furniture and adjacent bathroom, volume 42.5 m
, situated in
Vancouver. Dried samples of the viruses on plastic surfaces were transported in sterile
containers between the laboratory and the hotel room.
Inactivation of viruses by ozone gas on different surfaces: Since we
wanted to evaluate the effect of ozone gas on dried samples of virus we first
examined the ability of several representative viruses to retain significant infectivity
following the drying process. Most of the viruses showed up to 1 log
decrease in
infectivity as a result of the drying process itself. After this the dried films (of HSV,
influenza virus, FCV, poliovirus, and RV) showed similar decay curves, with a 50%
decrease (T ½) of 3-4 hours at room temperature. Thus in all cases there were more
than adequate amounts of infectious virus remaining after several hours, during which
experiments with ozone gas could be carried out. These decay curves were not
significantly affected by the presence of 10% serum (fetal bovine serum, FBS).
Similar findings on virus drying kinetics were reported recently (Terpstra et al 2007),
and these results confirm the general belief that infectious viruses can persist for long
times on inanimate surfaces.
Several viruses, representing different virus families and structural features, were then
treated with a single mobile ozone generator in the laboratory chamber, as described in
Materials and Methods. All viruses tested, HSV, influenza, MCV, FCV, and RV,
representing DNA and RNA viruses with and without membranes, showed similar
kinetics of virus inactivation on three hard surfaces, plastic, glass and stainless steel.
The T ½ values ranged from 5-8 hours, but there were no consistent differences
between the viruses or the surfaces. Examples for HSV (DNA virus with membrane),
influenza (RNA virus with membrane), and RV (RNA virus without membrane) are
shown in Fig.2. Rhinovirus (Fig 2c) was slightly more resistant than the other two
viruses. Nevertheless these results suggest that all or most viruses should be
susceptible to ozone gas.
Field tests at Ambient Humidity: Following successful inactivation of several
viruses in the laboratory experiments, we conducted tests in a large unoccupied
laboratory, volume 65 m
, with the aid of three portable ozone generators of the kind
used in the previous tests. Peak ozone level attained was 28 ppm, at an ambient RH of
40%, and total time of exposure, including rise and fall periods, was 60 min. The
results from two separate tests are combined in Table 1, and these indicate successful
inactivation of two log
or more infectious virus under these simulated field
conditions. Duplicate samples showed reasonable agreement, and the results were
unaffected by the position of the sample within the room. Thus prolonged exposure to
a fairly high dose of ozone gas at ambient humidity can result in two log’s inactivation
of several viruses; but in practice we would prefer a system giving greater efficacy.
Enhancement of virus inactivation by high humidity: We next examined the
possibility of improving virus killing by treating dried samples of several viruses with
ozone gas in the presence of high relative humidity. Preliminary laboratory
experiments indicated that the maximum enhancing effect was obtained by increasing
the ozone to the maximum level first followed by a burst of water vapour to increase
RH to greater than 70%, preferably >90%. However we did not have the capability of
testing the enhancing effect of graded doses of humidity.
Table 2 shows the effect of RH on the degree of inactivation of 3 different viruses
within a test office, 35.4 m
volume. Under these conditions, which involved much
more restricted exposure than the conditions used for Table 1, the degree of
inactivation was lower and more variable at ambient RH, but in all cases the
combination of ozone gas plus high RH consistently yielded substantial inactivation.
Therefore optimum efficacy of the ozone treatment requires the presence of high RH,
for at least several minutes.
Composition of virus samples: Based on these findings, we next conducted a
number of experiments with different viruses in the test office, which contained
standard office furniture. For this purpose we used a newly developed prototype ozone
generator, containing multiple ozone units, together with a built in catalytic converter
and fan (shown in Fig 1), and an accessory humidifier capable of generating a
humidity of more than 90% within 5 minutes. Details of the protocols are described in
Materials and Methods.
In this test system we were able to examine the effects of sample preparation and
composition, organic load, and sample location within the room. Wet and dry films of
viruses were found to be equally susceptible to the treatment regimen. Also the nature
of the surface on which samples were dried did not affect the result. Thus in addition
to the different hard surfaces mentioned above (glass, plastic and stainless steel, Fig
2), cotton and fabric surfaces gave results similar to plastic (not shown). Inoculum size
(10-1000 uL) and degree of dilution of the virus did not influence the result, nor did
the presence of cellular debris in the sample. For example influenza virus and Sindbis
virus in crude cell extracts and in clarified supernatants were equally susceptible
(more than 3 log inactivation in dried films treated with ozone in high humidity).
We also tested the effect of serum and blood products, since samples in the field, such
as tissues and corpses, and instruments used in dental and hospital clinics, might be
contaminated with such materials (Cristina et al 2008). However, as shown in Table 3,
the presence of whole human blood, or human and bovine serum components, did not
affect the efficacy of virus inactivation, in either dry (data shown) or wet samples of
Viral aerosols: Virus - containing aerosols, a potential problem in certain dental and
medical practices (Cristina et al 2008), were also tested by spraying known volumes of
FCV suspension into the test chamber in the presence or absence of ozone gas, and
collecting samples of condensate for virus assays. In comparison, similar amounts of
virus were sprayed into the chamber without ozone gas, and measured volumes
collected. This experiment was performed twice, resulting in retrieval of
approximately 1% of the sprayed virus each time, and inactivation of more than 99%,
as indicated in Table 4. Thus the ozone gas is also capable of efficiently killing
aerosol-borne virus.
Field tests with high humidity: A standard hotel room (volume 42.5 m
) was used
for the evaluation of the prototype ozone generator with accessory humidifier, using
influenza- and FCV as examples of viruses with and without membranes, respectively.
Known amounts of virus were dried onto glass slides, which were then transported to
the room for ozone and humidity treatment, using the protocol developed in the office
tests, above. Pairs of samples were placed in three different locations within the room,
including an adjacent bathroom. Treated and control (unexposed) samples were then
returned to the laboratory for reconstitution and assay. The Results are summarized in
table 5. Both viruses were substantially inactivated, and the location of samples within
the room did not affect the outcome.
Susceptible Viruses: Table 6 summarizes the viruses successfully inactivated, by 3
or more log
, and their relevance. As indicated, these viruses represent many different
families with a range of animal virus structures. Some of them have also been
suggested to be suitable surrogates for important viruses that are difficult to cultivate
in vitro or require special containment facilities (eg. Sindbis virus and yellow fever
virus for hepatitis C; These two viruses plus vesicular stomatitis viruses for HIV;
human influenza virus for avian influenza; Steinman, 2004). To date we have not
encountered an ozone- resistant virus.
The objective of this study was to develop a practical and efficient apparatus for
decontamination of confined spaces containing infectious viruses. Such an apparatus
could be very useful in hospitals and health care facilities, and other locations where
outbreaks are relatively common, such as cruise liners (Lawrence 2004). In addition
there are many other public and private buildings that could benefit from an
appropriate antiviral decontamination apparatus. Existing technologies are clearly
inadequate (McDonnell and Russell, 1999; Barker et al 2004; Sattar 2004).
Previous studies with ozone in water have proven its usefulness in commercial
laundries and food processing facilities (Kim et al, 1999; Shin and Sobsey, 2003;
Naitou and Takahara, 2006; 2008; Cardis et al 2007). However, in order to decrease or
eradicate virus contaminants in inaccessible locations, such as crevices, fixtures,
undersides of furniture, etc. it is necessary to utilize the efficient penetrating ability of
a gas. Since ethylene oxide is not considered an acceptable alternative (McDonnell
and Russell,1999), then gaseous ozone should be the best choice available.
A few studies have indicated the feasibility of ozone gas as an antiviral agent
(Carpendale and Freeberg, 1991; Wells et al 1991; Khadre and Yousef, 2002; Shin and
Sobsey, 2003; Cataldo, 2006; Lin and Wu, 2006). We verified this by means of
laboratory studies and several field trials in a large room. We then discovered that the
addition of a burst of high humidity, following the attainment of peak ozone level,
resulted in substantially greater reductions in virus infectivity, under a variety of
conditions. The precise mechanisms of action against virus are not understood;
however the broad oxidizing activity against many macromolecules (Cataldo 2006)
suggest that viral membranes, protein coats and nucleic acids could all be vulnerable.
Nevertheless the requirement of humidity for optimal efficacy indicates that hydroxyl
ions and possibly additional water-derived radicals could be involved, as suggested for
the aqueous environments (Lin and Wu 2006).
We developed the prototype apparatus to take advantage of the desired features based
on these experimental results (Fig 1,Viroforce 1000). The key features are: a battery of
ozone generators enclosed within the machine; a powerful catalytic converter to
convert ozone back to oxygen within minutes, allowing immediate entry to the
decontaminated premises; a circulating fan; built in remote control and programmable
functions. In addition we employ an accessory humidifier, which produces an
immediate cloud or mist of microscopic water droplets, without heating. We
demonstrated that this apparatus was capable of inactivating 3 log’s or more of many
different infectious viruses in rooms such as an office and a hotel room. We also
reported recently that the same apparatus worked efficiently in a cruise liner cabin to
inactivate norovirus (Hudson et al 2007).
To date we have successfully tested the apparatus in laboratory and field conditions
against 12 representative viruses, mostly human pathogens. Some of these viruses
(Table 6 legend) have also been promoted as valid surrogates for viruses that are
difficult or dangerous to cultivate and test by conventional techniques, such as
hepatitis C virus, HIV, avian influenza (Steinman, 2004).
The location of the test virus in the room was not a factor, a result that might be
expected considering the penetrability of the ozone gas, nor was the presence of blood
and serum products. The latter was an important result since the possibility of microbe
protection against ozone by organic films has been suggested (Serra et al 2003). In
addition, the presence of such contaminated materials has been suggested as a risk for
spread of infections in medical and dental practices (Cristina et al 2008). Another
possible factor, which has been shown to play a role in other liquid anti-microbial
applications (Sattar 2004; Malik et al 2006), is the presence of a porous surface such
as fabric or carpet in which the virus or other organism is embedded. This limitation
was not seen however in our experience with ozone gas against viruses or bacteria
(Hudson et al 2007; Sharma and Hudson 2008).
As a result of these studies, we believe that the apparatus we have developed, based on
the use of ozone gas and high humidity, has many potential applications wherever
efficient decontamination of rooms is required.
Abbreviations: DMEM, Dulbecco minimum Eagle medium; PBS, phosphate-
buffered saline; pfu, plaque-forming unit (one pfu = one infectious virus particle); RH,
relative humidity; viruses: Ad 3/11, adenovirus type 3/11; FCV, feline calicivirus;
HSV, herpes simplex virus type 1; MCV, mouse coronavirus; PV, poliovirus type 1
vaccine strain; RV 1A/14, rhinovirus type 1A/14; SINV,Sindbis virus; VSV, vesicular
stomatitis virus; VV, vaccinia virus; YFV, yellow fever virus,vaccine strain.
Key words: ozone, ozone gas, antiviral, decontamination, viruses, humidity, ozone
generator, catalytic converter, field trials
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Sample #
Fraction pfu
Average log
Exp #1: HSV
(herpes simplex)
Exp #1: HSV
< 0.001
Exp #1: HSV
Exp #2: HSV
Exp #2: RV
(rhinovirus 14)
Exp #2: PV
< 0.01
< 0.01
> 2.0
Viruses were dried onto glass slides and transported to a large test room, volume 65
, where they were treated with ozone from small generators, at ambient humidity
(40% RH) and temperature (20
C), for 1 hour; the peak level attained in both
experiments was 28 ppm. Following the treatment the samples were retrieved and
transported back to the laboratory for reconstitution and subsequent infectivity assays.
Control slides were not exposed to ozone. Complete details are described in Materials
and Methods.
Fraction pfu
+ 70% RH
< 0.0012
> 2.9
< 0.0017
> 2.8
Viruses were dried onto plastic surfaces and placed in the test office for ozone
treatment at ambient humidity (38% RH) or at elevated humidity (70% RH), in
separate tests on the same day. The prototype generator was programmed to deliver up
to 20 ppm for 20 min, with or without a burst of extra humidity, followed by catalytic
conversion of ozone to oxygen, as described in Materials and Methods. At the end of
the test, samples were retrieved for reconstitution and subsequent assays.
No ozone (pfu)
+ ozone (pfu)
2.0 x 10
3.2 x 10
+ bovine serum albumin 1:1
4.2 x 10
3.1 x 10
+ human serum 1:1
4.2 x 10
4.3 x 10
+ whole human blood 1:1
1.0 x 10
1.3 x 10
Samples of dried SINV on plastic surfaces, containing the supplements indicated, were
treated with ozone in the test office, as described in the legend for Table 2, with a
burst of high humidity (90% RH).
Virus titer, no ozone
Virus titer + ozone
Exp #1
< 10
> 2.48
Exp #2
Suspensions of FCV were prepared in PBS and sprayed into the laboratory test
chamber with or without 20 ppm ozone produced by a small generator. Samples of the
condensate were collected in 6-well trays, and their volumes and content of infectious
FCV were measured.
Influenza, log
FCV, log
> 3.9
> 3.9
Dried samples of the viruses were transported to the hotel, duplicate pairs were placed
in different locations within the test hotel room, and ozone treatment conducted as
described in Table 2, with an accessory humidifier, which gave a maximum RH of
95% . Following the treatment, samples were returned to the laboratory for
reconstitution and assay, together with unexposed controls.
(> 3 LOG
(+ or -)
Herpes simplex virus (HSV)
Representative herpes virus
Adenovirus types 3 and 11
(Ad 3,11)
Representative adenoviruses
Vaccinia virus (VV)
Representative pox virus
Influenza virus
(human strain H3N2)
Representative of human and
avian influenza viruses
Murine coronavirus (MCV)
Surrogate for SARS virus
Sindbis virus (SINV)
Surrogate for Hepatitis C virus
Yellow fever virus (YFV)
Surrogate for Hepatitis C virus
Vesicular stomatitis virus (VSV)
Rhabdovirus (ubiquitous in
vertebrates, invertebrates, plants)
Poliovirus (PV)
Enteric virus
Rhinovirus types
1A & 14 (RV 1A, 14)
Common cold viruses
Feline calicivirus (FCV)
Surrogate for Norovirus
No ozone-resistant viruses have been found
Sindbis virus and Yellow fever virus are in the same virus family as Hepatitis C virus,
and have been promoted as suitable substitutes in antiviral testing.
Influenza virus, murine coronavirus, Sindbis virus, Yellow Fever virus, and vesicular
stomatitis virus are all RNA viruses with membranes, with structures similar to HIV.
Therefore this combination could be considered as a suitable substitute for HIV in
antiviral testing.
FIG 1. Prototype Ozone Generator (Viroforce 1000)
The generator contains 8 corona discharge units, a powerful circulating fan,
and a catalytic converter to convert ozone back to oxygen after the treatment.
Part of the control panel is also visible. The unit is shown by itself and located
in the test hotel room.
Not shown is the accessory humidifier.
FIG 2. Kinetics of Inactivation of Viruses on Different Surfaces.
Multiple aliquots of each virus, in separate experiments in the laboratory, were
dried onto the different surfaces, and exposed to ozone gas (10 ppm) at
ambient humidity (45% RH). Periodically duplicate samples were removed for
reconstitution and freezing. They were subsequently thawed and assayed by
plaque formation on the appropriate cell lines.
FIG 1a
FIG 1b
... All 12 viruses tested, on different hard and porous surfaces, and in the presence of biological fluids, could be inactivated by at least 3 log10. 24 Another study analyzed the influence of different species of microorganisms, relative humidity, and ozone dose on the disinfection of surfaces using this gas. The authors concluded that the survival of microorganisms and the dose of ozone (ozone concentration times the exposure time) have an exponential relationship. ...
... Therefore, although this is indeed a limiting factor, it does not impede the use of ozone. 24 ...
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Objective Airborne particles are one of the most important factors in the spread of infectious pathogens and must be monitored in healthcare facilities. Viable particles are living microorganisms, whereas non-viable particles do not contain microorganisms but act as transport for viable particles. The effectiveness of ozone in reducing these particles in a non-controlled room and a controlled cleanroom using high-efficiency particles air (HEPA) filter was analyzed in this study. Materials and Methods Viable particles and non-viable particles sized 0.5 and 5 μm were quantified before and after ozonation in two different health environments: non-controlled (group 1) and controlled area, which was associated with a HEPA filtering system (group 2). Active air sampling using a MAS 100 was used to count the number of viable particles, while the number of non-viable particles/m3 was obtained following the manufacturer's recommendations of the Lasair III 310C system. Results Our results of the viable particles counting were not quantifiable and analyzed using statistical tests. Both groups showed a slight tendency to reduce the number of viable particles after ozonation of the environmental air. A statistically significant reduction of non-viable 5 μm particles after ozonation was observed in both groups (G1: p = 0,009; G2: p = 0,002). Reduction in the non-viable 0.5 μm particles after ozonation was observed only in group 2, associated with the HEPA filter. In group 1, after ozonation, a significant increase in 0.5 μm particles was observed, probably due to the breaking of 5 μm particles by ozone gas. Our results suggest that ozone gas can break 5 μm particles and, when associated with a HEPA filter, increases its effectiveness in removing 0.5 μm particles. Conclusion Considering that 5 μm particles are important in the air transport of microorganisms, their reduction in the environment can be a relevant parameter in controlling the dissemination of infections.
... Within this context, ozone has been a subject of growing interest [9,10] Ozone (O 3 ) is a molecular gas with three oxygen atoms bonded by high-energy covalent bonds, which makes ozone a powerful oxidizing agent and, therefore, a highly antimicrobial agent. Although it is mainly used for water treatment, it has also been proven to be highly effective at eliminating bacteria, fungi, and molds, and inactivating viruses, including the SARS virus, on surfaces and in aerosols suspended in the air [11][12][13][14][15]. Its efficiency depends on the treatment conditions (e.g., concentration, exposure time, temperature, and humidity) and material properties (e.g., surface reactivity and porosity) [7,10,11,[16][17][18]. ...
... All the supplies assessed were contaminated with the SARS-CoV-2 strain 2019-nCoV/ USA-WA1/2020, which was inactivated by heating at 65 • C for 30 min (ATCC ® VR-1986HK™, ATCC, Manassas, VA, USA) at 1 × 10 3 copies/µL. In all cases, the volume of the contamination drop was 10 µL (occupying a surface equivalent to 1 cm 2 ), corresponding to 1 × 10 4 copies, which was considered a reasonable amount of virus to remain stable on a surface for enough time to experimentally evaluate the virucidal activity of the procedure [7,13,28]. After that, the samples were allowed to dry in a laminar flow hood until treatment with the corresponding ozone conditions. ...
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(1) Background: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to cause profound health, economic, and social problems worldwide. The management and disinfection of materials used daily in health centers and common working environments have prompted concerns about the control of coronavirus disease 2019 (COVID-19) infection risk. Ozone is a powerful oxidizing agent that has been widely used in disinfection processes for decades. The aim of this study was to assess the optimal conditions of ozone treatment for the elimination of heat-inactivated SARS-CoV-2 from office supplies (personal computer monitors, keyboards, and computer mice) and clinical equipment (continuous positive airway pressure tubes and personal protective equipment) that are difficult to clean. (2) Methods: The office supplies and clinical equipment were contaminated in an area of 1 cm2 with 1 × 104 viral units of a heat-inactivated SARS-CoV-2 strain, then treated with ozone using two different ozone devices: a specifically designed ozonation chamber (for low–medium ozone concentrations over large volumes) and a clinical ozone generator (for high ozone concentrations over small volumes). SARS-CoV-2 gene detection was carried out using quantitative real-time polymerase chain reaction (RT-qPCR). (3) Results: At high ozone concentrations over small surfaces, the ozone eliminated SARS-CoV-2 RNA in short time periods—i.e., 10 min (at 4000 ppm) or less. The optimum ozone concentration over large volumes was 90 ppm for 120 min in ambient conditions (24 °C and 60–75% relative humidity). (4) Conclusions: This study showed that the appropriate ozone concentration and exposure time eliminated heat-inactivated SARS-CoV-2 RNA from the surfaces of different widely used clinical and office supplies, decreasing their risk of transmission, and improving their reutilization. Ozone may provide an additional tool to control the spread of the COVID-19 pandemic.
... However, even with these caveats, the differences for our results fall inside of the twofold range of data that is considered acceptable for serological assays (Reed et al. 2002;Wood and Durham 1980). Gaseous ozone dosages of 3-300 ppm⋅min have been found to be effective in neutralizing several families of viruses from sterile surfaces (Bri e et al., 2018;Maier et al., 2016;Hudson et al., 2009). But other viruses exhibit resistance to gaseous ozone treatment, with some retaining up to 80% infectivity after over 400 ppm⋅min of ozone. ...
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Due to the immense societal and economic impact that the COVID-19 pandemic has caused, limiting the spread of SARS-CoV-2 is one of the most important priorities at this time. The global interconnectedness of the food industry makes it one of the biggest concerns for SARS-CoV-2 outbreaks. Although fomites are currently considered a low-risk route of transmission for SARS-CoV-2, new variants of the virus can potentially alter the transmission dynamics. In this study, we compared the survival rate of pseudotyped SARS-CoV-2 on plastic with some commonly used food samples (i.e., apple, strawberry, grapes, tomato, cucumber, lettuce, parsley, Brazil nut, almond, cashew, and hazelnut). The porosity level and the chemical composition of different food products affect the virus's stability and infectivity. Our results showed that tomato, cucumber, and apple offer a higher survival rate for the pseudotyped viruses. Next, we explored the effectiveness of ozone in deactivating the SARS-CoV-2 pseudotyped virus on the surface of tomato, cucumber, and apple. We found that the virus was effectively inactivated after being exposed to 15 ppm of ozone for 1 h under ambient conditions. SEM imaging revealed that while ozone exposure altered the wax layer on the surface of produce, it did not seem to damage the cells and their biological structures. The results of our study indicate that ozonated air can likely provide a convenient method of effectively disinfecting bulk food shipments that may harbour the SARS-CoV-2 virus.
... Hudson et al. reported that the maximum anti-viral efficacy of ozone required a short period of high humidity (>90% relative humidity) after the attainment of the peak ozone gas concentration (20-25 ppm). Mouse coronavirus (MCoV) on different surfaces (glass, plastic, and stainless steel) and in the presence of biological fluids was inactivated by ozone by at least 3 log 10 in the laboratory and in simulated field trials [132,133]. Here, we summarized the data of the virucidal activity of ozone water (not gas) against SARS-CoV-2 due to the different experimental methods with other chemical disinfectants (Table 6). Hu et al. implied that an ozone concentration exceeding 18 mg/L could reduce vital SARS-CoV-2 to an undetectable level effectively within 1 min [134]. ...
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The pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has emerged as a serious global public health issue. Besides the high transmission rate from individual to individual, indirect transmission from inanimate objects or surfaces poses a more significant threat. Since the start of the outbreak, the importance of respiratory protection, social distancing, and chemical disinfection to prevent the spread of the virus has been the prime focus for infection control. Health regulatory organizations have produced guidelines for the formulation and application of chemical disinfectants to manufacturing industries and the public. On the other hand, extensive literature on the virucidal efficacy testing of microbicides for SARS-CoV-2 has been published over the past year and a half. This review summarizes the studies on the most common chemical disinfectants and their virucidal efficacy against SARS-CoV-2, including the type and concentration of the chemical disinfectant, the formulation, the presence of excipients, the exposure time, and other critical factors that determine the effectiveness of chemical disinfectants. In this review, we also critically appraise these disinfectants and conduct a discussion on the role they can play in the COVID-19 pandemic.
... De acordo com pesquisas, a desinfecção de esgoto doméstico com doses de 6 mg/L de ozônio por um período médio de 46 minutos, procedida em uma etapa posterior ao tratamento convencional por métodos biológicos de lodo ativado e químicos de coagulação, propiciou a redução (HUDSON et al., 2009;BLANCO et al., 2021). ...
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O SARS-CoV-2 é um novo tipo de coronavírus capaz de infectar humanos e causar a síndrome respiratória aguda grave COVID-19, uma doença que, devido ao seu alto índice de disseminação e fatalidade, tem causado enormes impactos no Brasil e no mundo. Estudos publicados por diversos pesquisadores indicaram a frequente detecção de fragmentos do SARS-CoV-2 em amostras obtidas de redes de esgoto ou de estações de tratamento dos mesmos. A presença do novo coronavírus nesses ambientes tem levantado a possiblidade da transmissão indireta da COVID-19 via rota fecal-oral, ou seja, por meio do contato com águas residuais contaminadas. Além disso, a presença do novo coronavírus nesses ambientes também tem levantado a possibilidade da disseminação do SARS-CoV-2 em animais domesticados e selvagens, assim, propiciar a propagação do patógeno em surtos futuros por meio de infecções cruzadas. Sabendo que inibir a propagação do SARS-CoV-2 através de matrizes aquáticas tem se demonstrado de grandíssima importância como controle da COVID-19, e que o descarte ou reutilização das águas residuais de forma segura tem dependido diretamente da eficácia dos processos de tratamento das mesmas, o objetivo desta revisão foi de descrever os métodos convencionais de inativação ou remoção do SARS-CoV-2 de esgotos, e tecnologias promissoras que poderiam ser utilizadas com estas finalidades. No artigo, foram destacados os mecanismos de ação das técnicas e também breves recomendações que visavam fomentar práticas eficientes e seguras caso implementadas.
... Since a contamination-free food supply chain is the first and foremost step of food processing it is essential to disinfect the food produced just after it is harvested. Reports have suggested that ozone due to strong virucidal activity is capable of inactivating the virus as well as bacteria (Hudson et al., 2009;Yaneva et al., 2022;Alimohammadi & Naderi, 2021). Considering this challenge, the Central Institute of Post harvesting Engineering and Technology (CIPHET), India, has developed an ozone-based fruit and vegetable washer-cumpurifier known as Ozo-C, which is a portable and economical machine (ICAR-CIPHET, 2020). ...
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The food processing industry is currently facing challenges in delivering safe, healthy, and high-quality food. Constant monitoring at each step of the supply chain of food is vital to resolve the issue of food contamination. To achieve this aim and to meet consumer prospects, the technologies promoting the concept of clean label food have been widely cherished. Ozonation is one such advanced technology that assists in maintaining food product quality and safety. Its manifold approach and zero-by-product production make it a promising food disinfectant technique. Ozone due to its oxidative property has been widely used in sanitizing, washing, odor removal, water treatment, and in equipment, fruits, vegetable, and meat processing disinfection. Ozonation in foods is done in such a way that no nutritional, sensory, and physicochemical characteristics are altered. In this review, an attempt is made to give an overview of the impact and contribution of ozone as a disinfectant in food processing while comparing it with conventional disinfectants and its overall application in the food industry.
... Hudson and colleagues evaluated the effect of concentration, exposure time, and relative humidity in a study using 12 viruses. This work showed a reduction of three orders of magnitude, concerning the initial virus titer, at a concentration of 25 ppm of ozone per 15 min exposure to >90% RH [70]. Another study suggested that ozone sterilization was more effective with no air movement (no fans) at low temperature and humidity than at high temperature and humidity [71]. ...
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Background: Health care-associated infections (HAIs) are a significant public health problem worldwide, favoring multidrug-resistant (MDR) microorganisms. The SARS-CoV-2 infection was negatively associated with the increase in antimicrobial resistance, and the ESKAPE group had the most significant impact on HAIs. The study evaluated the bactericidal effect of a high concentration of O3 gas on some reference and ESKAPE bacteria. Material and methods: Four standard strains and four clinical or environmental MDR strains were exposed to elevated ozone doses at different concentrations and times. Bacterial inactivation (growth and cultivability) was investigated using colony counts and resazurin as metabolic indicators. Scanning electron microscopy (SEM) was performed. Results: The culture exposure to a high level of O3 inhibited the growth of all bacterial strains tested with a statistically significant reduction in colony count compared to the control group. The cell viability of S. aureus (MRSA) (99.6%) and P. aeruginosa (XDR) (29.2%) was reduced considerably, and SEM showed damage to bacteria after O3 treatment Conclusion: The impact of HAIs can be easily dampened by the widespread use of ozone in ICUs. This product usually degrades into molecular oxygen and has a low toxicity compared to other sanitization products. However, high doses of ozone were able to interfere with the growth of all strains studied, evidencing that ozone-based decontamination approaches may represent the future of hospital cleaning methods.
... Study has shown how ozone can deactivate strains with or without virus coverage. 47 Some strains, such as herpes simplex virus type 1 and vesicular stomatitis virus decrease after receiving ozone. Significant showed in infectious particles in 15 minutes. ...
Atmospheric ozone is produced when nitrogen oxides react with volatile organic compounds. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome contains a unique N-terminal fragment in the Spike protein, which allows it to bind to air pollutants in the environment. 'Our approach in this review is to study ozone and its effect on the SARS-CoV-2 virus and patients with coronavirus disease 2019 (COVID-19). Article data were collected from PubMed, Scopus, and Google Scholar databases. Ozone therapy has antiviral properties, improves blood flow, facilitates the transfer of oxygen in hypoxemic tissues, and reduces blood coagulation phenomena in COVID-19 patients. Ozone has immunomodulatory effects by modulating cytokines (reduction of interleukin-1, interleukin-6, tumor necrosis factor-α, and interleukin-10), induction of interferon-γ, anti-inflammatory properties by modulating NOD-, LRR- and pyrin domain-containing protein 3, inhibition of cytokine storm (blocking nuclear factor-κB and stimulating nuclear factor erythroid 2-related factor 2 pathway), stimulates cellular/humoral immunity/phagocytic function and blocks angiotensin-converting enzyme 2. In direct oxygen-ozone injection, oxygen reacts with several biological molecules such as thiol groups in albumin to form ozonoids. Intravenous injection of ozonated saline significantly increases the length of time a person can remain hypoxic. The rectal ozone protocol is rectal ozone insufflation, resulting in clinical improvement in oxygen saturation and biochemical improvement (fibrinogen, D-dimer, urea, ferritin, LDH, interleukin-6, and C-reactive protein). In general, many studies have shown the positive effect of ozone therapy as a complementary therapy in the recovery of COVID-19 patients. All the findings indicate that systemic ozone therapy is nontoxic and has no side effects in these patients.
Milk cream is a fluid milk product comparatively rich in fat, in the form of an emulsion of fat‐in‐skimmed milk, obtained by physical separation from milk, widely used in the food industry. This study aimed to evaluate the consequences of ozone treatment exposure time on lipid oxidation of milk cream, using Peroxide Value (PV) and Thiobarbituric Acid Reactive Substances (TBARS) methods. Ozonation process significantly affected PV and TBARS value (P < 0.05), in particular, the lipid oxidation increased with the ozone exposure time. The colour characteristics (L‐, a‐, and b‐ values) of milk cream samples were also affected by ozone treatment (P < 0.05); the decrease of colour parameters could be related to carotenoids degradation due to ozone treatment. Therefore, although ozone treatment is an extremely useful method to reduce the microbiological load of foods, the duration of ozone treatment is a key factor to determine the capacity of the ozonation process to balance microbial and chemical quality of foods. This article is protected by copyright. All rights reserved
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Ozone has been used for surface disinfection to contain bacterial, fungal, mold, and certain viral infections; however, the use of ozone generated from nonthermal plasma devices have not been thoroughly investigated for surface disinfection. Here, we aimed to determine the impact of nonthermal plasma‐generated ozone (PGO) on the coronavirus. Human coronavirus 229E was exposed to PGO and its infectivity was evaluated. PGO exposure of approximately 7 ppm reduced the viral titer after 4 h. Our results indicate that PGO exposure not only reduces the expression of the viral nucleocapsid gene and spike glycoprotein levels but may also stimulate the expression of the antiviral response gene in host cells. These findings can thus be useful to support existing surface disinfection methods. HCoV‐229E virus, a surrogate of SARS‐CoV‐2, was exposed to cold plasma‐generated ozone for 4 h under ambient room conditions. A significant reduction of viral infectivity in host MRC‐5 cells was observed. Exposure to plasma‐generated ozone led to a reduction in the expression of viral nucleocapsid genes and a spike in glycoprotein levels. Also, enhanced expression of antiviral response genes was observed in host cells.
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The reactions of ozone with a series of biological macromolecules are reviewed. With proteins, ozone causes the oxidation or the ozonolysis of certain amino acid residues, for instance, tryptophan, tyrosine and cysteine. As a result of this attack the protein molecules undergo changes in their usual folding and binding ability and are denaturated as shown by polarimetric or chirooptical measurements and by the inhibition of their biological activity. In any case viscosimetric measurements show that the amide bond of proteins is resistant to the ozone attack and no chain scission phenomena occur as in the case of radiolysis of proteins. A special protein is hemoglobin which is characterized by a complex tetrameric structure where each of the 4 polypeptide chains binds a prosthetic haeme group having a porphyn structure. It is shown by a series of systematic studies on model molecules, on the isolated prosthetic groups, on isolated hemoglobin (methemoglobin) and on whole blood that the action of ozone is specifically directed toward the prosthetic haeme groups of this protein causing their fission into oxidized degradation products. Therefore, ozone is selectively bound by haeme groups of hemoglobin. RNA and DNA are very reactive with ozone in comparison to proteins. The ozone attack is directed mainly toward the nucleic acids. The stoichiometric ratio between ozone and RNA monomeric unit has been determined both polarimetrically and iodometrically. The complete collapse of RNA supramolecular structure occurs at a RNA/O3 molar ratio between 2.0 and 1.5. Also DNA appears to be highly reactive with ozone, reactivity which is similar to RNA in all aspects.
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Ozone has been proposed as an alternative oral antiseptic in dentistry, due to its antimicrobial power reported for gaseous and aqueous forms, the latter showing a high biocompatibility with mammalian cells. New therapeutic strategies for the treatment of periodontal disease and apical periodontitis should consider not only antibacterial effects, but also their influence on the host immune response. Therefore, our aim was to investigate the effect of aqueous ozone on the NF-κB system, a paradigm for inflammation-associated signaling/transcription. We showed that NF-κB activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IκBα proteolysis, cytokine expression, and κB-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-κB system, suggesting that it has an anti-inflammatory capacity.
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Since the early 1990s, the use of ozone in many commercial and industrial laundering applications has been evolving rapidly. Ozone allows washing to be conducted using cold water, thereby saving considerable heat energy and water consumption. Additionally, ozone enhances the wash process, resulting in a significant reduction in detergent dosage and number of rinses, thus saving water. Ozone/cold water cycles are gentler to fabrics, thus extending linen life. Finally, ozone/cold water laundering is beneficial for effluents, resulting in reductions in COD (chemical oxygen demand). Microorganisms are destroyed effectively in ozone-wash waters, and washing and drying cycles are shorter, thus saving labor. In this paper, the authors describe some specific case studies at commercial laundering installations in the UK, whereby the users of ozone have reaped major benefits, including enhanced microorganism kills/inactivation and significant cost savings.
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Bacterial infections continue to pose a threat to health in many institutional and communal settings, and epidemics are frequent. Current control measures are clearly inadequate; thus, there is a need for a simple, effective, and safe way to decontaminate surfaces. We evaluated the efficacy of a portable ozone-generating machine, equipped with a catalytic converter and an accessory humidifier, to inactivate 15 different species of medically important bacteria. An ozone dosage of 25 ppm for 20 minutes, with a short burst of humidity in excess of 90% relative humidity, was able to inactivate more than 3 log(10) colony-forming units of most of the bacteria, including Acinetobacter baumannii, Clostridium difficile, and methicillin-resistant Staphylococcus aureus, in both in a laboratory test system and simulated field conditions. In many cases, complete eradication was achieved. Dried and wet samples were equally vulnerable to the ozone. Inactivation of bacterial samples dried onto soft surfaces (eg, fabric, cotton, filter paper) were comparable with that observed for samples on plastic. The ozone generator can provide a valuable decontamination tool for the removal of bacteria in many institutional and communal settings, including hospitals and other health care institutions.
Ozone has the strongest oxidization effect after fluorine, and this property has been used in sterilization for food and processing plants in Japan. Moreover, there is no fear of toxic residues as with chlorine-based sterilizers and no hazardous trihalomethanes are formed. Based on these advantages, ozone has been used in water and air treatment for food products food materials and food processing plants. Use in the food processing is now increasing in Japan. This paper describes ozone sterilization, introducing sterilization systems and equipment applied to food in Japan.
Recent food putrefaction with new strains of microorganisms, such as Lactic acid bacteria and disinfectant-resistant strains of gram-negative bacteria, mold, yeast have increased interest in exploring different disinfection techniques for food sanitation. In Japan, food processing companies consider Lactic acid bacteria and gram-negative bacteria, mold, yeast, to be of greatest concern because of the severity and number of food putrefaction they cause. We supplied a laboratory machine that sterilizes for food packaging film and preformed cups using ozonated water and ozone gas. UV-ray sterilizing can be used for support. Ozone should be useful in reducing the degree of microbial contamination caused by inadequate disinfection against new resistant strains. There is growing tendency to use ozone in food industry as an effective means of disinfection without any additives. Many types application equipment have been developed. Based on the properties of ozone as a strong germicidal agent, conversion of factories to use ozone for sterilization of food packaging film is being implemented.
A device was designed to deliver a constant source of given concentrations of ozone to fluids containing human immunodeficiency virus type 1 (HIV-1). Ozone was found to inactivate HIV-1 virions in a dose-dependent manner. Greater than 11 log inactivation was achieved within 2 hours at a concentration of 1,200 ppm ozone. Similar concentrations of ozone had minimal effect on factor VIII activity in both plasma and immunoaffinity-purified preparations of factor VIII treated for the same time period. The data indicate that the antiviral effects of ozone include viral particle disruption, reverse transcriptase inactivation, and/or a perturbation of the ability of the virus to bind to its receptor on target cells. Ozone treatment offers promise as a means to inactivate human retroviruses in human body fluids and blood product preparations.
The inactivation of human immunodeficiency virus (HIV) and cytotoxic properties of ozone-treated serum and serum-supplemented media were examined. The titer of HIV suspensions in human serum was reduced in a dose-dependent manner when treated with total reacted ozone concentrations at a range of 0.5 to 3.5 micrograms/ml-1. Complete inactivation of HIV suspensions was achieved by 4.0 micrograms/ml-1 of ozone in the presence or absence of H-9 cells. In contrast, cellular metabolism, as measured by MTT dye cleavage, and DNA replication, as measured by BUdR incorporation, were enhanced in H-9 cells grown in media treated with quantities of ozone that completely inactivate HIV. The permissively HIV-infected cell line HXB/H-9 was cultured in ozone-treated media for six days with culture supernatants being sampled and assayed on alternate days for HIV p24 core protein. HIV p24 was reduced in all treated cultures compared to control cultures, with an average reduction of 46% [p24].
Acute exposure of humans to ozone results in reversible respiratory function decrements and cellular and biochemical changes leading to the production of substances which can mediate inflammation and acute lung injury. While pulmonary function decrements occur almost immediately after ozone exposure, it is not known how quickly the cellular and biochemical changes indicative of inflammation occur in humans. Increased bronchoalveolar lavage (BAL) fluid levels of neutrophils (PMNs) and prostaglandins (PGE2) have been reported in humans as early as 3 hr and as late as 18 hr after exposure. The purpose of this study was to determine whether a broad range of inflammatory mediators are elevated in BAl fluid within 1 hr of exposure. We exposed eight healthy volunteers twice: once to 0.4 ppm ozone and once to filtered air. Each exposure lasted for 2 hr during which the subjects underwent intermittent heavy exercise (66 liters/min). BAL was performed 1 hr after the exposure. Ozone induced rapid increases in PMNs, total protein, LDH, alpha-1 antitrypsin, fibronectin, PGE2, thromboxane B2, C3a, tissue factor, and clotting factor VII. In addition, there was a decrease in the recovery of total cells and alveolar macrophages, and decreased ability of alveolar macrophages to phagocytize Candida albicans. A comparison of these changes with changes observed in an earlier study in which subjects underwent BAL 18 hr after an identical exposure regimen indicates that IL-6 and PGE2 levels were higher 1 hr after exposure than 18 hr after exposure, fibronectin and tissue-plasminogen activator levels were higher 18 hr after exposure, and that PMNs, protein, and C3a were present at essentially the same levels at both times. These results indicate that (i) several inflammatory mediators are already elevated 1 hr after exposure; (ii) some mediators achieve their maximal levels in BAL fluid at different times following exposure. These data suggest that the inflammatory response is complex, depending on a cascade of timed events, and that depending on the mediator of interest one must choose an appropriate sampling time.