Whole Room Disinfection

Members only
Whole room disinfection
2010
R&D report no. 299
R&D report no. 299

Whole room disinfection
A Malinowska and J Holah
2010
Campden BRI 2010
Station Road, Chipping Campden, Gloucestershire, GL55 6LD. UK

Tel: 01386 842000
www.campden.co.uk
Information emanating from this company is given after the exercise of all reasonable care and skill in its compilation, preparation and
issue, but is provided without liability in its application and use.
Legislation changes frequently. It is essential to confirm that legislation cited in this publication and current at time of printing, is still in
force before acting upon it.
The information contained in this publication may not be reproduced without permission from the Publications Manager.
SUMMARY
To meet retailer, customer and consumer expectations, there are increasing demands within
the food industry for higher standards of control of microorganisms in food production
environments. This requirement for further reduction of pathogens and the identification of
persistent strains has led to a significant interest in the use of whole room disinfection
techniques to supplement routine cleaning and disinfection.
A range of whole room decontamination systems are available commercially, including
ultraviolet light, titanium dioxide and ultraviolet light, ozone, hydrogen peroxide vapour
(HPV) and ionisation, but these techniques have had little microbiological assessment for
their use in the food and drink industry.
This report describes work undertaken to investigate the efficacy of whole room disinfection
on microorganisms attached to surfaces both in the laboratory and in the factory
environment. The microorganisms assessed were Listeria monocytogenes, Pseudomonas
aeruginosa and Staphylococcus aureus, selected because of their known presence, and in
some cases persistence, in the food factory environment. Bacillus subtilis var. globigii was
also assessed with hydrogen peroxide to test its sporicidal activity. This work was also
undertaken to examine the effect of whole room disinfection techniques on surfaces placed
in different spatial orientations.
Chemical fogging using quality disinfectants was effective at reducing airborne microbial
populations by 2 to 3 log orders in 30 to 60 minutes and attached microorganisms on
horizontal surfaces by up to 6 log orders in 60 minutes, with minimal effect on vertical
surfaces and underneath equipment. The amount of chemical released by nebulisers is so
small (relative to chemical fogging), that the level of decontamination achieved on surfaces
is poor. However, the particle size of chemical produced appears to be small enough to
interact with surfaces of all orientations, over a longer time period in which the particles
remain airborne, so what decontamination it does achieve is approximately the same over
all surface geometries.
At low concentrations of (10 g/m3), the effect of HPV was much more pronounced on the
spores of B. subtilis var globigii than on the vegetative bacteria tested, with an approximate
6 log reduction of globigii spores being achieved. At concentrations of 20 g/m3, an
approximately 3 log reduction was achieved with P. aeruginosa, S. aureus and L.
monocytogenes. At this concentration of HPV, the vegetative microorganisms (all of which
are catalase positive) were all able to partially resist the concentration/volume of disinfectant
that they came into contact with. Spores, however, do not express catalase and therefore
have no effective defence mechanism. When the HPV concentration was increased to 40
g/m3, an increase in susceptibility was seen with P. aeruginosa and L. monocytogenes,
resulting in a reduction in excess of 4 logs for P. aeruginosa and 5 logs for L.

monocytogenes. This is in excess of the 4 log reduction required in the European Surface
Test (EN 13697) for surface disinfectants. S. aureus was significantly more resistant than
the other vegetative organisms and its log reduction was not increased at 30 or 40 g/m3.
For ozone, a relationship between ozone concentration and log reduction was established,
though the effect of ozone on each of the three vegetative strains tested was markedly
different with, at 20ppm, S. aureus being most resistant followed by P. aeruginosa and then
L. monocytogenes being most sensitive. A log reduction of >4 logs was achieved for
L. monocytogenes at an ozone concentration of 20ppm, which equates to the requirements
of the European Surface Test (EN 13697) for chemical disinfectants. A log reduction
approaching 4 logs was also achieved for P. aeruginosa. This would suggest that under the
conditions of the practical trials, ozone is more effective than HPV for vegetative
microorganisms.
For both HPV and ozone, there is little practical difference between the log reductions
achieved on horizontal, vertical and underneath surfaces and as such, they can effectively
penetrate every part of a room, including sites that might prove difficult to gain access to
with conventional liquids and manual disinfection procedures. The major disadvantage of
using gases, such as ozone and HPV, is the potential toxicity at high concentrations, which
precludes using them in areas where people are working. The techniques can therefore
only be used in areas that can be isolated and sealed off during the decontamination
process.
Field trials in which ozone was used as a periodic room decontaminant showed little effect
over 1-3 days using an ozone concentration of 8ppm, and the overall reduction in counts
after disinfection was probably less than 1 log order. After 3 days, however, the results for a
pizza manufacturer potentially showed a downward trend in the numbers of microorganisms
present, both before cleaning and after cleaning and disinfection, throughout the three day
period. This may be because for all areas in which ozone is used, oxidisable material within
the room may create an ozone demand which must be satisfied by oxidation before any
significant decontamination of microorganisms can occur. A dosage equivalent to at least
90 ppm for an hour (e.g. 30 ppm for 3 hours), as postulated from the laboratory studies
undertaken, may be required to produce a 5-6 log reduction in vegetative microorganisms
(e.g. L. monocytogenes) as a periodic room decontaminant.
In a 4 week field trial, there was no evidence that ozone was unable to maintain control of
the microflora of the food contact surfaces after disinfection. TVC counts after disinfection
for ozone treated food contact surfaces compared favourably to the post disinfection counts
of combined disinfectant approval trials conducted at other chilled food plants. No adverse
effects have been reported for ozone on the structure and fabric of the building since the
ozonation equipment has been installed. A daily ozone treatment of 8ppm for 30 minutes
may therefore be appropriate as an adjunct to or replacement of chemical disinfection,
though the use of ozone as a replacement for chemical disinfection must be appropriately
validated for each factory situation.
CONTENTS
Page No.
1.
BACKGROUND
2.
INTRODUCTION
3.
MATERIALS & METHODS
3.1
Laboratory trials
3.1.1
Chemical fogging
3.1.2
Hydrogen peroxide vapour
10
3.1.3
Ozone
11
4.
3.2
Field trials
12
3.3
Statistical analyses
18
RESULTS AND DISCUSSION
18
4.1
Pre-trial
23
4.2
Laboratory trials
25
4.2.1
Chemical fogging
25
4.2.2
28
4.2.3
Ozone
31
4.3
Field trials
36
5.
CONCLUSIONS
43
6.
REFERENCES
47
APPENDICES
Appendix 1
Media recipes
Appendix 2
Laboratory and field trial data
Appendix 3
Equipment manufacturer and contact details
1.
BACKGROUND
During manufacture, food can be exposed to microbiological cross-contamination from

surfaces and the air, which may give rise to food spoilage and safety issues. The traditional
approach to controlling such contamination has been to target specific sites within the
manufacturing environment with cleaning and disinfection regimes. The primary focus is
typically on food production equipment and much of the rest of the processing area, whilst
cleaned, is not routinely decontaminated. This targeted approach may have been sufficient
to maintain day-to-day control of contamination, but has not eliminated all of the organisms
responsible and, in some instances, microbial strains have become persistent in food
factories, surviving for several years. Previous research at Campden BRI assessed the
microbial flora of the high risk areas of five chilled food factories, all operating good practice
cleaning and disinfection regimes, and identified persistent strains of Listeria spp. and
Escherichia coli that had remained in the processing environment in excess of three years
(Holah et al., 2002; Holah et al., 2004). Clearly, if there was any loss of hygiene control in
these factories, these organisms would always be present and therefore could present a
constant risk to product safety.
In high risk food processing areas, thorough disinfection of surfaces is required in order to
reduce the numbers of microorganisms and to prevent transmission of these contaminants.
The belief is that regular use of novel disinfection techniques that can decontaminate the
whole of the area will reduce the number of environmental microorganisms in the production
areas; this may well improve the quality and safety of the food being produced, thereby
reducing wastage and increasing profitability.
2.
INTRODUCTION
There is now a desire to supplement traditional, targeted chemical disinfection with

alternative approaches which will control the processing area in its entirety, a technique that
can be termed as whole room disinfection. Novel disinfection techniques that are able to
disinfect whole areas have been implemented in the pharmaceutical and clinical sectors, but
there is limited information on the ability of these techniques to be applied to the food
processing environment.
The range of techniques designed for whole room disinfection is increasing, but those that
are commercially available include:
- chemical fogging
- hydrogen peroxide vapour
- ozone
- chlorine dioxide
- ultraviolet light
- titanium dioxide coating and ultraviolet light
- ionisation
The critical factors to address before using these techniques include: identifying areas
where the decontamination processes can be applied, health and safety issues related to
using the technique, any effect on the fabric of the equipment and the environmental
building materials, and the practical considerations related to their use in the food
processing environment.
There is also a need to understand how often a whole room disinfection method will be used
in the production area. The techniques can be used on a daily basis, after the routine
cleaning and disinfection procedure has been implemented or, as seen in some factory
environments, they can be used daily to replace the terminal disinfection step. There is also
the option to use a whole room disinfection technique as part of the periodic cleaning and
disinfection procedures which may occur monthly, quarterly or annually, or they may only be
used for decontaminating an area after a pathogen contamination incident.
The level of disinfection that the whole room disinfection systems can achieve also needs to
be determined, as some may achieve decontamination of all exposed room surfaces, such
as ceilings, walls, floors and food processing equipment, but others may include some
penetration into equipment to contact indirectly exposed surfaces. They may also provide
disinfection of the air in the area being treated. The advantages of using these techniques
are that for some, the decontamination process can be certified, providing documented
evidence that the procedure has taken place.
Chemical fogging, nebulisers, hydrogen peroxide vapour (HPV) and ozone (O3) were
studied in this work.
Chemical fogging
Applying chemical disinfectants to production areas as fogs or mists is a method that has
been used routinely in the food industry to control cross contamination. The purpose of
fogging a production area is to create and disperse a disinfectant aerosol to reduce the
numbers of airborne microorganisms and also to apply disinfectant to surfaces that may be
difficult to reach, such as overhead surfaces. Fogging is done by using either a static,
purpose built system in an area of a factory with strategically placed nozzles, or more
commonly by using a mobile unit. The equipment works by supersaturating the atmosphere
with a fog of disinfectant chemical.
The disinfectant fog is generated into the local atmosphere by charging the unit with
compressed air and forcing the disinfectant solution through dedicated nozzles, producing
particles in the range of 10 to 20 m. Research carried out by Burfoot et al. (1999)
demonstrated that fogging was most effective when using compressed air-driven fogging
nozzles producing fog droplets with a median diameter of between 10 and 20 m, as the
droplets in this size range dispersed well and settled within 45 minutes. An air velocity of
100 m s-1 was required at the nozzle for effective dispersal of the chemical. Larger particles
can be used if the air velocity is increased or fans are used to assist with the distribution of
the droplets. Under typical conditions, fogging is carried out for a minimum of 15 to 30
minutes to enable the fog to disperse and the chemical action to occur and after fogging, a
period of 45 to 60 minutes is required to allow the droplets to settle out of the air and onto
the surfaces, reducing the risk of operators inhaling the chemical droplets.
Disinfectants that can be fogged include quaternary ammonium compounds (QACs),
amphoterics and peracetic acid (PAA). Typically, a formulated QAC based disinfectant will
be used for fogging at concentrations between 1 and 3%. However, if the site has a
problem with spores, a QAC disinfectant will not be as effective and an option would be to
use a PAA based disinfectant at 1% concentration. PAA is ideal for fogging, due to its
natural breakdown into water and a low concentration of acetic acid and therefore many
applications do not require rinsing.
Areas of application
Chemical fogging is primarily carried out in high risk processing environments, particularly in
factories manufacturing cheese, salad, sandwiches, ready meals and cooked meats, and in
dairies. It is estimated that more than 50% of chilled food manufacturers conduct this
method of disinfection periodically, in conjunction with the routine cleaning and disinfection
routines. Within the production environment, applications include freezers, chillers, ripening
rooms, storage areas and process lines.
Due to its rapid degradation into innocuous by-products, decontamination with hydrogen
peroxide vapour (HPV) is a technique that has been widely used for disinfection of the
pharmaceutical environment, including clean rooms and production filling lines, and
therefore it may be an alternative to chemical fogging for the food industry. HPV is said to
have excellent material compatibility and is safe for use on a wide range of metals, including
stainless steel and aluminium, plastics such as polypropylene and polycarbonate, and other
materials, such as electronics. However, the efficacy of HPV is reduced by the presence of
organic matter; therefore, cleaning the area prior to use is essential. The disadvantage of
using HPV is the potential toxicity at high concentrations, which prevents the technique from
being used in areas where people are working. Therefore the decontamination process can
only be used in rooms that can be sealed off or quarantined for the duration of the treatment.
Mobile systems can be used throughout the factory environment, or areas can be equipped
with ports to which the equipment can be docked while the decontamination procedure is
carried out. The most commonly used aqueous solution of H2O2 is 30 or 35% w/w, which is
frequently evaporated to produce H2O2 concentrations ranging from 0.1 to 10 mg L-1
(0.00001 to 0.001%) depending on the exposure temperature, which ranges from 4 to 80oC.
The HPV is applied to the room in a heated carrier gas, initially air, and vapours from the
room are returned to the gas generator where further quantities of the H2O2 are evaporated.
As more vapour is introduced into a room, the pressure and concentration of the
peroxide/water vapour will increase. The gaseous state is therefore advantageous because
it ensures uniform contact with all surfaces, including horizontal and vertical surfaces and
cracks. The term 'dry fog' is sometimes used to describe the vapour with droplet sizes
below 10m. Hydrogen peroxide gas diffuses passively when introduced into a given area
and therefore constant movement of the gas is required to ensure that all surfaces are
contacted. This can be aided at atmospheric pressure by using fans or air-handling
systems, or by introducing a slight positive or negative pressure in the area being fumigated.
Hydrogen peroxide demonstrates a broad spectrum of efficacy against viruses, bacteria,
mycobacteria, fungi and bacterial spores. Its activity as a powerful oxidising agent is known
to damage cellular proteins, lipids and nucleic acids, and the increased reactivity of the
gaseous peroxide may be due to the greater presence of the short lived radicals and ions
that form. It is more effective against Gram positive than Gram negative bacteria; however,
the presence of catalase or other peroxidases, particularly in Gram positive bacteria, such
as Staphylococcus spp., allows increased tolerance, due to enzymatic degradation. Elkins
et al. (1999) carried out a study to assess the significance of catalase expression in the
protection of Pseudomonas aeruginosa against H2O2 and demonstrated that KatA catalase
was important for resistance of planktonic and biofilms of P. aeruginosa to H2O2, particularly
at high concentrations. The sporicidal concentration for HPV has been identified in the
range of 0.1 to 3.0 mg L-1 at room temperature (Meszaros et al., 2005).
Ozone
This chemical has been used for decades for water treatment, as it inactivates a wide range
of micro-organisms through oxidation, but the benefit of using ozone in the food industry is
that it is environmentally friendly, with any residual ozone spontaneously decomposing to
oxygen.
Ozone is a triatomic form of oxygen, which is unstable and breaks down into molecular
oxygen. The half-life for this reaction is approximately 20 minutes and this rapid process of
natural degradation is both an asset and a liability. On the plus side, it means that the
ozone disappears literally without a trace, leaving no chemical by-products, but on the minus
side, it means that ozone has a very short effective life. High reactivity, penetrability and
spontaneous decomposition into a non-toxic product make ozone a viable disinfectant for
use in food production areas.
Due to its reactive, unstable nature, ozone is produced at the point of use. Ozone
generators effectively pass air through a high-energy source within the equipment and the
resulting physicochemical reaction leads to the formation of ozone that can be used for area
or surface decontamination. Widely used high-energy sources include UV light,
electrochemical cells or corona discharge. A corona is formed by an electrical discharge
around a gas, which causes ionisation and consequently the formation of ozone. The
production of ozone is most effective in a temperature-controlled environment, since the
stability of ozone decreases as the temperature increases.
Microorganisms inherently vary in their sensitivity to ozone with factors such as temperature,
humidity and presence of chemicals; the amount of organic matter surrounding the cell also
greatly affects the degree of inactivation. At the concentrations typically used, ozone is an
effective bactericide and virucide, with greater resistance observed with mycobacteria and
bacterial spores. Effective sporicidal activity is only seen at high relative humidity (75 to
95%). Yeasts and moulds have been reported to have a wide range of resistance profiles
but are generally less resistant than bacterial spores.
Work by Fan et al. (2007) showed that the average time for a 2 log reduction of Listeria
innocua on solid media was 1.3 hours at 20oC, and 2.5 hours at 5oC at 100 nl L-1 ozone
concentration. Work by Taylor and Chana (2000) indicated a 2 log reduction in both
airborne and surface adhered Pseudomonas aeruginosa in 2 h when exposed to 2 ppm
ozone.
3.
MATERIALS & METHODS
3.1
Laboratory trials
The purpose of the laboratory trials was to develop methods to examine whether the
systems under assessment were able to decontaminate all surfaces, irrespective of
orientation, throughout the whole room. The laboratory trials protocol was based on the
European Norm surface disinfectant test method BS EN 13697: 2001 - Chemical
disinfectants and antiseptics - Quantitative non-porous surface test for evaluation of the
bactericidal activity and/or fungicidal activity of chemical disinfectants used in food,
industrial, domestic and institutional areas.
There are various types of chemical fogging, hydrogen peroxide vapour and ozone
techniques that are commercially available; representative techniques were used in this
study. Each whole room technique available, however, will differ slightly in its application,
such that there may be small differences in their overall performance. The results from the
techniques used in this study are thus specific to these techniques, but are generally
reflective of the performance of that technology.
Preparation of stock and working cultures
Cultures of Staphylococcus aureus (NCIMB 9518), Pseudomonas aeruginosa (NCIMB
10421) and Listeria monocytogenes (NCTC, NCI03 57-09) were maintained on Tryptone
Soya Agar slopes (TSA; Oxoid CM 131; Lab 305), stored at 4C, and were re-cultured from
beads every month to maintain viability. When a working culture was required, the culture
was sub-cultured onto a TSA slope and incubated for 24 hours at 37C. A first subculture
was used as the working culture and was recovered from the slope by adding 5g of sterile
glass beads (VWR International Ltd., BDH, 2.5-3.5 mm) and 9ml of diluent (BS - see
Appendix 1) to each slope. The slopes were then shaken gently to remove the culture from
the agar surface. The resulting suspension was then filtered through a funnel containing
sterile glass wool and eluted further with diluent to maximise recovery. The optical density
of the bacterial suspension was measured at 420nm (Spectrophotometer Libra S4,
Biochrom Ltd.) and calibration graphs of absorbance against viable count were used to
prepare a concentration of 108cfu ml-1. A 1ml volume of this bacterial suspension was
transferred into 9ml of diluent (10-1 dilution) for further dilution, as required.
Inoculation of surfaces
For each test, 25 stainless steel discs (2 cm diameter, Grade 2 B 1.4301 (EN 10088-1),
EN 10 088-2), previously sterilised, were inoculated with 0.05ml of 108cfu ml-1 test
suspension. The suspension was dried onto the discs at a temperature of 37C for
approximately 1 hour. The discs were allowed to equilibrate to room temperature before the
test was commenced. A total of 20 discs were treated and 5 discs were left untreated
(positive controls).
Microbial coverage
Epifluorescence microscopy was used to show the attachment and distribution of S. aureus,
P. aeruginosa and L. monocytogenes dried onto stainless steel discs. Three coupons were
inoculated with 0.05ml of 108cfu ml-1 test suspension of each test microorganism and dried
at a temperature of 37C for approximately 1 hour. The discs were allowed to equilibrate to
room temperature and then stained with Acridine orange (HD Supplies, UK) for 3 minutes.
The stain was then rinsed of using sterile distilled water (SDW). The discs were then left to
dry in the dark and analysed using a x100 oil immersion lens connected to a microscope
(Leica DM 2000) with UV light system (ebq 100) and Leica's Image Processing and Analysis
toolkit (Leica QWin) running under the industry standard Microsoft Windows environment.
Experimental set-up
The experiments were conducted in an aerobiology laboratory, 350 cm wide, 405 cm long
and 300 cm high (volume 43 m3). The 20 discs were positioned on metal stands throughout
the air laboratory in three orientations: horizontally, vertically and underneath the shelf, as
shown in Figure 1. Five discs were left untreated and processed at the same time as test
discs.
Figure 1 - The arrangement of test surfaces in the aerobiology laboratory
During experiments, all ventilation systems in the laboratory were switched off and the room
was effectively sealed to outside air movements. Any internal air currents during the trials
were created by the operation of the technology under test.
Disc analysis
After conducting the whole room disinfection process, the discs were aseptically transferred
into sterile plastic universal containers (Sterilin, diameter 4-5cm) containing 5g sterile glass
beads (diameter 2.5-3.5 mm) and 9ml diluent and 1ml sodium thiosulphate inactivator (see
Appendix 1). The containers were agitated on a horizontal surface for 1 minute to recover
the remaining bacteria into suspension. Each sample was serially diluted in diluent to 10-4
and plated out in duplicate using TSA. To validate the bacterial recovery process, each disc
was recovered from its container and rinsed with 10 ml sterile distilled water (SDW). Each
disc was then placed test side up on a pre-poured TSA agar plate and 0.1 ml SDW was
pipetted onto the disc and rubbed over the surface with a pipette tip for 1 minute. The discs
were then over poured with TSA agar. All plates were incubated at 37C for 48 hours.
The plates were then enumerated and the colony forming units (cfu) per test surface
calculated. From the test results and those recorded for the positive controls, the log
reduction in bacteria after each treatment was calculated.
3.1.1
Chemical Fogging
Two systems were assessed: a traditional fogging system (H&M Disinfection System Ltd.,
Total Hygiene Solution) and a dry mist system (Nebulair).
The chemical fog was applied using a drum top fogger (H&M Disinfection System Ltd ) using
2% disinfectant solution (B1878 Byosan concentrate) supplied by Byotrol. The fogging
system was connected to compressed air and adjusted to 4 bar working pressure. The
contact time was 40 minutes with an additional 1 hour to ventilate the room.
The Nebulair system from Mercatos used 'dry' mist technology to produce micro-particles
(size 1.22 m) of disinfectant that move in the air currents of the room, enabling the
disinfectant to contact all surfaces. Byosan A1616 (Byotrol) was used in different
concentrations: 10%, 20% and 50%. The Nebulair system was switched on for an hour
treatment and then the room was left for an hour to defog before re-entry.
The Cleanaer device is an aerosol technology that delivers very small quantities of liquid in
the form of charged droplets to the environments treated. The Cleanaer device uses an
electrospray technology that delivers small amounts of liquid into the air without tusing heat
or propellants. At the spray face of the device, an electric field creates fine droplets from a
spray capillary. The electrical field is produced by a high voltage, but there is very low
current drawn, so the power at the spray face is tiny (less than 10mW). The technology is
battery-powered. Droplets produced by the electrospray are partially discharged by the
device and are carried away from the spray face by mutual repulsion and air currents.
Four Cleanaer air aerosol technology battery-operated units were installed in the
aerobiology test area, at a height of ~190 cm, against the side wall. The units were
activated by leading electrical connections outside the room to an external on/off switch.
During the first trial, P. aeruginosa discs were exposed to the treatment for 3 hours. During
the second trial, S. aureus discs were exposed to 6 devices positioned against the walls
(~190 cm and ~60 cm height) for 5 hours. Electrostatically charged particles were released
from the liquid fed devices during approximately 83% of the actual time the devices were
working. One device used approx. 2 ml of the 2% biocide solution supplied.
3.1.2
All the trials were conducted using the proprietary Bioquell process. The Bioquell Clarus
R/R2 produced HPV which was subsequently micro-condensed onto a surface to give a high
concentration of H2O2. HPV was generated by dropping a measured volume of 30% w/w
liquid onto a vapouriser heated to 130C. The resulting HPV was injected until the air was
saturated, at which point hydrogen peroxide began to condense on a surface at a high
concentration (up to 70%).
The HPV decontamination cycle consisted of three phases, in a one step process:
(i) Conditioning: the vapouriser is heated to operational temperature.
(ii) Injection: HPV is injected for a defined period. HPV is delivered via a dual axis
distribution system to ensure a high velocity and even distribution throughout the room.
When the required amount of H2O2 has been injected, the enclosure is allowed to dwell with
no further injection to ensure adequate H2O2 exposure.
(iv) Aeration: The aeration equipment is activated, which catalyses the decomposition
process by passing the air/vapour mixture through an activated carbon filter and breaking
down the HPV to water vapour and oxygen.
The HPV concentration, temperature and relative humidity within the room were measured
by an instrumentation module and monitored by a control computer situated outside the
room, which provided real time feedback of the cycle progress.
Bioquell's hydrogen peroxide vapour equipment was placed in the aerobiology laboratory.
The room was then sealed with a sally tape supplied by Bioquell. The cycle usually lasted
for several hours. Two hydrogen peroxide concentrations were tested. 10g/m3 was used
for the first set of trials; the concentration was increased to 20 g/m3 for the second set of
trials. Additional one-off experiments were conducted using higher concentrations of 30 and
40 g/m3. All steps were monitored by a control computer situated outside the room.
10
3.1.3
Ozone
All the trials were conducted using the proprietary Steritrox patented process including,
where appropriate, their quench technology. The Radical Small Mobile Unit (UMB) from
Steritrox was a portable machine, which operated independently of mains electricity and was
positioned in the centre of the room. The operator selected the relevant setting from the
touch-screen menu and left the area. The machine auto cycled through all of the phases of
the decontamination cycle with constant visual alarms to communicate that it was in
operation. The ozone decontamination cycle consisted of three phases:
(i) Humidification: for maximum antimicrobial activity of ozone, the area was humidified to
70 to 90%.
(ii) Decontamination: the ozone vapour initially reacts with any volatile organic compounds
present in the atmosphere of the area being decontaminated, a process known as ozone
debt absorption. Having overcome this, the vapour concentration builds rapidly, to a test
concentration (which was 8 to 25 ppm).
(iii) Aeration: a biocidal quenching agent was used during the final phase of the cycle to
mop up the remaining ozone, leaving the room safe for immediate re-occupation.
Steritrox's ozone equipment was placed in the aerobiology laboratory. The room was then
sealed with a sally tape supplied by Steritrox. The cycle lasted from 40 minutes up to
several hours, depending on concentration and contact time. These trials were undertaken
with Steritrox's engineer in assistance.
Steritrox laboratory trials
Ozone trials were also undertaken at Steritrox's laboratory in Pershore, Worcestershire.
For the first trial, 25 stainless steel discs, previously sterilised, were inoculated with 0.05ml
108cfu ml-1 S. aureus test suspension. The suspension was dried onto the discs at a
temperature of 37C for approximately 1 hour. Then the discs were allowed to equilibrate to
room temperature before the test commenced. A total of 20 discs were treated and 5 discs
were left untreated (positive controls).
For the second trial, 10 stainless steel discs for each microorganism, previously sterilised,
were inoculated with 0.05 ml 108cfu ml-1 S. aureus, P. aeruginosa or L. monocytogenes test
suspension. The suspension was dried onto the discs at a temperature of 37C for
approximately 1 hour. Then the discs were allowed to equilibrate to room temperature
11
before the test was commenced. A total of 7 discs were treated and 3 discs were left
untreated (positive controls) for each microorganism.
All test and control surfaces were inoculated on the day of the trial in the Food Hygiene
Laboratory. After inoculation and drying, all samples were packed and transported to the
Steritrox facilities. After treatment, the samples were again packed and transported back to
Campden BRI for processing.
The experiments were conducted in a laboratory with a volume of 75 m3 and the discs were
positioned horizontally on a stainless steel table.
The ozone trials undertaken in both establishments can be summarised as follows:
(i) Laboratory trials at Steritrox
exposure to 25 ppm ozone for 90 minutes, testing against S. aureus only.
exposure to 8 ppm ozone for 30 minutes, testing against S. aureus, P. aeruginosa and L.
monocytogenes.
(ii) Campden BRI laboratory trials
exposure to 20 ppm ozone for 30 minutes, then manual ozone removal using extract
fans, against S. aureus and L. monocytogenes.
exposure to 20 ppm ozone for 30 minutes with natural decay to 15 ppm and manual
removal using extract fans, testing against S. aureus, P. aeruginosa and L.
monocytogenes.
Ozone dosage calculations
The dosage of ozone was calculated by multiplying the ozone concentration used in ppm
and the contact time in hours.
3.2
Field trials
The field trials were conducted in conjunction with representatives from Steritrox. Three
single-use ozone decontamination trials in factories were assessed. Those assessments
included representative factories of the RTE, poultry and dry food industry. In addition, one
4 week, continuous factory trial was completed in a sandwich factory.
The field trial methodology was based on a procedure for the approval of terminal
disinfectants for use in high-care areas of a major retailers food suppliers.
12
Swabs
A total of 10 swabs and 10 contact plates were taken at different sites within the
environment. The sites were chosen to represent flat, open, easily cleanable surfaces, and
those areas not expected to be cleaned easily; this included food contact surfaces and nonfood contact surfaces of a variety of materials of construction.
The swab samples were taken with sterile plastic applicators (Sterilin), using the method
described in the Campden BRI Manual of Hygiene Methods for the Food and Drink Industry
Guideline No. 45 (2003). The swabs were then placed in 9ml of Maximum Recovery
Diluent (MRD; LABM 103) and 1ml of sodium thiosulphate inactivator (See Appendix 1).
On receipt in the laboratory, the samples were vortexed using a Rotamixer (Hook and
Tucker Instruments Ltd.) for 30 seconds to resuspend the micro-organisms.
Total Viable Count (TVC)
A 1 ml sample of the swab resuspension fluid was taken and used to prepare a serial
decimal dilution series in MRD. Duplicate 1 ml aliquots were pour plated with Nutrient Agar
(NA; Oxoid CM3) and incubated at 30oC for 48 hours. The colonies were counted and the
results expressed as colony forming units (cfu) per swab.
Contact plates
Contact plates were taken using the method described in the Campden BRI Manual of
Hygiene Methods for the Food and Drink Industry Guideline No. 45 (2003), using 55 mm
contact plates (Sterilin) filled with NA to give a convex meniscus. Colonies appearing after
incubation at 30oC for 48 hours were counted and the results expressed as cfu per plate.
Controls
On each sampling day, portions of microbiological media were incubated at 30oC for 48
hours (NA) and enumerated as positive and negative controls.
(i) Factory 1: Poultry Factory
Test site: Ventstream chiller, room size 2300m 3.
The trial was conducted at the end of production. The environment was cleaned with
detergent and rinsed and then terminal disinfectant was replaced by ozone treatment: a
single, periodic ozone treatment.
13
Machine used: 2 x Radical Small Mobile Unit (UMB)

Ozone treatment: 5ppm 1hour + Quench
A total of 15 swabs and 15 contact plates (Table 1) were taken as detailed below. The sites
were sampled by Campden BRI staff, who took samples before cleaning, after cleaning and
after disinfection.
Table 1 - Swab and contact plate sites
Site Code
Sample site - product contact surfaces
Plastic shackle (outside)
Plastic shackle (inside)
White board
Back plate of shackle (bird contact)
Back of shackle
Stainless steel surface (external)
Stainless steel surface (internal)
Spray head (metal)
Plastic crate (internal)
10
Stop plastic button
Site Code
Sample site - Environmental samples
1E
Stainless steel panel (entrance)
2E
Plastic cladding
3E
Floor (concrete)
4E
Back of door
5E
Evaporator fin
(ii) Factory 2: Pastry manufacturer

Test site: Ice slurry room, room size 53m 3.
The trial was conducted at the end of production on two occasions. The environment was
cleaned with detergent and rinsed and the terminal disinfectant was replaced by ozone
treatment.
Ozone treatment: 8ppm for 40 minutes + quench
14
A total of 15 swabs and 15 contact plates (Table 2) were taken as detailed below.
The sites were sampled by Campden BRI staff, who took samples before cleaning, after
cleaning and after disinfection.
Site Code
Stainless steel vat (inside)
Green painted motor
Rod power motor
Stainless steel balance (top)
Underneath stainless steel table
Meat pump nozzle (stainless steel)
Inside stainless steel meat pipe
V-mag stainless steel exit pipe
V-mag stainless steel inside hopper
10
Plastic ice slurry pipes (outside)
Site Code
1E
Drain trap (side)
2E
Wall cladding
3E
Floor
4E
Top of gulley pot (drain)
5E
Floor to wall edging (concrete)
(iii) Factory 3: Pizza manufacturer

Test site: High care cook-house, room size 75m3.
The trial was conducted at the end of production on three occasions. The environment was
cleaned with detergent and rinsed and the terminal disinfectant was replaced by ozone
treatment.
Ozone treatment: 8ppm for 40 minutes + quench
A total of 15 swabs and 15 contact plates (Table 3) were taken as detailed below. The sites
were sampled by Campden BRI staff, who took samples before cleaning, after cleaning and
after disinfection.
15

Site Code
Tumbler (inside)
Shelf underneath tumbler
Red switch on/off (tumbler)
Glass mixer - blade
Glass mixer - bottom (inside)
Glass mixer - outlet
Stainless steel table-top
Glass mixer - panel
Stainless steel table - top
10
Blast Chill Room 18B door handle
Site Code
1E
Floor (stainless steel)
2E
Wall (behind glass mixer)
3E
Floor (red)
4E
Around drain seal to the floor
5E
Drain inside (drain's basket)
(iv) Factory 4: Sandwich manufacturer

Test site: High care area, room size 1080m3.
Ozone treatment: Routine end of production cleaning and disinfection. Gross solids
removal, rinse with 3ppm ozonated water, chemical application, rinse with 3ppm ozonated
water, seal room, disinfect with 8ppm for 30 minutes + quench.
Machine used (for gaseous treatment): Installed Radical Whole Room System (generation 1,
now superseded by generation 2).
The trial was 4 weeks in duration with sample sites sampled on 3 occasions each week.
These were on a Wednesday afternoon by Campden BRI staff, who took samples before
cleaning, after cleaning and the next morning after disinfection, and on Monday and Friday
mornings by the factory trained representative, who took samples after ozone disinfection
only (Table 4). Samples taken by Campden BRI staff were stored in the fridge and
processed as soon as possible. Samples taken by the factory representative were collected
by a courier in cool boxes and delivered to Campden BRI on the day, where they were
processed. Due to low microbial level, 'before cleaning' sites had to be changed over the
16
4 week trial (Table 5). In addition to food contact surfaces a number of environmental
surfaces were also sampled (Table 6).
Table 4 - Swab and contact plate sites - first set of samples
Site Code
Sample site
Floor, away from the ozone unit
Floor, away from the ozone unit
Flowa line (food contact surface)
Surface above the conveyor belt on Line 1 conveyor belt
Sandwich slicing table
Conveyor belt (away from the ozone unit) on Line 1 conveyor belt
Conveyor 2 table
Slicer - Grothe (long plate)
Mixer
10
Conveyor belt on butter machine

Table 5 - Swab and contact plate sites - second set of samples
Site Code
1
2
3
4
Sample site
Waste hatch handle (contact plate taken from door plate or the
widestby
part
of the handle
)
Floor
Conveyor
1
Goods in hatch handle (contact plate taken from door plate or the
widest
Grothe part
knobof the handle )
Microwave door handle - can you find different location (I think it is

covered
with plastic
during1)
ozone treatment)
Glove
dispenser
(by bag
Conveyor
Scraper blade holder (underneath the conveyor belt)
Flow rap wheel (Flowa line)
9
10
1e
Drain 2 (middle one)

Can opener (contact plate taken from the widest part of the can
opener)
Conveyor 2 table
2e
17
Table 6 - Swab and contact plate sites - environmental samples
3.3
Site Code
Sample site
Floor by conveyor belt 1
Drain 1
Cheese table
Boot (staff)
Floor by Grothe machine
Sink (Low Risk area)
Floor (Low Risk area)
Hatch frame (Low Risk area)
Wall (Low Risk area)
10
Drain (Low Risk area)
Statistical Analyses
The performance of the whole room disinfection techniques was assessed by statistical
analysis in a Minitab programme (version 15) using one-way analysis of variance (ANOVA).
A value of P>0.05 indicates that there is no significant difference between groups whilst
P<0.05 indicates that the groups were significantly different.
4.
RESULTS & DISCUSSION
The results are expressed as a series of statistical analyses and graphs. The trials included
laboratory experiments as well as factory field trials. In summary:Pre-trial for microbial coverage of stainless steel coupons
43 laboratory trials against L. monocytogenes, P. aeruginosa and S. aureus were
performed which included:
o 5 experiments using hydrogen peroxide vapour - H2O2 (10 g/m3)
o 12 experiments using hydrogen peroxide vapour - H2O2 (20 g/m3)
o 1 experiment using hydrogen peroxide vapour - H2O2 (30 g/m3)
o 1 experiment using hydrogen peroxide vapour - H2O2 (40 g/m3)
o 2 experiments using gaseous ozone - O3 (8 ppm)
o 3 experiment using gaseous ozone - O3 (25 ppm)
o 5 experiment using gaseous ozone - O3 (20 ppm with shorter dwell time)
o 11 experiments using gaseous ozone - O3 (20 ppm)
o 2 experiments using chemical fogging (H&M Disinfection Systems Ltd.)
o 2 experiments using aerosol technology (Atrium Innovations)
o 10 experiments using Nebulair system
18
4 field trials, with Steritrox support, were conducted:

o
o
o
o
4 week factory trial in sandwich manufacturer

I day trial in poultry factory
2 day trial in pastry manufacturer
3 day trial in pizza manufacturer
The overall results of the work, expressed as the mean log reduction of microorganisms
achieved, are shown in Table 7.
Each laboratory experiment at Campden BRI's laboratory involved testing of 20 stainless
steel discs (2 cm diameter) located around the testing room in different orientations
(horizontal, vertical and underneath) over different expose methods and times.
Each factory trial involved taking the swab samples from the designated locations before
cleaning, after cleaning and after room decontamination.
Table 7 - Descriptive statistic of log reduction of different microorganisms resulting
from the system used, its concentration and discs orientation
Chemical fogging - Byosan 2%
Microorganism
P.aeruginosa
S.aureus
Orientation
N Mean StDev Minimum
Median
Maximum
horizontal
5.13
0.53
4.18
5.26
5.96
underneath
1.94
2.16
0.43
0.70
5.26
vertical
1.69
1.35
0.84
1.21
4.42
horizontal
5.08
2.21
0.21
5.78
6.76
underneath
0.50
0.57
-0.08
0.43
1.30
vertical
0.76
0.86
0.17
0.38
2.44
19
Nebulair system - Byosan 10 %

Microorganism
P.aeruginosa
S.aureus
Orientation
Mean StDev Minimum
Median
Maximum
horizontal
16 0.80
0.38
0.32
0.73
1.59
underneath
12 0.92
0.66
0.16
0.69
2.48
vertical
12 0.74
0.54
0.15
0.69
2.10
horizontal
16 0.08
0.29
-0.84
0.09
0.53
underneath
12 0.58
1.02
-2.04
0.50
1.94
vertical
12 0.39
0.39
-0.05
0.28
1.16
Median
Maximum

Microorganism
P.aeruginosa
S.aureus
Orientation
Mean StDev Minimum
horizontal
24 0.52
0.36
-0.14
0.59
1.32
underneath
18 1.17
0.75
-0.15
1.21
2.44
vertical
18 0.78
0.67
-0.39
0.82
2.18
horizontal
0.20
0.47
-0.05
0.06
1.34
underneath
1.47
0.77
0.08
1.63
2.38
vertical
0.85
0.42
0.13
1.05
1.26
Orientation
Mean StDev Minimum
Median
Maximum
horizontal
16 0.42
0.46
0.01
0.27
1.95
underneath
12 1.25
0.81
0.05
0.99
2.70
vertical
12 0.73
0.44
0.13
0.61
1.34

Microorganism
S.aureus
Atrium Innovations aerosol technology - 2 % biocide

Microorganism
P.aeruginosa
S.aureus
Orientation
N Mean StDev Minimum
Median
Maximum
horizontal
0.56
0.77
-1.05
0.79
1.48
underneath
0.68
0.31
0.38
0.54
1.21
vertical
0.54
0.31
0.32
0.43
1.12
horizontal
0.24
0.14
-0.02
0.25
0.40
underneath
0.11
0.30
-0.40
0.21
0.37
vertical
0.01
0.44
-0.59
0.27
0.34
20
Hydrogen peroxide vapour - 10 g/m3

Microorganism
Orientation
Mean StDev Minimum
Median Maximum
B.subtilis var.globigii
vertical
5.83
horizontal
P.aeruginosa
S.aureus
5.83
5.83
5.83
12 1.75
0.89
0.76
1.33
2.77
underneath
1.38
1.07
-0.86
1.29
2.73
vertical
1.59
0.68
0.85
1.32
2.73
horizontal
28 1.34
0.50
0.59
1.33
2.52
underneath
21 1.61
0.90
0.76
1.62
5.11
21 1.41
0.59
0.78
1.27
2.79
vertical
Hydrogen peroxide vapour - 20 g/m
Microorganism
P.aeruginosa
S.aureus
L. monocytogenes
Orientation
horizontal
32 2.85
1.30
1.01
2.30
4.51
underneath
24 3.02
1.23
0.95
2.75
4.51
vertical
24 2.96
1.32
0.93
2.92
4.51
horizontal
32 2.44
1.26
1.06
2.10
6.48
underneath
24 2.26
0.74
1.14
2.12
3.55
vertical
24 2.48
1.36
1.23
2.07
6.48
horizontal
32 2.96
1.40
0.80
2.74
5.96
underneath
24 3.16
1.20
1.31
3.00
5.96
24 2.96
1.27
1.40
2.67
5.96
vertical
Hydrogen peroxide vapour - 30 g/m
Microorganism
P.aeruginosa
S.aureus
L. monocytogenes
Mean StDev Minimum
Median Maximum
Orientation
Mean StDev Minimum
Median Maximum
horizontal
3.44
0.213
3.29
3.44
3.59
underneath
3.20
0.55
2.81
3.20
3.59
vertical
3.59
0.00
3.59
3.59
3.59
horizontal
2.02
0.44
1.71
2.02
2.34
underneath
2.15
0.29
1.95
2.15
2.35
vertical
2.06
0.35
1.81
2.06
2.30
horizontal
4.86
0.21
4.71
4.86
5.01
underneath
5.49
0.00
5.49
5.49
5.49
vertical
4.43
0.08
4.38
4.43
4.49
21
Hydrogen peroxide vapour - 40 g/m3

Microorganism
P.aeruginosa
S.aureus
L. monocytogenes
Orientation
Mean StDev Minimum
Median Maximum
horizontal
4.65
0.00
4.65
4.65
4.65
underneath
3.50
0.65
3.04
3.50
3.95
vertical
4.13
0.74
3.61
4.13
4.65
horizontal
2.37
0.03
2.36
2.37
2.39
underneath
2.65
0.30
2.44
2.65
2.86
vertical
2.59
0.12
2.50
2.59
2.68
horizontal
5.39
0.00
5.39
5.39
5.39
underneath
5.39
0.00
5.39
5.39
5.39
vertical
4.89
0.71
4.39
4.89
5.39
Maximum
Ozone - 8 ppm
Microorganism
Orientation
Mean StDev Minimum
P.aeruginosa
horizontal
0.86
0.77
0.41
Media
n
0.61
S.aureus
horizontal
0.65
0.09
0.55
0.66
0.81
L. monocytogenes
horizontal
0.49
0.22
0.15
0.47
0.75
Orientation
Mean StDev Minimum
Maximum
horizontal
31 4.07
1.11
1.61
Media
n
4.55
underneath
23 3.56
1.29
1.46
3.85
5.30
vertical
23 2.42
0.98
1.15
2.35
4.89
horizontal
32 1.24
0.50
0.71
1.12
3.48
underneath
24 1.76
0.25
1.36
1.72
2.29
vertical
24 1.32
0.29
1.01
1.19
2.12
horizontal
24 4.39
1.15
1.44
5.04
5.24
underneath
18 4.22
1.16
1.87
4.99
5.24
vertical
18 4.35
1.31
1.81
5.04
5.24
Maximum
2.57
Ozone - 20 ppm (~ 50 ozone/hours)

Microorganism
P.aeruginosa
S.aureus
L. monocytogenes
5.30
Ozone - 20 ppm (~ 20 ozone/hours)

Microorganism
S.aureus
L. monocytogenes
Orientation
horizontal
24 0.97
0.60
0.35
Media
n
0.84
underneath
17 1.73
0.98
0.90
1.40
3.89
vertical
18 1.05
0.25
0.77
1.03
1.67
horizontal
16 1.62
0.47
0.65
1.59
2.69
underneath
11 1.94
0.42
1.16
2.07
2.41
vertical
12 1.25
0.30
0.84
1.25
1.66
Mean StDev Minimum
3.06
22
Ozone - 25 ppm
Microorganism
Orientation
S.aureus
horizontal
60 2.07
4.1
Mean StDev Minimum

1.78
-0.08
Media
n
1.14
Maximum
6.15
Pre-trial
All epifluorescence images of P. aeruginosa, S. aureus and L. monocytogenes indicated that

the cultures were attached as a monolayer to the stainless steel discs. Example images of
P. aeruginosa, S. aureus and L. monocytogenes are shown in Figures 2a-c respectively. No
evidence was seen of clumping of cells in a vertical direction that would have limited
penetration of chemical or gaseous disinfectants.
Figure 2a Distribution of P. aeruginosa dried onto stainless steel discs
23
Figure 2b Distribution of S. aureus dried onto stainless steel discs
Figure 2c Distribution of L. monocytogenes dried onto stainless steel discs
24
4.2
Laboratory trials
4.2.1
Chemical fogging
Exposure of P. aeruginosa and S. aureus to chemical fogging at a concentration of

2% B1878 Byosan concentrate (courtesy of Byotrol).
Each experiment involved the testing of 20 stainless steel discs (2 cm diameter) located
around the testing room in different orientations (horizontal, vertical and underneath); log
reductions are shown in Figure 3.
95% CI for the Mean

Orientation
horizontal
underneath
v ertical
7
6
Log reduction
5
4
3
2
1
0
Organism
P.aeruginosa
S.aureus
Figure 3 - Effect of chemical fogging on microbiological log reduction

depending on orientation
The results achieved are typical for a chemical fog and indicate that decontamination is
strongest on horizontal surfaces (particularly at lower levels), on which the majority of the
disinfectant aerosols settle.
A good level of disinfection is achieved on horizontal surfaces (5 logs), which is above that
required for surface disinfectants as defined by the European Standard surface disinfectant
test EN 13697, in which a 4 log reduction is required to pass the standard.
25
Exposure of P. aeruginosa and S. aureus to Nebulair System at concentration of 10%,

20% and 50% using Byosan disinfectant
95% CI for the Mean

1.2
Organism
P.aeruginosa
S.aureus
Log reduction
1.0
0.8
0.6
0.4
0.2
0.0
Concentration (%)
10
20
50
Figure 4 - Effect of Byosan on P. aeruginosa and S. aureus when nebulised

at 3 concentrations in the Nebulair System
The degree of microbial reduction by the Nebulair system, using the same biocide as for
chemical fogging, was very poor in comparison, with less than one log order of
decontamination being achieved. In general, the concentration of Byosan used also
appeared to have little effect on log reduction, though there may be some increase in log
reduction between 10 and 20% Byosan for S. aureus. This may relate to the actual quantity
of disinfectant available for disinfection. For chemical fogging, approximately 10 litres of
Byosan was released into the room during the fogging cycle, whereas only ~120ml of
Byosan was released into the room during the Nebulair System cycle (1L lasted for
approximately 8 hours).
26
95% CI for the Mean

2.5
Orientation
horizontal
underneath
v ertical
Log reduction
2.0
1.5
1.0
0.5
0.0
Concentration (%)
10
20
S.aureus
50
10
20
P.aeruginosa
50
Figure 5 - Effect of Byosan on P. aeruginosa and S. aureus with respect to their

orientation when nebulised in the Nebulair System
Exposure of P. aeruginosa and S. aureus to aerosol technology at a concentration of
2% biocide (courtesy of Atrium Innovation)
The Atrium Innovation technology achieved a small, 0.5 log reduction in P. aeruginoasa and
a marginal, approximately 0.1 log reduction in S. aureus. As for the Nebulair System, only a
small quantity of chemical, 125 ml, was aerosolised during the trial . Orientation did not
have an effect on decontamination, although the low log reductions achieved would probably
mask any differences.
The effect of orientation of the test samples on decontamination by the Nebulair System
Atrium Innovation technology (Figures 5 and 6) suggests that, unlike chemical fogging,
decontamination by nebulisation is not affected by surface orientation. This may be
because the smaller particles generated by the nebulisers (10-20m for fogged particles as
against 1.22 m for nebulised particles [Nebulair System]) are more able to adhere to, and
27
thus have an effect upon, vertical and underneath surfaces. Smaller particles also remain
airborne longer (their settlement rate is less) and, therefore, have more chance to interact
with non horizontal surfaces.
1.25
Orientation
horizontal
underneath
v ertical
1.00
Log reduction
0.75
0.50
0.25
0.00
-0.25
-0.50
Organism
P.aeruginosa
S.aureus
Figure 6 - Effect of aerosol technology on microbiological log reduction

depending on orientation
4.2.2
Exposure to Hydrogen Peroxide Vapour at concentration of 10 g/m3, 20 g/m3 , 30 g/m3

and 40 g/m3 courtesy of Bioquell
Each experiment at 10 g/m3 and 20 g/m3 involved the testing of 20 stainless steel discs (2
cm diameter) located around the testing room in different orientations (horizontal, vertical
and underneath), whilst each experiment at 30 g/m3 and 40 g/m3 involved the testing of 6
stainless steel discs. Log reductions are shown in Figure 7.
28
95% CI for the Mean

6
Organism
B.subtilis v ar.globigii
L.monocy togenes
P.aeruginosa
S.aureus
Log reduction
5
4
3
2
1
0
Concentration
10 g/m3
20 g/m3
30 g/m3
40 g/m3
Figure 7 - Effect of H2O2 vapour on microbiological log reduction

at different concentrations
The results in Figure 7 show that microorganisms respond differently to HPV and that there
was a significant difference between the logarithmic microbial reductions of spores and
vegetative bacteria (p=0.003) at a concentration of 20g/m3 and below. An approximate 6 log
reduction of globigii spores was achieved at 10g/m3, which is equivalent to that required for
spores in the decontamination of aseptic equipment.
A relatively poor log reduction of approximately 1.5 logs was achieved for P. aeruginosa and
S. aureus at 10g/m3, the default concentration for the HPV equipment, though this was
substantially increased to an approximately 3 log reduction when this concentration was
doubled to 20g/m3. There was little difference, however, in the log reductions achieved
between the vegetative microorganisms tested, with S. aureus being slightly more resistant.
When the concentration was increased to 30 and 40g/m3, different survival rates between
the vegetative microorganisms was seen. L. monocytogenes was seen as the least
resistant microorganism and its survival was reduced with these increasing concentrations,
though there was no significant difference (P=0.9091) in survival at 30 or 40 g/m3. The
survival of P aeruginosa also was reduced with concentration, though the log reduction at 40
g/m3 was only marginally significantly different to that at 20 g/m3 (P=0.0804). There is no
significant difference in the susceptibility of S. aureus with increasing HPV concentration
29
above 20g/m3 (p=0.8499 at 30g/m3 and P=0.9837 at 40 g/m3). The results for 30 and 40
g/m3 HPV relate, however, to a mean of only 6 discs per microorganism, whilst those at 10
and 20 g/m3 were the mean of 30-80 discs per microorganism.
Figure 8 shows the effect of surface orientation on the decontamination of P. aeruginosa,
S. aureus and L. monocytogenes by HPV at 20g/m3. No statistical differences were seen
between orientations for all organisms (P=0.828, P=0.884 and P=0.780 respectively).
20 g/m3, 43 min cycle, 60 min dwell time

4
Orientation
horizontal
underneath
v ertical
Log reduction
0
Organism
L.monocytogenes
P.aeruginosa
S.aureus
Figure 8 - Effect of H2O2 vapour on microbiological log reduction

depending on orientation at 20 g/m3
30
4.2.3
Ozone
Exposure to ozone at concentrations 8 ppm, 20 ppm and 25 ppm - courtesy of

Steritrox
The tests conducted at Campden BRI used a Radical Small Mobile Unit (UMB) and involved
the testing of 20 stainless steel discs (2 cm diameter) per experiment which were located
around the testing room in different orientations (horizontal, vertical and underneath) for:>20 ppm for 30 min - total time: ~4 hrs (no quench)
>20 ppm for 30 min - total time: ~3.5 hrs (no quench)
The test conducted at the Steritrox laboratory, Birlingham, Worcestershire, used a Meditrox
MDX50 machine and involved the testing for:7 stainless steel discs per microorganism located in horizontal orientation. 8ppm for
30 min. - total time: ~70 min from door close to door open + quench
20 stainless steel discs per trial located in horizontal orientation. 25ppm for 90 min total time: ~ 2 - 2.5h from door close to door open + quench
The log reductions achieved in all ozone trials are summarised in Figure 9.
5
Organism
S.aureus
L.monocytogenes
P.aeruginosa
Log reduction
0
Concentration
8 ppm
20 ppm
25 ppm
Figure 9 - Effect of gaseous O3 on microbiological log reduction

at different concentrations
31
The results in Figure 9 show two trends. Firstly, there is a clear relationship between ozone
concentration and log reduction, with the log reduction for S. aureus ranging from 0.7 logs at
8ppm, to 1.5 logs at 20ppm and 2.1 logs at 25ppm. Secondly, the effect of ozone on the
three vegetative strains tested is markedly different with, at 20ppm, S. aureus being
statistically (P=0.000) and practically most resistant, followed by P. aeruginosa, and
L. monocytogenes being most sensitive.
The effect of sample orientation on log reduction at an ozone concentration of 20ppm is
shown in Figure 10.
Orientation
horizontal
underneath
v ertical
Log reduction
0
Organism
L.monocytogenes
P.aeruginosa
S.aureus
Figure 10 - Effect of gaseous O3 on microbiological log reduction

depending on orientation at 20 ppm long cycle
There is no statistical difference in orientation for L. monocytogenes (P=0.896), though the
orientation log reductions are statistically different for both P. aeruginosa (P=0.000) and
S. aureus (P=0.000). The results for S. aureus, in which a greater log reduction is shown for
the underneath surface, are probably due to test variance. The results for P. aeruginosa are
more varied, with a lower reduction on vertical than underneath surfaces. The reason for
this is not clear, but may be due to air circulation patterns in the laboratory. In any case,
there is little evidence to suggest that ozone is not able to penetrate to all surfaces,
irrespective of their orientation.
32
25
100%
Key
Ozone
Humidity
90%
85%
15
80%
10
75%
Humidity (RH)
Ozone Concentration (ppm)
95%
20
70%
65%
0
0:00 0:30
1:00 1:30
2:00 2:30
Time
3:00 3:30
4:00
60%
23.09.09
Figure 11 - Ozone concentration and humidity levels at 20 ppm treatment (short cycle)
During the trials it became clear that the effect of ozone on log reductions was a
combination of concentration and contact time. When ozone concentration is plotted against
time, the effect of forced ozone removal from the atmosphere following ozonation to 20ppm
(Figure 11) and natural decay following ozonation to 20ppm (Figure 12) can easily be seen.
The amount of ozone in the air that the microorganisms are exposed to following the applied
20ppm ozone concentration is much greater when the ozone is left to decay naturally than
when the quench cycle is initiated. The quench cycle can be applied when the ozone
concentration drops to 8ppm and a typical profile of the reduction on ozone concentration in
the air following quenching can be seen in Figure 13.
The area under the line in the concentration versus time plots can be described as the
ozone dosage. The ozone dosage can be calculated for all of the ozone experiments and is
shown, along with the log reduction achieved, for L. monocytogenes in Table 8.
33
95%
25
Ozone C onc.
Humidity
20
85%
15
80%
75%
10
Humidity
Ozone Concentration (ppm)
90%
70%
5
65%
60%
0
0:00 0:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 4:30
Time
11.11.09
Figure 12 - Ozone concentration and humidity levels at 20 ppm treatment (long cycle)
Figure 13 - Ozone concentration and humidity levels at 8 ppm treatment

(courtesy of Steritrox)
34
Table 8 - Relationship between dose and log reduction for L. monocytogenes
Trial
Ozone dosage
(ppmhour)
Mean log
reduction
65.35
3.51
42.26
4.92
25.64
1.84
24.72
1.38
22.07
4.55
5.01
0.49
A graph of ozone dosage versus log reduction can then be plotted and the regression line
calculated for each microorganism. The data for L. monocytogenes are shown in Figure 14.
Whilst it can be seen that the accuracy of the regression line is not good, the concept of
using the line to predict the ozone dose required to produce a given log reduction of a target
microorganism can be seen. Further work would be required to build a sufficiently robust
model to validate the approach.
6
42.26
Mean log reduction
22.07
65.35
3
25.64
24.72
5.01
0
0
10
20
30
40
50
60
Ozone dosage (ppm hour)
70
80
90
Figure 14 - Relationship between ozone dose and log reduction for L. monocytogenes
35
4.3
Field Trials
The TVC results from the swab points before cleaning, after cleaning and after disinfection
for the 3 field trials using ozone as a periodic decontaminant are shown in Figure 15a and b
(poultry factory food contact and environmental samples), Figure 16a and b (pastry
manufacture food contact and environmental samples) and Figure 17a and b (pizza
manufacture food contact and environmental samples). Raw data for these three trials are
shown in Appendix 2.
Sample
site
1
2
3
4
5
6
7
8
9
10
Log
3
Before cleaning
After cleaning
After disinfection
Figure 15a - Effect of gaseous O3 treatment on microbiological log reduction of food

contact samples within a poultry factory
36
Sample site
1S. steel upright (entrance)
2 Plastic cladding
3 Floor (concrete)
4 Back of door
5 Ev aporator fin
8
7
Log
6
5
4
3
2
Before cleaning
After cleaning
After disinfection
Figure 15b - Effect of gaseous O3 treatment on microbiological log reduction of

environmental contact samples within a poultry factory
The results from the single trial in the poultry factory (Figures 15a and b) are varied with
perhaps 5 of the 10 food contact sampling sites showing a reduction after cleaning with the
other sites showing no change or even an increase in counts. Reductions after disinfection
are clearer for the environmental samples. If an overall reduction in counts is observed for
all samples, it is probably less than 1 log order.
37
3.5
Sample
site
1
2
3
4
5
6
7
8
9
10
3.0
Log
2.5
2.0
1.5
1.0
f
Be
e
or
cle
g
in
an
1
r
te
Af
c le
g
in
an
Af
r
te
f
sin
di
n
tio
ec
f
Be
re
c le
g
in
an
r
fte
cle
g
in
an
Af
r
te
fe
si n
i
d
n
io
ct

contact samples within a pastry manufacture facility
Sample site
Drain trap (side)
Wall cladding
Floor
Top of gulley pot (drain)
Floor to wall edging
7
6
Log
5
4
3
2
1
0
r
fo
Be
cle
g
in
an
1
r
te
Af
c le
g
in
an
te
Af
ti o
ec
f
n
is i
rd
Be
re
fo
cle
g
in
an
le
rc
tf e
g
in
an
Af
te
nf
isi
d
r
n
tio
ec

environmental samples within a pastry manufacture facility
38
9
Sample
site
1
2
3
4
5
6
7
8
9
10
8
7
6
Log
5
4
3
2
1
0
f
Be
e
or
c le
g
in
an
r
te
Af
cle
g
in
an
Af
r
te
1
f
sin
di
n
tio
ec
f
Be
e
or
cle
g
in
an
r
te
Af
cl
ng
ni
a
e
te
Af
2
f
s in
di
n
tio
ec
f
Be
e
or
cle
g
in
an
A
r
fte
cle
g
in
an
Af
r
te
3
f
sin
di
n
tio
ec

contact samples within a pizza manufacture facility
8
7
6
Sample site
Floor (S.steel)
Wall
Floor
Around drain seal to the floor
Drain inside (drain's bask et)
Log
5
4
3
2
1
0
1
1
1
2
2
2
3
3
3
n
n
n
ng
ng
ng
ng
ng
ng
tio
tio
tio
ni
ni
ni
ni
ni
ni
c
c
c
a
a
a
a
a
a
e
fe
e
fe
e
fe
le
le
le
cl
in
cl
in
cl
in
rc
rc
rc
e
is
e
is
e
is
r
e
r
e
r
e
d
d
d
t
t
t
r
r
r
fo
fo
fo
Af
Af
Af
te
te
te
Be
Be
Be
Af
Af
Af

environmental samples within a pizza manufacture facility
39
The results for the pastry factory over the two day trial (Figure 16a) are difficult to interpret
as the food contact surface counts appear to increase after cleaning and then decrease after
disinfection with ozone. The counts on the food contact surfaces are, however, relatively
low and small differences in recovery efficiency by the swabbing technique on such low
numbers may mask real trends. The counts on environmental surfaces (Figure 16b) are
higher than on food contact surfaces and may show a reduction after disinfection,
particularly on the second day.
The results for the food contact surfaces of the pizza factory over the three day trial (Figure
17a) show that, on each individual day, average counts are lower after cleaning and then
lower still after disinfection. The results could also show a downward trend for the numbers
of microorganisms present both before cleaning and after cleaning and disinfection
throughout the three day period. This downward trend is perhaps more clearly shown with
the environmental samples (Figure 17b).
The TVC swab results from the 4 week trial in the sandwich manufacture facility in which
ozone was used in lieu of a chemical disinfectant are shown for food contact surfaces in
Table 9 and for environmental surfaces in Table 10.
Table 9 TVC counts for food contact surfaces before cleaning, after cleaning and
after ozone disinfection within a sandwich manufacture facility
SWAB RESULTS (Mean log)
Food Contact surfaces
Before
cleaning
After
cleaning
After
disinfection
Flowa line
Surface above the conveyor belt on
Line
1
Sandwich
slicing table
2.84
0.70
1.17
2.88
0.70
1.29
3.45
1.57
1.22
Conveyor belt on Line 1
1.44
2.22
1.18
Conveyor 2 table
2.52
1.49
1.15
Slicer - Grothe (long plate)
3.07
0.70
1.27
Inside mixer
0.88
2.14
1.52
0.94
0.70
1.56
Can opener
1.18
1.18
0.70
Grothe blade wheel
0.70
Cheese table
3.76
4.70
1.70
Sink (LR)
2.53
5.70
2.07
Mean log
2.32
1.98
1.29
40
Table 10 - TVC counts for environmental surfaces before cleaning, after cleaning and
after ozone disinfection within a sandwich manufacture facility
SWAB RESULTS (Mean log)
Environmental samples
Before
cleaning
After cleaning
After disinfection
Floor
4.94
3.19
2.82
Floor
5.82
4.70
2.28
Waste hatch handle
1.00
cont
0.70
Floor by conveyor 1
4.69
3.25
0.70
Goods in hatch handle
1.18
0.70
0.70
Grothe knob
3.39
1.00
0.70
Microwave door handle
1.65
cont
0.70
Glove dispenser
3.27
0.70
0.70
Scraper blade holder

Flow rap wheel (Flowa
line)
Drain 2
2.94
2.94
0.70
1.81
1.81
3.55
4.81
4.81
2.85
Floor by conveyor 1
5.81
4.3
1.59
wip door handle
1.48
Drain 1
4.86
4.71
4.57
Boot (staff)
4.78
4.48
5.87
Floor by Grothe machine
4.62
5.05
4.12
Floor (LR)
3.52
1.18
2.18
Hatch frame (LR)
4.35
0.70
5.88
Wall (LR)
0.70
0.70
1.30
Drain (LR)
5.13
3.94
1.92
Mean log
3.65
2.83
2.27
The mean counts of all samples before cleaning, after cleaning and after disinfection for
both food contact and environmental surfaces for the 4 week field trial are shown in Table
11. As a comparison, the average data from 10 field trials of 8 weeks duration undertaken in
chilled food plants and in which chemical disinfectants were tested for approval by a major
retailer are also shown.
41
Table 11 A schematic comparison of chemical disinfection and O3
Pre
Post
After
cleaning cleaning disinfection
Average of counts per swab from

10 separate, 8 week duration
disinfectant trials:
4.73
2.80
1.30
Average of counts per swab from 4

week duration trials using ozone
as a disinfectant (food contact
surfaces):
2.32
1.98
1.29
Average of counts per swab from 4

week duration trials using ozone
as a disinfectant (Environmental
samples):
3.65
2.83
2.27
The results in Table 9 show that the TVC count decreased after cleaning and again after
disinfection. After disinfection, for all food contact surfaces, TVC counts were very low,
though this is correlated to the pre-cleaning results. TVC counts on environmental surfaces
(Table 10) were reduced after cleaning and again after disinfection, though were higher than
on food contact surfaces and were more variable. Variability of TVC counts was dependent
on the count prior to cleaning.
The results in Table 11 show that for food contact surfaces, the counts after disinfection
compare favourably to the post disinfection counts of the combined disinfectant approval
trials. Ozone was thus seen to maintain control of the microflora of the food contact
surfaces.
During the 4 week trial no adverse effects were observed on the structure and fabric of the
building. Indeed, the management of the factory also report no adverse effects since the
ozonation equipment was installed.
42
5.
CONCLUSIONS
The results achieved for Byosan during chemical fogging are typical for quality disinfectants
in that good decontamination is achieved on horizontal surfaces but little is achieved on
other surface orientations. These findings are in agreement with those of Campden BRI and
the Silsoe Research Institute which undertook a MAFF funded project in 1988 (Burfoot et al.,
1999). This work suggested that fogging is effective at reducing airborne microbial
populations by 2 to 3 log orders in 30 to 60 minutes and horizontal surfaces up to 6 log
orders in 60 minutes, with minimal effect on vertical surfaces and underneath equipment.
Chemical fogging is therefore useful for the periodic decontamination of the air and as an
automated method to apply disinfectants to accessible, food processing equipment surfaces.
It will have little or no effect, however, on ceilings and overhead structures.
As the amount of chemical released by the Nebulair system and Cleanaer technology is so
small, the level of decontamination on surfaces it achieves is relatively poor. However, the
particle size of chemical they produce appears to be small enough to interact with surfaces
of all orientations, so what decontamination it does achieve is approximately the same over
all surface geometries. Smaller particles will also have longer contact times in the vicinity of
the coupon surfaces, which could lead to particle/surface interactions, because their
settlement rate will be slower. There may be a role for nebulisers in low level, continuous
decontamination of food production areas, if the quantity of disinfectant it produces satisfies
Health and Safety requirements with respect to operatives exposure to the chemical via
breathing the droplets.
At low concentrations of HPV (10 g/m3), the effect of HPV was much more pronounced on
spores of B. subtilis var. globigii than on the vegetative bacteria tested, with an approximate
6 log reduction of globigii spores being achieved. With the vegetative bacteria, a
relationship was established between log reduction and concentration, with an
approximately 3 log reduction being achieved for P. aeruginosa, S. aureus and
L. monocytogenes at 20g/m3. Hydrogen peroxide at high concentrations is considered a
good disinfectant and in parallel trials (results not shown), a 5 log reduction in vegetative test
microorganisms according to the methodology of the disinfectant suspension test BS EN
1276, was achieved at a hydrogen peroxide concentration of 10-15% (30% hydrogen
peroxide is vapourised in the Bioquell units tested). It may be proposed, therefore, that at
the lower concentrations of HPV used in the trials (10-20 g/m3), P. aeruginosa, S. aureus
and L. monocytogenes (all of which are catalase positive) were all able to partially resist the
concentration/volume of disinfectant that they came into contact with.
When the HPV concentration was increased to 30 and 40 g/m3, an increase in susceptibility
was seen for P. aeruginosa and L. monocytogenes, resulting in a log reduction in excess of
4 logs for P. aeruginosa and 5 logs for L. monocytogenes at 40 g/m3. This is in excess of
43
the 4 log reduction required in the European Surface Test (EN 13697) for surface
disinfectants.
S. aureus was significantly more resistant than the other vegetative organisms P.
aeruginosa and L. monocytogenes, and B. subtilis var. globigii spores and its log reduction
was not increased at 30 or 40 g/m3. The reason for this resistance is unclear, but may be
due to the tendency of S. aureus cells to clump together, which may resist chemical
vapours. No statistical differences in log reductions were seen between orientations for all
microorganisms tested.
HPV systems may be able to control pathogens such as Listeria in the environment at high
concentrations (e.g. 40 g/m3), though further trials are needed to support this. They do,
however, offer an excellent option for the control of spore forming bacteria.
With ozone, as with HPV, there is a relationship between concentration and log reduction,
though the effect of ozone on each of the three vegetative strains tested was markedly
different with, at 20ppm, S. aureus being most resistant followed by P. aeruginosa, and L.
monocytogenes being most sensitive. Whilst there appeared to be statistical differences in
the log reductions achieved at different sample orientations, these are not thought to be
practically different.
A reduction of >4 logs was achieved with L. monocytogenes at an ozone concentration of
20ppm, which equates to the requirements of the European Surface Test (EN 13697) for
chemical disinfectants. A reduction approaching 4 logs was also achieved with P.
aeruginosa.
The results suggest that, for each microorganism tested, it could be possible to describe a
relationship between ozone concentration and exposure time that can be described as an
ozone dosage. A graph of ozone dosage versus log reduction can then be plotted and the
regression line calculated for each microorganism. From the tentative data in Figure 13 for
L. monocytogenes, extrapolation suggests that a 6 log reduction could be achieved for L.
monocytogenes with a dosage of approximately 90 ppm hours. This equates, for example,
to an exposure of 10 ppm ozone for 9 hours or 30ppm ozone for 3 hours. Whilst this is
theoretical, if it were to be possible to achieve a 6 log reduction of L. monocytogenes on all
surfaces regardless of orientation in a food processing area (i.e. a treatment to the
environment equivalent to the cooking process of the food product), this would provide a
very powerful technique for contamination control. Again, further trials would be required to
substantiate this dosage in practice.
As an overall conclusion from the laboratory trials, vapours and gases (as typified by HPV
and ozone) have several advantages as they can effectively penetrate every part of a room,
44
including sites that might prove difficult to gain access to with conventional liquids and
manual disinfection procedures. The major disadvantage of using gases, such as ozone, is
the potential toxicity at high concentrations, which precludes using them in areas where
people are working. The technique can therefore only be used in areas that can be isolated
and sealed off during the decontamination process.
The food contact and environmental surface results for the three field trials in which ozone
was used as a periodic room decontaminant showed little effect over 1-3 days and an
overall reduction in counts after disinfection was probably less than 1 log order. This is
perhaps not surprising as when 8ppm ozone was tested under laboratory conditions (Figure
9), a reduction of less than1 log order was observed for the three strains tested under the
same ozone application conditions (8ppm, 30min).
After 3 days, however, the results for the pizza factory could show a downward trend for the
numbers of microorganisms present on food contact and environmental surfaces, both
before cleaning and after cleaning and disinfection, throughout the three day period. Ozone
generation equipment manufacturers have postulated that when ozone is first applied to a
cleaned room, there is a mass of organic material that creates an ozone demand which
must be satisfied by oxidation before any significant oxidation of microorganisms can occur.
In essence, this is no different from the effect of organic matter on traditional chemical
disinfectants, e.g. a chlorine organic break point in water treatment. The ozone demand will
be affected by ozone concentration and if the applied ozone concentration is low, this ozone
demand may require an extended time period before it is fully oxidised.
What is clear is that 8ppm ozone for 30 minutes is insufficient for periodic, whole room
decontamination and a dosage of approximately 90ppm/hours as postulated from the
laboratory studies may be required. Given that the ozone demand of a factory is likely to
exceed that of the relatively clean aerobiology laboratories in which the practical trials were
undertaken, however, the necessary dosage may well exceed this figure.
In the 4 week field trial, after disinfection, for all food contact surfaces, TVC counts were
very low, though this is correlated to the pre-cleaning results. TVC counts on environmental
surfaces were also reduced after cleaning and again after disinfection, though were higher
than on food contact surfaces and were more variable.
The counts after disinfection for ozone treated food contact surfaces compare favourably to
the post disinfection counts of the combined disinfectant approval trials. In the disinfectant
approval trials, only sites with TVC counts of >105/swab were chosen, which is in excess of
the approximately 103/swab in the ozone trial. It might be argued, therefore, that the low
count after disinfection in the ozone trial is due to the low count prior to cleaning. It could
also be argued, however, that the sandwich factory is little different from any other high care
45
factory and that the low TVC counts before cleaning (and thus after disinfection) are due to
the continual application of ozone every night, reducing the plant's environmental bioburden.
The TVC counts post disinfection do, however, show that over the 4 week period of the trial,
there was no evidence that ozone was unable to maintain control of the microflora of the
food contact surfaces and thus could undertake the role of a chemical disinfectant.
During the 4 week trial no adverse effects were observed by ozone on the structure and
fabric of the building. Indeed, the management of the factory have also reported no adverse
effects over the time the ozonation equipment has been installed. The plant did not contain
any natural rubber, however, which is known to be affected by ozone. A nightly treatment of
8ppm for 30 minutes may thus be appropriate as an adjunct or replacement of chemical
disinfection, a concentration which is a balance off effectiveness and ensuring that the
effects of ozone on the structure and fabric of the building are minimised.
Any use of ozone as a replacement for chemical disinfection must, however, be
appropriately validated for each factory situation.
46
6.
REFERENCES:
Andersen, B.M., Banrud, H., Boe, E., Bjordal, O. & Drangsholt, F. (2006) Comparison of UV
C light and chemicals for disinfection of surfaces in hospital isolation units. Infection Control
& Hospital Epidemiology, 27(7), 729-734.
Brown, K. (ed.) (2005) Guidelines on air quality standards for the food industry. Second
edition. Guideline No. 12, Campden BRI.
Burfoot, D., Hall, K., Brown, K. & Xu, Y. (1999) Fogging for the disinfection of food
processing factories and equipment. Trends in Food Science & Technology, 10 (6-7), 205210.
Czarneski, M.A. & Lorcheim, P. (2005). Isolator decontamination using chlorine dioxide gas.
Pharmaceutical Technology, 29(4), 124-133.
Czarneski, M.A. & Poisson P. (2005) Chlorine dioxide gas decontamination of a blow/fill/seal
machine. Controlled Environments Magazine.
EN 1276:1997 Chemical disinfectants and antiseptics Quantitative suspension test for the
evaluation of bactericidal activity of chemical disinfectants used in food, industrial, domestic
and institutional areas Test methods and requirements (phase 2/step 1)
http://www.bsigroup.com/
EN 13697:2001 Chemical disinfectants and antiseptics Quantitative non-porous surface
test for the evaluation of bactericidal and/or fungicidal activity of chemical disinfectants used
in food, industrial, domestic and institutional areas Test methods and requirements without
mechanical action (phase 2/step 2) http://www.bsigroup.com/
Elkins, J.G., Hassett, D.J., Stewart, P.S., Schweizer, H.P. & McDermott, T.R. (1999)
Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen
peroxide. Applied & Environmental Microbiology, 65(10), 4594-4600.
Eylath, A. S., Madhogarhia, E., Rivera, E., Lorcheim, P., Czarneski, M. (2003) Successful
sterilization using chlorine dioxide gas: part two - cleaning process vessels. BioProcess
International, 1(8), 2-4.
Fan, L., Song, J., McRae, K.B., Walker, B.A. & Sharpe, D. (2007) Gaseous ozone treatment
inactivates Listeria innocua in vitro. Journal of Applied Microbiology, 103, 2657-2663.
47
Fichet, G., Comoy, E., Duval, C., Antloga, K., Dehen, C., Charbonnier, A., McDonnell, G.,
Brown, P., Lasmezas, C.I. & Deslys, J-P. (2004) Novel methods for disinfection of prioncontaminated medical devices. The Lancet, 364, August 7th.
Health & Safety Executive (1983) Ozone: health hazards and precautionary measures. HSE
Guidance Note - EH 38.
Health & Safety Executive (2005) Table 1: List of approved workplace exposure limits. HSE
- EH 40.
Holah, J., Bird, J. & Hall, K. (2002) The microbial ecology of high risk, chilled food factories;
evidence for persistent Listeria spp. and Escherichia coli strains. R&D Report No.165,
Campden BRI.
Holah, J., Bird, J. & Hall, K. (2004) Listeria monocytogenes and Escherichia coli in high risk,
chilled food factories; where do they come from? R&D Report No.199, Campden BRI.
Huang, Z., Maness, P-C., Blake, D.M., Wolfrum, E.J., Smolinski, S.L. & Jacoby, W.A. (2000)
Bactericidal mode of titanium dioxide photocatalysis. Journal of Photochemistry &
Photobiology A: Chemistry, 130(2-3), 163-170.
Hudson, J.B., Sharma, M. & Petric, M. (2007) Inactivation of Norovirus by ozone gas in
conditions relevant to healthcare. Journal of Hospital Infection, 66, 40-45.
Jeng, D. K. & Woodworth, A. G. (1990) Chlorine dioxide gas sterilization under square-wave
conditions. Applied & Environmental Microbiology, 56(1), 514-519.
Katara, G., Hemvani, N., Chitnis, S., Chitnis, V. & Chitnis, D.S. (2008) Surface disinfection
by exposure to germicidal UV light. Indian Journal of Medical Microbiology, 26, 241-242.
Kim, J.-G. & Yousef, A.E. (2000) Inactivation kinetics of foodborne spoilage and pathogenic
bacteria by ozone. Journal of Food Science, 65(3), 521-528.
Knapp, J.E., & Battisti, D.L. (2001) Chlorine dioxide (Chapter 11). Disinfection, Sterilization
and Preservation. Edited by S.S. Block. Lippincott Williams and Wilkins, Philadelphia, 215
228.
Kuhn, K.P., Chaberny, I.F., Massholder, K., Stickler, M., Volker, B.W., Sonntag, H.G. &
Erdinger, L. (2003) Disinfection of surfaces by photocatalytic oxidation with titanium dioxide
and UVA light. Chemosphere, 53(1), 71-77.
48
Maatta, J., Piispanen, M., Kymalainen, H.R., Uusi-Rauva, A., Hurme, K.R., Areva, S.,
Sjoberg, A.M. & Hupa, L. (2007) Effects of UV radiation on the cleanability of titanium
dioxide coated glazed ceramic tiles. Journal of the European Ceramic Society, 27 (1), 101108.
Maness, P-C., Smolinski, S., Blake, D.M., Huang, Z., Wolfrum, E.J. & Jacoby, W.A (1999)
Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing
mechanism. Applied & Environmental Microbiology, 65(9), 4094-4098.
McDonnell, G., Grignol, G. & Antloga, K. (2002) Vapour phase hydrogen peroxide
decontamination of food contact surfaces. Dairy Food & Environmental Sanitation, 22(11),
868-873.
Meszaros, J.E., Antloga, K., Justi, C., Plesnicher, C. & McDonnell, G. (2005) Area
fumigation with hydrogen peroxide vapor. Applied Biosafety, 10 (2), 99-100.
Rosenblatt, D. H., Rosenblatt, A., A. & Knapp, J., E. Use of chlorine dioxide gas as a
chemosterilizing agent. US Patents 4504442 (1985) & 4681739 (1987).
Seo, K.H., Mitchell, B.W., Holt, P.S. & Gast, R.K. (2001) Bactericidal effects of negative air
ions on airborne and surface Salmonella Enteritidis from an artificially generated aerosol.
Journal of Food Protection, 64,113116.
Silsoe Research Institute (1998) A practical guide to the disinfection of food processing
factories and equipment using fogging. MAFF Advanced & Hygienic Food Manufacturing
LINK Programme.
Taylor, J. & Chana, D. (2000) The evaluation of ozone for airborne and surface disinfection.
R&D Report No.109, Campden BRI.
Wintner B., Contino, A. & O'Neil, G. (2005) Chlorine dioxide, Part 1 A versatile, high-value
sterilant for the biopharmaceutical industry, BioProcess International, 3(11), 42-46.
Vohra, A., Goswami, D.Y., Deshpande, D.A. & Block, S.S. (2005) Enhanced photocatalytic
inactivation of bacterial spores on surfaces in air. Journal of Industrial Microbiology &
Biotechnology, 32, 364-370.
49
APPENDIX 1
To enumerate organisms from tested and control surfaces TSA (BS) agar was used for all
microorganisms. Sodium thiosulphate inactivator was used as a resuspension solution to
stop any carry-over of the chemical reaction, Reference BS EN 1276, REC-FH-025.
Table Sodium Thiosulphate Inactivator
Formula
Sodium Thiosulphate
Deionised water
5g
1000ml
Table Tryptone Soya Agar TSA (BS) - CM0131

Formula
Pancreatic digest of casein
Enzymatic* digest of soya bean
Sodium chloride
Agar
pH 7.3 0.2 @ 25C
gm/litre
15.0
5.0
5.0
15.0
Table Diluent (BS)

Formula
Tryptone, pancreatic digest of casein (LABM MC5)
1g
Sodium Chloride
8.5g
Deionised water
make up to 1000ml
APPENDIX 2
Raw microbiological sampling data from three, periodic ozonation field trials
Poultry factory
BEFORE CLEANING
AFTER CLEANING
Product Contact Surfaces
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
TVC
TVC
TVC
log
log
log
Count/plate
(cfu/swab) cfu/swab (cfu/swab) cfu/swab (cfu/swab) cfu/swab
1. Plastic shackle (outside - U)
7.15E+04
4.85
1.19E+04
4.08
4.75E+02
2.68
2. Plastic shackle (inside)
1.91E+06
6.28
2.32E+04
4.37
1.56E+04
4.19
17
3. White board
8.20E+04
4.91
5.90E+03
3.77
5.60E+02
2.75
14
4. Back plate of shackle (bird contact)
3.75E+05
5.57
1.53E+04
4.18
4.90E+02
2.69
18
5. Back of shackle
7.00E+06
6.85
4.30E+06
6.63
1.58E+06
6.20
50
6. Stainless steel surface (external)
1.75E+08
8.24
2.43E+03
3.39
9.60E+06
6.98
7. Stainless steel surface (internal)
3.52E+08
8.55
1.23E+05
5.09
1.15E+07
7.06
8. Spray head (metal)
1.80E+06
6.26
8.05E+04
4.91
1.07E+05
5.03
9. Plastic crate (internal)
1.08E+05
5.03
9.25E+04
4.97
1.43E+07
7.16
>300
10. Stop plastic button

Environmental Samples
4.35E+06
6.64
4.40E+06
6.64
2.24E+05
5.35
17
1E. Stainless steel upright (entrance)
1.63E+08
8.21
5.70E+07
7.76
7.39E+06
6.87
>300
2E. Plastic cladding
1.53E+08
8.18
4.40E+04
4.64
1.05E+02
2.02
3E. Floor (concrete)
1.31E+08
8.12
1.02E+07
7.01
4.26E+06
6.63
>300
4E. Back of door
4.35E+04
4.64
1.78E+06
6.25
7.30E+02
2.86
50
5E. Evaporator fin
3.43E+06
6.54
4.33E+06
6.64
4.65E+05
5.67
118
Pastry manufacturer 1st day
BEFORE CLEANING
AFTER CLEANING
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
1. Stainless steel vat (inside)
TVC
TVC
TVC
log
log
log
Count/plate
<5
<0.70
<5
<0.70
<5
<0.70
0
2. Green painted motor
<5
<0.70
1.00E+01
1.00
<5
<0.70
3. Rod power motor
1.00E+01
1.00
9.00E+01
1.95
<5
<0.70
4. Stainless steel balance (top)
<5
<0.70
1.40E+02
2.15
<5
<0.70
5. Underneath stainless steel table
<5
<0.70
1.21E+03
3.08
9.35E+02
2.97
6. Meat pump nozzle (s.steel)
6.50E+01
1.81
8.95E+02
2.95
<5
<0.70
7. Inside stainless steel meat pipe
2.00E+01
1.30
5.00E+00
0.70
<5
<0.70
8. V-mag stainless steel exit pipe
1.40E+03
3.15
8.00E+01
1.90
5.00E+00
0.70
9. V-mag stainless steel inside hopper
<5
<0.70
9.50E+01
1.98
5.00E+00
0.70
10. Plastic ice slurry pipes (outside)

1E. Drain trap (side)
5.00E+00
<0.70
3.65E+02
2.56
<5
<0.70
2.50E+06
6.40
2.00E+01
1.30
<5
<0.70
2E. Wall cladding
5.00E+00
0.70
6.50E+01
1.81
<5
<0.70
3E. Floor
1.62E+07
7.21
9.40E+04
4.97
3.20E+04
4.51
65
4E. Top of gulley pot (drain)
7.50E+06
6.88
6.00E+03
3.78
2.90E+02
2.46
16
5E. Floor to wall edging (concrete)
7.25E+03
3.86
9.65E+04
4.98
1.13E+04
4.05
45
Pastry manufacturer 2nd day
BEFORE CLEANING
AFTER CLEANING
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
1. Stainless steel vat (inside)
TVC
TVC
TVC
log
log
log
Count/plate
5.00E+00
0.70
5.00E+00
0.70
1.00E+01
1.00
0
2. Green painted motor
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
3. Rod power motor
8.70E+02
2.94
1.50E+01
1.18
5.00E+00
0.70
4. Stainless steel balance (top)
5.00E+01
1.70
1.93E+03
3.29
5.00E+00
0.70
5. Underneath stainless steel table
2.50E+01
1.40
3.50E+01
1.54
7.00E+01
1.85
6. Meat pump nozzle (s.steel)
2.00E+01
1.30
3.45E+02
2.54
4.80E+02
2.68
7. Inside stainless steel meat pipe
1.50E+01
1.18
5.00E+00
0.70
5.00E+00
0.70
8. V-mag stainless steel exit pipe

9. V-mag stainless steel inside
hopper
10. Plastic ice slurry pipes (outside)
5.00E+00
0.70
8.00E+01
1.90
5.00E+00
0.70
5.00E+00
0.70
9.00E+01
1.95
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
1E. Drain trap (side)
6.40E+03
3.81
1.30E+04
4.11
6.85E+03
3.84
61
2E. Wall cladding
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
3E. Floor
1.55E+07
7.19
7.80E+03
3.89
1.87E+03
3.27
45
4E. Top of gulley pot (drain)
3.72E+03
3.57
1.13E+05
5.05
7.00E+01
1.85
10
5E. Floor to wall edging (concrete)
2.74E+04
4.44
7.65E+02
2.88
9.00E+01
1.95
37
Pizza factory 1st day
BEFORE CLEANING
AFTER CLEANING
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
1. Tumbler (inside)
TVC
TVC
TVC
log
log
log
1.55E+02
2.19
1.50E+01
1.18
5.00E+00
0.70
2. Shelf underneath tumbler
8.50E+03
3.93
6.80E+02
2.83
1.50E+01
1.18
3. Red switch on/off (tumbler)
2.00E+01
1.30
2.40E+03
3.38
7.50E+02
2.88
27
4. Glass mixer - blade
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
5. Glass mixer - bottom (inside)
2.65E+02
2.42
2.50E+01
1.40
5.00E+00
0.70
6. Glass mixer - outlet

7. S.steel table-top (next to glass
mixer)
8. Glass mixer - panel
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
4.00E+01
1.60
2.50E+01
1.40
5.00E+00
0.70
1.32E+03
3.12
5.00E+00
0.70
5.50E+01
1.74
9. S.steel table - top (next to tumbler)
1.36E+08
8.13
5.00E+00
0.70
5.00E+00
0.70
10. Blast Chill Room 18B door handle

1E. Floor (S.steel)
7.05E+02
2.85
2.50E+01
1.40
5.00E+00
0.70
1.63E+07
7.21
4.65E+04
4.67
7.45E+04
4.87
59
2E. Wall (behind glass mixer)
2.05E+02
2.31
5.00E+00
0.70
5.00E+00
0.70
3E. Floor (Red)
4.90E+06
6.69
2.39E+06
6.38
5.15E+05
5.71
>3.00e+02
4E. Around drain seal to the floor
5.10E+07
7.71
1.32E+07
7.12
1.89E+05
5.28
>3.00e+02
5E. Drain inside (drain's basket)
4.80E+05
5.68
7.75E+04
4.89
5.00E+00
0.70
Count/plate
0
Pizza factory 2nd day
BEFORE CLEANING
AFTER CLEANING
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
TVC
TVC
TVC
log
log
log
Count/plate
1. Tumbler (inside)
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
4.60E+02
2.66
7.75E+03
3.89
5.00E+00
0.70
12
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
1.90E+02
2.28
5.00E+00
0.70
5.00E+00
0.70

mixer)
5.00E+00
0.70
4.00E+01
1.60
5.00E+00
0.70
6.50E+01
1.81
4.50E+01
1.65
5.00E+00
0.70
1.25E+02
2.10
5.00E+00
0.70
5.00E+00
0.70
9.25E+02
2.97
3.50E+01
1.54
5.00E+00
0.70
11

2.00E+01
1.30
1.30E+05
5.11
8.00E+03
3.90
1E. Floor (S.steel)
7.50E+03
3.88
7.90E+03
3.90
4.60E+03
3.66
5.00E+00
0.70
5.00E+00
0.70
5.00E+00
0.70
3E. Floor (Red)
3.27E+05
5.51
4.35E+05
5.64
3.36E+06
6.53
11
5.15E+05
5.71
2.47E+05
5.39
6.75E+04
4.83
11
1.97E+03
3.29
3.00E+01
1.48
5.00E+00
0.70
Pizza factory 3rd day
BEFORE CLEANING
AFTER CLEANING
AFTER
DISINFECTION
CONTACT
PLATES
AFTER
DISINF.
TVC
Log10
TVC
Log10
TVC
Log10
Count/plate
1. Tumbler (inside)
9.00E+01
1.95
<5
<0.70
<5
<0.70
5.90E+02
2.77
5.00E+00
0.70
<5
<0.70
7.00E+01
1.85
3.65E+02
2.56
<5
<0.70
13
<5
<0.70
<5
<0.70
<5
<0.70
2.40E+02
2.38
5.00E+01
1.70
5.00E+00
0.70

mixer)
<5
<0.70
3.50E+01
1.54
<5
<0.70
1.00E+01
1.00
5.00E+00
0.70
2.50E+01
1.40
145
2.27E+03
3.36
1.00E+01
1.00
<5
<0.70
<5
<0.70
5.00E+00
0.70
5.00E+00
0.70

3.50E+02
2.54
8.85E+02
2.95
8.75E+02
2.94
77
1E. Floor (S.steel)
1.67E+05
5.22
2.02E+03
3.31
5.25E+02
2.72
41
<5
<0.70
2.97E+03
3.47
9.00E+01
1.95
3E. Floor (Red)
3.90E+05
5.59
6.05E+04
4.78
1.31E+03
3.12
95
4.55E+05
5.66
1.08E+04
4.03
6.50E+01
1.81
46
1.10E+04
4.04
5.50E+02
2.74
5.50E+01
1.74
33
APPENDIX 3
Whole room
disinfection technique
Chemical fogging
Equipment
Manufacturers/Suppliers
H&M Disinfection Systems Ltd.
18 Dalby Court
Gadbrook Business Centre
Northwich
Cheshire
CW9 7TN
Tel: +44 (0) 1606 49845, Fax: +44 (0) 1606 330231
Email: sales@hm-dis.com, www.hm-dis.com
Mercatos Ltd.
Hill Top Road
Ambleside
Cumbria
LA22 9EQ
Tel: +44 (0) 845 6025586
Email: info@mercatos.co.uk, www.mercatos.co.uk
Hydrogen peroxide
vapour
Ozone
Atrium Innovation Ltd.

Pipe House, Lupton Road
Wallingford,
OX10 9BT, UK
Tel: +44 (0) 1491 838326, Email: info@cleanaer.com
www.cleanaer.com / www.aerstream.com
Bioquell UK Limited
52 Royce Close
West Portway
Andover
Hants
SP10 3TS
Tel: +44 (0) 1264 835 835, Fax: +44 (0) 1264 835 836
http://www.bioquell.co.uk
Steritrox Ltd.
The Old Stables
Upper End
Birlingham
Worcestershire
WR10 3AA
Tel: +44 (0) 1386 751800
http://www.steritrox.com; http://www.radical.gb.com

Whole Room Disinfection

Caricato da

Informazioni sul documento

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Whole Room Disinfection

Caricato da

Copyright:

Formati disponibili

Members only

Whole room disinfection

R&D report no. 299

R&D report no. 299

Campden BRI 2010

Station Road, Chipping Campden, Gloucestershire, GL55 6LD. UK

resulting in a reduction in excess of 4 logs for P. aeruginosa and 5 logs for L.

MATERIALS & METHODS

Hydrogen peroxide vapour

RESULTS AND DISCUSSION

Hydrogen peroxide vapour

Laboratory and field trial data

Equipment manufacturer and contact details

During manufacture, food can be exposed to microbiological cross-contamination from

There is now a desire to supplement traditional, targeted chemical disinfection with

MATERIALS & METHODS

Figure 1 - The arrangement of test surfaces in the aerobiology laboratory

Hydrogen peroxide vapour

Machine used: 2 x Radical Small Mobile Unit (UMB)

Sample site - product contact surfaces

Plastic shackle (outside)

Plastic shackle (inside)

Back plate of shackle (bird contact)

Stainless steel surface (external)

Stainless steel surface (internal)

Spray head (metal)

Plastic crate (internal)

Stop plastic button

Sample site - Environmental samples

Stainless steel panel (entrance)

(ii) Factory 2: Pastry manufacturer

Sample site - product contact surfaces

Stainless steel vat (inside)

Green painted motor

Rod power motor

Stainless steel balance (top)

Underneath stainless steel table

Meat pump nozzle (stainless steel)

Inside stainless steel meat pipe

V-mag stainless steel exit pipe

V-mag stainless steel inside hopper

Plastic ice slurry pipes (outside)

Sample site - Environmental samples

Drain trap (side)

Top of gulley pot (drain)

Floor to wall edging (concrete)

(iii) Factory 3: Pizza manufacturer

Table 3 - Swab and contact plate sites

Sample site - product contact surfaces

Shelf underneath tumbler

Red switch on/off (tumbler)

Glass mixer - blade

Glass mixer - bottom (inside)

Glass mixer - outlet

Stainless steel table-top

Glass mixer - panel

Stainless steel table - top

Blast Chill Room 18B door handle

Sample site - Environmental samples

Floor (stainless steel)

Wall (behind glass mixer)

Around drain seal to the floor

Drain inside (drain's basket)

(iv) Factory 4: Sandwich manufacturer