The action of ozone generated from a small domestic device was examined with a view to using it in clinical isolation units accommodating immuno suppressed patients. Over a six-hour period in an average size room the device did not generate sufficient ozone to suppress bacterial and fungal growth. A useful bactericidal action, against a variety of human pathogens was achieved with ozone concentrations between 0.3 to 0.9 ppm. Bactericidal ozone concentrations are close to the limit permitted for human exposure however and further experiments are indicated.
Ozone killing action against bacterial and fungal
species; microbiological testing of a domestic ozone
A DYAS, BJ BOUGHTON, BC DAS
From the Departments of Microbiology and Haematology, Queen Elizabeth Hospital, Edgbaston,
SUMMARY The action of ozone generated from a small domestic device was examined with a view
to using it in clinical isolation units accommodating immunosuppressed patients. Over a six-hour
period in an average size room the device did not generate sufficient ozone to suppress bacterial
and fungal growth. A useful bactericidal action, against a variety of human pathogens was
achieved with ozone concentrations between 0-3 to 0-9 ppm. Bactericidal ozone concentrations
are close to the limit permitted for human exposure however and further experiments are indicated.
Ozone has well documented bactericidal properties
which are of use in the decontamination of uninhabited
“bioclean” areas.’ 2 The purpose of the present
study was to determine the effectiveness of an inexpensive
domestic ozone generator in maintaining an
ultra clean environment for immunosuppressed
Material and methods
The Miles and Misra technique of bacterial enumeration3
was modified as follows. Peptone water suspensions
were made from plate cultures of E coli,
Proteus sp, Pseudomonas aeruginosa, Serratia sp,
three strains of Staphylococcus aureus, Candida
albicans and Aspergillus fumigatus. Subsequent tenfold
dilutions were made in sterile water. Two separate
20 Al drops of each dilution were then delivered
on to each of two predried plates. Blood agar was
used, except for Proteus sp where cysteine lactose
electrolyte deficient (CLED) agar was used and for
Aspergillus fumigatus where Sabouraud’s agar was
used. One plate was exposed to ozone for 4 h, the
duplicate being placed in an ozone free environment.
After 4 h all plates were incubated overnight
at 37°C and the colonies counted using those dilutions
which gave discrete colonies on both plates.
All experiments were performed in duplicate.
Ozone was produced using a small “Airbracer”
Accepted for publication 31 May 1981
produced by Coronair Ltd, Effingham, Surrey. The
machine has a constant output of ozone and variable
concentrations were produced by operating it in different
test volumes. The main experiments used
either a cupboard (0.087 m3) or a hospital room
(66 m3). In the cupboard, the Airbracer was
switched on at the beginning of the experiment and
in the hospital room it was switched on two hours
prior to commencing the experiments. Ozone concentrations
were monitored using the Bendix 8002
analyser (Bendix Instruments, Lewisbury, West
In the smaller space (0.87 x 10-2/m3), ozone concentrations
varied between 03 and 0 9 ppm. These
levels were reached within 10 min of switching on
the machine. In the hospital room experiments,
ozone was undetectable (despite an analyser
sensitivity of 0-001 ppm).
The Table shows the CFUs surviving exposure to
0*3-0*9 ppm of ozone for 4 h. At this concentration
ozone effectively inhibited the growth of all the bacterial
species and Aspergillus fumigatus. Candida
albicans was comparatively resistant however.
In additional experiments using plates exposed to
ozone 0 9 ppm for 4 h prior to bacterial inoculation,
it was possible to show that the effect of ozone on
the agar was not bactericidal.
In the hospital room experiments at concentra-
Ozone killing action against bacterial and fungal species
Range ofconcentrations ofseven bacterial and two fungal species (dispensed in 20 p1 volumes) exposed to 0*3-0-9 ppm
ozone for 4 h. The Table shows the number ofcells surviving such treatment
Concentrations before ozone exposure (CFUslml)
106 los lO4 103 102
Staph aureus 5000 150 75 – –
Staph aureus (penicillin-resistant) * 450 75 – –
Staph aureus (flucloxacillin-resistant) * * 25 –
E coli _0 –
Ps aeruginosa – – * 125 50 Serratia sp – * 150 50 –
Proteus – 50 25 25
Aspergillus fumigatus – – 800 – –
Candida albicans – – * 25 50
The results are a mean of two experiments.
tions of ozone below 0-001 ppm, no bactericidal
effect was seen with any of the organisms tested.
Fig. 1 shows further experiments in which Staph
aureus was exposed for 0-4 h to 0-9 ppm ozone. The
bactericidal effect was mos; rapid within the first two
hours of exposure but thereafter, the killing rate was
Our studies have demonstrated that when operating
in a small space, the generator produced bactericidal
concentrations of ozone (in the order of 1 ppm).
However, its use in a hospital single room produced
undetectable concentrations of ozone, and no bactericidal
effect could be demonstrated. Previous
workers have demonstrated that the minimum bactericidal
concentration of ozone is 1 ppm,4 whilst
other studies have suggested values between 20 ppm
and 40 ppm.’ 2 Broadwater and colleagues5
described bacterial killing by ozone as an “all or
none” effect but Hamelin and Chung4 showed a
progressive effect occurring over 1 ppm. In our
studies, 1 ppm ozone was sufficient to reduce the
numbers of most of the species tested by greater
Hibben and Stotzkey6 examined the effect of
ozone on fungal spore germination. Compared with
bacteria, they found fungi to be less sensitive.
Aspergillus spp, were found to be of intermediate
sensitivity and required 1-1-5 ppm for six hours to
inhibit germination. Our own experiments showed
Aspergillus sp and C albicans to be less sensitive to
It is disconcerting however that in low ozone concentrations,
mutations have been reported. Hammelin
and Chung described the appearance of four
mutant strains of E coli growing in 0*1 ppm ozone.4
Our work was carried out to examine the possible
use of this small ozone generator to decontaminate
rooms containing immunosuppressed patients.
Ozone, at high concentration, has been used to disinfect
rooms prior to patient occupation,2 but these
concentrations are toxic to man.78 Mortality rates
are increased in exercising mice exposed to 0-3 ppm
ozone for three hours and then challenging them
with an aerosol of Strep pyogenes.9 In mice exposed
1 2 3 4
The number ofsurviving Staph aureus exposed to ozone,
0-3 to 0-9 ppm for 0-4 h.
to 2*5 ppm for five hours and allowed to inhale
Staph aureus, the rate of phagocytosis of pulmonary
bacteria slowed compared with controls.’0 In addition
Peterson and colleagues” studied polymorph
function in healthy male human volunteers exposed
to 0*35-042 ppm ozone for 4 h. They found that, at
72 h after exposure, both bacterial killing and
phagocytosis were moderately but significantly
We conclude that our domestic ozone generator
did not produce bactericidal concentrations of ozone
when operating in a hospital room. Higher and bactericidal
ozone concentrations may have unacceptable
toxic side effects to man but this, like the effect
on airborne bacteria remains to be established.
We would like to thank Coronair Ltd for the loan of
their Airbracer and Dr Alistair Kerr, Department of
Chemistry, Birmingham University for the loan of
the ozone analyser. Dr B Boughton is Senior Lecturer
to the Leukaemia Research Fund.
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Dyas, Boughton, Das
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Hibben CR, Stotzky G. Effects of ozone on germination of fungus
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Requests for reprints to: Dr BJ Boughton, Department of
Haematology, Queen Elizabeth Hospital, Edgbaston,
Birmingham B15 2TH, England.