Evaluation of an Ozone Cabinet for Disinfecting Medical ...1262996/FULLTEXT01.pdf · Datum Date 2018 -11 -05 Avdelning, institution Division, Department Department of Physics , Chemistry

Linköping University | Department of Physics, Chemistry and Biology Master thesis, 30 hp | Educational Program: Physics, Chemistry and Biology

Spring and Autumn term 2018 | LITH-IFM-A-EX—18/3583--SE

Evaluation of an Ozone Cabinet for Disinfecting Medical Equipment

Ida Ljungberg

Examinator, Thomas Ederth Tutor, Maria Lerm

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2018-11-05

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Department of Physics, Chemistry and Biology

Linköping University

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Evaluation of an Ozone Cabinet for Disinfecting Medical Equipment

Författare Author

Ida Ljungberg

Nyckelord Keyword

Sammanfattning Abstract

The spreading of infection is a significant and well-known problem in all healthcare environments today. The most prevalent ways that infection spreads are either by direct contact between two individuals where one has an infection, or with an

intermediate person or object as an infection carrier. This thesis aims to evaluate a method that could operate to disinfect the

type of medical equipment which is not suited to be disinfected by the commercially existing methods.

In keeping with the long term goal of preventing the spread of infection, this project evaluates an ozone cabinet according to its

antimicrobial properties and investigates if the cabinet is suited to work as a disinfectant for some chosen test objects. The

objects were borrowed from different hospital institutions at Motala Lasarett and the antimicrobial effect was evaluated

according to the reduction of colony forming units (CFUs) of samples taken from the object's surfaces after the treatment.

The results show that the ozone cabinet is not able to kill bacterial spores (Geobacillus stearothermophilus), but could be very efficient at killing living bacteria. Concentration setting 4 (56 ppm) in combination with a treatment period of at least 40 minutes proves bacterial reductions varying between 83-98 %. Nevertheless, the sources of error are numerous and there is a great variation between identical runs which indicates that more studies need to be performed in order to obtain clearer results.

.

Master of Science in Applied PhysicsEvaluation of an Ozone Cabinet for Disinfecting Medical

Equipment

Ida Ljungberg

LITH-IFM-A-EX—18/3583—SE

Supervisors: Maria LermIKE, Linköping University

Magnus StridsmanClinicum Test and Innovation, Region Östergötland

Martin BerglundMedicinsk teknik, Region Östergötland

Magnus RobergVårdhygien, Region Östergötland

Examiner: Thomas EderthIFM, Linköping University

Department of Physics, Chemistry and BiologyLinköping University SE-581 83 Linköping, Sweden

Copyright © 2018 Ida Ljungberg

AbstractThe spreading of infection is a significant and well-known problem in all healthcare en-vironments today. The most prevalent ways that infection spreads are either by directcontact between two individuals where one has an infection, or with an intermediate per-son or object as an infection carrier. This thesis aims to evaluate a method that couldoperate to disinfect the type of medical equipment which is not suited to be disinfectedby the commercially existing methods.

In keeping with the long term goal of preventing the spread of infection, this projectevaluates an ozone cabinet according to its antimicrobial properties and investigates ifthe cabinet is suited to work as a disinfectant for some chosen test objects. The objectswere borrowed from different hospital institutions at Motala Lasarett and the antimicro-bial effect was evaluated according to the reduction of colony forming units (CFUs) ofsamples taken from the object’s surfaces after the treatment.

The results show that the ozone cabinet is not able to kill bacterial spores (Geobacillusstearothermophilus), but could be very efficient at killing living bacteria. Concentrationsetting 4 (56 ppm) in combination with a treatment period of at least 40 minutes provesbacterial reductions varying between 83-98 %. Nevertheless, the sources of error are nu-merous and there is a great variation between identical runs which indicates that morestudies need to be performed in order to obtain clearer results.

AcknowledgmentsFirst, I would like to thank Clinicum Test and Innovation for giving me the opportu-nity to do this Master Thesis project. I would like to thank Magnus Stridsman for givingme a great introduction to the project and supporting me along the way with great en-couragement. Special thanks to Martin Berglund for helping me with all the problemsthat arose during this project and always making time for me and supporting me. Thisproject would never have been possible without your help. Also, thank you to MagnusRoberg for being a source of guidance and your interest in my work.

I would also like to thank Maria Lerm for valuable opinions, feedback and providingme with a lab for my work. Also, thank you to Thomas Ederth for examining this work.

Finally, thank you to Sepideh Kamrani for teaching me how to work in the lab andadvising me from a biomedical analyst’s point of view.

Linköping, October 2018Ida Ljungberg

Contents1 Introduction 1

1.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Problem statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2 Theory 42.1 Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1.1 Ozone as a disinfectant . . . . . . . . . . . . . . . . . . . . . . . . 42.1.2 Ozone production . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.1.3 Current research . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2 The ozone cabinet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.2.1 Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.2.2 Previous bacteria tests in the ozone cabinet . . . . . . . . . . . . 9

2.3 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.3.1 Spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.4 Colony forming units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.5 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.5.1 Hematin agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.5.2 UTI agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.5.3 Blood agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.6 Spore specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.7 Dry disinfection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.7.1 Hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . 132.7.2 UV light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.7.3 Ethylene oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.7.4 Gamma radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.7.5 Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.8 Degrees of cleanliness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.9 Defining disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.10 The test objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.10.1 Blood pressure cuff . . . . . . . . . . . . . . . . . . . . . . . . . . 182.10.2 Drug pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.10.3 X-ray neck collar . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.10.4 Transportation bag . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Materials 22

4 Method 234.1 Specimen collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234.2 Obtaining data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244.3 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

4.3.1 Antimicrobial efficiency . . . . . . . . . . . . . . . . . . . . . . . . 254.3.2 ANOVA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5 Results 275.1 Spore specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.2 ANOVA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.3 Mean and standard deviation . . . . . . . . . . . . . . . . . . . . . . . . 315.4 Antimicrobial efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335.5 Disinfection with Meliseptol . . . . . . . . . . . . . . . . . . . . . . . . . 365.6 Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375.7 Visual effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

6 Discussion 396.1 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

6.1.1 Spore specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396.1.2 ANOVA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396.1.3 Mean and standard deviation . . . . . . . . . . . . . . . . . . . . 406.1.4 Antimicrobial efficiency . . . . . . . . . . . . . . . . . . . . . . . . 416.1.5 Disinfection with Meliseptol . . . . . . . . . . . . . . . . . . . . . 436.1.6 Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446.1.7 Visual effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

6.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456.3 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

7 Conclusion 49

Appendices 50

Appendix A Specimen collection and analysis -a description 50

Appendix B ANOVA 53

Appendix C Data 57

Appendix D Data summary 73

Linköping University i

Abbreviations

Abbreviation DefinitionANOVA Analysis of VarianceBI Biological carrierBPC Blood Pressure CuffCFU Colony Forming UnitCPAP Continuous Positive Airway PressureCxDy Concentration setting x in combination with duration setting y

(x = 1,2,3,4), (y = 1,2,3,4,8)eSwab Copan Liquid Amies Elution SwabsNC X-ray Neck CollarUTI Urinary Tract InfectionUVR Ultraviolet Radiation

Master Thesis report

Linköping University 1

1 IntroductionThis thesis work has been carried out at Clinicum test and innovation, Region Östergöt-land and examined at IFM, theDepartment of Physics, Chemistry and Biology at LinköpingUniversity. The purpose of the project was to investigate if an ozone cabinet could be usedto disinfect some different types of medical equipment. There is no previously publishedliterature covering this, however, there is literature that describes the effect of ozone ingeneral as a disinfectant.

1.1 Motivation

The most common way of infection spreading within Sweden’s healthcare institutionstoday is through intermediate contact spreading when the infection is spreading from oneperson to another through a third person or object. [1] Various pieces of medical equip-ment which are frequently used in hospitals today are not being cleaned or disinfected.This could be due to their material which might be complicated to clean or disinfect withthe techniques that are available today. The equipment might also be of a complicatedstructure with many cavities and joints which could be challenging to clean properly. Ifit were possible to use an ozone cabinet to disinfect some of these objects, this mightreduce the spread of infections in Sweden’s healthcare institutions.

1.2 Background

Elozo Oy is a company stationed in Finland which produces different solutions for saferand healthier surroundings. Among their many products are different kinds of ozonecabinets. The ozone cabinets are sold commercially and are utilized mainly for removingodors from fire fighter’s clothes, theater costumes or smelly mattresses, rugs or othertextiles. [2] One of the ozone cabinets produced by the company, Elozo D800 CleaningSystem (see Figure 1), was donated to the Medical Technology department at LinköpingUniversity Hospital, Region Östergötland. It is of interest for Elozo Oy to expand theareas of use for the cabinet and it is of interest for the hospital to find new disinfectionmethods to reduce the spread of infections.



Figure 1: The ozone cabinet (Elozo D800 Cleaning System) [3]

1.3 Problem statement

This thesis work investigated the possibility of ozone being used as a disinfectant fordifferent objects that are hard to disinfect with other methods that are standard withinhospitals in Sweden. The objects were chosen due to their different materials and shapesto better understand what objects are suitable for being disinfected in the cabinet. Thequestions that this thesis work aimed to answer are the following:

- Can a blood pressure cuff, a drug pump, an X-ray neck collar and a transportationbag for Continuous Positive Airway Pressure (CPAP) and similar devices be disinfectedby being placed in the ozone cabinet?

- What concentrations of ozone in combination with what duration of treatment hasthe best effect on each of those different objects, respectively?

This project also investigated the effect that the ozone cabinet had on bacterial spores.Another question that was answered in this study was, therefore:

- Does the ozone cabinet have the ability to kill bacterial spores (Geobacillus stearother-mophilus)?

If time allowed, the following question was aimed to be answered by this thesis work:

- Can a CPAP, a mattress cover, an emergency room pillow cover, ambulance ECGelectrodes or a sling lift be disinfected by being placed in the ozone cabinet?

Before this thesis work, the antimicrobial efficiency of the cabinet was unknown. This



raised an additional question to extend the work if the cabinet was found not to be suit-able for disinfection purposes. In this case, the thesis would examine the efficiency of theexisting cleaning procedure for each of the chosen objects by finding an answer to thefollowing question:

- How well do the currently used cleaning methods work for a blood pressure cuff, adrug pump, an X-ray neck collar, a transportation bag for CPAP (and similar) devices,a CPAP, an emergency room pillow cover and ambulance ECG electrodes (if they arebeing cleaned by the hospital staff)?

1.4 Limitations

The project was limited to testing only the objects listed in Section 1.3 and no other.The project was also limited to testing the ozone cabinet. A comparison would be madeto the existing disinfection methods for the individual objects only if applicable and ifthere was time or if the ozone cabinet did not exhibit satisfying levels of antimicrobialproperties.

Another limitation of this project was the number of objects that could be tested. Ifthe objects were hard to access that might affect the number of tests that could be per-formed and so that will have an influence on the statistical validity of the results. Theproject was also limited to the material that is available from Vårdhygien and the lab timeat IKE, the Department of Clinical and Experimental Medicine at Linköping University.



2 TheoryThis chapter contains the theory related to this project along with a description of theobjects that will be investigated and the current method used to disinfect them.

2.1 Ozone

Ozone is a chemical compound consisting of three oxygen atoms put together, formingO3. Ozone exists naturally in the atmosphere and is a colorless (or blue like) gas. [4] Theozone in the atmosphere is generated by oxygen, organic compounds or nitrogen oxidesthat are catalyzed by UV radiation. [5] Ozone has many different areas of use and can,for example, be used to sterilize bottled water, disinfect wastewater and eliminate odors.Ozone is also used as an antimicrobial agent in food processing and can be utilized in firerestoration. [6] The ozone molecule can be visualized in Figure 2.

Figure 2: The ozone molecule [7]

Ozone has, among many other hazards, the ability to cause and intensify a fire, causeskin irritation, eye irritation, respiratory irritation and may be fatal if inhaled. [5]

2.1.1 Ozone as a disinfectant

Ozone is an unstable molecule that will give away an oxygen atom and form O2 at anygiven opportunity. Ozone can work as a disinfectant since it will oxidize molecules thatbuild up the bacteria and thereby destroy them in the process when the ozone moleculegets reduced and gives away an atom. This process, by which ozone eliminates bacteria,is called an oxidative burst. When the ozone comes in contact with the bacterial cellwall, this process will take place and generate a hole in the cell wall. [8] The burst orig-inates from a reaction between the ozone molecules and the double bonds of the lipidsthat constitute the cell membranes. [9] This will lead to the bacteria having difficultiesmaintaining their structure at the same time as more ozone molecules create more holes.This will eventually lead to the bacteria not being able to keep their content and henceit will die. [8] The ozone will also enter the cells after a hole is created and oxidize thenucleic and amino acids, which also leads to cell lysis. [9]

Since the ozone will give away an oxygen atom in this reaction, this means that thebyproduct after the process is oxygen (O2), which is completely harmless. This indicatesthat there are no harmful byproducts that are produced in the process, which is very



favorable. [10] When using this technique, the only instrument required is the cabinetin addition to a power outlet to provide the cabinet with electricity. There should alsobe an extraction pipe to pump out any indwelling ozone molecules that might still bepresent after the process.

2.1.2 Ozone production

Ozone can be produced in several ways, for example (as mentioned in section 2.1) throughUV radiation in the atmosphere. The ozone cabinet used in this thesis work utilizes amethod called the corona discharge to produce ozone. The corona discharge is the processwhen a high voltage is applied to an oxygen gas flow, which splits the O2 molecules intoindividual oxygen atoms (see Figure 3). When these individual O atoms get in contactwith naturally occurring O2, the two will collectively form O3 molecules. [11]

Figure 3: The corona discharge [11]

2.1.3 Current research

There is a great deal of research regarding the usage of ozone as a disinfectant but mostof it is in relation to smaller laboratory experiments. By reading about the applica-tions for the ozone cabinet at the vendor’s website, the only thing that is mentionedfor hospital use is for eliminating odor from mattresses and bed linen and nothing con-cerning disinfecting medical equipment. [2] However, regarding the smaller experiments,there is a large number of articles published regarding the usage of ozone as a disinfectant.

There are studies showing that ozone can be used to reduce the number of bacteriain the tooth root canal. In a study from 2013, researchers found that 82 % of all thebacteria (Streptococcus mitis and Propionibacterium acnes) in the tooth root canal wereeliminated as a result of being treated with ozone gas for 40 seconds. The ozone concen-tration used in this study was 2100 ppm (± 10 %). [12]



Another study from 2015 shows that corrugated tubes (used for tracheostomized pa-tients) that have been in contact with non-intact skin and need high-level disinfectioncould be exposed to ozone gas and successfully be disinfected. The study used an ozonegenerator which produced 33 mg/L of ozone and treated the tube for 15 minutes. Theresult showed that 99.99 % of all bacteria (Pseudomonas spp., Alcaligenes spp., Acineto-bacter, Klebsiella spp., Citrobacter, and Enterobacter) died from the treatment. [13]

Most studies that are published on ozone as a disinfectant show that the research fo-cus lies in disinfecting wastewater and foods, even though there are a few that focus ondisinfecting medical equipment (such as the one above). There are no scientific studies onozone cabinets’ effect on medical equipment, which means that this project will includenew research.

This project will also investigate the effect that the ozone cabinet has on bacterial spores.Several articles describe how ozone can successfully kill spores in liquid samples. For ex-ample, in a study from 2014, it was proved that a 40-minute treatment (when a bubblingstream of ozone of 5.3 mg/L was applied to a liquid) induced a 99 % reduction of A.acidoterrestris spores. [14] Several other articles describe how ozone in combination withsome other disinfectant can prove to be sporicidal. One example is a study from 2011where a combination of 80 ppm ozone together with a 3 % hydrogen peroxide vaporproved a 99.9999 % reduction of Bacillus subtilis spores. In this study, the spores weredried onto steel discs or cotton gauze pads and the treatment lasted for 30 to 90 minutes.[15] The number of published research papers that covers the sporicidal properties ofgaseous ozone alone are very few but they exist. An article published in 2006 describeshow ozone showed successful results in killing Bacillus subtilis spores. The treatmentwas made using a very high concentration of ozone (1500 ppm) which yielded a 99.9 %reduction of the spores after a 4-hour exposure. In this study, the spores were dried ontoa glass surface prior to ozone treatment. [16]

2.2 The ozone cabinet

The ozone cabinet that will be used in this project is the Elozo D800 Ozone CleaningSystem, see Figure 1. The cabinet has a volume of 758 L and is made from stainlesssteel components. It contains an ozone generator, three fans (that circulate the ozone airinside the cabinet) and supportive electronics. [17] Its specifications can be read in thetable below.



Table 1: Table of specifications for the ozone cabinet [17]

SpecificationsApprovals Electricity safety IEC-60335-1

EMC-tests EN 55022EN 610004-2,-3,-4,-5-6 ja -11

CE-certified

Name Elozo D800 Ozone Cleaning System

Voltage Uin 220-240 VAc 1Imax 5.3 A

Power consumption Pmax 0.35 kW/h

Ozone production 100 % power 1500 mg/h

Safety Door safety / auto stop mechanismElectromagnetic lock mechanismConnection to a ventilation system required

Elozo Oy recommend that the cabinet is used in a clean and dry environment wherethe temerature is 15-25 °C and the relative humidity is lower than 50 %. [18] Bothtemperature and humidity might have an effect on the final result since they both canaffect bacterial growth. [19] The temperature and humidity inside the cabinet cannot bechanged from the outside, but they will both be measured and used as a potential errorsource if the values are not within the limits set by the manufacturer.

2.2.1 Settings

The cabinet offers different settings for the treatment duration in four steps; 20, 40, 60 and120 minutes. The cabinet also admits different settings for ozone concentration, namedstep 1, 2, 3 and 4. All settings can be set at the cabinet’s instrument panel which can beseen in Figure 4. The power settings correspond to maximum concentrations according toTable 2. The concentrations were measured by Elozo Oy during the 60 minute programwith an industrial ozone analyzer (Teledyne model 465L). The values are approximateand are affected by the external suction and how many items are being placed in thecabinet. It is also affected by how many microorganisms exist on the surfaces of theobjects. Upon request, Elozo Oy made further measurements of the shorter programs toinvestigate whether the ozone concentration would reach the same levels as the one hourprogram. The results show that the 20-minute program yields maximum ozone levels18-35 % lower than the one hour program. The maximum ozone concentration from the20-minute program can be visualized in the middle column in Table 2.



Table 2: Maximum concentrations of the 20-minute and one hour program, respectively

Power setting Conc at 20-minute program Conc at 1-hour program1 24 ppm 37 ppm2 32 ppm 48 ppm3 42 ppm 54 ppm4 46 ppm 56 ppm

Figure 4: The instrument panel

The Elozo ozone cabinet produces ozone without considering the current ozone concentra-tion inside the cabinet. The cabinet takes in surrounding air and produces ozone pulsesevery 10 minutes. This can be seen in Figure 5.



Figure 5: Measurement of the ozone concentration in the cabinet during the one hourprogram

2.2.2 Previous bacteria tests in the ozone cabinet

The Finnish company SeiLab Oy have done some testing on one type of bacterium andmold in the ozone cabinet to investigate how well they survive in the cabinet. The testswere done in collaboration with Elozo Oy, and was made with the D-series ozone cabinetson hospital textiles. The organisms tested were Listeria monocytogenes (causes stomachflu, infections, and meningitis) which can be discovered in foods and animals and thesecond was mold (causes asthma and respiratory infections) which can be found in moistinteriors and old structures. The results from the experiments were that a treatmentperiod of 58 minutes for Listeria monocytogenes and 2 hours for mold resulted in a 100% elimination of the bacteria or fungi (mold). [20] The concentration settings were notmentioned in the report.

2.3 Bacteria

Bacteria exist everywhere and their main function is to break down organic substances.The bacterial cell contains a single-stranded DNA molecule which flows around in thebacterial cytoplasm. The cytoplasm consists of water, protein, fat, and carbohydrates.Along with these substances, ribosomes are also present in the cytoplasm to control thereplication of the cell. The bacterial cell membrane consists of phospholipids and pro-teins but their appearance differ depending on if it is a gram-positive or gram-negativebacterium. Gram-positive cells have one cell membrane while gram-negative cells have adouble membrane. The bacteria also have a cell wall to help it stay together and improvethe strength of the cell. The appearance of bacteria can be very different as they can, forexample, take the shape of a globe, a rod or be spring shaped. [21]



When an infection has occurred, the infectious subject has broken through the protectivetissue (our skin) somewhere on our body. This could happen through a wound, by eatingor drinking something, by inhalation, or through mucous membranes. [21]

2.3.1 Spores

When bacteria live under environmental conditions that are not beneficial (too warmsurroundings, in the presence of chemicals or lack of nutrition), some bacteria speciesmight transform into spores as a way to survive. The DNA in the cell encapsulatesitself and becomes much more resilient to environmental stresses. When this happens,the cytoplasm, cell membrane, and cell wall dry up together and create a waterproofand heat resistant shell around the DNA strand. The bacterial cell can survive as aspore for a long time (up to several years) until the spore is exposed to a more beneficialenvironment. Then it has the ability to go back to its normal cell structure. The sporestructure makes bacteria much more resistant to disinfection. To obtain a trustworthydisinfection it is therefore very beneficial to use a method that is able to kill spores aswell as vital bacteria. [21]

2.4 Colony forming units

Bacteria are present everywhere and are measured in colony forming units (CFUs) sincethey form colonies when inoculated onto different agar plates. Everybody has CFUs allover their bodies but a person with an infection or some kind of skin disease has a muchhigher CFU quota. These persons are therefore more likely to transmit bacteria to others,which might cause a spread of infection. [22]

The colony forming unit is a measure of bacterial colonies that are present in a sam-ple. CFUs are measured in CFU/mL (or CFU/g for solid samples or CFU/area forsurface samples) and they reveal the amount of viable bacterial cells in a sample. [23]After taking a sample, the diluted specimen is placed evenly on the surface of an agarplate before being incubated for a certain time. After this, the bacterial colonies can becounted with the naked eye. By doing this before and after the objects are placed in theozone cabinet, it is possible to measure a change in CFUs to see if the method is effective.

Different bacteria have different life spans and according to Sepideh Kamrani, biomedicalanalyst at Region Östergötland, the bacteria used in this project have life spans varyingbetween one day to several months. It could be of great value to determine the bacterialspecies that will be grown in this project to see how long they would survive without theozone treatment. However, this is something that will not be done in this project due tolimited time and resources.



2.5 Agar

To grow bacteria, the sample taken from the test subject is transferred to a growthmedium which is left for incubation in a heated cabinet for one to two days. This willprovide the medium with optimal conditions for maximal bacterial growth. Differentbacteria prefer different mediums (that provide specific nutrients) which means that itis possible to control which species are grown. [21] In this project, three different agarplates will be used as a growth medium for the bacteria. The reason for these specificones is that they provide growth platforms for the most common bacteria that are beingspread through indirect contact within healthcare environments. The three agar typeswill be presented below along with the bacteria which can be characterized using them.These three different types of agar plates provide growth mediums for both gram positiveand gram negative bacteria, which is beneficial for this study since it is possible that theyreact differently to ozone.

2.5.1 Hematin agar

Hematin agar is a non-specific growth medium which is used to isolate fastidious bacteria.The hematin agar is a brown agar with a heated blood additive. [21] The bacteria thatcan be cultivated on the hematin agar are Neisseria gonorrhoeae, Neisseria meningitidis,Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae. [24] Theycan cause diseases such as gonorrhea, meningitis, pneumonia, and tonsillitis. [21] Each ofthe bacteria can be characterized by studying the shape, color, and size of the colonies.

2.5.2 UTI agar

Urinary Tract Infection (UTI) agar is used to identify the main microorganisms thatcause urinary tract infections. The prepared UTI agar is a white opaque gel placedin Petri dishes. The bacteria which can be observed and characterized using this agarare Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aerugi-nosa, Proteus mirabilis, and Staphylococcus aureus. [25] The different bacteria can all beindividually identified according to their appearance (color, size and shape of colonies).

2.5.3 Blood agar

Blood agar is an agar with blood additive which most bacteria can grow on. [21] Thebacteria species that grow on blood agar are Streptococcus pneumonia, Streptococcuspyogenes, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. [26] Thesebacteria species can, for example, cause pneumonia, tonsillitis, scarlet fever, impetigo,sepsis, severe wound infections and muscle infections.



2.6 Spore specimens

Spore specimens are often used as a control or reference for sterilization processes. Abiological carrier (BI) is used and loaded with a known concentration of spores. In thisproject and in many other, Geobacillus stearothermophilus is used. The reason behindthis choice is that this bacterium is highly resistant to environmental stresses and providesa good test that does not depend on what material it grows on. The BIs are loaded witha large number of microbial populations compared to the amounts which are present onitems in hospital environments, which means that if the disinfection method is capableof killing the spores, it is a good disinfection method in general. [27]

In this project, Geobacillus stearothermophilus spores are inoculated onto stainless steeldiscs (BIs) which are placed in permeable Tyvek envelopes. The spores are inoculatedon the inside curves of the discs prior to placement in the Tyvek envelopes. The materialof the envelopes allows a flow of gas to pass through its walls, which will make the ozonereach the disc when it is placed in the ozone cabinet. [28] The stainless steel capsules canbe seen from two angles in Figure 6, and the Tyvek envelope (which contains a disc) canbe seen in Figure 7.

Figure 6: Stainless steel disc

Figure 7: A spore specimen in the Tyvek envelope [28]

2.7 Dry disinfection methods

The most common way of sterilizing hospital equipment is by steam sterilization with anautoclave. The technique rapidly kills 100 % of all bacteria and spores and is inexpen-sive, nontoxic and easy to use. The only disadvantage is that the contaminated objects



subjected to this form of sterilization must be able to withstand heat and moisture. [29]

One of the advantages of working with ozone is that the objects do not have to beresistant to moisture and heat. The medical equipment that is sensitive to heat andmoisture can be hard to disinfect by these procedures and because of this, some of themost commonly used dry disinfection methods will be presented in this section.

2.7.1 Hydrogen peroxide

Hydrogen peroxide, H2O2, is a compound of great interest since its byproducts are com-pletely harmless (water and oxygen). Hydrogen peroxide has the ability to producehydroxyl radicals, OH, which react with lipids, DNA, and proteins. [30] Studies havealso shown that the hydroxyl radicals carry strong oxidative properties which can degradenucleic acids, enzymes, and cell membrane components. [31] This means that there areseveral ways that the gas can cause injuries to bacterial cells which might kill them, andthereby clean or disinfect a contaminated object.

Hydrogen peroxide has advantages including its byproducts being non-hazardous, a rel-atively short disinfection period (items can be sterilized in 80 minutes or less) and is nottemperature dependent. Another benefit of using the gas is that the treated objects donot have to be ventilated after the process. [32] Hydrogen peroxide is a gas of great inter-est due to its favorable properties and there are constantly new studies being published inthis field. For example, there is research carried out by Vårdhygien, Region Östegötlandthat examines how an entire room can be disinfected by a single device that exudes thegas. However, there are also disadvantages of employing the gas, including that it hasshown to not possess the same antimicrobial effects when it comes to textile materialswhich are recommended to be disinfected in other ways. [33] The gas is also flammable,can cause eye damage and should not be inhaled due to its toxicity, which could makehydrogen peroxide a gas difficult to handle. [34]

2.7.2 UV light

Ultraviolet radiation (UVR) mainly induces two different photoproducts (cyclobutanepyrimidine dimers and 6–4 photoproducts) when it comes in contact with a DNA helix.The cyclobutane pyrimidine dimer is a result of UVR induced bindings between twoadjacent thymine or cytosine nucleobases. In the case of thymine, the thymine moleculeincludes an aromatic heterocyclic compound that double binds to its neighboring thyminethrough bonds between the two carbon atoms at the fourth and fifth position, respectively(see Figure 8). The same process applies to two adjacent cytosine molecules. [35]



Figure 8: Cyclobutane pyrimidine dimer [35]

The 6-4 photoproducts (Figure 9) are a result of UV radiation that induces a bindingbetween the sixth carbon atom in the thymine aromatic heterocyclic compound and thefourth carbon atom in an adjacent cytosine molecule. The same process can take placebetween two adjacent cytosine molecules. [35]

Figure 9: 6-4 photoproduct [35]

The formation of these products will break the bindings between the base pairs in thehelix and instead form bonds to other nucleotides on the same strand, see Figure 10.If the damage is not repaired, the new bindings might disrupt DNA transcription andreplication processes. This can lead to errors in the reading of genetic code and causemutations and death. [36, 37]



Figure 10: UVR causing a bond between same stranded base pairs [38]

UV light sources have been used for decontamination purposes within healthcare envi-ronments for the last 60 years but have become of great interest especially during thepresent time due to the excessive use of mobile phones and tablets in today’s hospitalcare. [39] Since these devices are not originally made for use in this environment, theyare not suited for being disinfected with the liquid disinfectants that are most commonlyused. The usage of UVR has been shown to be a good alternative for disinfecting planesurfaces (glass or PPE), such as for smartphones and tablets. [40] New research in thisfield is published continuously and it is of great interest to hospitals to prevent infectionspreading by broadening the spectrum of materials that can be disinfected with UV lightin a convenient manner. [41]

Since UV light can split O2 molecules to create single O atoms and thereby create ozone(for example in the atmosphere), this should be something that also happens when UVRis used as a disinfectant if there is normal air surrounding the test environment. However,no scientific articles have been published to cover this matter. Nonetheless, there havebeen many articles describing the effect of UVR in combination with ozone when themethods are used simultaneously to increase the antimicrobial effect of the treatment.[42, 43]

2.7.3 Ethylene oxide

Ethylene oxide has a strong alkylation reaction (reaction of giving away an alkyl group)with nucleic acids and functional proteins existing in cells. By exposing contaminatedequipment to this gas, the alkylation reaction disrupts the normal cell activity of the bac-terial cells in the equipment and can thereby disinfect the object since the denaturationcould lead to cell death. [44]

Ethylene oxide is a gas with bactericidal and fungicidal properties and is effective againstmost materials. [45] The main advantage of using ethylene oxide as a disinfectant is



that it can be used on materials that are sensitive to both heat and moisture withoutdestroying the material. [46] However, when exposed to humans, the gas causes irritationin the eyes, throat, and skin and also leads to feelings of nausea and vomiting. A chronicexposure can lead to the development of leukemia, cancer of the pancreas, stomach can-cer, and non-Hodgkin lymphoma. [45] In addition to the health hazards connected tothis method, the process of disinfecting materials with ethylene oxide is expensive andtime-consuming (one treatment can take up to six hours). Ethylene oxide is also a gasthat is known to be flammable, a serious risk to be considered when using this gas. Dueto the fact that many materials absorb the gas, there is a need for the products to beleft in air for some time after the process to remove superfluous ethylene oxide molecules.[32, 46]

2.7.4 Gamma radiation

Gamma rays are high energy photons which bombard the object that is going to be dis-infected. This causes a displacement of electrons within the material of the object whichcreates free radicals that help in breaking chemical bonds. These radicals can breakthe bonds between the base pairs in the DNA helix of bacterial cells which will hinderreproduction and cause apoptosis. In this manner, gamma radiation can be used as adisinfectant since the rays break down DNA in bacterial cells that contaminate the object.

Medical devices that are made of polymers are however not suited for this method sincethe crosslinking of polymers change when they are being exposed to gamma radiation.This can change the tensile strength, impact strength and elongation at break. Metalsare normally not affected negatively by gamma radiation, but metals in contact withpolymers have shown to corrode when being exposed to the process. Because of this, thecomposition of materials in the object needs to be considered before being irradiated. [47]The advantages of using gamma radiation is that the process does not leave any residueswhich need to be removed afterward. [47] It is also independent of external temperatureand pressure and has a deep penetration depth. [32]

2.7.5 Formalin

Formaldehyde (CH2O) is a frequently used compound in disinfection procedures. Thechemical reacts with free amino groups in the nucleosides and forms different derivativeswhich breaks down the DNA strands. [48] The water-based solution of formaldehyde iscalled formalin and has been proven to be an efficient killer of bacteria, viruses, sporesand tuberculosis bacteria. It is used in vaccine production, sterilization procedures ofsurgical instruments and disinfection of entire patient rooms. [49]

However, exposure to low-level formaldehyde can cause respiratory issues and skin ir-



ritation. Ingestion of formaldehyde might be fatal, which means that the substance mustbe handled carefully and that human exposure should be limited in as great extent aspossible. [49]

2.8 Degrees of cleanliness

There are different degrees of cleanliness that are defined by Vårdhandboken, which isa service provided by Sweden’s county councils and regions. There are three degrees ofcleanliness for medical equipment; sterile medical equipment, highly clean medical equip-ment and clean medical equipment. The sterile medical equipment refers to the objectsthat penetrate the skin or are in contact with different body fluids. These objects haveto be completely free from microorganisms to be classified as sterile, which they are ifthe probability of finding a microorganism on the object is less than one in a million.The highly clean medical equipment are the objects that come in contact with mucousmembranes or damaged skin but do not penetrate it. Highly clean medical equipment isdefined as in the case when the probability of finding a microorganism on the object isless than one in a thousand. The final degree of cleanliness, clean medical equipment, areobjects that normally do not come in contact with damaged skin or mucous membranes.[50] There is no defined limit of the probability of finding a microorganism on objects inthis category (clean medical equipment).

The objects that are going to be investigated in this project all belong to the last category,clean medical equipment. These objects are not supposed to be used on open woundsbut might be used on patients that have infections. The main reason behind infectionsbeing spread in hospital environments is through physical contact. This can be both indirect contact with an infected patient or with an intermediate person or object. Thiscould be through a third person, clothes or medical equipment. [1] Because of this, it isimportant to clean or disinfect the equipment when needed and maybe even make it aroutine to clean/disinfect them periodically to prevent diseases from spreading betweenpatients or from a patient to someone in the hospital staff.

2.9 Defining disinfection

There are no set values available that describe what classifies an object as disinfected.However, according to SS-EN ISO 15883-1:2009, which is a standard set for washer-disinfectors, disinfection is defined as "reduction of the number of viable microorganismson a product to a level previously specified as appropriate for its intended further handlingor use". There are no standards published that covers this for non-washer disinfectors,but by applying the same standard to the ozone cabinet it is possible to state that thelevel of disinfection depends on the type of equipment and its areas of use. Because ofthis, it is not possible to prove that the ozone cabinet can work as a disinfectant when



it reaches a certain level of CFU reduction since the object and its areas of use haveto be taken into consideration. This means, for example, that an object which belongsto the category of highly clean medical equipment (see Section 2.8) will be classified asdisinfected when the probability of finding a microorganism on the object is less than onein a thousand.

Since the objects that are investigated in this project all belong to the category of cleanmedical equipment, there are no specific levels that classify the objects as disinfected.However, it is still of interest to examine how a high degree of disinfection can be reachedfor both current and future applications. According to Thomas Wilhelmsson, hygienenurse at Landstinget Värmland, a disinfection method does not have to be sporicidal tobe declared a disinfection method. [51] This would mean that even though the ozonecabinet might not be able to kill bacterial spores, it could still be a good disinfectionmethod if it would present a great reduction of living bacteria.

2.10 The test objects

This Section will contain a description of all of the individual test objects, their areas ofuse and how they are currently being cleaned or disinfected.

2.10.1 Blood pressure cuff

A blood pressure cuff (BPC) is a device that is used to measure the blood pressure ofpatients for many various reasons. It is done frequently in both emergency rooms, healthcenters and routine visits to the doctor. The cuff is used to check the blood pressure ofa patient by strapping the inflatable cuff around the upper arm while pumping in theair into the cuff to make it shut off the blood flow in the arm. When this happens, thepressure should be lowered slowly in order to measure the blood pressure. By using astethoscope, the systolic (during a heartbeat) and diastolic (between heartbeats) pressurecan be measured this way. A blood pressure measurement with either too high or toolow values can be a first indication that a person is ill or that something in the body isincorrect. The blood pressure cuff used in this study can be seen in Figure 11.



Figure 11: The blood pressure cuff

The BPC which is used in this project is produced by the company Boso and is of themodel CA01. The blood pressure cuff is hard to clean today due to its arm wrappingcomponent which is of a textile-like material. The strap is often made out of velcro whichalso is a material which is hard to clean with the conventional cleaning methods. TheBPC is a device that only comes in contact with intact skin and is not normally cleanedor disinfected at all if there are no stains of blood or other body liquids visible on it.However, to prevent infection from spreading these kinds of devices should be cleaned ordisinfected periodically even though the contamination is not noticeable with the nakedeye.

2.10.2 Drug pump

The drug pumps are constructed to pump all kinds of drugs, suspensions, blood or nutri-tion to patients. In the cavity to the right (see Figure 12), a syringe with the requesteddrug is placed. In the settings of the pump’s software system, the doctor or nurse canspecify the pump’s flow rate. The device then pumps the drug through a tube into thepatient’s bloodstream. The pumps are used by one patient at a time but are transferredfrom patient to patient frequently (often daily). [52]



Figure 12: The drug pump

The pumps used in this project are produced by Fresenius Kabi and the model is AgiliaSP MC. According to Martin Berglund, medical technician at Motala Lasarett, the drugpumps are currently cleaned with Meliseptol Foam pure but due to their many joints,they could be hard to clean properly.

2.10.3 X-ray neck collar

The thyroid is a very sensitive organ to X-ray exposure and the need for X-ray collars(see Figure 13) is therefore high in clinics which uses radiation as a treatment method.[53]

Figure 13: The X-ray collar

The X-ray collar used in this project is produced by MAVIG, a company that producesX-ray Protection and Medical Suspension Systems. The collar is made out of a materialcalled ComforTex HPMF (High Performance Medical Fabric), which according to CarolaLarsson (Area Manager at MAVIG, Nordic and Baltic Countries) should withstand beingdisinfected with ozone. Larsson describes the material as a woven microfiber fabric with



incorporated carbon fiber to make it non-electrostatic. The material is coated to preventfluids and bacteria to penetrate the fabric and reach the inner material, which is a leadrubber and has an unknown reaction to ozone. The X-ray neck collar is only cleaned bythe hospital staff in the case of it having an unpleasant smell. If this is the case, the neckcollar is cleaned with a liquid disinfectant and a moist cloth.

2.10.4 Transportation bag

The transportation bag that is investigated in this project is not being disinfected orcleaned at all by the hospital staff. This bag, which can be seen in Figure 14, is used totransport CPAP devices that are used by patients in their home environment. There aremany similar bags that are utilized to transport many other kinds of medical equipmentfor the same purpose. The bags are used over and over again and can be used by manydifferent patients.

Figure 14: The transportation bag

The bag is made of a textile fabric and has zippers made for closing and opening. Thebag is not suitable to be cleaned by the currently used methods and is thereby transferredfrom patient to patient without any cleaning procedure at all.



3 MaterialsOf the instruments used in this project, the primary one was the ozone cabinet (ElozoD800 Cleaning System). To measure the temperature and the humidity in the cabinet,a combined thermometer and hygrometer (Extech Instruments RHT10) was used. Tomeasure the ozone concentration outside the cabinet, an ozone meter (Aeroqual 500) wasemployed. The device was equipped with a sensor head suited to measure ozone con-centrations between 0 and 0.15 ppm. All of these were provided by Motala Lasarett andClinicum Test and Innovation, Region Östergötland.

To take samples of the bacteria on the objects, Copan Liquid Amies Elution Swabs(eSwabs) were used along with spore specimens (Apex Biological Indicators), originallymanufactured for testing with gaseous hydrogen peroxide. The eSwabs and the sporespecimens were provided by Vårdhygien and Clinicum Test and Innovation. The inocu-lation was done at a hygiene lab located at Linköping University Hospital. The bacteriawere inoculated onto three different agar plates; hematin, UTI, and blood agar.

The test objects, which all are presented in Section 2.10 were provided by Motala Lasarettand were temporarily borrowed from the hospital environment for testing.



4 MethodThis Section will describe how the specimen collection was performed, how the data wasobtained and analyzed, and finally how the statistical analysis was performed.

4.1 Specimen collection

The specimen collection was performed by swabbing the objects with eSwabs both beforeand after the object had been treated with ozone. The eSwabs were transported to a labfor inoculation and bacterial growth to examine the amount of CFUs that were presentbefore and after the cleaning process. The specimen collection process can be seen inFigure 15.

Figure 15: Specimen collection with eSwab [54]

When the first test was performed, all of the objects were marked with a permanentmarker to create six equally sized spots. All the marked parts covered the same areaand included equal amounts of complicated structures (zippers, seams, joints or cavities).The objects were touched by the author of this project and by some of the staff at theMedical Technology department to provide some extra contamination to the objects. Thecontamination was done carefully and all of the marked pieces on the objects were con-taminated in the same way. The swabbing was done in a standardized (to the greatestextent possible) way by applying the same (experienced) force to the swabs and movingthem in the same way during the same time period. The first marked spot was swabbedwith an eSwab and transported to a hygiene lab for inoculation, incubation and bacte-rial growth. This sample worked as a reference to know how much bacteria was presentbefore the treatment. This was done for all objects before they were placed in the ozonecabinet. After 20 minutes (duration 1) the cabinet was opened and the second spot wasswabbed and the sample was sent to the lab. This was repeated when the objects hadbeen treated for 20, 40, 60, 120 and 240 minutes. There were also one or two spore spec-imens placed in the cabinet for each of the setting combinations. The spore specimenswere also transported to a lab for analysis.

Due to the results from the spore specimens (presented in Section 5.1), there was aneed for another control test in this project; nose control samples. These samples weretaken with eSwabs directly from a nostril and then placed in the cabinet together withthe other objects. This supplied results that were not dependent on the different mate-



rials and also gave significantly higher numbers of CFUs and thereby a more apparentreduction. Additionally, the nose control samples were shown to be advantageous whenexamining the standard deviation from these tests since these numbers were much smallerthan for the tests done on the different objects.

Out of all the test objects it is only the drug pump that is currently cleaned period-ically when used in hospitals. The currently used cleaning procedure (spraying withMeliseptol Foam pure and wiped with paper) was tested and evaluated in the same wayas the ozone treatment.

4.2 Obtaining data

The eSwabs and spore specimens were transported to a hygiene lab at Linköping Uni-versity Hospital and were inoculated onto three different types of agar plates (hematin,UTI, and blood agar). The agar plates were then incubated for 48 hours in 35-37 °C.After this time, the CFUs could be counted in all of the agar plates. By studying thenumber of CFUs both before and after the ozone treatment it was possible to draw someconclusions regarding the effect of the treatment. Figure 16 shows an example of thebacterial growth from two different samples (one taken before ozone treatment and onetaken after ozone treatment).

Figure 16: Example of CFUs on an UTI agar plate where the left has been inoculatedwith a sample that has not been treated with ozone, and the right has been inoculatedwith an equivalent sample that has been treated with ozone

To obtain data from the spore specimens, they were placed in a control liquid and storedin a heating cabinet for 40-48 hours at a temperature of 56 °C. The control liquid (pro-prietary formulation of soybean casein digest medium) is a pH indicator (bromocresolpurple) which shifts into a yellow color if the sample is positive. [55] A positive samplemeans that the spores have not been completely eliminated and that there still are livingspores in the sample. A positive result does not say anything about how many sporeswere killed, only that it was not 100 %. If the sample is negative (100 % elimination ofspores), the liquid will retain its purple color.



The whole process of transporting the specimens to reading out the results is furtherdescribed in Appendix A. This approach was used at all times during this project.

4.3 Statistical analysis

There were several tests under the same conditions (same kind of object, same duration,and concentration of ozone). For these tests, there was an average value calculated forthe number of CFUs before and after the treatment. The mean value, x, was calculatedaccording to the following formula:

x= 1n

n∑j=1

xj =x1 +x2 + . . . +xn

n. (4.1)

Where n is the number of tests. The standard deviation could then be calculated foreach of the measurements that contributed to the mean value calculations. The stan-dard deviation, s, was calculated before and after treatment according to the followingformula:

s=√√√√ 1n−1

n∑j=1

(xj −x)2. (4.2)

These values were calculated using Python.

4.3.1 Antimicrobial efficiency

Since there is no official documentation regarding the definition of a disinfected object,as many of the settings as possible will be tested and each evaluated according to theirantimicrobial efficiency. There is no percentage number that is considered to be a limitfor successful disinfection which means that it will be hard to state whether the methodis working or not. However, the antimicrobial efficiency will give a percentage numberof the reduction of CFUs which the treatment produces. The bacteria specimens fromthe different objects will be inoculated onto three different types of agar plates (hematinagar, UTI agar, and blood agar) and the number of CFUs will be counted on each ofthem (both before and after the ozone treatment). To investigate if the cabinet can workas a disinfectant or not, the following equation will be used:

Antimicrobial efficiency = (x0 −x1)+(y0 −y1)+(z0 − z1)x0 +y0 + z0

·100 % (4.3)



Where x0 and x1 are the number of CFUs on the hematin agar before and after theozone treatment, y0 and y1 are the number of CFUs on the UTI agar before and after thetreatment and z0 and z1 are the number of CFUs on the blood agar before and after thetreatment. This equation gives the antimicrobial efficiency of the cabinet (the reductionof CFUs) as a percentage value. This equation was used for all different objects and alldifferent duration and ozone concentration settings to find the most optimal settings forall objects.

The tests were run as many times as possible to gain statistical validity to the results. Fortests that were done at least three times, equation 4.1 and 4.2 could be used to calculatethe mean reduction of CFUs along with the standard deviation between the differentruns.

4.3.2 ANOVA

There were two different two-way ANOVA (analysis of variance) tests performed. Thefirst, ANOVA for different objects, explains if the ozone cabinet works differently ondifferent objects or not. The second, ANOVA for different bacteria, explains if the cabinetworks differently on different bacteria. The theory behind the ANOVA tests can be readin Appendix B. The conditions for the tests are also mentioned there. The software usedto analyze this was SPSS and the level of significance was set to α = 0.05.



5 ResultsThe results from the spore specimens, the statistical analysis, the CFU quantificationand the antimicrobial efficiency will be presented in this Section.

5.1 Spore specimens

A total of 19 spore specimens that were placed in the ozone cabinet at the highestconcentration all showed positive results (not eliminating 100 % of the spores). Thespecimens were left in the cabinet for 20 to 240 minutes.

5.2 ANOVA

This Section provides the results from the ANOVA tests, done for both different agar anddifferent objects. The effects of interest are "Agar" and "Object" and the results show thatthere is no significant difference in how the ozone cabinet works for the different objectsor the different agar. The ANOVA tests were done for all concentration and durationsettings with data from Appendix C.

Table 3: Results from the ANOVA test for different agar (concentration 4)

Feature Settings Effect F(2,66) pCFUs C4D8 Time 5.278 0.024

Agar 0.321 0.726Time*Agar 0.320 0.727


Agar 0.326 0.722Time*Agar 0.313 0.733

CFUs C4D3 Time 7.570 0.007Agar 0.336 0.716Time*Agar 0.329 0.720



Agar 0.315 0.731Time*Agar 0.177 0.838



Table 4: Results from the ANOVA test from different objects (concentration 4)


Object 1.390 0.282Time*Object 1.384 0.248

CFUs C4D4 Time 5.665 0.029Object 1.616 0.221Time*Object 1.580 0.229






Agar 0.042 0.959Time*Agar 0.039 0.962
















Agar 0.279 0.757Time*Agar 0.006 0.994
















Agar 0.276 0.760Time*Agar 0.375 0.688














5.3 Mean and standard deviation

Since there is no significant difference in the ozone cabinet’s effect on the different objectsor different bacteria, the mean and standard deviation of CFUs were calculated with datafrom all objects and all agar together. The results (plotted in Figure 17-20) are based ondata from Appendix D. The mean and standard deviation are presented in four separatebar plots that describe each of the four concentration settings. The plot to the left showsthe average number of CFUs before and after ozone treatment for all objects. The plotto the right shows the average number of CFUs before and after ozone treatment on thenose control samples. The error bars show the standard deviation. The x-axis describesthe five different durations (20, 40, 60, 120 and 240 minutes) and the y-axis shows thenumber of CFUs. Note the different scales on the y-axes. The plots describing all objectsare based on data from at least 12 runs and the plot describing the nose control samplesare based on data from 3 runs.



Figure 17: Average number of CFUs before and after ozone (concentration 4)






5.4 Antimicrobial efficiency

The antimicrobial efficacy of the ozone cabinet was calculated according to equation 4.3.The average reduction of CFUs for each of the objects will first be presented separatelyin Figure 21-24. Further down in this section is a presentation of the average reductionof CFUs on all objects (not separated) in Figure 25. Below this, the result from thenose control samples can be visualized in Figure 26. The y-axis describes the reductionof CFUs as a percentage value and the x-axis shows the treatment duration in minutes.The error bars show the standard deviation. The plots describing all objects are basedon data from at least 12 runs and the plot describing the individual objects and the nosecontrol samples are based on data from 3 or 4 runs.

Figure 21: Mean reduction of CFUs on the blood pressure cuff



Figure 22: Mean reduction of CFUs on the drug pump

Figure 23: Mean reduction of CFUs on the X-ray neck collar



Figure 24: Mean reduction of CFUs on the transportation bag

Figure 25: Mean reduction of CFUs on all objects together



Figure 26: Mean reduction of CFUs on the nose control samples

5.5 Disinfection with Meliseptol

The drug pump was contaminated and disinfected with Meliseptol pure foam spray threetimes. The mean and standard deviation of the number of CFUs before and after treat-ment can be seen in Figure 27.

Figure 27: Average number of CFUs before and after Meliseptol spray

The mean average reduction of CFUs was 97.8 ± 3.0 %. The pump was swabbed im-mediately after being disinfected with the Meliseptol spray, and the treatment time wastherefore set to one minute (which is the approximate time it takes to wipe the pump).The average reduction of CFUs with this method was incorporated in Figure 22 in or-der to be visually compared to the ozone disinfection. The result can be seen in Figure



28, where the reduction of CFUs due to the Meliseptol spray treatment is representedthrough the black line.

Figure 28: Mean reduction of CFUs on the drug pump when using ozone and Meliseptolpure foam separately

5.6 Variation

The contamination was done manually and presented large variations in the number ofCFUs that were counted between the different runs. To prove this difference, the numberof CFUs on the blood agar plates for all C4D8 (concentration 4 and duration 8) runs willbe presented in Table 11 below.

Table 11: CFUs on the blood agar, data from C4D8 runs

Object Run Beforeozone

Afterozone

BPC 1 6 02 41 03 88 0

Pump 1 19 02 58 23 17 1

NC 1 11 02 384 43 24 0

Bag 1 1 02 248 43 344 5



5.7 Visual effects

The effect that the ozone had on the different objects has not been evaluated in thisstudy, however, there were clear visual effects on two of the objects. The ozone had anegative effect (which could easily be seen) on the blood pressure cuff and the X-ray neckcollar. Figure 29 shows the blood pressure cuff after all the ozone treatments. The metalring that works to tighten the cuff has become rusty from the ozone.

Figure 29: The blood pressure cuff after all ozone treatments

The X-ray neck collar was also affected by the ozone. The lead rubber inside the collarhas crumbled apart as a result from being treated with ozone. This can be seen in Figure30, where the outer material was cut open to visualize eventual effects.

Figure 30: The X-ray neck collar after all ozone treatments



6 DiscussionThis Section includes a discussion regarding the results, the methods and sources of errorand ends with suggestions for future work.

6.1 Results

The discussion about the results has been divided into seven parts covering the sameheadlines that were included in the Results Section.

6.1.1 Spore specimens

The ozone cabinet was proven not to have sporicidal properties due to its inability tokill the spore specimens even with the toughest treatment. According to previous stud-ies (presented in Section 2.1.3), ozone can be sporicidal at higher concentrations whichindicates that the ozone cabinet does not provide high enough concentrations of ozoneto kill spores. The study mentioned in Section 2.1.3, which proved a 99.9 % reduction ofspores, used an ozone concentration of 1500 ppm for 4 hours. This is much higher thanthe maximum ozone concentration in the Elozo cabinet (56 ppm). When comparing thesetwo, it is reasonable to conclude that the cabinet does not provide sufficient ozone levelsto be sporicidal. However, the spore specimens used in this study only gave a negativeresult (proving a treatment to be sporicidal) if all spores in the specimen were killed.This means that it is not possible to know if the cabinet killed many spores but not all,or if the cabinet did not kill a single one.

However, according to Section 2.9, a disinfection method does not have to be sporici-dal in order to be accepted. Hence the ozone cabinet could still be used as a disinfectorif it is able to reduce the number of living bacteria to a satisfying level.

6.1.2 ANOVA

The results from the ANOVA tests (Table 3-10) show that there is no significant differ-ence in how the ozone cabinet works for the different objects or the different agar. Thiswas tested for all concentration and duration settings and is thereby valid for all possiblesettings of the cabinet. However, this does not prove if the materials are suited for ozonetreatment since the effect on the materials themselves have not been examined. Also,this does not state anything regarding how the cabinet affects other materials that werenot tested in this project. Since there was no significant difference in how the cabinetworks for different agar (i.e. different bacteria), this means that the cabinet is efficientagainst both gram positive and gram negative bacteria, which is very advantageously forthis method.



Another conclusion that is possible to draw from looking at the results from the ANOVAtests is that the time (before and after ozone) makes a significant difference for the higherconcentrations and makes a smaller difference as the concentration gets lower. For exam-ple, concentration 1 does not prove a significant difference in the number of CFUs whenlooking at the time (before and after ozone) for any of the duration settings. This impliesthat the ozone cabinet is more effective at killing bacteria at higher ozone concentrations.

6.1.3 Mean and standard deviation

The mean and standard deviation of the numbers of CFUs are presented in Section 5.3.It is evident that there are large error bars in these plots, corresponding to the standarddeviations of the measurements. The reasons for these large uncertainties could be many.For example, it is impossible to contaminate all the objects with the same amount of bac-teria every time since this is not something that is possible to control. It also depends onhow the sample was taken (pressure on eSwab, duration of swabbing and area swabbed).It could also be affected by the temperature and humidity in the cabinet, the temper-ature during transportation and variations in the inoculation process. As mentioned inSection 2.2, Elozo Oy recommend that the cabinet is used in an environment where thetemperature is 15-25 °C and the relative humidity is lower than 50 %. These values werenot always within these limits and the relative humidity was more often above 50 %,which could have affected the results. Regarding the inoculation process, it is impossibleto inoculate the exact same volume of bacteria solution to all agar plates since this isdone manually. It could also depend on incubation time and readout mistakes. Someof the agar plates had an extremely large number of CFUs which could not be countedmanually and were therefore estimated. Sometimes, the CFUs had grown together whichalso made the readout difficult.

Even though the error bars are significant, it is still possible to see the trend in these plots.It is most clear for the nose control samples, which could be due to the fact that the errorsources here are fewer. This is because the sample was easier to take and provided a muchhigher average number of CFUs per sample. All plots (except the nose control samples atconcentration 1) show a distinct decrease in CFUs after ozone treatment. Concentration4 (Figure 17) shows a great decrease in the number of CFUs after ozone (> 96 % reduc-tion) for 40 minutes and longer treatment periods while a 20-minute treatment provesa decrease of around 43 %. The nose control samples have a larger decrease but provethe same trend. Figure 18 shows that all objects treated with ozone of concentration 3have a CFU reduction that becomes more clear with longer duration periods. The nosecontrol samples have varying results (duration 2 stands out with only an average of 50% reduction), however, this is due to one single measurement that showed an increase ofCFUs of 178 %. The other measurements of this setting prove a reduction similar to thoseof C3D1 and C3D3 which is a reason for the large error bar. The bar plots correspond-



ing to concentration 2 settings (Figure 19) all show a decrease in the number of CFUsafter ozone but at various levels. The objects have a larger average reduction for shortertreatments (C2D1 and C2D2), but the reduction is quite similar and lies around 50 % forthe longer treatment periods. The nose control samples show a much clearer reduction(all above 89 %). The plots corresponding to concentration 1 (Figure 20) shows varyingresults. The number of CFUs on the objects decreases after ozone treatment for all du-ration settings, but the nose control samples show a large increase (up to 130 %) in thenumber of CFUs after ozone treatment for three of the five settings. This is a quite clearindication that concentration 1 does not provide ozone levels high enough to kill bacteria.

In conclusion, these results show that concentration 4 and concentration 3 both prove ahigh reduction in the number of CFUs after ozone treatment. It is possible to see thatthe reduction increases with longer treatment periods even though the standard devia-tions are large. Concentration 2 also provides a reduction of CFUs for all settings, buta smaller reduction (at least for the test objects) compared to the higher concentrationsettings. Concentration 1 proves an increase of CFUs for three of the five nose controlsamples, which indicates that this concentration is too low to be bactericidal.

6.1.4 Antimicrobial efficiency

The first four plots in Section 5.4 describe the mean reduction of CFUs on the differ-ent objects. Starting with the blood pressure cuff (Figure 21), it is possible to see thatconcentration 4 reaches a high level of disinfection (89.5-98.6 % reduction of CFUs) attreatment periods of 40 minutes or more. The error bars for these measurements are verysmall (varying between 1.8 and 7.2 %), which makes the result trustworthy. Concentra-tion 3 also shows a large reduction at the one and four-hour treatments (96.1 and 100% reduction, with a standard deviation of 3.6 and 0.0 % respectively). Concentration 2proves results similar to concentration 3, but with increasing error bars which is a signthat the treatment could be less efficient. Concentration 1 has very large error bars foralmost all data points, which makes it hard to conclude anything about the effect thistreatment has on the blood pressure cuff.

Figure 22 shows that both concentration 4 and concentration 3 yield a very high level ofdisinfection (> 93.9 %) for the drug pump after treatment periods starting at 40 min-utes. The error bars for these measurements are very small (< 6.5 %) which reinforces thestatement of these settings working well. Concentration 2 and concentration 1 both havelarger error bars and lower reduction levels (except for the longest treatment period),which could indicate that these concentrations do not provide sufficient levels of ozonefor the intended purpose.

The mean reduction of CFUs on the X-ray neck collar can be visualized in Figure 23. A



great reduction of CFUs was obtained after the two and four-hour program with a mini-mum reduction of 97.7 % and a maximum standard deviation of 2.7 % for the two highestconcentration settings. All concentrations also show a large reduction after a 40-minutetreatment even though concentration 1 and 3 have larger error bars. Concentration 2shows to have good results as well but is a bit below concentration 3 and 4 at highertreatment periods.

The transportation bag has results similar to the other objects when studying concen-tration 4 settings (large reduction for all treatments starting at 40 minutes, but bestafter two or four hours). The other concentration settings indicate lower CFU reductions(except for concentration 3 after 4 hours of treatment). This indicates that concentration4 provides the highest and most unvarying reduction of CFUs on the transportation bag.

When comparing the four objects, it is possible to draw the conclusion that the CFUreduction is similar concerning all objects. Differences can be seen when looking at the40-minute treatments, where the drug pump and neck collar have very high levels ofreduction at both concentration 3 and 4. The blood pressure cuff and the transporta-tion bag, however, do not reach the same level of reduction for the same concentrations.This could be due to the fact that the objects have different surfaces. The pump andthe neck collar has more plane surfaces that could be easier for the ozone to reach andthereby easier to disinfect. The blood pressure cuff and transportation bag both havewoven (and rougher) surfaces that could create a larger surface and thereby more areasfor the bacteria to live, as well as harder for the ozone to reach. It is possible that thiscould be a reason for the difference between the results, but these are only speculations.Another, and more probable, explanation would be that there is not enough data behindthese plots. Each of the data points is only based on information from three or four runs,which due to many error sources could be very approximate. It is reasonable to believethat a larger number of identical runs would lead to data that evens out the differencesbetween the objects.

Due to the fact that there was no significant difference between how the ozone treatedthe different objects, there was a plot created that described the mean reduction of CFUson all objects together. This plot can be seen in Figure 25. This plot clearly describesthat concentration 4 gives the largest reduction of CFUs for all duration settings startingat 40 minutes. Concentration 4 yielded a reduction of 89.3 % (± 21.4 %) after the 40-minute treatment and a reduction of 98.2 % (± 2.6 %) after a four-hour treatment. Thereason for not reaching a 100 % CFU reduction on all four-hour treatments could be thatthe bacteria on the objects form spores due to the unfavorable environment inside thecabinet. Since the cabinet does not have the ability to kill bacterial spores, this could bea reason for not obtaining a higher reduction rate. It could also be due to the fact that



the cabinet might produce ozone levels too low to unquestionably kill all bacteria. Sincethe bacterial species were not characterized, it is also possible that some of the speciesare harder for the cabinet to kill. However, due to the results from the ANOVA tests, itis more reasonable to believe that the explanation for not reaching a 100 % reduction isdue to too low ozone levels or the formation of spores.

The last plot from Section 5.4 (Figure 26) describes the mean reduction of CFUs onthe nose control samples. This plot shows a high level of antimicrobial activity for con-centration 2, 3 and 4 at one-hour treatments or longer. The same could probably besaid for 40-minute treatments where the big offset in the C3 curve is due to one sin-gle measurement which proved a 178.8 % increase in CFUs. Due to the small amountof data available (each data point is a result from three runs only), this feature is prob-ably something that would not be visible in a similar study with larger datasets available.

The conclusion that can be drawn from analyzing all these plots is that the ozone cabinetdoes have the ability to reduce the number of living bacteria on the objects’ surfaces. Theresults also show that the bactericidal properties of the cabinet increase with higher ozoneconcentration and longer treatment duration. Since concentration 4 induces the largestreduction of CFUs on all objects after 40 minutes or more, this is the concentration thatshould be used if this cabinet were used as a disinfection method. The difference in ozoneconcentration between the different power settings is not that great which means thatconcentration 4 should not noticeably affect the materials in a negative way more thanthe lower concentrations. Another reason for using concentration 4 is that the ozoneis not expensive to produce since it is generated from ambient air, which means that ahigher ozone concentration does not become more expensive to use. When using concen-tration 4, a treatment duration of 40 minutes yielded an 89.3 % reduction (± 21.4 %)on average, while a four-hour treatment yielded a 98.2 ± 2.6 % reduction on average. Itwould be a great advantage to have short treatment periods if this were used at hospitals.However, the tests performed in this thesis proved much better results for the two-hour(94.2 ± 15.7 % reduction) and four-hour treatments. The large standard deviations onthe shorter treatments prove that more studies have to be performed to gain more dataand more accurate results. By obtaining more data it should be more apparent howwell the shorter treatment periods actually work and it would thereby become easier torecommend one specific combination of concentration and duration settings for optimalresults (a great reduction of CFUs in combination with the shortest treatment periodpossible).

6.1.5 Disinfection with Meliseptol

The drug pump was disinfected with Meliseptol foam pure spray in order to compare theozone treatment to the method used in hospitals today. This treatment was performed



three times and gave a very stable reduction of CFUs. The result proved a mean CFUreduction of 97.8 % with a small standard deviation of 3.0 %. Even though the datacollection is small, this method seems to be very reliable according to the results.

The main advantage of using this method compared to the ozone treatment is the shorttreatment duration. The Meliseptol spray only requires a treatment duration of oneminute. This is a huge difference compared to the ozone cabinet which needs at least 40minutes (see Figure 28) to prove a reduction in the same range. The same number ofdatasets (three complete datasets) were obtained in both experiments but the Meliseptoltreatment proved results with much smaller error bars, which also indicates a more reli-able treatment.

Due to the prominent advantages of using the Meliseptol pure foam spray, there is no usefor further investigations with the ozone cabinet for this particular piece of equipment.However, information was obtained that proved that the drug pump had a material whichsuccessfully could be disinfected with ozone. Other medical equipment in the same (orsimilar) material which cannot be disinfected with the Meliseptol spray might, therefore,be disinfected with ozone.

6.1.6 Variation

Table 11 explains the variation between three equivalent runs for all objects. Since thereis no significant difference in how the cabinet treats different bacteria, the results in thistable comes from one of the agar (blood) to make it legible. When studying this table, itis the column called "Before ozone" that is of interest. This column describes the num-ber of CFUs that were present on the objects’ surfaces before ozone treatment on threedifferent occasions. The number of CFUs vary between 1 and 384, which is a big spanwhen looking at numbers in this range. This table works to illustrate the great variationbetween different runs as a way to explain the large difference between t

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Evaluation of an Ozone Cabinet for Disinfecting Medical ...1262996/FULLTEXT01.pdf · Datum Date 2018 -11 -05 Avdelning, institution Division, Department Department of Physics , Chemistry