Ozolle: Sciellce & Ellgilleerillg, 31: 369-378 Copyright © 2009 [nternational Ozone Association [SSN: 019[-9512 print /1547-6545 online 00[: 10.1080/019[9510903155816

W Taylor & Francis ~T~Y!llrE.frJnci!GrOlJr

Quantification of the Bactericidal, Fungicidal, and Sporicidal Efficacy of the JLA Ltd. Ozone Laundering System

Kyle Allison,1 Jacqueline Hook,2 Dick Cardis,2 and Rip G. Rice3

1MGS Laboratories, Science Centre, Egham, Surrey, United Kingdom 2 JLA Limited, Ripponden, West Yorkshire, United Kingdom 3RICE International Consulting Enterprises, Sandy Spring, Maryland, USA

The objec/il'e o//liis study I\'{IS /0 demolls/ra/e the bac/er­icidal, jilllgicidal, alld sporicidal ejJlcacy 0/ /lle ill-use call­cell/ratioll 0/ ozolle ill SOllllioll gelleraled COlllilluOIISly by Ille JLA Limiled OTEX I'alidaled ozone laullderillg system againsl a rallge o/I)1Jical microorgallisms associaled lI'ilil laullderillg applicaliolls. Tile sludy IIsed tile JLA HC 100 lallllderillg macliille as tile cOlltaillmellt I'essel. Test microbial sllspellsiolls were ill traduced directly illlo the drum o/the lallllderillg macllille cOlltailling a IWOII'II lerel o/lI'aler ill Ille presellce 0/ soluble ozolle. Usillg stalldard microbiologi­cal teclllliqlles, tile 11111 lIbel's 0/ sl/J'l'ivillg bacteria, mold spores or baclerial spores lI'ere delermilled, alld reductiolls ill viable cOllllls were calculated. Tllr> folloll'illg test orgall­isms lI'ere employed: Pseudomonas aeruginosa, Methicillin­Resistant Staphylococcus aureus (MRSA), Enterococcus faecal is, E. coli, Acinetobacter baumannii, Aspergillus niger (spores), and Clostridium difficile (spores). All tests used mlliliple COlllacttimes 0/0, 1,3,5,7,11 and 15 minutes at 20 °C±2 0c. Lerels 0/-:;'0.25 mg/ L o/dissolved ozone had minimal effeci on tile bacleria. Lel'els 0/ >0.45 mg/ L ozone slloll'ed good biocidal actil'it)', except agaimt Acinetobacter bmllllannii, lI'11icil showed strong resist({/Ice to ozolle. Agaillst jilllgal spores, dissolred ozolle lerels 0/ > 1.2 mg/ L were reqllired 10 oblain a 2.8 log redllctioll. For Clostridium diffieile spores, a dissolred OlOlle lel'el betll'eell 0.90 and 1.20 mg/L gare >1.6 log redllctioll lI'itil no discemable spore reeolw)'. In erel), illstallee, Ille cOlltrol tests (110

ozone) recoI'ered lerels of microorganisms Illat prol'('(1 that the aclioll 0/ Ille lalil/deril/g machine lias I/O al/timicrobial acti!'it)" al/d thai the log redllctiol/s obtail/ed are sole/)' due 10 the eHeels of (lissolred ozone.

Keywords Ozone, Ozone Laundering, Microorganism Kills, JLA Ltd. Ozone Laundering System, Laundry Applica­tions, Pseudomonas aen/gil/osa, Methicillin-Resistant

Recei vcd 4/23/2009; Accepted 7/ I /2009 Address correspondence to Rip G. Rice. RI CE International

Consulting Enterprises, 1710 Hickory Knoll Road, Sandy Spring, MD 20860, USA. E-mail: RGRice4ozone@aol.com

Staphylococcus awells (M RSA), El/lerococCHS Jacca­lis, E. coli, II cilletobacter bWI/JWllllii, Aspergilllls I/iger, Closlridium di[jicile

STUDY OBJECTIVE

The objective of this study was to demonstrate the bactericidal, fungicidal and sporicidal efficacy of the in­use concentration of ozone in solution generated continu­ously by the OTEX validated ozone disinfection genera­tor, against a range of typical organisms associated with la undering applica tions. The study used the JLA HC I 00 washing machine as the containment vessel.

The test method used was a Kill Time assay, whereby the test microbial suspension was introduced directly into the drum of the washing machine containing a known lcvel of water in thc prcsence of soluble ozone. At various intervals, an aliq uot of test mixture was transferred to a neutralizing solu tion to inactivate or supprcss any bacter­icidal, fungicidal or sporicidal activity, Using standard microbiological techniques, thc numbcr of surviving bac­teria, mold spores or bacterial spores was dctermined and a reduction in viable count calculated. The study was performed by MGS laboratories Ltd., an independent contract testing laboratory. Thc OTEX Validated Ozonc Disinfection Generator and JLA HC 100 washing machines were installed and programmed by JLA representatives.

EXPERIMENTAL TESTS AND METHODS (ALLISON, 2007)

Preparation of Test Organisms The organisms listed in Table I were used in the study.

Efficacy of the JLA Ltd. Ozone Laundering System September-October 2009 369

TABLE 1. Test Organisms Used in the Quantitative Suspension Test

Organism

PselidolllOllas aerllgillosa MRSA Ellterococclls faccalis Escherichia coli Acinetobacter ball/lIC11/llii Aspergilllls niger (spores) ClostridiulII di/ficite (spores)

1 Manassas, VA,USA.

Source

ATCC t

NCTC2

NCTC NCTC

NCIMB 3

ATCC ATCC

2 National Collection or Type Cultures, Salisbury, UK. J l3ucksbur11. Scoiland .

PREPARATION OF BACTERIA

Number

\5442 12493

775 10418 12457 16404 9689

Bacteria for testing were cultured onto Tryptone Soy Agar (TSA) for 24 hours before being suspended 111

Tryptone Soya broth for a further 24 hours.

Preparation of the Mold Spore Suspension The centers of 10 SDA plates were inoculated from the

required slope and mold allowed to grow until the entire plate had sporulated (this was usually not less than 7 days) . The plates were harvested under laminar flow by pipetting 2 mL R&P onto the surface of the agar plate, and gently spreading and scraping the diluent over the surface of the plate using a sterile spreader so as to remove as many spores as poss ible without removing the fungal basa l layer of agar.

The resulting suspension of spores/R&P was placed onto a sintered disc 40 micron filter with vacuum flask, and a vacuum was generated to pull the liquid and spores through into the fla sk below. To aid filtration , the sintcred disc was flushed vigorously before reapplying vacuum.

R&P aliquots of 9 mL were added to the residue, stirred, and steps 3 and 4 repeated until the liquid emerging below the disc was no longer black with spores. The resulting suspen­sion of spores/R&P then was placed into McCartney tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was decanted off and the spores re-suspended in purified water. The centrifugation process was repeated until the solution in which the spores were suspended was clear. The suspension was enumerated prior to use.

Immediately before use, the spore suspension was microscopically examined to confirm that less than I in 10 fields of view contained mycelial fragments or germi­nating spores. If more than I in 10 fields of view

contained mycelial fragments steps 3- 8 were repeated. If more than I in 10 fields of view contained germinating spores, the suspension was discarded.

PREPARATION OF BACTERIAL SPORES

A Culti-loop T1\t of Clostridillm di/fici/e was transferred to 100 mL of cooked meat medium and incubated anae­robically at 37°C (98.6 OF) fo r not less than 10 days. The resulting broth was transferred to McCartney tubes and centrifuged at 2000 rpm for 20 minutes. The supematant was decanted off and the spores resuspended in SO/SO ethanol and purified water and left for 20 minutes. The centrifugation process was repea ted and the supematant disca rded, followed by resuspension of the spores in puri­fi ed water. Prior to use, the spore suspension was micro­sco pically examined to confirm tha t less than I in 10 fields of view contained germinating spores.

PREPARATION OF THE DISINFECTANT SOLUTION

Ozone was generated in the drum of the JLA HC-IOO washing machine using the connected OTEX Validated Ozone Genera tor.

Test Parameters

• All tests using bacteria were performed in the presence of the growth medium.

o The fungicida l and sporicidal tes ts were per­fo rmed without the presence of the growth medium.

• Multiple ozone contact times of 0, I, 3, 5, 7, II and IS minutes were used in all tests.

• All testing was performed at 20 °C±2°C (68 °F±3.5 OF).

o Ozone levels were determined using the Ozone CHEMets® test method (CHEMetrics, Inc., Calverton, V A, USA).

o Control tests without ozo ne were conducted for comparison.

o Total viable counts or the water source were per­formed to determine if it contained any contam­inating organisms that would affect the result.

Test Method In order to carry out the efficacy tests, the following

washing machine program was used:

Program Details Cycle Time - min Dip Level Wash Action

Program I: cold wash Detergent volumes

370

30 30 cm volume = 68 L ambient 12 sec wash; 3 sec stop time No detergent in lise

K. Allison et a l. September-October 2009

The JLA HC 100 was turned on and allowed to fill with fresh wa ter. Ozone then was injected into the water via a sparger in the machine once a safe water level had been reached. After the fina l specified water level had been reached, the test microbial solution was introduced directly into the drum via the soap box. This was flushed through with water taken from the drum via the sampling poin t.

After 30 seconds of mixing, the sampling point was allowed to clear for 15 seconds, and then an aliquot was taken in a steri le container. I mL of the sample then was transferred to 9 mL of neutralizer and allowed to stand for 5 minutes. This was then diluted down to a countable range suitable for the organism under test and I mL duplicate pour plates were prepared using the appropriate agar. This first sample was classed as T = 0 and provided the data for the microbial load of the test.

Subsequent samples were taken from the washing machine drum in the same manner after I , 3, 5, 7, I I and 15 minutes. At the same time interva ls, the dis­solved ozone levels were measured using the Ozone CHEMets® test met hod that employs DDPD chemis­try. A sample is treated with an excess of potassium iodide. Ozone oxidizes iodide ion to iod ine. The iodine then oxidizes DDPD, a methyl-substituted form of DPD (N,N-diethyl-p-phenylenediamine) , to form a purple-colored species in di rect proportion to the ozone concentration. Results are expressed in ppm (par ts per million) . The bacteria l plates were incubated at 30- 35 °C (86-95 OF) for 24-48 hours and the funga l plates at 20-25 °C (68- 77 OF) for 2- 5 days prior to enumeration.

Control tests also were performed without ozone, to determine if the cycle had an effect on antimicrobial activity. These tests were conducted prior to the OTEX tests to ensure that no residua l ozone could affect the resu lt.

CALCULATION OF RESULTS

Determination of Microbial Load The aliquot taken at T = a was used to determine the

starting cfu/mL of the test, from which the log reduction of viable cells could be calculated. The di lution(s) offering a satisfactory plate count range between 30 and 300 cfu were identified. N then was calculated using the following equation:

N = c/(nd)

Where:

N is cfu/mL of the test inoculum c is the sum of the colonics counted on the plate set takcn

intu m:cuullt

n is the number of plates taken into account

d is the dilution factor corresponding to the di lution taken into account

Determ ination of Antimicrobial Efficacy A di lution factor of log I was applicable. The average plate count was calcu lated; where the

result = 0.5, this was recorded as I. Where the result = TNC, this was recorded as > 300. Log lo of the average then was calcula ted .

Then (log N -1) - (log test) = log reduction. Where the log (test) is "-" the log reduction was recorded as "greater than" (».

SUMMARY OF LOG REDUCTIONS

Summary results of 7 tests are given in the following tables. Appendix A contains detailed resu lts tables for the various tests.

Test No. l : Organism: Pseudolllollas aerugillosa

Time Ozone mins ppm

a 0.050 I 0.075 3 0.250 5 0.350 7 0.400 II 0.400 15 0.450

Time Ozone nuns ppm

0 0.050 I 0.075 3 0.250 5 0.350 7 0.400 II 0.400 IS u.4SU

Test Control

Log Log Log Log Recovery Reduction Recovery Reduction

6.4 6.3 0.0 6.4 0.0 6.3 0.0 6.3 0.1 6.3 0.0 5.5 0.9 6.3 0.0 3.4 3.0 6.3 0.0

<2.0 > 4.4 6.3 0.0 <2.0 > 4.4 6.3 0.0

Test No.2: Organism: MRSA

Test Control

Log Log Log Log Recovery Reduction Recovery Reduction

5.7 5.8 -0. 1 4.5 1.2

< 1.0 > 4.7 <1.0 > 4.7 <1.0 > 4.7 < 1.0 > 4.7

5.8 5.8 5.8 5.8 5.7 5.7 S.B

0.0 0.0 0.0 0.1 0. 1 0.0

Efficacy of the JLA Ltd. Ozone Laundering System September-October 2009 371

Test No.3: Organism: Ellterococcus/aecalis

Test Control

Time Ozone Log Log Log Log mins ppm Recovery Reduction Recovery Reduction

0 0.050 6.0 6.1

I 0.100 5.8 0.2 6.0 0.1

3 0.250 5.5 0.5 6.0 0.1

5 0.450 2.2 3.8 6.0 0.1

7 0.450 1.8 4.2 6.0 0.1

II 0.450 1.6 4.4 6.0 0.1 15 0.450 1.3 4.7 6.0 0.1

Test NO.4: Organism: Escherichia coli

Test Control

Time Ozone Log Log Log Log n1lns ppm Recovery Reduction Recovery Reduction

0 0.050 5.5 5.8 0.075 5.6 - 0.1 5.9 -0.1

3 0.250 5. I 0.4 6.0 - 0.2

5 0.350 3.5 2.0 5.7 0.1

7 0.400 2.1 3.4 5.9 -0.1

II 0.400 1.6 3.9 5.8 0.0 15 0.450 < 1.0 > 4.5 5.8 0.0

Test No.5: Organism: Acilletobacter ballll/allllii

Test Control

Time Ozone Log Log Log Log mins ppm Recovery Reduction Recovery Reduction

0 0.050 5.5 5.9

I 0. 100 4.7 0.8 5.9 0.0

3 0.250 4.9 0.6 6.0 -D. I

5 0.450 3.8 1.7 6.0 -0.1

7 0.450 3.5 2.0 6.0 - 0.1

II 0.450 3.2 2.3 6.0 -0.1 15 0.'150 .... "7 2.S 6.0 - 0.1 i.. I

372 K. Allison et al.

Test No. 6: Organism: Aspergillus lIiger (spores)

Test Control

Time Ozone Log Log Log Log mins ppm Recovery Reduction Recovery Reduction

0 0.450 3.8 3.8

0.550 3.8 0.0 3.7 0.1

3 0.700 3.0 0.8 3.7 0.1

5 1.000 1.7 2.1 3.7 0.1

7 1.200 1.0 2.8 3.7 0.1

II 1.500 1.0 2.8 3.7 0.1

15 1.800 < 1.0 > 2.8 3.7 0.1

Test No.7: Organism: Clostridiulll dif/icile (spores)

Test Control

Time Ozone Log Log Log Log mins ppm Recovery Reduction Recovery Reduction

0 0.400 2.6 2.6

0.500 2.4 0.2 2.6 0.0

3 0.750 2.0 0.6 2.6 0.0

5 0.900 1.0 1.6 2.6 0.0

7 1.200 < 1.0 > 1.6 2.6 0.0

II 1.400 < 1.0 > 1.6 2.6 0.0

15 1.200 < 1.0 > 1.6 2.6 0.0

DISCUSSION OF RESULTS

Using the OTEX Validated Ozone generator, a con­tinualreductioll of Pseudomollas ael'llgil/osa cells occurred as the levels of dissolved ozone increased. After 5 min­utes, the dissolved ozone levels had reached 0.350 ppm and achieved a Log 0.9 reduction of viable cells. An increase of 0.050 ppm ozone gave an increase in Log reduction of 2.1. After II minutes, the dissolved ozone level had reached 0.450 ppm and achieved a Log reduc­tion of > 4.4. This result was maintained after 15 minutes, with no discern ible recovery of organism. When testing ozone against MRSA, the same increase of dissolved ozone gave a mllch fa ster Log kill. After 5 minutes, the dissolved ozone level reached 0.350 ppm and gave a Log reduction or >4.7.

September-October 2009

The viable cells of Ellterococcus /aecalis gave a more gradual decline over the 15 minute contact time, suggest­ing a greater resistance against ozone than the previous two organisms. After 5 minutes, dissolved ozone levels reached 0.450 ppm and the Log reduction achieved was 3.8. Dissolved ozone remained at the same level for the rest of the test and the degree of biocidal activity increased to Log 4.7 after 15 minutes; however, viable cells of E. /aecalis still were recovered.

Testing of OTEX generated ozone at 0.450 ppm against E. coli gave a >4.5 Log reduction of viable cells after 15 minutes with no discernable recovery of cells. As with the previous three organisms, most of the biocidal efficacy occurred after dissolved levels of ozone reached >0.250 ppm.

The Log reduction of Acil1etobacter baulIlal1l1ii cells was much lower than with the other four bacteria. After 15 min utes, dissolved levels of ozone had reached 0.450 ppm, but the log reduction had only reached 2.8 of the potential 4.5 Log reduction. A study by Chamul et al. (2002), found that "Of the Gram­negative microorganisms, Acinetobacter baulI1al1l1ii was found to be one of the most resistant to ozone at low concen tra tions."

It should be noted that the tests conducted against all the above bacteria were performed in the presence of the growth medi um (Tryptone Soya Broth). It is common knowledge that ozone is readily absorbed by organic materials. This means that the dissolved ozone had to overcome this absorption before it could have a biocidal effect. Both of the spore-forming organisms were tested without growth medium; instead they were introduced into the washing machine drum suspended in sterile water.

In the case of OTEX-generated dissolved ozone against Aspergillus niger spores, the system showed a gradual increase in biocidal efficacy under conditions more applicable to the final use of the system. Dissolved ozone levels reached 1.80 ppm after 15 minutes, giving a Log reduction of >2.8 viable spores.

There are limitations of generating enough A. l1iger spores to return a significant inoculum when dosed into the drum. Because of these limitations, it is diffi­cult to tell if the decrease in viable spores is gradual, due to the fact that some spores are morc susceptible than others, or if the system would have demonstrated lower level biocidal efficacy with an increase of organic matter that a higher yield of spores would have produced .

Due to the difficulty in obtaining ClostridiulIl d!r(icile spores, the starting challenge in 68 liters of water was again low. In the presence of no interfering substancc, the OTEX system was able to generate very high levels of dissolved ozone. After I minute, the dissolved ozone concentration had reached 0.500 ppm, which g~1\'C a Log 0.2 reduction of ClostridiulIl d!r(icile. After 3 minutes,

dissolved ozone reached 0.750 ppm, giving a Log reduc­tion of 0.6. After reaching 1,20 ppm of dissolved ozone after 7 minutes, no discernable ClostridiulIl difjici/e spores were recovered, giving a reduction of viable spores of > 1.6 logs.

In every instance, the control tests recovered levels of organisms that provc that the action of the washing machine itself (without ozone being added) has no anti­microbial activity and that the log reductions of the tests are due to the dissolved ozone alone.

CONCLUSIONS

o In the case of the bacterial tests, the addition of the growth medium reduced the levels that the dissolved ozone could reach.

o Levels of 0.250 ppm of dissolved ozone had minimal effect 011 the bacteria. Levels >0.450 ppm showed good bactericidal activity, except against Acilletob({cter b({/liJ/({llllii.

o Against fungal spores, dissolved ozone levels of> 1.20 ppm are required to attain a 2.8 Log red uction.

o For Clostridilllll difj/cile spores, a dissolved ozone level of between 0.900 ppm and 1.20 ppm gave> 1.6 log reduction with no discernible spore recovery.

REFERENCES

Allison, K. "Quantitative Suspension Testing Using thc OTEX Validated Ozone Generator and JLA HelOO (10 kg) Washing Machinc", MGS Laboratorics, Science Centre, Coopers Hill Lane, Egham, Surrey, TW20 OLB, UK, 2007.

Chamul, R.S., M. Reed, and J.L. Silva, "Chiller Water Treatmcnt rrom Channel Cat fi sh (/cla/I/l'/ls plII/flall/s) Processing Plants with Ultrasound, OWlle, and Pulsed Light", Mississippi State University, l\'l ississippi State, MS. 2002.

APPENDIX A, DETAILED RESULT SHEETS

In this Appendix, tables of detailed experimental data results are presented for the seven organisms studied.

Date Test Performed Product Test Temperature Urganism

10 /05 / 2007 Ozone 20 °C±2 °C l'sel({/Ol/lUl/aS ael'lIgiliosa

Efficacy of the JLA Ltd. Ozone Laundering System September-October 2009 373

Control Test

Plate Count

Contact Timc, min Dilution Plate I Platc 2 Average Cfu/mL Log Log Red uction

a -4 187 169 178 1.8 x 106 6.3 0.0 I -4 176 176 176 1.8 x 106 6.3 0.0 3 -4 193 174 184 1.8 x 106 6.3 0.0 5 -4 192 183 187 1.9 x 106 6.3 0.0 7 -4 198 164 181 1.8 x 106 6.3 0.0 II -4 185 180 183 1.8 x 106 6.3 0.0 15 -4 177 175 176 1.8 x 106 6.3 0.0

Test: Disinfection Level: MAX Pressure (psi): 5 SCFH: 4.25 Ozone Activation: 56 seconds Level Stop: 2 minutes 24 seconds

Contact Time, min

a I 3 5 7 II IS

Control Test

Contact Time, min

a I 3 5 7 II 15 ~~.-..:-~ ... ...:........,-,- .

374

Ozone Level, ppm Dilution

0.05 -4 0.075 - 4 0.25 -4 0.35 -4 0.40 -2 0.40 -2 0.45 -2

Date Tcst Performed Product Test Temperature Organism

Plate 1

200 199 198 28 26

Nil Nil

Plate Count

Dilution Plate I Plate 2

-4 64 52 -4 63 60 -4 69 62 -4 54 62 -4 52 58 -4 55 57 -4 55 59

Plate Count

Plate 2 Average Cfu/mL

191 195 184 36 29

Nil Nil

Average

58 62 66 58 55 56 57

196 2.0 x 106

197 2.0 x 106

191 1.9 x 106

32 3.2 x 105

28 2.8 x 103

<1 < loa <I < 100

10 / as / 2007 Ozonc 20 °C±2 °C MRSA

Cfu/mL

5.8 x 105

6.2 x 105

6.6 x 105

5.8 x 105

5.8 x 105

5.6 x 105

5.7 x 105

~.---,~~.:.-:...-..>.~".:; ...........

K. Allison et al. September-October 2009

Log Log Reduction

6.4 6.4 0.0 6.3 0.1 5.5 0.9 3.4 3.0

<2.0 >4.4 <2.0 >4.4

Log Log Reduction

5.8 0.0 5.8 0.0 5.8 0.0 5.8 0.0 5.7 0.1 5.7 0.1 5.8 0.0

Test: Disinfection Level: MAX Pressure (psi): 5 SCFH: 4.25

Contact Time, min Ozone Level, ppm Dilution

0 I 3 5 7 I I 15

Control Test

0.05 - 4 0.075 -4 0.25 -3 0.35 -I 0.40 -I 0.40 - I 0.45 -I

Date Tcst Performed Product Tes t Temperature Organism

Ozone Activation: 56 seconds Level Stop: 2 minutes 24 seconds

Plate I

54 58 33 Nil Nil Nil Nil

Plate Count

Plate 2 Average

57 55 35 Nil Nil Nil Ni l

56 57 34

< I < I < I < I

17 / 05 / 2007 Ozone 20 DC±2 DC

Cfu/mL

5.6 x 105

5.7 x 105

3.4 x 104

< 10 < 10 < 10 < 10

Ell terococcus Iaecalis

Log Log Reduction

5.7 5.8 -0.1 4.5 1.2

< 1.0 > 4.7 < 1.0 > 4.7 < 1.0 > 4.7 < 1.0 > 4.7

Plate Count

Contact Time, min Di lution Plate I Plate 2 Average Cfu/mL Log Log Reduction

0 -4 134 107 121 1. 2 x 106 6.1 I - 4 98 101 100 1.0 x 106 6.0 0.1 3 - 4 105 105 105 1.1 x 106 6.0 0.1 5 -4 112 102 107 1.1 x 106 6.0 0. 1 7 - 4 110 113 11 2 1.1 x 106 6.0 0.1 I I - 4 100 116 108 1. 1 x 106 6.0 0.1 15 - 4 10 1 112 107 1. 1 x 106 6.0 0.1

Test: Disinfection Level: MAX Pressu re (psi): 5 SCFH: 5.0 Ozone Activation: 58 seconds Level Stop: 2 minutes 36 seconds

Plate Count

Contact Time, min Ozonc Level, ppm Dilution Plate I Plate 2 Average Cfu/mL Log Log Rcduction

0 0.05 - 4 100 87 94 9.4 x 105 6.0 I 0.10 - 4 63 54 59 5.9 x 105 5.8 0.2 3 0.25 -4 35 30 33 3.3 x 105 5. 5 0.5 5 " 0.45 - I 17 15 16 1.6 x 102 2.2 3.8 7 0.45 - I 7 7 7 7.0 X 101 1.8 4.2 I I 0.45 - I 4 4 4 4.0 X 10 1 1.6 4.4 15 0.45 -I 3 2 2.0 X .10 1 1.3 4.7

. - - -- -- . - -

Efficacy of the JLA Ltd. Ozone Laundering System September-October 2009 375

Date Test Performed 10/05/2007 Product Ozone Test Temperature 20 °C±2 °C Organism Escherichia coli

Control Test

Plate Count

Contact Time, min Dilution Plate I Plate 2 Average Cfu/mL Log Log Reduction

0 -4 69 65 67 6.7 x 105 5.8 I -4 94 68 81 8.1 x 105 5.9 -0.1 3 -4 91 90 91 9.1 x 105 6.0 -0.2 5 - 4 45 55 50 5.0 x 105 5.7 0.1 7 -4 71 79 75 7.5 x 105 5.9 -0.1 II -4 54 63 59 5.9 x 105 5.8 0.0 15 -4 67 73 70 7.0 x 105 5.8 0.0

Test: Disi nfection Level: MAX Pressure (psi) : 5 SCFH: 4.25 Ozone Activation: 56 seconds Level Stop: 2 minutes 24 seconds

Contact Time, min

0 I 3 5 7 II 15

Control Test

Contact Time, min

0 I 3 5 7 I I 15

376

Plate Count

Ozone Level, ppm Dilution

0.05 -3 0.075 -3 0.25 -3 0.35 -2 0.40 - I 0.40 - I 0.45 - I

Date Tes t Perfo rmed Product Test Temperature Organism

Plate I

327 400 136 31 13 4

Ni l

Plate Count

Dilu tion Plate I Plate 2

-4 85 93 -4 85 83 - 4 110 118 -4 112 86 - 4 110 97 - 4 121 104 -4 109 I II

Plate 2 Average Cfu/mL

34 1 334 3.3 x 105

365 383 3.8 x 105

128 132 1.3 x 105

34 33 3.3 x 103

15 14 1.4 x 102

4 4 4.0 X 101

Nil Nil < 10

17 / 05 / 2007 Ozone 20 °C±2 °C AciJlelobacler bmllJlwlI1ii

Average Cfu/mL

89 8.9 x 105

84 8.4 x 105

11 4 1.1 x 106

99 9.9 x 105

104 1.0 x 106

113 1.1 x 106

110 1.1 x 10(' = .... =

K. All ison et al. September- October 2009

Log Log Red uction

5.5 5.6 -0. 1 5. 1 0.4 3.5 2.0 2.1 3.4 1.6 3.9

< 1.0 > 4.5

Log Log Reduction

5.9 5.9 0.0 6.0 -0.1 6.0 - 0.1 6.0 -0.1 6.0 -0.1 6.0 -0.1

Test: Disinfection Level: MAX Pressure (psi) : 5 SC FH: 5.0

Contact Time, min Ozo ne Level, ppm Dilution

0 I 3 5 7 I I 15

Control Test

0.05 -4 0. 10 -3 0.25 -3 0.45 -2 0.45 -2 0.45 - I 0.45 - I

Date Test Performed Product Test Temperat ure Organism

Ozone Activation: 58 seconds Level Stop: 2 minutes 36 seconds

Plate I

33 48 75 66 31

158 48

Plate Count

Plate 2

29 45 70 62 34

142 42

Average Cfu/mL

31 3.1 x 105

47 4.7 x 104

73 7.3 x 104

64 6.4 x 103

33 3.3 x 103

150 1.5 x 103

45 4.5 x 102

09/ 05 / 2007 Ozone 20 °C±2 °C Aspergillus niger

Log Log Red uction

5.5 4.7 0.8 4.9 0.6 3.8 1.7 3.5 2.0 3.2 2.3 2.7 2.8

Plate Count

Contact Time, min Dilution Plate I Plate 2 Average Cfu/mL Log Log Reduction

0 -2 57 58 58 5.8 x 103 3.8 I -2 55 56 56 5.6 x 103 3.7 0.1 3 -2 57 49 53 5.3 x 103 3.7 0.1 5 -2 50 58 54 5.4 x 103 3.7 0.1 7 - 2 59 49 54 5.4 x 103 3.7 0.1 I I -2 60 51 56 5.6 x 103 3.7 0.1 15 -2 39 50 45 4.5 x 103 3.7 0.1

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Test: Disinfection Level: MAX Pressu re (psi): 5 SCFH: 4.50 Ozone Activation: 55 seconds Level Stop: 2 minutes 25 seconds

Plate Count

Contact Time, min Ozone Level, ppm Dilution Plate I Plate 2 Avcrage Cfu/mL Log Log Red uction

0 0.45 -2 60 65 63 6.3 x 103 3.8 I 0.55 -2 57 68 63 6.3 x 103 3.8 0.0 3 0.70 - I 119 103 I I I 1. 1 x 103 3.0 0.8 5 1.00 - I 3 6 5 5.0 X \0' 1.7 2.1 7 1.20 - \ 2 Ni l I 1.0 x 10 ' 1. 0 2.8 1\ !.SO - \ 2 Nil I 1.0 x 10' 1. 0 2.8 15 1.80 - I Nil Ni l Nil < 10 < 1.0 > 2.8

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Efficacy of the JLA Ltd. Ozone Laundering System September-October 2009 377