About ozone part 3

Figure 3. Data obtained during 16-min test on water that contained C. difficile spores at 75/C (Microsearch Laboratories Ltd., May 15, 2004).

Test #2. Testing of Four OTEX Laundering Cycles – Microsearch Labs - Nov. 8, 2004

Four ozone laundering cycle studies (Test Codes) of various garments were conducted and the challenge organisms (S. aureus MRSA strain and C. difficile) recovered and analyzed post-washing. Cycle 1 (Test Code 1) is a heavy washing for foul and infected, heavily soiled clothing. This cycle also has a sluice cycle (high wastewater level flush). The machine fills up with cold water, does a wash action, and continuously drains through an overflow. This sluice cycle is followed by a normal wash cycle. Cycle 2 (Test Code 2) is for lightly soiled sheets and towels.

Cycle 3 (Test Code 3) is for delicate items, such as personal clothing and woolens. Cycle 4 (Test Code 4) is a rewash cycle used for oil/grease stained articles. With this cycle, 50/C water is used to emulsify the oils and aid washability.

Figure 4. EU Suspension Test conducted on OTEX ozone laundry water with C. difficile spores (Microsearch Laboratories Ltd., May 15, 2004).

The amount of ozone is constant for each washing programme. The difference between cycles is that the more heavily soiled items require more detergent, which destroys some of the ozone. It is important to know that satisfactory microorganism kills can be attained by the four washing cycles, regardless of the degree of soil.

A control untreated batch also was tested for these microorganisms in duplicate. Results are listed in Table 1. All ozone launderings resulted in > 5-logs kill (>99.999%), whereas washing without ozone (Controls) gave <99.999 % kill.

Test #3. – Microsearch Labs – MRSA Contamination of Nurses Uniforms Test – 2004

Microsearch Laboratories carried out comparative tests on nurses’ uniforms impregnated with a strain of the superbug MRSA (methicillin-resistant Staphylococcus aureus). This microorganism is being detected with increasing frequency in USA hospitals and care homes (TIME Magazine Archive, 2006).

The care labels of nurses’ uniforms commonly carry the recommendation that they should be washed at 40ºC (104°F). Therefore, one test was carried out using a conventional 40ºC wash cycle (without ozone). A second test was carried out with an OTEX (cold water) cycle.

Figure 5 is a photograph showing the MRSA microorganism which had been impregnated onto a membrane. The membranes were implanted into the garments prior to the uniforms undergoing any laundry process.

Figure 5. MRSA impregnated onto membrane. Microsearch Labs, 2004.

Figure 6 shows the residual MRSA culture on the recovered membrane after having been washed at 40ºC (104°F). Figure 7 shows the absence of residual MRSA culture on the recovered membrane after an OTEX ozone-laundering cycle.

Figure 6. MRSA on membrane after 40°C wash.

Figure 7. Absence of MRSA after OTEX.

Results – These results indicate that a greater than log-8.0 reduction (99.999999%) in MRSA was obtained on populations of garments washed by the OTEX process. The average log reduction achieved by the 40ºC (104°F) wash was only 3.3 (99.93%). To clarify, the reduction of MRSA achieved by the OTEX procedure was greater than log-8.0. Microsearch personnel were unable to isolate any survivors from the OTEX treated garments. (Microsearch Laboratories, 2004).

Test #4. Antimicrobial Efficacy of the OTEX Process at 60% Ozone Output Against

Escherichia Coli - Microsearch Labs, April 29, 2005

A validation trial was conducted to determine the antimicrobial activity of an OTEX treatment at 60% of the maximum ozone output of the OTEX system against Escherichia coli. In this trial, E. coli was added as liquid culture directly to the input flow of a JLA washing machine. This culture was added in sufficient volume to produce a contamination level of the order of log-7 cells/mL.

This work and the reconsideration of the optimum operating ozone level was prompted by confounding adverse evidence produced during a third party evaluation during which poor log kill data was obtained for E. coli. The initial aim in this trial was to produce evidence of a baseline

log kill potential with E. coli as a direct contaminant of wash waters with no additives running at ambient temperature, then to demonstrate the effect of ozone under identical conditions.

An ambient temperature wash trial was conducted which contained no additives and which was of 20 minutes duration. Estimates of the E. coli levels in the wash water were obtained by the analysis of samples collected at 3, 10 and 20-minute intervals. In an identical wash program after the first sample was recovered (i.e., 3 minutes) the OTEX device was activated and thereafter produced a continuous charge of ozone at 60% of the maximum available ozone output. Subsequent sampling occurred as described above.

Each trial was preceded by a hot sanitizing wash and rinse cycle. Data obtained are reported in Table 2 and Figure 8.

Table 2.	OTEX Revalidation Trial 60% Ozone Output Treatment
		T = 3 min		T = 10 min	T = 20 min
		E. coli		E. coli	E. coli
Treatment		Cfu/mL		Cfu/mL	Cfu/mL
Ambient Wash
No Ozone No		9.30E+07		8.40E+07	6.20E+06
Additives
Ambient OTEX
Only 60%
Ozone Output		7.80E+07		< 1	< 1
Wash

Figure 8. E. coli kill data for OTEX at 60% of the available ozone output vs ambient wash treatments. Microsearch Labs, April 29, 2005.

In the control experiment with no additives or ozone treatment, these data show an E. coli log-reduction of approximately 1 log cycle during the 20-minute wash period. During the treatment with ozone E. coli could not be recovered after the initial dosing period. In fact by the ten-minute

mark, these data indicate that a 7-log reduction was obtained corresponding to 7 minutes of

ozone dosing at 60% of the maximum available output.

Test #5. OTEX Bacteriological and Viral Investigation: OTEX Laundry System

Solution Test (OTEX Report Sept. 2005)

A laboratory investigation was carried out with the objective of providing documentary evidence of the bactericidal and virucidal activity of the OTEX system at ambient temperature against thermal disinfection (75°C = 167°F)) wash processes. The work was carried out on 1 July 2005 at JLA’s R & D Technical Laboratory, Ripponden, West Yorkshire, UK. The microorganisms and viruses employed (Table 3) were independently prepared by Microsearch Laboratories Ltd. for testing.

Table 3. Solution Challenge Test Organisms

Microorganism		cfu/mL
Staphylococcus aureus		1.3E+08
Pseudomonas aeruginosa		3.1E+09
Candida Albicans		3.1E+08
Escherichia coli		5.2E+08
Streptococcus faecalis		5.0E+08
Aspergillus niger		3.1E+08
Clostridium difficile		4.2E+08
Clostridium perfringens		9.2E+08
Campylobacter jejuni		6.0E+08
Aeromonas mixed species		8.2E+08
Actinobacter sps		4.3E+08
Lactobacilli sps		3.9E+08
Virus particle	Particles/mL
Lambda phage	3.8E+24
FCoVA	2.6E+24
Saccharomyces virus ScV-L-BC	3.1E+23
Vibrio phage fs1	2.6E+28

The four virus particles selected for testing represent both single and double strand RNA and DNA, which is the structure of the vast majority of all virus types.

Program Details and Test Conditions – Tests were carried out using an extended sluice program in a JLA model HW164 (16 k dry weight) washing machine. No detergent was employed during this series of tests. Details are tabulated below.

Tests were conducted with water temperatures at both ambient, i.e., as supplied, and at 75°C (167°F), which is above the recommended thermal disinfection temperature of 71°C (160°F). Domestic supply water was employed with a water hardness of 60 ppm CaCO₃ for all tests.

Program Details:	Cycle Time (mins)		Temp		Wash Action
			(°C)
Program 1: Cold	30		Ambient
Sluice					12 sec wash/3
Program 2: Thermal	30		75°C		sec stop time
Sluice
Detergent Volumes		No Detergent in use.

A single unit OTEX system was employed and was maintained at the following settings throughout the trial with the exception of the control test with no ozone:

Ozone Concentration Setting	8 (highest)
Pressure	5 psi
Flow Rate	3.5 cfh

Test samples were taken from the wash drum throughout the wash cycle to determine the concentration of dissolved ozone in the water. This was measured by using the Chemets method, which employs DPD chemistry. Dissolved ozone levels increased from 0.2 ppm at the start to 0.6 ppm after 15 minutes, with samples being taken at 3, 7, 11, and 15 minutes of washing.

Data obtained are presented in Figures 9-14. Figures 9, 10 and 11 show results of bacterial sampling at ambient temperature - no ozone (control), 75°C (167°F = thermal washing), and ambient temperature with ozone (OTEX), respectively. Note that without ozone and at ambient temperature (Figure 9), only small amounts of bacterial kills were obtained. With thermal washing (Figure 10), three strains of bacteria remained at significant levels even after 15 minutes. But with ozone at ambient temperature (Figure 11), no bacteria were present after 3 minutes of washing.

Figure 9. Test results - control @ ambient temperature - no ozone – bacteria.

Figure 10. Test results - Thermal @ 75°C - no ozone – bacteria.

Figure 11. OTEX System - Ambient Water Temperature - bacteria.

Figures 12, 13 and 14 show similar results of virus and phage sampling at ambient temperature - no ozone (control), 75°C (167°F = thermal washing), and ambient temperature with ozone (OTEX), respectively. Note that without ozone and at ambient temperature (Figure 12), only small amounts of viral inactivations were obtained. With thermal washing (Figure 13), viral

inactivations were obtained after 5 minutes., and the same results were obtained with ozone at ambient temperature (Figure 14) after 5 minutes (but at lower costs).