CFR Pro 500 Ozone Assisted Case Study

University of Minnesota Case Study
Microbiological Efficacy of CFR’s Ozone Assisted Cleaning System

University of Minnesota
School of Public Health, Division of Environmental & Occupational Health
Donald Vesley, PhD; Andrew Streifel, MPH; David Downey

1. SUMMARY OF STUDIES
CFR’s ozone assisted cleaning system, the Altra 400 OZ SP, has been evaluated by the University of Minnesota School of Public Health. The results of this evaluation have proven that the Altra 400 OZ SP is an effective carpet cleaning system. The tank study demonstrated the disinfecting power of ozone in the tank against a broad spectrum of microorganisms. The laboratory experiments that involved cleaning a carpet swatch with a heavy microbial load resulted in reductions ranging from 91 % to 96 %. Cross contamination to a clean carpet swatch was less than 1.1 %. The Altra 400 OZ SP, as compared to a typical carpet extractor, is more effective in reducing the level of a broad spectrum of microorganisms on carpet and in eliminating microbial cross contamination. Air sampling has shown that the Altra 400 OZ SP aerosolizes significantly fewer microorganisms than a typical carpet extractor. Overall, the Altra 400 OZ SP has consistently shown superior microbiological efficacy over a typical carpet extractor.

The evaluation included a laboratory phase and a field test phase. The laboratory phase consisted of a tank study and a carpet cleaning study. The tank study evaluated the Altra 400 OZ SP’s in tank reduction of microorganisms. The carpet cleaning study involved microbiological monitoring of the Altra 400 OZ SP and a typical carpet extractor. The laboratory experiments were performed using three different microbial challenges. These included Staphylococcus epidermidis, Cladosporium sp. fungi, and Bacillus subtilis spores. Staphylococcus epidermidis is a gram-positive bacteria that is common in areas occupied by humans. cladosporium is the most common fungus found in outdoor air. Bacillus subtilis spores are very resistant to environmental stress. The field test phase also involved microbiological monitoring of the Altra 400 OZ SP and a typical carpet extractor. The data presented in the following report are the results of the laboratory phase and the field test phase of the research plan.

The tank study examined the effectiveness of ozone in reducing the level of microorganisms in the Altra 400 OZ SP. Microorganisms were added directly into the tank. The vacuum, pump, and ozone were turned on and samples were collected from the tank at various time intervals. Ozone levels in the tank water were measured during the experiments. Approximate D values (time required for 90 % reduction) were derived for the three microorganisms used in the experiments.

The carpet cleaning experiments were carried out in a vertical laminar airflow cleanroom so that the room could be purged quickly between experiments and to eliminate ambient microorganisms for air sampling purposes. Microorganisms were seeded (sprayed) evenly onto a 3 ft. x 4 ft. new, biocide free carpet swatch. The seeded carpet concentrations represented a very high load, unlikely to be seen in a carpet under ambient conditions. The cleaning system then made two passes over the entire carpet surface. Carpet samples were taken before and after seeding the carpet and after cleaning the carpet. In order to test for microbial cross contamination, each seeded carpet test was immediately followed by cleaning a new nonseeded carpet swatch with the machine that had cleaned the seeded carpet. Carpet samples were taken before and after cleaning the nonseeded carpet. Also during the experiments, air samples were collected with a High Volume Casella. Samples were collected before and while cleaning both the seeded and nonseeded carpet swatches.

The field tests were carried out on a carpeted floor of a typical office corridor at the University of Minnesota. Each trial consisted of two 5 ft. x 20 ft. areas of carpeted corridor. One area was cleaned with the Altra 400 SP OZ and the other with a typical carpet extractor. Surface sampling was done with contact plates before, after, and an hour after cleaning. Before cleaning samples were taken from 20 randomly selected sites. After cleaning and an hour after cleaning samples were collected from adjacent but mutually exclusive sites to the before cleaning samples. Air samples were obtained with an Andersen 2-stage sampler. Air samples were collected before cleaning, while cleaning, and an hour after cleaning. Ozone levels in the air were monitored during the Altra 400OZ SP trials.

2. LABORATORY TANK STUDY

A. METHODS

The Altra 400 OZ SP was evaluated using three different microbial challenges: Staph epidermidis, Cladosporium, and Bacillus subtilis spores. Cultures of these organisms were added to 1500 ml of sterile, buffered, distilled water (SBDW) to provide 104-106 cfu/ml (colony forming units per milliliter).

The Altra 400 OZ SP was filled with 12 gallons of distilled water (at a temperature of approximately 25 degrees C), and the head of the system was placed on the floor. The 1500 ml of SBDW/microorganisms were poured into the tank. This provided 103-105 cfu/ml (colony forming units per milliliter) in the tank. The solution was then thoroughly mixed and a 100 ml sample was collected as a baseline count. All samples were placed in bottles containing sodium thiosulfate to neutralize the ozone. The vacuum, pump, and ozone were turned on, and samples were collected from the tank at various time intervals. The time intervals depended on the microorganism being tested. Also, the level of ozone in the tank water was monitored with every sample.

Following the trials, the tank samples were plated. Plate Count Agar was used for bacterial sampling and Inhibitory Mold Agar (IMA) for fungal sampling. Bacterial plates were incubated at 36 degrees C for 48 hours and fungal plates were incubated at 25 degrees C for 72 hours before counting.

B. RESULTS

These results are the means of two trials completed with each microorganism. More detailed results are included in the appendix. D value = time required for a one log (90 %) reduction in the number of viable organisms.

D value

Staph epidermidis 1.7 min
Cladosporium 12.1 min
Bacillus subtilis 33.8 min

C. DISCUSSION

The tank study has demonstrated the disinfecting power of ozone against a broad spectrum of microorganisms. The level of ozone in the tank water ranged from 0.0 ppm to 0.1 ppm. The increasing D value from Staph epidermidis to Bacillus subtilis was predictable due to the increasing resistance of the microorganisms. The D values for these three microorganisms prove that the Altra 400 OZ SP is generating a sufficient amount of ozone to destroy even the most resistant microorganisms.
3. LABORATORY CARPET CLEANING STUDY

A. METHODS

These experiments were carried out in a vertical laminar airflow cleanroom so that the room could be purged quickly between experiments and so ambient microorganisms could be eliminated for air sampling purposes. The CFR Altra 400 OZ SP and a typical carpet extractor were evaluated using three different microbial challenges: Staph epidermidis, Cladosporium, and Bacillus subtilis spores. Cultures of these organisms were added to 1500 ml of sterile, buffered, distilled water to provide 104-106 cfu/ml (colony forming units per milliliter). The same methods were followed for both the CFR Altra 400 OZ SP trials and the typical carpet extractor trials. The few exceptions to this are mentioned where necessary.

Before trials began, the cleaning system being used was filled with distilled water (at a temperature of approximately 25 degrees C). The Altra 400 OZ SP was filled with 12 gallons or the typical carpet extractor was filled with 3-4 gallons. No detergent was added. For each trial, two new, biocide free carpet swatches (3 ft. x 4 ft ) were used. The carpet swatches were taped to two separate sheets of plywood (each approximately 4 ft. x 5 ft. ). Another sheet of plywood was placed on the floor of the cleanroom, behind the carpet swatch, to create a smooth and level surface on which to clean. This sheet of plywood was enclosed in a new plastic cover before each trial to eliminate the possibility of contamination.

One of the carpet swatches was placed in the cleanroom, and three carpet plugs (each 20.3 cm2) were cut from it at randomly selected locations, diluted in 100 ml SBDW and manually shaken. Also, two surface samples were collected adjacent to each plug sample, for a total of six surface samples. These surface samples were collected for comparison and to validate the contact plate method for field testing. Nunc contact plates filled with Dey Engly (D/E) Neutralizing Agar were used to collect these surface samples. The microbes were then sprayed (approximately 500 ml) evenly onto the carpet swatch (“Seeded Carpet”). Plug and contact plate samples (“After Seeding” samples) were collected adjacent but mutually exclusive to the “Before Seeding” samples. The cleaning system then made two passes over the entire carpet surface. During all trials, the variable speed controller on the Altra 400 OZ SP was set at three. Immediately after cleaning the carpet, plug and contact plate samples (“After Cleaning” samples) were collected adjacent to the previous samples. After moving the “Seeded Carpet” up against the wall, the second carpet swatch (“Nonseeded Carpet”) was brought into the cleanroom. Three carpet plugs were cut from randomly chosen locations, and two contact plate samples were collected adjacent to each plug sample (“Before Cleaning” samples). The machine then made two passes over the entire carpet surface. After cleaning the carpet, plug and contact plate samples (“After Cleaning2” samples) were collected adjacent to the samples taken before cleaning.

Air sampling was carried out with a High Volume Casella (700 L/min). The sampler was placed in a corner of the cleanroom on top off a Hepa filtered box which contained the sampler’s pump. During CFR trials, two minute samples were collected “Before Cleaning” and “While Cleaning” both the “Seeded” and “Nonseeded” carpet swatches. During the typical carpet extractor trials, only half minute samples were collected in an attempt to get countable “While Cleaning” samples. While air sampling, the cleanroom was not on. Plate Count Agar was used in the air sampling plates for all of the trials. Also, during each CFR trial, the level of ozone in the air was monitored using an Eco SensorTM ozone detector.

During the Altra 400 OZ SP trials, tank samples were collected at various points to monitor the level of ozone and the number of microorganisms in the tank water. Also of importance in the Altra 400 OZ SP trials was that the system received a 5 minute warm-up once turned on and it was turned off (as was the typical carpet extractor) between carpet swatches (except in two of the trials).

Following the work done in the cleanroom, the plug and tank samples were plated. Plate Count Agar was used for bacterial sampling and Inhibitory Mold Agar (IMA) for fungal sampling. Bacterial plates were incubated at 36 degrees C for 48 hours and fungal plates at 25 degrees C for 72 hours before counting.
B. CFR ALTRA 400 OZ SP RESULTS

Two trials were completed with each microorganism.

PLUG SAMPLES

These results are the means and standard deviations of two trials completed with each microorganism. % Reduction - after cleaning “Seeded Carpet” % Transfer - from “Seeded Carpet” to “Nonseeded Carpet”
% Reduction % Transfer*
Staph epidermidis 95.34 (+/-0.72) <0.01 -0="" 0="" br="">Cladosporium 88.68 (+/-10.86) <0.10 -0="" 0="" br="">Bacillus subtilis 92.05 (+/-1.17) <1.05 -0="" 07="" br="">* % Transfer = [count after cleaning nonseeded carpet / (count
after seeding seeded carpet - count after cleaning seeded carpet)] x 100

AIR SAMPLES

Samples were collected for two minutes with a High Volume Casella (700 L/min). Any blanks in the data represent contaminated plates or samples that were not collected. Cfu/m3 (colony forming units per cubic meter) represents the total colony forming units including nontest organisms. Normal ambient air levels in human occupied indoor spaces are approximately 35-50 cfu/m3.

Before Cleaning While Cleaning
Staph epidermidis
Trial I
Seeded Carpet ---- ----
Nonseeded Carpet ---- ----
Trial 2
Seeded Carpet ---- ----
Nonseeded Carpet ---- ----
Cladosporium
Trial I
Seeded Carpet ---- ----
Nonseeded Carpet ---- ----
Trial 2
Seeded Carpet 9.19 cfu/m3 28.62 cfu/m3
Nonseeded Carpet 4.24 cfu/m3 37.80 cfu/m3
Bacillus subtilis
Trial I
Seeded Carpet 9.89 cfu/m3 55.82 cfu/m3
Nonseeded Carpet 7.77 cfu/m3 30.03 cfu/m3
Trial 2
Seeded Carpet 18.02 cfu/m3 42.75 cfu/m3
Nonseeded Carpet 11.31 cfu/m3 25.80 cfu/m3

C. TYPICAL CARPET EXTRACTOR RESULTS
Three trials were completed with each microorganism.

PLUG SAMPLES

These results are the means and standard deviations of three trials completed with each microorganism. % Reduction - after cleaning “Seeded Carpet” % Transfer - from “Seeded Carpet” to “Nonseeded Carpet”
% Reduction % Transfer*
Staph epidermidis 81.97 (+/-6.30) <7.02 -10="" 92="" br="">Cladosporium 91.84 (+/-1.19) <2.14 -2="" 78="" br="">Bacillus subtilis 53.54 (+/-30.64) 29.10 (+/-29.07)
* % Transfer = [count after cleaning nonseeded carpet / (count after
seeding seeded carpet - count after cleaning seeded carpet)] x 100

AIR SAMPLES

Samples were collected for half a minute with a High Volume Casella (700 L/min). Any blanks in the data represent contaminated plates or samples that were not collected. Cfu/m3 (colony forming units per cubic meter) represents the total colony forming units including nontest organisms. The approximate value of > 4.0 x 103 cfu/m3 = too numerous to count (TNTC). Normal ambient air levels in human occupied indoor spaces are approximately 35-50 cfu/m3.
Before Cleaning
cfu/m3 While Cleaning
cfu/m3
Staph epidermidis
Trial 1
Seeded Carpet 9.19 > 4.0 x 103
Nonseeded Carpet --- ---
Trial 2
Seeded Carpet 0 4.0 x 103
Nonseeded Carpet --- ---
Trial 3
Seeded Carpet 1.4 x 101 1.8 x 103
Nonseeded Carpet 5.7 x 100 4.3 x 102
Cladosporium
Trial 1
Seeded Carpet 5.2 x 101 >4.0 x 103
Nonseeded Carpet --- ---
Trial 2
Seeded Carpet 3.4 x 101 > 4.0 x 103
Nonseeded Carpet --- ---
Trial 3
Seeded Carpet 1.7 x 101 1.7 x 103
Nonseeded Carpet 1.4 x 101 1.4 x 103
Bacillus subtilis
Trial I
Seeded Carpet 3.7 x 101 4.0 x 103
Nonseeded Carpet 4.6 x 101 4. 8 x 102
Trial 2
Seeded Carpet 2.6 x 101 7. 8 x l02
Nonseeded Carpet 101 x l01 2. 8 x I02
Trial 3
Seeded Carpet 1.1 x 101 > 4.0 x 103
Nonseeded Carpet 2.6 x 101 2.9 x 102

D. ALTRA 400 OZ SP vs TYPICAL CARPET EXTRACTOR

Comparisons of the percent reduction after cleaning a seeded carpet swatch, the percent transferred from a seeded carpet swatch to a nonseeded carpet swatch, and air sampling results from while cleaning the seeded carpet swatches. These results are the means of the trials completed with the Altra 400 OZ SP and a typical carpet extractor.

% Reduction

% Transfer*

Air Sampling While Cleaning Seeded Carpet cfu/m3
CFR Typical CFR Typical CFR Typical
Staph epidermidis 95.34% 81.97% < 0.01% < 7.02% 40.81** > 3.3 x 103
Cladosporium 88.68% 91.84% < 0.10% < 2.14% 28.62 > 3.2 x 103
Bacillus subtilis 92.05% 53.54% < 1.05% 29.10% 49.29 > 2.9 x 103
* % Transfer = [count after cleaning nonseeded carpet / (count after
seeding seeded carpet - count after cleaning seeded carpet)] x 100.
** Data taken from two trials not included in this report (44.52 cfu/m3 and 37 10 cfu/m3).

E. DISCUSSION

The Altra 400 OZ SP as compared to the typical carpet extractor, is more effective in reducing the level of microorganisms on carpet and in eliminating microbial cross contamination. Air sampling results are also in favor of the Altra 400 OZ SP. While using the Altra 400 OZ SP the level of microorganisms in the air remained below or within the range of normal ambient air levels. However, while using the typical carpet extractor the level of microorganisms in the air was significantly higher than normal ambient air levels. The air sampling results for the Altra 400 OZ SP are approximately 75 times less than the typical carpet extractor results.

The level of ozone in the air while cleaning with the Altra 400 OZ SP should not be a concern. When the tank lid was closed during operation the level of ozone in the air was not detectable.

4. FIELD STUDY

A. METHODS

Field trials were carried out on the first floor corridors of the Boynton Health Services building at the University of Minnesota - Twin Cities. The floor was vacuumed a day before each trial so that “Before Cleaning” surface samples would be countable (and not too numerous to count). All trials were performed while the floor was unoccupied, and all doors leading into the corridors were closed during the trials, eliminating ventilation into the hallways. Each trial consisted of two 5 ft. x 20 ft. areas (A and B) of carpeted corridor. In each trial, the CFR system was used first to clean area A. The typical carpet extractor was then used to clean area B which was located in a different corridor. The two areas were separated as such to eliminate the possibility of aerosols from one machine contaminating the other area being tested. Nunc contact plates filled with D/E Neutralizing Agar were used to surface sample 20 (only 18 in Trial 5) randomly selected sites. “After Cleaning’’ and the “Hour After Cleaning” samples were taken from adjacent but mutually exclusive sites to the “Before Cleaning” samples.

Air samples were collected “Before Cleaning,” “While Cleaning,” and an “Hour After Cleaning.” Air samples were obtained with an Andersen 2-stage sampler (28.3 L/min). The sampler was placed on a cart and located in the middle of the trial area. The two stages of the Andersen sampler were cleaned with alcohol before each sample. Samples were collected for 5 minutes “Before Cleaning” and an “Hour After Cleaning.” ‘’While Cleaning” air samples were collected for 8 to 10 minutes depending on the time required to clean the trial area. Plate Count Agar was used for air sampling.

Each machine made only one pass over the area to be cleaned. The variable speed controller on the Altra 400 OZ SP was set at level 3 during each trial. The typical carpet extractor was pulled at a speed comparable to the speed of the CFR system.

While cleaning with the Altra 400 OZ SP, the level of ozone in the air was monitored using two Eco SensorsTM ozone detectors. One detector was taped on the side of the machine near the vacuum exhaust port (carpet level) and the other was taped on the top of the machine, in close proximity to the operator (operator level)

The Altra 400 OZ SP was filled with 12 gallons of distilled water (at a temperature of approximately 25 degrees C) and the typical carpet extractor was filled with 6 gallons of distilled water. Twelve ounces (1 ounce/gallon of water) of CFR’s O-Z CleanTM was added to the Altra 400 OZ SP tank and six ounces was added to the typical carpet extractor’s cleaning tank before each trial. There was not a warm-up period for the CFR machine during the trials except for a ten minute warm-up before cleaning in Trial 5.

The contact plates and air sampling plates were incubated at 36 degrees C for two days before counting.

B. SURFACE SAMPLING RESULTS

These results are the mean and standard deviation for each set of samples. The mean of the means is also presented.
Before Cleaning
cfu/plate After Cleaning
cfu/plate Hour After Cleaning
cfu/plate
CFR
1 10.15 (+/-6.02) 15.35 (+/-8 67) 17.55 (+/-10.69)
2 19.00 (+/-4.67) 20.90 (+/-22.05) 11.10 (+/-11.83)
3 19.65 (+/-5.15) 25.60 (+/-2.50) 15.05 (+/-2.67)
4 19.50 (+/-6.25) 25.25 (+/-2.95) 14.35 (+/-2.03)
5 13.22 (+/-6.85) 22.78 (+/-9 43) 10.11 (+/-9.13)
Mean 10.30 13.98 9.63
TYPICAL
1 17.40 (+/-3.38) > 78.80 (+/-53.77) > 80.45 (+/-53.35)
2 17.30 (+/-4.77) > 62.65 (+/-59.84) > 64.21 (+/-62.86)
3 11.90 (+/-5.18) > 36.60 (+/-24.74) > 52.70 (+/-50.17)
4 18.40 (+/-3.10) > 29.95 (+/-28.67) > 26.50 (+/-28.60)
5 13.33 (+/-4.64) > 22.50 (+/-2406) > 20.39 (+/-19.85)
Mean 9.67 > 46.10 > 48.85

C. AIR SAMPLING RESULTS

Samples were collected with an Andersen 2-stage sampler (28.3 L/min). “Before Cleaning” and an “Hour After Cleaning” samples were collected for 5 minutes. “While Cleaning” samples were collected for 8 to 10 minutes depending on the time required to clean the trial area. These results are lower or within the expected ambient air levels in a normal office corridor (approximately 35-50 cfu/m3) because of the controlled nature of the experiment (the floor was unoccupied and there was no ventilation into the corridors during the experiments). Cfu/m3 = colony forming units per cubic meter.

Before Cleaning
cfu/plate After Cleaning
cfu/plate Hour After Cleaning
cfu/plate
CFR
1 0 3.89 14.13
2 0 3.89 0
3 0 3.89 0
4 14.13 11.66 0
5 14.13 0 7.07
Mean 5.65 4.67 4.24
TYPICAL
1 0 23.67 0
2 0 17.67 7.07
3 7.07 39.92 14.13
4 14.13 31.09 0
5 0 63.59 7.07
Mean 4.24 35.19 5.65

D. OZONE MONITORING RESULTS

Two ozone detectors were taped to the Altra 400 OZ SP during the field trials. One on top of the machine (operator level) and the other on the side of the machine near the vacuum exhaust port (carpet level). These results are the means and standard deviations of the five trials. In determining the mean and standard deviation, the highest recorded levels of ozone while cleaning were used.
Operator Level (ppm) Carpet Level (ppm)
CFR 0.08 (+/-0.l8) 0.004 (+/-0.01)

E. DISCUSSION

In every aspect of the field tests, the Altra 400 OZ SP was the superior machine. The Altra 400 OZ SP aerosolized significantly fewer microorganisms than the typical carpet extractor. The “While Cleaning” air sampling results for the Altra 400 OZ SP are approximately seven times less than the typical carpet extractor results. The surface sampling results are all in favor of the Altra 400 OZ SP. Probably the most important surface sampling numbers in this study are those that were collected an “Hour After Cleaning.” It is expected that when a dirty carpet is cleaned microorganisms and dirt are going to be raised to the surface. This explains why the “After Cleaning” numbers are higher than the “Before Cleaning” numbers. In all trials but one, the Altra 400 OZ SP “Hour After Cleaning” numbers are lower than the “After Cleaning” numbers. This suggests that the ozone being pulled through the carpet may have an effect on those organisms left in the carpet after cleaning.

In the majority of the Altra 400 OZ SP trials, ozone in the air was undetectable. Even the higher recorded ozone levels, extrapolated to a time-weighted average (TWA for a normal eight hour workday) and a short-term exposure limit (STEL-15 minute), are significantly lower than the Occupational Safety and Health Administration’s (OSHA) permissible exposure levels (TWA = 0.1 ppm and STEL = 0.3 ppm).