Evaluation of the air disinfection potential of the AlRsterildevice
Testing by
Dr. Louise Fletcher
Area of expertise: Waste Management; aerobiology environmental micrologiology
CONTENTS
1.Air Disinfection potential
1.1 Objectives of the Study
1.2 Test microorganisms
1.3 Culture preparation
1.4 Preparation of the nebuliser
1.5 Preparation of the air samplers
1.6 The test enclosure
1.7 Air disinfection experimental methodology
1.8 Enumeration of the bacteria in the impingers
1.9 Results and discussion
1. AIR DISINFECTION POTENTIAL
1.1 Objectives of the Study
The objective of the experiments was to determine the efficacy of the device in terms of its ability to reduce the concentration of viable microorganisms in the air in a one cubic metre test enclosure.
1.2 Test microorganisms
The surface tests were carried out using pure cultures of three microorganisms as follows:
Staphylococcus aureus – ATCC6538
Escherichia coli – ATCC10536
Aspergillus fumigatus (isolated from green waste compost)
1.3 Culture preparation
The S. aureus and E. coli cultures were prepared by using the cultiloops to inoculate 50ml of sterile nutrient broth (Oxoid, UK). The broths were then incubated at 37°C for 24 hours and shaken at 100rpm. The A. fumigatus stock was prepared by inoculating sterile malt extract agar plates and incubating at 40°C for 48 hours. After incubation the plates were washed with sterile ringer’s solution to harvest the fungal spores.
1.4 Preparation of the nebuliser
Initial tests carried out using the 3-jet nebuliser as per the PHE report (15/046 A) failed to yield significant colony counts and therefore the decision was taken to use the 6-jet nebuliser in order to increase the concentration of airborne microorganisms in the air inside the enclosure. The nebuliser was autoclaved and prior to the start of each test it was filled with 50ml of the test culture.
1.5 Preparation of the air samplers
The air samples were taken with six AGI-30 samplers which were weighed and then filled with 30ml of ringer’s solution and autoclaved. The flow rate for the air samples was 12l/min.
1.6 The test enclosure
The tests were carried out inside a 1 cubic metre enclosure that was made from aluminium laboratory scaffold covered in a double sheet of heavy duty plastic. During the experiments the plastic sheeting was sealed with tape to ensure no leakage of bioaerosols into the test chamber. The device was placed in to the enclosure together with the nebuliser and six AGI-30 impingers. A fan was placed underneath the outlet of the nebuliser to ensure the microorganisms stayedin suspension. The arrangement can be seen in the photographs below.
PLACEMENT OF THE DEVICE, NEBULISER AND IMPINGERS IN THE ENCLOSURE
ARRANGEMENT OF THE SIX IMPINGERS
LOCATION OF THE FAN UNDER THE OUTLETFROMTHE NEBULISER
1.7 Air disinfection experimental methodology
Prior to the start of the experiment the enclosure, nebuliser and impingers were prepared as described above and were placed into the enclosure as illustrated in the photographs above. The enclosure was then sealed and the fan and the device switched on and operated for 2 hours. After 2 hours the nebuliser was switched on and operated for 5 minutes.
1.8 Enumeration of the bacteria in the impingers
After the end of the test the impingers were taken into the laboratory and weighed to determine the volume of ringer’s solution in the samplers. Then using aseptic techniques an aliquot of 0.1ml from each impinger was plated out onto two sterile tryptone soya agar for E. coli and S. aureus and onto malt extract agar for the A. fumigatus. The E. coli and S. aureus plates were incubated at 37°C for 24 hours and the A. fumigatus for 48 hours at 40°C. After incubation the number of colonies on each plate was counted and was multiplied by 10 and then by the volume of liquid in each impinger (determined by the weights) to determine the number in the sampler. This was then multiplied up to determine the concentration per cubicmetre.
1.9 Results and discussion
The results from the tests can be seen in Table 1 below which shows the mean concentrations after 5 minutes and 60 minutes together with the calculated percent reduction. After 60 minutes the airborne microorganisms are undetectable in the air inside the enclosure when Airsteril technology
is in operation.
Testing by
Dr. Louise Fletcher
Area of expertise: Waste Management; aerobiology environmental micrologiology
CONTENTS
1. Surface disinfection potential
1.1 Objectives of the Study
1.2 Test microorganisms
1.3 Culture preparation
1,4 Preparation of stainless steel squares
1.5 Surface experiment methodology
1.6 Enumeration of the bacteria on the steel squares
1.7 Results
1. SURFACE DISINFECTION POTENTIAL
1.10 Objectives of the Study
The objective of the experiments was to determine the efficacy of the device in terms of its ability to reduce the concentration of viable microorganisms on stainless steel surfaces.
1.11 Test microorganisms
The surface tests were carried out using pure cultures of two microorganisms as follows:
Staphylococcus aureus – ATCC6538
Escherichia coll – ATCC10536
Clostridium difficile
1.12 Culture preparation
The S. aureus and E. coll cultures were prepared by using the cultiloops to inoculate 50ml of sterile nutrient broth (Oxoid, UK). The broths were then incubated at 37°C for 24 hours and shaken at 100rpm. After incubation the culture was enumerated and then used to inoculate the surfaces. The C. diffcile culture was prepared by inoculating a 50ml sterile nutrient broth (previously purged with nitrogen gas to remove any oxygen before autoclaving) with a small aliquot of stock laboratory culture and incubating for 24 hours and shaken at 100rpm. Because C. difficile is an anaerobic bacteria the culture was prepared in sealed Wheaton bottles and inoculated using a sterile syringe through the rubber septum
1.13 Preparation of stainless steel squares
Prior to use the stainless steel squares were placed in a beaker and washed in detergent for 1 hour after which they were rinsed with deionised water. Each square was then washed individually for 10 seconds with deionised water to make sure there was no residual detergent. The squares were then autoclaved at 121°C for 15minutes.
1.14 Surface experiment methodology
The tests were undertaken using previously decontaminated stainless steel squares prepared as outlined above. The squares were inoculated respectively with 5(El of a pure culture of S. aureus, E. coll C. difficile. The squares were placed into a laminar flow microbiological cabinet until the inoculum had completely dried. While the squares were being prepared the ventilation system in the chamber was switched on and operated at 12 AC/hr to purge the air inside the chamber after which it was switched off. The device was placed into the chamber along with a small support for the squares which was within 1m of the device and the device was switched on and operated for 2 hours before the start of the test. After drying, 15 of the squares inoculated for each microorganism were placed into the aerobiological chamber and 5 were retained for immediate enumeration. The test squares were then exposed to the test device and at 8 hours, 24 hours and 48 hours 5 squares for each microorganism were removed from the chamber.
INOCULATED STEEL SQUARES
POSITIONING OF THE DEVICE AND STEEL SQUARES IN THE AEROBIOLOGY CHAMBER
1.15 Enumeration of the bacteria on the steel squares
After exposure the squares were removed from the chamber and prepared for analysis. The surface of each square was swabbed using a sterile swab soaked in sterile ringer’s solution. The end of the swab was then snapped offand placed into a small bottle containing 10mlof sterile ringer’s solution. Each bottlewas then shaken for 30 minutes and vortexed for 1 minute. The solution was then diluted as required and plated out onto sterile tryptone soya agar plates. Allthe plates were then incubated for 24 hours at 37° C after which the total number of colonies on each plate were counted. The colony counts were then used to calculate the concentration of microorganisms in the 1 Omlof ringer’s solution and therefore the number of microorganisms recovered from each square surface. The counts from the five replicate steel squares were then used to determine the mean concentration with and without exposure to the device and this data was used to determine the mean reduction as a percentage.
1.16 Results
Table 1 shows the results obtained during the surface exposure experiment carried out using E. coll. The initial mean concentration on the steel squares was 10280 cfu which was reduced to 2100 cfu after exposure for 8 hours, 240 cfu after 24 hours and 10 cfu after 48 hours. This represents a reduction in the number of E. coll on the steel squares after 8 hours, 24 hours and 48 hours of
79.6%, 97.7% and 99.9% respectively.
TABLE 1 RESULTS OF THE SURFACE EXPOSURE EXPERIMENT CARRIED OUT USING E. COLl
As can be seen in Table 2 a similar trend was observed with S. aureus with an initial mean concentration of S. aureus on the steel squares slightly higher than that of E. coll at 14360 cfu. This was reduced to 1810 cfu after exposure for 8 hours, 217 cfu after 24 hours and 84 cfu after 48 hours. This represents a reduction in the number of S. aureus on the steel squares after 8 hours,
24 hours and 48 hours of 87.4%, 91.1% and 99.5% respectively.
TABLE 2 RESULTS OF THE SURFACE EXPOSURE EXPERIMENT CARRIED OUT USING S. AUREUS
Table 3 shows the results of the surface exposure experiment carried out using C. difficile. The initial mean concentration of C. difficile on the steel squares slightly lower than that of either the E. coll or the S. aureus at 9460 cfu. This was reduced to 810 cfu after exposure for 8 hours, 180 cfu after 24 hours and 40 cfu after 48 hours. This represent a reduction in the number of C. difficile on the steel squares after 8 hours, 24 hours and 48 hours of 91.44%, 98.1% and 99.6% respectively.
