Physical and Biological Efficiency Testing of ImpactAir Microbiological Air Sampler Using Techniques Described in ISO
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1 Physical and Biological Efficiency Testing of ImpactAir Microbiological Air Sampler Using Techniques Described in ISO
2 The work presented herein was performed by the Biosafety Investigation Unit of Public Health England (PHE). This document has been prepared using the original report (Report No. 15/020) provided to Pinpoint Scientific and is not to be taken as an endorsement or recommendation by PHE. Public Health England Microbiology Services Porton Down Salisbury SP4 0JG Tel: Page 2 of 28
3 Contents Contents 3 About Public Health England Biosafety Investigation Unit 4 Executive summary 5 Introduction 6 Materials and Method 8 Samplers 8 Test Micro-organisms 12 Microbiological Assays 12 Test Environments 13 Test Procedures 15 Results 18 TABLE 1: The experimentally determined sizes of particles containing Bacillus atrophaeus spores generated by the STAG from aqueous solutions of KI in 80% ethyl alcohol and the associated physical efficiency of ImpactAir for each particle size 18 TABLE 2: Collection of airborne Bacillus atrophaeus spores in the PHE Environmental Room 20 TABLE 2 (Continued) 21 TABLE 2 (Continued) 22 TABLE 2 (Continued) 23 TABLE 2 (Continued) 24 TABLE 3: Mean Mass Diameter determined by collection with the Cascade impactor 25 TABLE 4: Biological efficiency of ImpactAir compared with the Casella slit sampler. 26 Discussion 27 References 28 Page 3 of 28
4 About Public Health England Biosafety Investigation Unit The Public Health England Biosafety Investigation Unit at Porton Down has been carrying out independent evaluations of infection control interventions in laboratories, health care, containment, workplace and domestic settings for over twenty years. Their expertise is in air and water microbiology applied to nosocomial, pharmaceutical and containment situations. They have developed and offer standard techniques for the determination of the efficacy of filters and air disinfection units, the performance of safety cabinets, sealed centrifuges, rotors and air samplers. They are also able to assess liquid and gaseous disinfectants and the microbial air quality of healthcare facilities, workplaces and other environments. The Biosafety Investigation Unit provides specialist bespoke research, testing and evaluation services for commercial customers that delivers independent analysis and reports. However as a public sector body they are not able to endorse any particular products or recommend them for use by the NHS or others. Page 4 of 28
5 Executive summary The physical efficiency of the ImpactAir air sampler for collecting small bacteria-laden particles of various sizes has been compared with membrane filter samplers using the techniques described in ISO The samplers were operated simultaneously in a controlled room where they were challenged with airborne bacteria. Uniform sized particles of different diameters containing bacterial spores were generated into the room. The results showed that the ImpactAir sampler was more effective than the reference samplers for smaller particles up to 6.5 micron, and as effective as the reference samplers for the largest particles of 10.5 micron. When compared with using a paired t test, it was found that for the range of 0.8 to 2.8 micron the impact air was significantly more efficient than the test sampler. The biological efficiency of the ImpactAir sampler was compared to that of the Casella slit sampler, a commonly used reference sampler. The biological efficiency was measured as the comparative efficiency of collection of Staph epidermidis, a common human-associated clean room contaminant, and the extremely aerostable B. atrophaeus spore. The biological efficiency measured was found to be 125% that of the Casella sampler and when compared using a paired t test there was no significant difference, indicating the sampler is effective at sampling bacteria-laden particles, without undue loss of viability. Page 5 of 28
6 Introduction Determination of the microbiological quality of air is vital in a number of sites such as areas where pharmaceuticals and medical devices are manufactured, operating theatres and other critical areas in hospitals and food processing facilities. In many cases only sparse concentrations of airborne micro-organisms are present in these locations and this means that large volumes of air (1m 3 ) have to be sampled to collect sufficient numbers of micro-organisms for a proper quantitative assessment to be made. Accurate measurement of microbial contamination of air is also dependent on obtaining a representative sample from the air and limiting any losses that may occur between the sampler and the assay system. Losses can occur, either due to a failure of the sampler to capture particles containing micro-organisms (physical loss) or due to inactivation of viable micro-organisms during collection so that formation of visible colonies on agar surfaces will not occur (biological loss). The ImpactAir sampler is a slit impactor type of instrument based on the principle described by Bourdillon (1) in which air is aspirated through a single slit above a rotating plate. The resulting air stream containing microbial particles is directed onto the agar surface of the plate, as it rotates. The system employs an integrated sensor to maintain the critical slit to agar distance and the constant rotation of the plate ensures that once particles have been collected, the surface is moved away from the desiccating effects of the airstream, helping maintain viability. When the pre-set sampling cycle is completed the plates are removed and incubated. Viable organisms which form visible colonies are then counted. Efficient removal of particles containing micro-organisms from the air and their collection onto medium for identification often depends on the sizes of the particles. At present no sampling system or device has been considered a suitable reference method to which other samplers can be compared. ISO (2) recommends using a membrane filter sampler as the standard method. In filtration systems, accurately measured volumes of air are drawn through filter material of low pore size so that all particles containing micro-organisms are deposited by impaction and interception. Provided that the micro-organisms are resistant to desiccation by drawing air through the filter material, Page 6 of 28
7 this simple method can be used as a standard method by which the physical sampling efficiencies of other samplers can be determined. Bacterial spores of Bacillus atrophaeus NCTC are selected as the challenge micro-organisms because they are known to remain viable in aerosols and are not easily inactivated by desiccation during collection on membrane filters. They also form very characteristic orange colonies which are easily identified after overnight incubation. The number of bacteria collected on membrane filters can be counted by simply placing the filters on an agar surface and incubating. The physical efficiency of the ImpactAir sampler to collect airborne particles of various sizes can therefore be determined by comparison with the membrane filtration samplers operating side-by-side. This has been carried out in a controlled environmental chamber by generating the bacterial spores in particles of uniform size. The sizes of the particles containing the bacterial spores generated are determined using a cascade impactor (3) which fractionates the particles of different sizes during collection. The biological efficiency of a sampler is a measure of how effectively it can collect microorganisms on an agar plate in such a way as the micro-organism will subsequently form a colony. The biological efficiency depends on many factors, including the microorganism used, how it is grown and aerosolised, what it is aerosolised from and the stresses endured during the sampling process. ISO suggests that the common human derived environment contaminant Staphylococcus epidermidis is used as an indicator of biological efficiency. In this study the biological efficiency of the ImpactAir sampler is compared to that of the reference Casella slit sampler. To measure the biological efficiency, the ratio of recovery of Staph. epidermidis to the aerostable B. atrophaeus was determined for the ImpactAir and Casella sampler. Page 7 of 28
8 Materials and Method Samplers ImpactAir ImpactAir air samplers (figure 1) were provided by Pinpoint Scientific TM (Serial Numbers Proto 1, Proto 2 and Proto 3). The samplers were operated as described in the Manual provided, operating at 30 litres min -1. The flow rate was confirmed prior to testing using calibrated TSI 4040 mass flow meter (Serial No , UR 36401, Cal Date ). Sampler Serial No. Proto 1 was used for undertaking the physical efficiency tests and samplers Serial No. Proto 2 and Proto 3 were used for the Biological efficacy tests. Figure 1. ImpactAir sampler Page 8 of 28
9 For the physical tests, total counts were recorded after 24 hours incubation. However, for the biological efficiency tests described below, a two stage incubation process was used, with the plates being initially examined after a reduced incubation time, allowing for the identification of the separate species of colonies, prior to any possible overgrowth by the quicker growing B. atrophaeus. The plate were then re-incubated to allow any further growth and re-counted after a day s incubation. Membrane Filter Samplers These consist of aluminium membrane filter holders (figure 2) each incorporating an 80mm diameter, 3µm pore size Sartorius gelatine filter (Sartorius Stedim Biotech, part no ALK). The filters were mounted on a sterile back-up support and connected to a vacuum pump to provide a measured flow rate of ca. 30 litres min -1. The actual flow rates were determined and recorded before each set of tests using a calibrated TSI 4040 mass flow meter (Serial No , UR 36401, Cal Date ). After sampling, the membrane filters were carefully removed and placed on an agar growth medium with the exposed side facing upwards and incubated at 37 (±2) C for at least 18 hours. Figure 2. Membrane filter sampler Page 9 of 28
10 Cascade Sampler The four-stage Cascade sampler (3) (figure 3) was used to determine the mass mean diameters of the aerosols generated. The sampler was fitted with four microscope slides which were ground down to a width of 25mm in order to fit the impactor. The slides were decontaminated in a 10% sodium hypochlorite solution, washed with distilled water, dried in an oven and then autoclaved at 131 o C for 11 minutes. The slides were covered with a gel made up of a mixture of 5g Bovine Skin Gelatin (Type B Sigma) and 10ml of glycerol made up to 100ml with distilled water. This solution was heated to 120 C twice in an oven before it was applied thinly to the microscope slides. After sampling, the gel was dissolved in 5ml of warm sterile distilled water, diluted and spread on Tryptone Soya Agar plates (Biomerieux). The sampling rate was controlled by placing a 17.5 litres min -1 critical orifice between the sampler and the vacuum source, the flow being confirmed using a TSI 4040 mass flow meter (Serial No , UR 36401, Cal Date ). Figure 3. Cascade Sampler The recoveries from each stage of the sampler were calculated using the assay method above and from this the cumulative percentage loading on each stage was calculated. This data was then plotted on a log-log scale against the calculated d-50 cut-offs for each stage (the particle size at which 50% of the particles are collected). A linear regression was drawn to best fit the plot using SigmaPlot 12.0 and from this Page 10 of 28
11 the Mass Mean Diameter (MMD) can then be read as the particle size corresponding to the 50% point of loading. Casella slit sampler A calibrated low volume Casella slit sampler (Casella, London) was operated at 30 litres min -1 for two minutes during the biological efficiency testing. TSA plates (Becton Dickinson) were used in the sampler for all of these tests. The flow was calibrated using a TSI 4040 mass flow meter (Serial No , UR 36401, Cal Date ). Figure 4. Casella sampler Page 11 of 28
12 Test Micro-organisms Bacillus atrophaeus Suspensions of washed B. atrophaeus spores (NCTC 10073) in distilled water were prepared (4). This suspension was the source of the B. atrophaeus used in the physical and biological efficiency testing. Suspensions of spores (ca. 1 x 10 5 colony forming units (cfu) per ml) in 0%, 0.007%, 0.07%, 0.7% and 7% of potassium iodide (KI) in 80% aqueous ethanol were prepared for use in the physical efficiency testing. Staphylococcus epidermidis Staph. epidermidis (NCTC 11047) was grown up in liquid TSB-F (Biomerieux) at 37 o C (±2) in a static incubator for 24 (± 1) hours. All assays with Staphylococcus epidermidis were carried out on Tryptone Soya Agar plates. Microbiological Assays Media Media Item Batch Number Manufacturer Expiry Date TSA 150mm for test samplers TSA 90mm for reference samplers Gelatine Filter Becton Dickinson Becton Dickinson Sartorius Nutrient Broth Biomerieux Sterile distilled water Versol Microbiological assays All plates used in the samplers were incubated at 37(±2) C for the required length of time before counting the colonies. Page 12 of 28
13 Test Environments Environmental room The environmental room has a volume of 20m 3 with an optional flow of a horizontal clean air supplied through a bank of HEPA filters (figure 5). Microbial aerosols were generated in still air in the chamber as described below. At the end of the sampling period, the room was vented by supplying a horizontal flow of clean air for 10 minutes. After re-setting of the samplers another microbial cloud containing bacterial spores was generated and the experiment was repeated. The ability to flush the room with clean air to remove airborne organisms allows a large number of bacterial aerosol challenges to be carried out over relatively short periods. Figure 5. Test chamber A spinning top aerosol generator (STAG, figure 6) was used to produce an aerosol of controlled particle size containing bacterial spores (dependent on rotational speed of the spinning disc) (5). After formation, the particles reduced by evaporation to a size related to the solid content (KI) of the suspension aerosolized. The STAG Mark 2 (Bristol Industrial and Research Associates Ltd, Portishead, Bristol, UK) was used to generate particles of different sizes containing viable B. atrophaeus spores. This was done by preparing suspensions (ca. 1 x 10 5 cfu per ml) of the spores in 0-7% (w/v) solutions of potassium iodide (KI) in 80% aqueous ethanol. The suspensions were injected into the STAG, operating at ca. 48,000 rpm, using a peristaltic pump. The size of the particles produced depended on the concentration of the KI in the suspension. The liquid associated with the generated droplets evaporates completely in less than 1 second at Page 13 of 28
14 the test relative humidity (ca %). The concentration of spores in the suspension was low enough to ensure that the vast majority of particles were mono-dispersed. Figure 6. Spinning top aerosol generator (STAG) Class III Cabinet The biological efficiency testing carried out with Staph. epidermidis was undertaken in a Class III microbiological safety cabinet (internal volume 0.865m 3, figure 7) to allow for greater control of the mixing and concentration of the test aerosol. Modified ports were used to allow the supply of compressed air to the nebuliser. The cabinet generates 6 air changes a minute when the fan unit is operated and is supplied with HEPA filtered air. This allowed the aerosol generated to be rapidly removed after each test. Figure 7. Class 3 safety cabinet Page 14 of 28
15 A three jet Collison nebuliser (6) (figure 8) operating at a pressure of 26 pounds per square inch (psi) was used to generate the mixed microbial aerosol for the biological efficiency testing. The spray suspensions were made up using a stock 10 5 sterile aqueous dilution of the B. atrophaeus spore suspension and suitably diluted recent liquid culture of Staph epidermidis. Figure 8. Three jet Collison nebuliser Test Procedures Physical Efficiency The aerosols were generated from the various spore suspensions by the STAG Mark 2 as described above. The samplers were arranged in a semi-circle at a distance of 1 metre from the STAG in the environmental room. The STAG was 15 cm off the floor, placed above a small fan unit. Above the STAG a larger secondary fan was also operated to ensure effective distribution of the aerosol throughout the test chamber. The heads of the samplers were 0.8 metres from the floor. The nebulisation, sampling regimes and the fan speed were chosen after a number of preliminary experiments were carried out. Conditions were chosen so that a reasonable Page 15 of 28
16 number of colony forming units ( per plate) would be produced following incubation. The nebulisation was started remotely five seconds before the sampling process was started. The nebulisation and sampling times were two minutes. The plate counts obtained respectively with the tested sampler and membrane filter samplers are standardized in colony forming units per cubic meter in order to calculate the physical efficiency. When the Cascade impactor was used, the flow rate was controlled at 17.5 litres min -1 by the insertion of a critical orifice in the line between the sampler and the vacuum source. Biological Efficiency The ImpactAir sampler, a Casella slit sampler and the Collison nebuliser were placed in the cabinet. A mixed suspension of B. atrophaeus and Staphylococcus epidermidis was made up immediately prior to the experiment and 30 ml of this suspension was placed into the Collison nebuliser. The samplers were loaded with the agar plates and the cabinet fan unit was switched off. A small fan was used to mix the air within the cabinet while the cabinet ventilation system was switched off. The Collison nebuliser was operated at 26 psi for two minutes, starting and finishing 20 seconds before and after the samplers, respectively. The samplers were operated for a total of two minutes after this the cabinet was vented. During initial studies the plates were removed from the samplers and stored at 4(±2) C until the end of the working day when they were moved to a 37(±2) C incubator and incubated for no more than 17 hours before the orange colonies of B. atrophaeus (BA) and white colonies of Staph. epidermidis (SE) were counted separately. These studies showed the organisms to be slow growing. Therefore for all test runs the plates were removed from the samplers and placed in a 37(±2) C incubator and incubated overnight before the orange colonies of B. atrophaeus (BA) and white colonies of Staph. epidermidis (SE) were counted separately. The plates were further incubated for between 5 and 6 hours to allow for any further colonies to develop. Page 16 of 28
17 Due to the inherent variability encountered with the Staph. epidermidis, it is normal to undertake runs over a number of days to ensure that effective counts can be achieved. Each day s runs are then combined to give the overall results. The comparative biological efficiency of the ImpactAir sampler for sampling Staph. epidermidis (SE) was calculated as follows: - Biological Efficiency = Ratio of SE BA sampled by the ImpactAir Sampler Ratio of SE BA sampled by the Casella Sampler x 100 Page 17 of 28
18 Results The results of the physical tests are summarised in Table 1 and Figure 9 and shown in more detail in Table 2a-2e. Mean mass diameter determinations are shown in Table 3. The biological results are shown in Table 4. TABLE 1: The experimentally determined sizes of particles containing Bacillus atrophaeus spores generated by the STAG from aqueous solutions of KI in 80% ethyl alcohol and the associated physical efficiency of ImpactAir for each particle size Percentage KI in suspension Mass mean diameter determined experimentally by Cascade impactor (microns) % Efficiency of ImpactAir 0.0 <1 (theoretical 0.8 micron)* *As the washed spores have been aerosolised from 80% alcohol w/c distilled water, aerodynamic particle size can be assumed to be that of the naked spore - ca. 0.8 micron. Page 18 of 28
19 Figure 9. Physical Efficiency of the ImpactAir Sampler for a Range of Particle Sizes % Efficiency Particle size (Micron) Page 19 of 28
20 TABLE 2: Collection of airborne Bacillus atrophaeus spores in the PHE Environmental Room (a) Bacillus atrophaeus spores aerosolized from 80% aqueous ethanol Test N O cfu per m 3 collected by ImpactAir Filter 1 Filter 2 % efficiency to filter average ImpactAir Average Standard Deviation Page 20 of 28
21 TABLE 2 (Continued) (b) Bacillus atrophaeus spores aerosolized from 0.007% KI in 80% aqueous ethanol Test N O cfu per m 3 collected by ImpactAir Filter 1 Filter 2 % efficiency to filter average ImpactAir Average Standard Deviation Page 21 of 28
22 TABLE 2 (Continued) (c) Bacillus atrophaeus spores aerosolized from 0.07% KI in 80% aqueous ethanol Test N O cfu per m 3 collected by ImpactAir Filter 1 Filter 2 % efficiency to filter average ImpactAir Average Standard Deviation Page 22 of 28
23 TABLE 2 (Continued) (d) Bacillus atrophaeus spores aerosolized from 0.7% KI in 80% aqueous ethanol Test N O cfu per m 3 collected by ImpactAir Filter 1 Filter 2 % efficiency to filter average ImpactAir Average Standard Deviation Page 23 of 28
24 TABLE 2 (Continued) (e) Bacillus atrophaeus spores aerosolized from 7% KI in 80% aqueous ethanol Test N O cfu per m 3 collected by ImpactAir Filter 1 Filter 2 % efficiency to filter average ImpactAir Average Standard Deviation Page 24 of 28
25 TABLE 3: Mean Mass Diameter determined by collection with the Cascade impactor Percentage KI in suspension Stage Cumulative % of total Mass mean diameter (MMD) = Mass mean diameter (MMD) = Mass mean diameter (MMD) = Mass mean diameter (MMD) = 10.7 Page 25 of 28
26 TABLE 4: Biological efficiency of ImpactAir compared with the Casella slit sampler. TEST Casella ImpactAir Total Count cfu cfu ratio cfu cfu ratio Ba Se Se/Ba Ba Se Se/Ba % Efficiency Average Standard Deviation Page 26 of 28
27 Discussion The results showed that the ImpactAir sampler was more effective than the reference samplers for smaller particles up to 6.5 micron, and as effective as the reference samplers for the largest particles of 10.5 micron. When compared with using a paired t test, it was found that for the range of 0.8 to 2.8 micron the ImpactAir was significantly more efficient than the reference samplers, and for the other particle sizes it was as efficient as the reference samplers. This makes the sampler highly efficient across the range of particle sizes tested. The biological efficiency of the ImpactAir sampler was compared to that of the Casella slit sampler, a commonly used reference sampler. The biological efficiency was measured as the comparative efficiency of collection of Staph epidermidis, a common human-associated clean room contaminant, and the extremely aerostable B. atrophaeus spore. The biological efficiency measured was found to be 122% that of the Casella sampler and when compared using a paired t test there was no significant difference, indicating the sampler is effective at sampling bacteria laden particles, without undue loss of viability. The combination of high physical efficiency and good biological recovery reflects the advantages of rotating plate slit samplers compared to sieve or fixed slit sampler types. The rotating plate allows for high impaction velocities, whilst minimising the time in which the captured particles are exposed to the direct airflow. The reference filter samplers do not use impaction to capture the test aerosol and hence the particle capture is inherently less stressful. However, the filter membrane is not an ideal media to then maintain the viability of the captured particles and the greater than 100% efficacy of the ImpactAir may reflect the limitations of the reference sampler system. Page 27 of 28
28 References 1. BOURDILLON, R.B., LIDWELL, O.M. and THOMAS, J.C. (1941). A slit sampler for collecting and counting airborne bacteria. Journal of Hygiene, Cambridge 41, ISO , Cleanrooms and associated controlled environments-- Biocontamination control, Part 1: General principles and methods. 3. MAY, K.R. (1945). The cascade impactor: An instrument for sampling coarse aerosols. Journal of Scientific Instruments, 22, SHARP, R.J., SCAWEN, M.D. and ATKINSON, A. (1989). Fermentation at downstream processing of Bacillus. In "Bacillus" Edited by Colin R Harwood, Plenum Publishing Corporation. 5. FOORD, N. and LIDWELL, O.M. (1975). Airborne infection in a fully air-conditioned hospital. II. Transfer of particles between rooms resulting from the movement of air from one room to another. Journal of Hygiene, 75, MAY, K R (1973). The Collision nebuliser: Description, performance and application. Aerosol Science, 4, Page 28 of 28
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