Viable air sampling is a key part of an Environmental Monitoring program for controlled environments where aseptic products are manufactured. Both passive settle plate and active air sampling methods are used to ensure defined classification levels are met for each cleanroom. The use of a 90 mm petri plate using Tryptic Soy Agar (TSA) for passive air sampling method for 4 hour exposure is defined in Annex 1, while surface monitoring uses a 55 mm plate typically using Tryptic Soy Agar with Lecithin, Polysorbate 80 (TSA with LP80). The use of two separate media types and two different plate sizes requires maintaining both materials, increasing QC testing resources. The below study describes the use of contact plates called Growth Cassettes™ for both air applications in Environmental Monitoring with data showing their equivalence to traditional methods.
Materials and methods
Settle plate method
One TSA Petri plate and one Growth Cassette were placed side by side at 49 sample sites and exposed for approximately 4 hours. Since the plate size is different between Petri plates and Growth Cassettes (90mm Petri plate vs 57 mm Growth Cassettes), samples were also collected using one TSA Petri plate and two Growth Cassettes side by side for each sample site. When these two Growth Cassettes were used for each Petri plate, samples were collected from 50 low bioburden environment and 50 high bioburden environment. After 5-day incubation and manually counting, the results were compared between Petri plates and Growth Cassettes.
Active air sampling method
Air samples in the volume of 1000L were collected from the same sample site over 3 different days in high bioburden environment. Six samples were collected on the first day of sampling, 6 samples on the second day, and 9 samples on the third day for each Petri plate and Growth Cassette. Using a MAS-100 air sampler samples were collected in duplicate with one 90mm Petri plate and one Growth Cassette side by side. After incubation and counting the results between Growth Cassettes and Petri plates were compared.
Settle plate comparison
Table 1: Settle plate result of Growth Cassettes vs petri plate.
|Growth Cassette CFU Total||Petri Plate CFU Total|
|Single Growth Cassette used||72||148|
|Two Growth Cassettes used||570||489|
When only one Growth Cassette was used to sample each site, a total of 72 CFU was recovered from all cassettes whereas a total of 148 CFU was recovered from all Petri plates. This is equivalent to only 48.6% microbial growth recovery for Growth Cassettes compared to the standard Petri plates.
Table 1 shows the result for Growth Cassettes and Petri plates where two Growth Cassettes were used at each site. This was compared to the results obtained from using only one cassette.
Two Growth Cassettes results were combined from each sample site to calculate the CFU count for Growth Cassettes. Then the sum of counts from all of sample sites were calculated. A total of 489 CFU was recovered from Petri plates and 570 CFU from Growth Cassettes. Therefore, about 116% growth recovery was observed for Growth Cassettes when two cassettes are used.
Active air comparison
A total of 1726 CFU was observed from 21 petri plates and 1393 CFU from 21 Growth Cassettes. As shown in Figure 2, both plates have a relative high or low count at the same sites, with Growth Cassettes recovering 80.7% of growth from petri plates.
Discussion and conclusion
For settle plate method the results clearly indicate two Growth Cassettes should be used for each settle plate sample site (116% recovery vs. 48.6%). Since the surface area of a Petri plate is about 2.5 times greater than Growth Cassettes the lower percent recovery is expected when only one cassette is used. As shown in Figure 1 when two Growth Cassettes are used for each site most data points are very close to the regression line and there is a strong linear relationship between Petri plates and Growth Cassettes for both clean and dirty areas.
For the active air sampling method, Growth Cassette recovery compared to Petri plates was 80.7%. This is very high recovery considering the surface area of Growth Cassette is less than half of a Petri plate. This indicates that Growth Cassettes used in active air sampling give the same data as Petri plates regardless of the size difference due to the sample volume of air being focused on the smaller surface area.
Both passive and active air sampling methods showed comparable recovery for microbial growth for Growth Cassettes and the standard Petri plates. In summary one contact plate sized agar plate can be used for all EM applications thus minimizing the QC test time required in the laboratory.
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