Peer Review Article | Open Access | Published 7th January 2025
To Determine the Microbial Recovery from Different Surfaces Using a Standard Contact Plate Sampling Method
T Eaton, K Capper, M Ward, C Tobin, J Bright - AstraZeneca, Macclesfield, UK | EJPPS | 294 (2024) | https://doi.org/10.37521/ejpps29402 | Click to download pdf
Summary
The most appropriate sampling method to be utilised by 55 mm diameter tryptone soya agar (with neutralisers) contact plates to recover naturally occurring surface microbial contamination from hard surfaces has previously been determined. This sampling method was used to investigate the recovery from different surfaces, typical of those that are routinely sampled in a cleanroom. These were a polyester garment, stainless steel, cleanroom latex gloves, workstation barrier EDPM gauntlets and cleanroom goggles copolyester lens. The surfaces had been contaminated with microbe-carrying particles (MCPs) dispersed from people following exposure within heavily populated environments, and is more representative of the contamination that is found within cleanrooms. It was determined that the polyester garment, latex gloves and EPDM barrier gauntlet all had a recovery efficiency of around 70% and the copolyester lens goggles and stainless steel tray had a higher recovery of efficiency of around 80%.
Key words: Microbiological surface contamination, contact plates, RODAC plates, microbe-carrying particles (MCPs), environmental monitoring, recovery efficiency.
1. INTRODUCTION
For sterile products manufacturing, it is a requirement of Annex 1 of the European Union Guide to Good Manufacturing Practice (EU GGMP)¹ that microbiological monitoring of cleanrooms includes the use of 55 mm diameter contact plates for sampling defined surface locations. The Guide includes limits to be applied to this monitoring and the expectation is that supporting data for the recovery efficiency of the sampling method, and for differing surfaces, should be available.
Typically, circular RODAC (replicate organism detection and counting) plates (55 mm diameter, 24 cm² surface area) containing nutrient agar (between 15.5 and 16 ml) are used for sampling surfaces that are relatively flat. They are poured to give an agar meniscus that protrudes just above the rim, and are based upon plates that were originally reported on in 1964². It has previously been determined that an appropriate manual sampling method is to roll the contact plate media over the surface, using firm pressure for 1 second which recorded significantly higher efficiencies than just a single 1 second press of the media onto the surface with firm pressure³. Viable particles removed from the surface adhere to the agar and the lidded plates are then incubated and the number of colony forming units (CFUs) and types of micro-organisms recovered are reported, and the results expressed as the number of CFU per plate.
The material and finish of the surfaces to be sampled are reported to be one of many factors that influence the recovery efficiency⁴. Consequently, investigation of the recovery associated with different cleanroom surfaces was evaluated using the sampling method previously determined to be the most appropriate. Surfaces that are routinely sampled within a pharmaceutical cleanroom were chosen and these were a polyester garment, stainless steel, cleanroom latex gloves, workstation barrier ethylene propylene diene monomer (EPDM) gauntlets and cleanroom goggles copolyester lens. Actual surfaces from within the cleanroom were not utilised as these surface concentrations are too low and accurate results are less likely be obtained. In order to ensure the surfaces had sufficient microbial contamination, they were exposed in a microbiological testing laboratory that was used on a daily basis by numerous people and continually contaminated throughout the exposure period with naturally occurring microbe-carrying particles (MCPs), predominantly dispersed from personnel in relatively large numbers. The use of these naturally occurring MCPs is representative of the majority of the microbes recovered from cleanroom environments and also avoids issues resulting from the utilisation of standard commercial test organisms and a carrier medium to deposit suspensions of micro-organisms onto the test surfaces. Upon evaporation of the carrier medium, sub-micron unicellular microbes are distributed across the surfaces which are not representative of the larger MCPs present in cleanrooms and which have an average aerodynamic diameter of about 8 μm to 12 μm⁵ ⁶ ⁷. On contact, these unicellular microbes may be transferred to the plate with different efficiencies compared to the larger size naturally occurring MCPs.
After exposure, 20 separate locations on each type of surface were sampled and then, without any cleaning or disinfection of the surface at this location, the same location was immediately re-sampled with a second plate. Following incubation of the plates, the number of recovered CFUs were counted and the recovery efficiencies for each surface determined from the two samples and an overall average efficiency then calculated for each surface.
2. METHOD TO DETERMINE SURFACE MICROBIAL COLLECTION EFFICIENCES OF DIFFERENT SURFACES
Contact plates and incubation conditions
All plates utilised were Becton Dickenson, BD BBL™ IC-XT Trypticase™ Soy Agar medium with lecithin and polysorbate 80 surface neutralising agents, 55mm diameter RODAC™ LL. The plates have locking lid features and are gamma irradiated and sealed in triplicate polythene bags, sourced from an approved supplier and are routinely tested for their ability to recover microbial contamination. Although for the purpose of this investigation, Beckton Dickinson plates were used, the results are expected to be relevant to other similar contact plates. Following sampling, all plates were immediately and simultaneously incubated, in the same validated incubator, at 30- 35⁰c for 5 days and the number of microbial colonies counted and identification of the organisms from all plates completed.
Sampling methods
The following method of contact plate sampling was used;
Rolling the plate over the surface in a single motion, lasting 1 second, with firm force.
All of the sampling was performed by the same person.
Surface materials
The following test materials were utilised, and all had been sterilised by gamma radiation, with the exception of the tray which was sterilised by dry heat and are fully representative of their cleanroom conditions;
1. Polyester cleanroom garment
2. Stainless steel tray (316L, Ra surface finish 0.5 μm)
3. Cleanroom latex gloves
4. Workstation barrier EPDM gauntlet
5. Cleanroom goggles copolyester lens
Exposure of testing surfaces and sampling locations
The test materials were exposed for 5 days on a dedicated bench within a busy microbiological testing laboratory. The test materials and the subsequent sampling locations are shown in figures 1, 2 and 3.
Sampling procedure
Due to space limitations, the programme of work was completed over two separate trials. For trial 1, following the 5 day exposure period, from each of the ten sample locations for each of the five test materials, a first sample (sample A) was taken using the defined procedure, and then a second sample (sample B) was immediately taken at the exact same location without any cleaning or disinfection of the surface. The plates were labelled with the material type, the sampling location (1 to 10), and with the first (A) or second (B) sample reference. For trial 2, the whole procedure was repeated using a fresh set of identical test materials (plates labelled 11 to 20) to provide an overall total of 100 sampling locations and 200 plates (40 for each type of test material).
Determination of recovery efficiency
A mathematical model is described which may be used to assess the efficiency and consistency of a surface sampling method⁸. This is based upon multiple and two stage sequential sampling and the two stage sampling is a convenient method if the counts on the surface following the second sampling are relatively low. The recovery efficiency for the two stage sampling can be determined using equation 1.
Equation 1
Recovery efficiency (%) = [1 – (B /A)] x 100
Where,
B = total count from second sample
A = total count from first sample
3. TEST RESULTS
The results for the testing and the recovery efficiencies for the polyester garment, stainless steel tray and goggles copolyester lens are shown in table 1. The results for the latex gloves and the EPDM barrier gauntlets are similarly shown in table 2. Microbes on plates that had been contaminated previously in the same environment were subject to Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry identification³. This confirmed the majority to be the expected Gram positive skin microbes and typical environmental microbes. Species of Staphylococcus, Micrococcus and Bacillus were most commonly identified along with fewer Microbacterium and single isolates of other organisms such as Dermacoccus and Okibacterium.
Table 1 Plate counts and recovery efficiencies for the polyester garment, the stainless steel trays and goggles copolyester lens.
Note
a. Combined bacteria and mould counts
Table 2 Plate counts and recovery efficiencies for latex gloves and EDPM barrier gauntlet
Note
a. Combined bacteria and mould counts
4. DISCUSSION OF RESULTS
Shown in Table 3 is a summary of the results for all of the surface materials. For simplicity, the bacteria and mould counts have been combined as compared to the overall total bacteria count of 6379, there are only 13 total mould counts which is too few to estimate the sampling efficiencies for moulds alone. Although there are likely to be differences in the recovery efficiency of moulds compared to bacteria, fundamentally due to differences in their sizes, the low mould counts are only a small proportion (0.2%) of the total counts. These will have negligible influence on the average recovery efficiency calculations and may also anyway be (rarely) recovered from a cleanroom environment.
Table 3 Summary of test results
Notes
a. Combined bacteria and mould counts
b. Average of all of the individually calculated recoveries
It can be seen from table 3 that, although all of the five different test materials were simultaneously exposed in the same environment for each of the two trials, there were varying first contact plates (samples A) recorded counts. The stainless steel trays recorded the highest (1384) and both the polyester garment and the latex gloves recording the lowest (789). This could be due to actual variations in the amount of microbial contamination deposited during the exposure period, although previous investigations³ confirmed that exposure in this environment resulted in a relatively even distribution of contamination throughout, or it may also be an indication of the actual plate recovery for the surface sampled. However, as each surface is subjected to an identical second (B) sample at the exact same position, this will address both of these considerations and provide confidence that the results are an accurate reflection of the actual recoveries associated with each surface material.
Shown in table 3 are the average recovery efficiencies which are 82%, 80%, 69%, 69% and 66% for the goggles copolyester lens, stainless steel tray, latex gloves, EPDM barrier gauntlets and the polyester garment respectively. These were determined for combined bacteria and mould counts and to avoid bias from the samples that had the higher counts, the average efficiency was calculated from all of the individual twenty efficiencies. This shows that for one of the polyester garment samples (sample reference 7), the count recorded for the second sample was higher than for the first sample and the calculated recovery efficiency is a value less than zero, which is not possible. This ‘impossible efficiency’ is likely to be related to the acknowledged errors and inconsistencies that are associated with the microbiological testing methods. However, as this single result has no noteworthy influence to the overall calculated recovery efficiency, it has been retained. Shown in figure 4 are the individual recovery efficiencies for each surface material and with reference to the average recovery values that are shown in table 3, it appears that the five surface materials partition into two groups, with the polyester garment, latex gloves and EPDM barrier gauntlet with a lower recovery efficiency than the copolyester lens goggles and stainless steel tray. This can be better seen in figure 5 which shows the recovery efficiency data as boxplots. Comparing the recovery efficiencies across several groups would usually use a one-way analysis of variance, but this test assumes that the data within each group has a Normal (Gaussian) distribution, and that the variability of this distribution is the same for all of the groups. In view of the outliers, these assumptions do not hold with this dataset. The more appropriate test is a non-parametric version of one-way analysis of variance, called the Kruskal-Wallis test. This gives a test statistic of 26.00 which, when compared to a chi-squared distribution on four degrees of freedom (the number of groups minus one), gives a highly significant p-value of <0.0001. This supports the suggestion that not all of the surfaces have the same recovery efficiency with polyester garment, latex gloves and EPDM barrier gauntlet with a lower recovery efficiency of about 70% and the copolyester lens goggles and stainless steel tray with a higher recovery of efficiency of about 80%.
It is interesting to note that the work completed to determine the most appropriate sampling method, that was used for this investigation, recorded an average recovery efficiency of 53%³ and was related to the contamination recovered from ‘Trespa’ (a synthetic resin used for tables and benches) and vinyl (floor) surface materials. This recovery is less than that determined for the surface materials used for this investigation but is another indication that the recovery efficiency is related to the surface characteristics of the materials subjected to the sampling. Further investigation of the surface topography associated with these different surface materials would provide further information to help to understand the influence that the surface finish has on the microbial recovery efficiency.
5. CONCLUSIONS
The recovery of naturally occurring surface microbial contamination with sterile tryptone soya agar (with neutralisers) 55 mm diameter RODAC plates has been evaluated for five different surface materials. The evaluation utilised the most appropriate sampling method that had previously been determined¹, and all sampling was completed by the same technician. It was shown that the polyester garment, latex gloves and EPDM barrier gauntlet all had a recovery efficiency of around 70% and the copolyester lens goggles and stainless steel tray had a higher recovery of efficiency of around 80%. To understand the influence that the surface finish has on the recovery efficiency, further investigation of the surface topography associated with these different surface materials would be useful. The surface materials utilised for this investigation are typical of those that are routinely sampled within pharmaceutical cleanrooms and the use of naturally occurring microbe-carrying particles (MCPs) is representative of the majority of the microbes recovered from such cleanroom environments. Consequently, it is concluded that the microbial recovery efficiencies determined for this investigation are representative of the efficiencies associated with the surface concentrations that are reported for sampling within a pharmaceutical cleanroom.
References
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Authors
T Eaton¹ , K Capper¹, M Ward¹, C Tobin and J Bright¹
¹ AstraZeneca, Macclesfield, UK
T Eaton, K Capper, M Ward, C Tobin and J Bright AstraZeneca, Macclesfield, UK
Corresponding Author: Tim Eaton, Sterile Manufacturing Specialist
AstraZeneca,
UK Operations,
Silk Road Business Park,
Macclesfield
Cheshire. SK10 2NA
England
Email: tim.eaton@astrazeneca.com
Telephone: +44(0) 1625 514916
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