Evaluation of Three Different Contact Plate Methods for Microbial Surface Sampling of Naturally Occurring Human Borne Microbial Contamination

Abstract

The ability of irradiated 55 mm diameter tryptone soya agar contact plates to recover naturally occurring surface microbial contamination, using three different manual sampling procedures, was investigated. The investigation was completed by sampling hard surfaces contaminated with microbe-carrying particles (MCPs) dispersed from a person within heavily populated environments. This is more representative of the contamination that is found within cleanrooms and avoids issues resulting from the utilisation of standard commercial test organisms to distribute unicellular microbes onto surfaces, which are likely to be transferred to the plate with different efficiencies compared to the naturally occurring MCPs. It was determined that rolling the contact plate media over the surface, using firm pressure for either 1 or 5 seconds recorded little differences in the mean recovery efficiencies (53% and 48% respectively). However, both recorded significantly higher efficiencies than just a single 1 second press of the media onto the surface with firm pressure (16%). 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 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 1964 ². They are easy to use and require minimal training and the full area of the nutrient agar is rolled over the surface to be sampled, facilitated by the convex media profile, or they can be applied to the surface for a few seconds ensuring uniform and steady pressure with no rolling action. Viable particles removed from the surface adhere to the agar and the lidded plates are then incubated and examined and the number of colony forming units (CFU) and types of micro-organisms recovered are reported and the results expressed as the number of CFU per plate. The recovery efficiency will be affected by many factors, including the sort of plate media used, the type of micro-organisms present on the surface and the material and finish of the surfaces ³ to be sampled. It will also be influenced by the contact area of the plate media with the surface being sampled and may be influenced by the associated duration of this contact, both of which are associated with the plate sampling method. To reduce variations associated with these two parameters, there are commercially available plate holding devices which can be utilised for the sampling that apply the same pressure each time and also have an indicator of the specified contact time, that can be set for different durations. However, the surface sampling is typically a manual procedure that does not utilise such devices and the application method and contact time are reliant on the person undertaking the sampling and hence this is likely to be more variable. In order to determine a convenient manual sampling procedure that can be routinely used to provide more consistency of practice, three different methods were tested. Surfaces associated with three different areas, each used on a daily basis by numerous people, were utilised. These surfaces are continually contaminated with naturally occurring microbe-carrying particles (MCPs), predominantly dispersed from personnel, in relatively large numbers, and are representative of the majority of the microbes recovered from cleanroom environments. Actual cleanroom surfaces were not utilised as the surface concentrations are too low and accurate results less likely be obtained. The use of these naturally occurring MCPs 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 which have an average aerodynamic diameter of about 8 μm to 12 μm ⁴ ⁵ ⁶. On contact, these unicellular microbes may be transferred to the plate with different efficiencies compared to the larger size naturally occurring MCPs, for which the efficiency is likely to be greater. Bench and floor surfaces in laboratory and amenities areas, contaminated with MCPs, in sufficient numbers (typically greater than 20 MCPs) that could be used for the comparison of the three sampling methods, were identified from a preliminary study. From each of three adjacent but separate positions at 20 different locations, two sequential samples were taken from the exact same place using the three different sampling methods. Following incubation of the plates, the number of recovered CFUs were counted and the recovery efficiencies for each sampling method determined from the two samples, for each location, and an average efficiency then calculated. The resultant data was reviewed in order to determine if the recovery of surface microorganisms was influenced by the sampling method and the type of surfaces sampled and to also provide recommendations for the most appropriate sampling procedure for routine use to help to ensure consistency of practice.