David Jones and Nathan Storie share the results of a study on environmental monitoring
Environmental monitoring (EM) is a critical aspect in pharmaceutical manufacturing. The testing of air and surface samples is typically performed via the labour-intensive traditional method with a serial incubation. Dependent upon the testing method selected, the test time can vary - taking as long as seven days. Automated, non-destructive rapid testing methods can reduce the resource requirements, provide a considerable reduction in time to results and preserve any growing colonies for identification.
The Growth Direct System is one example of such a technology. The study described here was performed in order to identify a representative turnaround time for environmental monitoring using this system versus the traditional method.
The Growth Direct system uses the same principles and procedures as the compendial method.The technology automates the traditional visual plate counting method by using sensitive digital imaging that can detect growing colonies of as little as 100 cells. The method detects microscopic colonies well before they become visible to the naked eye by capturing their intrinsic cellular auto-fluorescent signal. To detect growing colonies, the system performs multiple image analysis of the sample, thereby tracking increases in the intensity of auto-fluorescent signals. This allows the system to distinguish growing colonies from debris. The ubiquitous nature of cellular auto-fluorescence in biological systems including microorganisms permits the system to detect the same broad range of species as are detected by the visual colony counting method. Additionally, the imaging method does not harm the cells and is thus non-destructive, so any micro-colonies present can be grown into visible colonies for microbial identification after completion of the assay.
A newly constructed clean room was sampled on two occasions using both the Growth Direct EM cassettes loaded into the Growth Direct System and the traditional contact plates placed in an incubator. Initial sampling was performed pre-disinfection and then additional sampling was performed again post-disinfection. This two-stage sampling allowed for various levels and types of microbial contamination to be captured and compared at identical sites under soiled as well as clean conditions. Sampling was executed by the traditional environmental monitoring method - collecting floor and wall surface samples using both the traditional contact plate and the Growth Direct EM cassettes and, collecting air samples using the SAS microbial air sampler.
The numbers of colonies detected by the rapid microbiological method (RMM) was comparable to or better than the visual count at the end of the assay.
Fig. 1 presents various detection curves for samples tested. The conventional EM incubation scheme is seven days - four days at 22.5°C followed by three days at 32.5°C. Using the automated rapid method the captured organisms were allowed to grow out at 22.5°C, then moved to 32.5°C and once again allowed to grow out. As seen in Fig. 1, plate growth plateaus at 22.5°C before moving to 32.5°C, at 48 hours (red vertical line). The incubation profile required will depend on the flora found in the facility under test. In this site example a 96-hour incubation scheme can be used. This amounts to a three-day reduction in incubation time. An evaluation of the growth characteristics is recommended as part of the validation process.
Microorganism identification
Because this method is non-destructive, the colonies could be picked following the test run for 16S identification.
This RMM can be used to generate equivalent data to the traditional test with no requirement for changing action or alert levels. The automated, rapid technology allows for the faster enumeration of micro-colonies, reducing the time to results. The faster time to results allows a clean room facility to be commissioned more quickly than can be performed with traditional methods. Using RMMs can achieve substantial cost and time savings when compared to the traditional environmental monitoring method.
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David Jones is director technical services and Nathan Storie is validation specialist at Rapid Micro Biosystems in Boston, USA.
