Evaluation of an Instantaneous Microbial Detection System in Controlled and Cleanroom Environments

ABSTRACT

The ability of an instantaneous microbial detection system (IMD-A) to monitor microbial populations in environmental air was evaluated. The IMD-A results were compared with results from conventional environmental air monitoring methods. The comparisons were carried out in controlled microbial barrier test chambers and in cleanroom environments. Additionally, microbial populations in environmental air in an unclassified environment were evaluated using the IMD-A and the all-gas impingement (AGI) method coupled with ScanRDI. In 1-m³ and 150-m³ controlled-barrier test chamber studies the mean recoveries with the IMD-A were equal to or greater than the mean recoveries obtained with the Anderson air sampler at various concentrations. The mean microbial recoveries obtained using the AGI were higher, but in the same order of magnitude, as those recovered by IMD-A. In classified environments, microbial recoveries from the SAS air sampler were substantially lower than microbial counts detected by the IMD-A. There were reasonable correlations of microbial recoveries between the IMD-A and the SAS air sampler results in cleanroom environments. Mean microbial recoveries from environmental air in an unclassified environment were similar in the IMD-A and AGI methods coupled with ScanRDI analysis. These results suggest that the IMD-A has the potential to reliably and instantaneously evaluate microbial populations in environmental air to provide a valuable technique for biopharmaceutical manufacturing.

ENVIRONMENTAL MONITORING METHODS

Examining the microbial content of air is a key component of environmental monitoring in pharmaceutical cleanroom environments. Overall environmental air monitoring also includes evaluating the particulate content of the air. Typically, particulate content at 5.0 and 0.5 µm levels is measured using total particulate monitoring systems such as the Climet, PMS, Royco, Lighthouse, APC units, or similar systems.

Evaluating microbial content in environmental air involves both active and passive air monitoring. Active microbial content in environmental air is typically evaluated using SAS, MAS, RCS, Mattson Garvin, Anderson air, liquid impinger, or SMA air sampling systems. Active air monitoring often involves the use of a device in which microorganisms from a known volume of air are captured on media plates, or alternatively, air is aspirated into a liquid and the microorganisms in the liquid are captured on a membrane filter and transferred to media plates to evaluate growth. Viable passive air is evaluated by the plate-count method using settling plates. Results from the microbial monitoring of the environmental air are typically not obtained until 3–5 days after sampling.

Table 1. Statistical evaluation of 1-m3 microbial barrier test chamber data from the IMD-A and an Anderson air sampler—Bacillus atropheus (spores)

That need to wait for several days to accommodate microbial growth before acquiring monitoring data has been a major limitation of conventional environmental monitoring methods. Over the past decade, several rapid culture- and nonculture-based methods have been developed to provide much faster turnaround for microbial data.¹

One promising nonculture-based rapid method receiving increased attention in recent years involves the ScanRDI system (Chemunex, France).² In this semiautomated system, the total number of viable organisms is determined by filtering samples through a membrane and labeling cells using a nonfluorescent substrate that diffuses across the cell membrane. This labeling differentiates between viable and dead cells based on the presence or absence of esterase activity and intact cell membranes. Only viable cells with membrane activity are able to cleave the dye and retain the fluorescent label. These viable microbial cells are quantified by scanning and counting using laser cytometry. Although the ScanRDI system offers the advantage of rapid evaluation of microbial populations, it is fairly specialized and does not allow for real-time detection of microbial populations. An ideal system for microbial air monitoring in pharmaceutical cleanroom environments would require little or no sample preparation or manipulation and would provide environmental microbial air monitoring data in real time.