Optimal microbial sampling approaches for pharmaceutical environments include surface, passive air, and active air monitoring methods.
Sponsored Content by Particle Measuring SystemsReviewed by Aimee MolineuxApr 30 2026 Several methods are used for microbiological sampling in pharmaceutical manufacturing facilities. Each of these procedures yields a result based on the formation of microbial colonies on the surface of a collection medium , but the specific instruments and techniques differ between them.
Because the results of any individual aspect of sampling do not constitute evidence of asepsis, all elements of sampling should be employed together to support sterility assurance, as specified in the current edition of EU GMP Annex 1: 2022.1 9.1 The site’s environmental and process monitoring program forms part of the overall CCS and is used to monitor the controls designed to minimize the risk of microbial and particle contamination. It should be noted that the reliability of each of the elements of the monitoring system when taken in isolation is limited and should not be considered individually to be an indicator of asepsis.
When considered together, the results help confirm the reliability of the design, validation, and operation of the system that they are monitoring. EU GMP Annex 1: 2022 More specific rules, as well as surface and passive monitoring equipment , are covered in the regulation document.2 For all sampling procedures, it is critical to ensure that the sample appropriately reflects the environmental microbiological conditions. This is often assessed and evaluated in terms of collection efficiency.
The primary focus of this study is active air sampling, but there are sampling features for each method of sampling that assure optimal collection efficiency: Active air sampling Collection efficiency for active air samplers is divided into two components: physical and biological collection efficiency. This article will evaluate the factors that are likely to influence the collection efficiency of these devices.
The primary technique driving collection efficiency is microbial impaction on the collection media; however, additional functions must also be considered. Impaction The BioCapt® Adjustable Height impactor is designed to maximize physical efficiency and can be adjusted to a variety of heights, allowing for the use of diverse media and pour depths without compromising the design's excellent biological efficiency. Figure 1. Diagram of Biocapt Adjustable Height Impactor.
Image Credit: Particle Measuring Systems W=width of impactor opening H=height above media S1 =Trajectory - No impaction S2 =Trajectory - Impaction When the impaction trajectory is large in comparison to the nozzle width , the microorganism will affect the collection media. The Stokes number quantifies the relationship between the particle's trajectory and the width of the slit , where the trajectory is determined by the particle's relaxation time and the fluid velocity.
The Stokes number is a dimensionless number that characterizes particles moving in a fluid : in this application, the Stk number captures the effect of inertial impaction on the media. A particle with a low Stokes number follows the air streamline, whereas a particle with a high Stokes number continues on its original course and impacts the media .
The number Stk50 represents a 50% physical collection efficiency and is given when 50% of particles are collected, while the other 50% remain entrained in the airflow and pass through without being collected. Furthermore, because the slit is wider than the particle, the particle can enter the aperture from multiple points along the plane of entrance; each entry point affects the particle's trajectory.
The height between the slit opening and collecting media influences collection efficiency and is proportional to slit width . The Reynolds number is another dimensionless variable that depicts airflow as a ratio of inertial and viscous forces in the sampler's air and distinguishes between turbulent and unidirectional flow inside the impaction zone. The Reynolds number is mostly determined by the flow rate employed and the sampler's physical dimensions.
Because slit width and airflow are constant, they can be normalized for efficiency calculations. The height is a variable that depends on the media used, and differences in plate dimensions and pour depths result in variation in this parameter, affecting impaction performance .
In the BioCapt® Adjustable Height Microbial Air Sampler Impactor and MiniCapt® Mobile Microbial Sampler, impaction occurs when the agar is 0.5–1.5 mm from the bottom surface of the impactor top plate, with an ideal height of one mm . The bottom plate has a rotary adjustment: one full revolution moves the plate height by 1 mm, and each ‘click’ represents a 0.1 mm change.
Setting the plate to the correct height allows for optimal collection efficiency across diverse media types. Physical d50 efficiency results The d50 value is the cutoff size at which 50% of particles in the airstream are predicted to impact and 50 % to pass through; this value corresponds to the Stk50 number. There are three techniques for calculating d50 efficiency based on physical efficiency test results.
Source: Particle Measuring Systems U is the air velocity through the instrument, r is the radius of the streamline’s curvature, p is the density of the sampled air, d is the equivalent particle diameter, C is the Cunningham correction factor , and n is the viscosity of room air .3 40 is the constant factor for air viscosity , Dh is the equivalent hydraulic diameter of air inlet nozzle in mm, and U is the impact velocity in m/s.
For a circular opening, the equivalent hydraulic diameter is the hole diameter, whereas for a rectangular slit, the equivalent diameter is equal to approximately twice the width of the slit.3 The d50 physical collection minimum values applied: Source: Particle Measuring Systems The MiniCapt® Mobile Microbial Air Sampler, MiniCapt® Remote and Pro Microbial Air Samplers, BioCapt® Microbial Impactor, and Biocapt® Single Use Microbial Impactor all share the same design. The design parameters and testing were completed in accordance with ISO 14698-1:2003 and EN 17141:2020 standards.
The conformity testing to this standard was carried out by CAMR of Porton Down, UK, and is documented in document n° 670/00. Isokinetic sampling In unidirectional situations, there is an optimal airflow rate required to serve as sheathing air. Historically, this flow rate was specified at 90 feet per minute, equivalent to roughly 0.45 meters per second.
Isokinetic Sampling must successfully sample the air without disrupting airflow patterns to obtain the most accurate representation of the particles entrained within that airflow. Isokinetic sampling operates on the following principles: When sampling with an incorrect velocity into the sampling probe, anisokinetic sample conditions influence the data from a total count and particle distribution standpoint, since more or less particles might be collected under these conditions. Figure 2. Isokinetic Sampling Diagram.
Image Credit: Particle Measuring Systems Sub-isokinetic sampling: occurs when the velocity of the air surrounding the sample probe exceeds the velocity of the air entering it. Heavy particles will continue to migrate in the straight down unidirectional flow line near the probe’s edges because their momentum carries them in. Lighter particles in those flow channels will form part of the sheath layer and pass through/ around the probe. This leads to oversampling of larger particles.
Super-isokinetic sampling: occurs when the velocity of the air surrounding the sample probe is lower than the velocity of the air entering it. Heavy particles at the probe’s edges will move in a straight, unidirectional flow line past the probe due to momentum. Lighter particles along these flow lines will be drawn into the probe, leading to an undersampling of bigger particles.
As shown in Figure 2, the probe has a single point of entry and is designed to efficiently dissect the air supply for sampling from the overall volume of air used in the flowing air supply. It has a sharp edge . Figure 3. Typical Isokinetic Sample Probe Design and Biocapt/Minicapt Sample Head Design .
Image Credit: Particle Measuring Systems There are some significant distinctions between the inlet design of an isokinetic probe and that of a microbial impactor. Source: Particle Measuring Systems The comparison shows that microbial samplers are intended to optimize impaction and accommodate compatible media plates. The vast flat surfaces created by the housing, as well as the spaces between the impactor inlets, will serve as impaction planes for particles that are not pulled through the impactor and onto the media surface.
The wider collection plane relative to the flow rate promotes the collection of bigger particles, which are commonly associated with free-floating microorganisms in the air. The collection plane is located within the sampler's lip, preventing extraneous sample exhaust.
The only way to perform isokinetic sampling with a standard microbiological sampler is to use an external isokinetic sample probe designed for the instrument’s flow rate and accompanying air velocity; however, this increases losses due to the movement of large particles through the transit tubes. The direction of EU Annex 1 is: "9.22 Where aseptic operations are performed, microbial monitoring should be frequent using a combination of methods such as settle plates, volumetric air sampling, glove, gown, and surface sampling .
The method of sampling used should be justified within the CCS and should be demonstrated not to have a detrimental impact on grade A and B airflow patterns.
" Installing a MiniCapt® Mobile instrument in unidirectional airflow has no effect on the airflow pattern within the chamber; as the exhaust is external to the process, there is no additional concern about particle loading of the expelled air post-sample. Figure 4. Visualization of Airflow With Minicapt Mobile Instrument.
Image Credit: Particle Measuring Systems Desiccation of media Desiccation of localized airflow can be accelerated with increasing instrument flow rate, as with settle plates, when the media is exposed to active sampling: the higher the flow rate, the faster the desiccation rate. The BioCapt Single Use impactors have been shown to efficiently collect data from an environment for up to 4 hours .
Traditional, high-flow-rate instruments require only a 10-minute sample to collect a cubic meter ; any longer will result in increased desiccation and reduced growth promotion of test isolates. Using the BioCapt Single Use units for continuous sampling follows EU GMP Annex 1 requirements. The Growth Promotion Test, carried out after incubation, shows a good recovery rate and a good condition of the media’s appearance.
False positive identification The BioCapt impactor's unique radial pattern enables the identification of microbes that reside within the impaction zone's geometry and are thought to have entered via the intake. The overall swept area of the impactor is only 35 mm2, compared to a total area of media on the plate of 6300 mm2, equivalent to 0.5 %.
The use of Biocapt® Single Use devices reduces the risk of secondary contamination from plate manipulation, as the agar is well protected by the device. Figure 5. Collection Profiles of Biocapt Plate and Generic Plate. Image Credit: Particle Measuring Systems Summary Environmental monitoring to assess sterility assurance is a multistep process, and relying on a single factor is considered inconclusive.
Microbial sampling is a function of environmental monitoring, and in critical, high-risk, Grade A environments, being able to accurately differentiate a zero count from a single count requires empirical quantitative data due to the fact that sterility cannot be determined because zero is a virtual concept; all of the air in all areas cannot be tested. No single data point can reliably determine pollution levels, leading EU Annex 1 to require continuous microbiological sampling in Grade A regions.
To extend the sample durations, settle plates can be used, although they are qualitative and not the best technique for separating zeros and ones. The BioCapt Single Use product has been proven and validated for extended periods of up to 4 hours .
To guarantee collection efficiency, the BioCapt and MiniCapt products were validated in accordance with ISO 14698:1, ISO 14698:2, and ISO/EN 17141. Collection and impaction efficiency are the most important criteria influencing sample accuracy, and the BioCapt has the highest level of collection efficiency, both physical and biological, even over long sample durations, without imposing undue stress on the media or isolates.
The sample is collected within the process zone of a filling line, close to the important process functions identified in site-based Environmental Monitoring Risk Assessments, without disrupting airflow patterns, and exhausted externally in accordance with Annex 1 and ISO 14698. References Other references used Acknowledgments Produced using materials originally authored by Giulia Artalli and Mark Hallworth, Product Line Manager Microbiology and Sterility and Senior GMP Scientist, Life Sciences at Particle Measuring Systems.
About Particle Measuring Systems Particle Measuring Systems has 35 years of experience designing, manufacturing, and servicing microcontamination monitoring instrumentation and software used for detecting particles in air, liquid, and gas streams, as well as molecular contamination monitoring. Specific applications include cleanroom monitoring, parenteral sampling, filter and in-line testing in deionized water and process chemicals, and point-of-use monitoring of inert gases and in-situ particle monitoring. Specialty monitoring includes parts cleanliness testing with a highly automated solution.
Particle measuring systems Whether you want to protect a product or meet industry requirements, such as ISO 14644, USP 797, or GMP, Particle Measuring Systems offers a wide range of particle counters and molecular monitors to meet your needs. With 35 years of experience, it has proven reliability to support your application. Particle counters Protect your product with PMS' reliable particle counters.
PMS has airborne, portable, and liquid particle counters for a wide variety of applications, including DI water, chemicals, and cleanroom monitoring. Compare particle counters or learn how to monitor your cleanroom or product by reading our papers. Molecular contamination monitors Molecular contamination causes costly problems for high-value products, production processes, and equipment surfaces. PMS has solutions for both Airborne Molecular contamination and Surface Molecular contamination .
With parts-per-trillion limits of detection, real-time sampling, NIST traceable calibrations, and various data analysis packages, you can monitor in confidence. Gas detectors If you need gas detectors for process control or continuous emissions monitoring, Particle Monitoring Systems can help. Get real-time, reliable results with its ammonia, hydrogen fluoride, and chlorine detectors for worker protection, CEMs, and pollution monitoring.
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