By Laurent Berliet & Karlin Gardner
Key Takeaway
Population verification is intended to confirm consistency with the manufacturer's certified population, not to establish a new population value. According to ISO 11138, results between 50% and 300% of the certified population confirm the manufacturer's stated value, which must continue to be used in subsequent calculations.
Introduction
Biological indicators (BIs) are essential tools for demonstrating the effectiveness of sterilization and decontamination processes. Consisting of a defined population of highly resistant reference microorganisms, they are used to evaluate spore log reduction (SLR) and, consequently, the sterility assurance level (SAL) of a given cycle.
Standards and regulatory reference, including ISO, USP, the European Pharmacopoeia, and EU GMP Annex 1, require or recommend incoming verification testing to confirm that BIs comply with the specifications certified by the manufacturer. This verification primarily focuses on population, strain identity, and purity, even when the BI supplied with a certificate of analysis is compliant with ISO 11138-1.
After addressing purity in Spore News No. SN095-V1, Biological Indicator Purity Testing: Key Requirements & Best Practices for Users, this issue focuses on population verification, a topic that is often misunderstood. Exact enumeration of spores inoculated onto an individual BI is not achievable, and variability is inherent to both inoculation and analytical recovery. Therefore, the certified population of a BI lot is based on an estimate used to establish an average value. This most probable number estimate remains highly reliable provided that a rigorous and controlled methodology is followed.
The objective of this Spore News is to clarify the concept of BI population, explain the principles behind its estimation, and review the applicable regulatory framework. It also aims to define what users should actually verify, how results should be interpreted, and which best practices help ensure reliable and compliant data.
Manufacturer Responsibilities Regarding Population
Manufacturers are required to certify the population inoculated onto each BI, suspension, or other standardized format. Since production lots may contain a large number of units, sometimes several thousand, manufacturers must also ensure population homogeneity throughout the entire lot. In addition, they must demonstrate that this population remains stable throughout the product’s shelf life.
These three requirements, certification, homogeneity, and stability, are fundamental to ensuring that users receive products performing as expected. It is therefore essential to ensure that suppliers rigorously conduct these controls.
At Mesa Labs, all production lots are tested before release. Population determinations are performed by several experienced technicians using samples collected at different stages of the manufacturing process and from across the entire lot. The results are then analyzed, including calculation of an average value, to obtain the most representative estimate possible of the actual population. This approach also allows verification of lot homogeneity while minimizing operator-to-operator variability.
In general, although some variation in population within a lot cannot be completely excluded (it is evident that not every BI will contain exactly the same number of spores), our manufacturing processes and quality controls provide a high level of confidence in the certified population and its potential intra-batch variability, which generally remains relatively close to the certified population level.
Additionally, Mesa Labs conducts extensive stability studies and maintains an ongoing monitoring program at regular intervals to ensure consistent performance throughout the shelf life of its entire BI product range.
Verification Expected from the User Upon Receipt
Contrary to a common misconception, user population verification is not intended to re-determine the exact population value of a lot, but rather to confirm compliance with the manufacturer’s stated specifications. The result itself has no intrinsic value; it simply needs to fall within the limits defined by applicable standards to demonstrate that the BI provides the required challenge.
Regulatory Framework and Interpretation of Results
According to ISO 11138-7:2019, the population measured during verification of the certified population should fall between 50% and 300% of the nominal population declared by the manufacturer.
These broad acceptance limits are not necessarily indicative of lot heterogeneity, but rather reflect the potential variability associated with population determination methods. Numerous variables can contribute to differences in the measured value.
It is therefore recommended, and indeed necessary, to consult the manufacturer to ensure that the methods used are consistent with those employed during product certification, as procedural differences can significantly influence the results.
Accordingly, any measured value within this range validates the user verification, without necessarily representing the exact true population:
ISO 11138-7:2019
Sterilization of health care products - Biological indicators - Part 7: Guidance for the selection, use and interpretation of results
11.2 Nominal population of test organism
11.2.1 ISO 11138-1 requires the manufacturer to report the nominal population of test organisms of each biological indicator as part of the labelling information. The requirements are given in the standards for the minimum number of microorganisms on a biological indicator or inoculated carrier to ensure a minimum resistance of the indicator. When tested, the population should be between 50 % and 300 % of the nominal population. The BI population as labelled by the manufacturer should be used in all calculations following verification. The biological indicator manufacturer should be consulted to ensure that the same techniques and procedures are used, because variations in testing procedures can affect the population determination results. ISO 11138-1 requires the manufacturer to provide this information on request.
Once this verification has been completed, lot conformity is established and the certified value provided by the manufacturer must be used for all subsequent calculations.
This point is particularly important for populations close to 10⁶ spores. It is possible to obtain a result below 10⁶ while remaining compliant with the acceptable range. For example, a measured value of 9 × 10⁵ for a certified population of 1.2 × 10⁶ corresponds to 75% recovery and is therefore compliant.
As stated in ISO 11138-1:2017, any result between 50% and 300% validates the verification, even if the measured value falls below the minimum population specification. In such cases, the original population is considered confirmed.
ISO 11138-1:2017
Sterilization of health care products - Biological Indicators - Part 1: General Requirements
6.3.2 Population verification shall be achieved when results fall within 50% to 300% of the manufacturer’s stated nominal population. Confirmation test results of the population determined by end users or manufacturers during stated shelf life may meet the 50% to 300% range but could fall below the minimum population specification as defined in this document. In these cases, the original population is considered to be verified if the confirmation test results are within the 50% to 300% range.
Why Is Population Verification Inherently Variable?
Population verification relies on an indirect statistical estimation.
In practice, only 30 to 300 colonies are actually counted, representing an extremely small fraction of the total BI population, often less than 0.01% for a population around 10⁶ spores.
Numerous factors throughout the process can influence spore recovery and the resulting count without calling into question the actual population of the biological indicators including:
- Spore recovery efficiency
- Sample homogenization
- Transfer quality
- Choice of diluent
- Culture medium used
- Equipment utilized
- Equipment temperature conditions
- Incubation conditions
- Operator experience
The accumulation of these variations, even when individually minor, is the most common cause for out-of-specification results, most frequently leading to recoveries below 50%, even though the actual lot population remains compliant.
This phenomenon is comparable to counting participants at a public demonstration. Depending on the method used (aerial photography, ground counts, sampling-based estimates), the resulting figures may vary significantly even though the actual number of participants has not changed. Similarly, measurement parameters in the laboratory are often underestimated, even though they explain a large proportion of the variability observed.
Mesa Labs Recommendations
To obtain reliable and meaningful results, Mesa Labs recommends that users:
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Consider population testing as a consistency verification, not a recertification process.
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Strictly follow the manufacturer’s procedure without unjustified local modifications.
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Use the equipment, consumables, and culture media recommended by the manufacturer.
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Maintain rigorous control of spore recovery procedures.
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Compare results with historical trends rather than relying on a single result.
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Contact the manufacturer in the event of unexpected or questionable results.
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Consider a counter analysis by an independent third-party laboratory using the manufacturer’s method exactly as written when additional investigation is required.
How Can You Assess Whether Your Methodology Is Established and Reproducible?
Users must critically evaluate their own practices rather than assume their methodology is flawless.
In theory, a population verification should result in a recovery close to 100%. While not every BI will contain the certified population down to the exact last digit, lots are generally highly homogeneous, and the certified value remains the most probable estimate because it is derived from multiple tests performed on randomly selected BIs by experts on a routine basis.
When implementing a new BI reference, users should therefore perform preliminary testing to ensure that the method is both controlled and reproducible.
If, for example, the end user’s recoveries consistently fall between 80% and 120% of the certified population across multiple tests, the method can reasonably be considered under control. However, when recoveries fall outside this range, or when substantial variability is observed, it may indicate that certain parameters are affecting recovery performance and that the method could be improved.
Users should not be satisfied with recoveries of only 50% to 70%, even though such results may technically meet acceptance criteria, because the risk of obtaining results below the 50% threshold increases due to normal analytical variability.
Conclusion
Population verification is not intended to re-determine the number of spores present in a BI. Its purpose is to confirm that the received product is consistent with the characteristics certified by the manufacturer.
ISO standards explicitly recognize the inherent variability of this measurement and provide a framework for interpretation through the acceptance criterion of 50% to 300% of the nominal population.
Understanding this principle is essential:
A compliant result confirms the certified population; it never replaces it.
Strict adherence to the manufacturer’s method remains the best way to obtain reliable, comparable, and regulatory-compliant results.
Additional Resources
- Spore News SN075 – End User or Third-Party Population Verification
- Spore News SN014 – Impact of Culture Media on Biological Indicator Spore Recovery
- Spore News SN022 – Heat Shock / Heat Activation
- Spore News SN095-V1 - Biological Indicator Purity Testing: Key Requirements & Best Practices for Users
- Population Test Instructions TS-401 to TS-407
- ISO 11138-1:2017 – Sterilization of Health Care Products – Biological Indicators – Part 1: General Requirements
- ISO 11138-7:2019 – Biological Indicators – Part 7: Guidance for Selection, Use and Interpretation of Results
- USP <1229.5> – Biological Indicators for Sterilization
- European Pharmacopoeia 5.1.2 – Biological Indicators and Related Microbial Preparations
- EU GMP Annex 1 – Manufacture of Sterile Medicinal Products
SN098-V1