Validation of Microbiological Methods - What do I need to bear in Mind?

When it comes to the suitability of microbiological testing and analysis procedures in the field of microbiological quality control or, where applicable, quality assurance, a number of questions come up, e.g.

• When do I need to validate a method?
• In which cases is this not fully necessary and a suitability test is sufficient?
• How do I proceed with validation, and where can I find guidelines or information on this?

1. Suitability Test

The European Pharmacopoeia provides an initial guide in Ph. Eur. 1. General Notices on the subject of validation of pharmacopoeial methods:
"The test methods given in monographs and general chapters have been validated in accordance with accepted scientific practice and current recommendations on analytical validation. Unless otherwise stated in the monograph or general chapter, validation of the test methods by the analyst is not required.."

However, this does not exempt the analyst from the obligation to demonstrate the suitability of the method used for the intended purpose, i.e. to carry out a suitability test. This means that a suitability test of a microbiological pharmacopoeial method is carried out to demonstrate that the standardised procedure (e.g. according to Ph. Eur. or in accordance with the United States Pharmacopoeia (USP)), is suitable for the intended purpose in one's own laboratory and in the presence of the specific product. Classic areas of application include, for example, testing for sterility or the determination of the total microbial count (TAMC/TYMC).

In the case of the "Microbial Enumeration Test according to Ph. Eur. 2.6.12", it states:
"The ability of the test to detect micro-organisms in the presence of product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.."

The Ph.Eur. also provides information on the expected results:
"When verifying the suitability of the membrane filtration method or the plate-count method, a mean count of any of the test organisms not differing by a factor greater than 2 from the value of the control defined in 4-5-2 in the absence of the product must be obtained. When verifying the suitability of the MPN method the calculated value from the inoculum must be within 95 per cent confidence limits of the results obtained with the control."

If one wishes to establish such a pharmacopoeial method, it is necessary to identify and take into account a whole range of characteristics of the respective test procedure, e.g.

• the composition of the article to be tested
• the complexity of sample preparation
• the reagents required to carry out the procedure
• the laboratory equipment required to carry out the procedure
• the laboratory environment

2. Validation of new/alternative methods

But what if one wishes to use a method other than those listed in the Pharmacopoeia, because the methods mentioned are not applicable to one's own product or because another method yields faster or better results, etc.?

In such a case, a full validation must of course be carried out. The following documents provide helpful information on the requirements and implementation

• Ph. Eur. chapter 5.1.6
• Ph. Eur. chapter 2.6.7
• Ph. Eur. chapter 2.6.21
• USP chapter <1223>
• USP chapter <1227>
• USP chapter <1113>
• PDA Technical Report #33
• PDA Technical Report #50

In line with the analogous requirements of the USP and also the Ph. Eur., the process usually begins with an initial validation/primary validation, which is often carried out by or in collaboration with the manufacturer of the respective method. This serves to characterise the underlying detection principle.

Further validation begins with the definition of internal requirements in the form of a URS (User Requirement Specification), also known as a specification document. Building on this, a risk-benefit analysis must be carried out. This serves to evaluate the advantages of the alternative method over the pharmacopoeial method and to identify any potential new risks. A carefully prepared risk analysis forms the basis for determining the validation parameters and ensures that the requirements defined in the URS are met. In accordance with established practice, this process comprises the four qualification phases DQ, IQ, OQ and PQ. The actual validation in the strict sense then takes place as part of the PQ and generally involves the evaluation/testing of the following parameters:

Table: Validation parameters: - Not required + Required 1 May be used as an alternative to LOD where appropriate 2 May be necessary in some cases

A whole range of different methods are now in use, offering benefits for the pharmaceutical industry, for example:

• Achieving faster results (time-to-result), leading to quicker release of production batches.
• Improved process understanding/IPC and real-time monitoring, enabling a rapid response to deviations
• Automation and the resulting reduction in manual errors

Some examples of methods in use:

• ATP bioluminescence: Rapid sterility test
• Solid-phase cytometry: Rapid bioburden test
• rDNA 16S sequencing: Identification of microorganism
• PCR (conventional or real-time): Rapid detection

When using alternative methods, it is also important to demonstrate equivalence with existing pharmacopoeia methods, e.g. in the form of a parallel test using the alternative method and the pharmacopoeia method. The result must be equivalent. In the case of a microbial count, for example, this can be demonstrated by the absence of any statistically significant difference between the results of the two methods. An improvement in a quantitative test could, for instance, be higher sensitivity.

It is precisely this higher sensitivity or lower selectivity that can pose a challenge with modern methods. The difficulty with modern methods that detect 'all viable counts' is that they detect significantly more microorganisms than the classic pharmacopoeia methods, which, for example, due to the partly selective properties of the culture media used and the individual requirements of the microorganisms, always have gaps in their growth-promoting properties, i.e. not all microorganisms grow into visible colonies on the respective culture media. Even on standard/universal media such as Case agar, various microorganisms such as Haemophilus spp. or Neisseria spp. grow poorly or not at all. The same applies to a whole range of fungi.

Literature and Sources:

• Ph.Eur. 5.1.6 Alternative Methods for Control of Microbiological Quality, currently under Revision - a Draft General Chapter was published in Pharmeuropa 37.2,
• Ph.Eur. 2.6.7 Mycoplasmas, General Chapter, Adopted 2025, in force April 2026
• Frequently asked questions on chapter 2.6.12 Microbiological examination of non-sterile products: microbial enumeration tests
• United States Pharmacopoeia (USP), chapter <1223>, USP chapter <1227>, USP chapter <1113>;
• PDA Technical Report #33; Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods
• PDA Technical Report #50, Alternative Methods for Mycoplasma Testing

In addition to the documents mentioned above, there is also a wide range of publications on the validation of alternative methods, e.g.:

• A Systematic Approach for the Evaluation, Validation, and Implementation of Automated ColonyCounting Systems, Sven Deutschmann, Bill Carpenter, Caroline Duignan, Chris Knutsen, Joanny Salvas, Lisa Wysocki, Lucile Plourde, Lynn Johnson and Wolfgang Eder, PDA Journal of Pharmaceutical Science and Technology, Vol. 76, Issue 6, November/December 2022
• Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea, Sven M. Deutschmann, Holger Kavermann, Yvonne Knack, Science Direct, Biologicals, Volume 38, Issue 2, March 2010, Pages 238-248
• Rapid Sterility Test Systems in the Pharmaceutical Industry: Applying a Structured Approach to their Evaluation, Validation and Global Implementation, Sven Deutschmann, Mousumi Paul, Marja Claassen-Willemse, Jonas van den Berg, Pieta IJzerman-Boon, Viviane Grunert da Fonseca, Ellen Brunbech, Lynn Johnson, Chris Knutsen, Lucile Plourde, Joanny Salvas, Philip Villari, Lisa Wysocki and Margit Franz-Riethdorf PDA Journal of Pharmaceutical Science and Technology January 2023
• Validation of Milliflex® Quantum for Bioburden Testing of Pharmaceutical Products, Oliver Gordon, Marcel Goverde, Alexandra Staerk and David Roesti, PDA Journal of Pharmaceutical Science and Technology May 2017, 71 (3) 206-224

Training courses dealing with microbiological issues relating to quality control and quality assurance are available on the GMP Compliance website.