This is a long-lasting – and potentially never‑ending – debate, which is often misunderstood, if not underestimated, by non-microbiologists, as we are dealing with the heterogenous distribution of live microorganisms in different physiological states with interfering food commensal microflora.
Did we find the solution? Certainly not. But, at least, some demonstrative points of evidence were discussed to help with technical assessments of microbiological schemes and assess its fitness for purpose (or not).
Culture-based vs. non-culture‑based: are we there yet?
One of the present hot topics in food microbiology is the ongoing switch from culture-based (or Pasteurian for the French) microbiology, to non-culture-based (eg, molecular techniques). This is expected to help solve the issue of viable but non-culturable cells. 
It should not be forgotten however that ‘culturability’ of cells remains necessary in broth media for enrichment. Agar-based media are no longer used in these new microbiological methods, yet culture-based microbiology (and its limitations) is certainly not obsolete.
Techniques such as PCR, loop mediated isothermal amplification (LAMP) and flow cytometry show promise, but will pose challenges during their validation according to ISO 16140-2:2016. This is because they will be compared to ‘full’ culture‑based techniques – which is still the case for most ISO reference standards.
The debate about pour plate versus MPN (most probable number) in broth is ongoing, and now these new methods have introduced a further point of difference to the discussion. Hence the reason that this point is far from settled: while colony forming units (CFU) are most widely used, how does the method compare with the Active Fluorescent Unit (AFU) for cytometry or genomic copies per volume for quantitative PCR?
Sample preparation has a major influence on the analytical result that should not be underestimated in this change of techniques; particularly in challenging food matrices for molecular techniques such as high phenol and sterol matrices like cocoa.
Implementation of a validated method or testing for new food matrices of validated food categories
ISO 16140 Part 3 has finally been published (January 2021); until now, user laboratories were only required under ISO 17025 to ensure its capacity to correctly perform the method. The ‘how’ was not provided (as it is a general standard), which led, particularly in microbiology, to endless discussion between laboratories, its customers, and the method supplier to prove how/if procedures were carried out properly.
The ISO 16140 Part 3 now provides the how for every analytical laboratory in microbiology to establish its capacity of implementation and fitness of purpose of the method for the food products of interest. Most accredited laboratories already had a methodology in place and did not wait for the publication of the ISO 16140 Part 3 to perform the verification. However, for the first time in standardisation there is now a protocol in place recognised by all the stakeholders of the microbiological analytical chain, yet the biggest challenge in implementing the protocol will still be mimicking the physiological state in real condition.
While this is partly outlined in ISO 16140 Part 2, it remains a debated point for Part 3. Although it may not be considered as important a step as Neil Armstrong’s (on the moon for humanity), it will certainly be helpful to have a resolution in the near future.
Standard or proprietary method?
The method one uses is a key component of the global value and relevance of the analytical process. As a tool’s purpose can evolve over time, so too can overall technicity and science. However, the cost of implementation in laboratories should not be underestimated. A call of tender takes time and resources. As such, analytical laboratories are not always looking for change, provided the current method proves satisfactory for both the laboratory and customer.
When considering changing, the return on investment should be reviewed; not only the cost of the method itself, but the training of personnel, and eventually, the upgrade of premises for example, in case of implementation of molecular techniques, lab layout reorganisation is needed, or else a high-throughput method might have limits on the incubation capacity.
In a regulated and highly standardised world, change does not happen overnight, nor does it happen without good reason. It must be motivated by one, if not all, stakeholders of the analytical chain. But when it does happen, this job will ultimately fall in the hands of the analytical lab and will likely require technical support.
Collaboration
The capacity to perform the analytical testing regime is the responsibility of the laboratory; consequently, it must ensure both the throughput of the chosen methods and the training of personnel.
As for capability, this is something to consider in partnership with the analytical supplier. A tool used for the wrong purpose won’t be very helpful. Thus, in order to avoid this pitfall, the laboratory team is expected to discuss the technical pros and cons of the proposed media and/or methods, especially when deciding to use an alternative proprietary method.
Cost efficiency is also a vital factor to consider, but it should not be the first (or indeed only) criteria of consideration. The technical capability of the analytical supplier should not be underestimated either; after all, who knows best if not the supplier?
Quality of service in terms of technical training, implementation support, and follow-up should be key points to consider in a call of tender and when using the method in the laboratory. Cost discussions will come at some point, but if this is the only interaction there is with the analytical supplier, the laboratory (whether internal or third party) is not demonstrating particular expertise and can be reasonably challenged for the relevance of its testing regime.
The ISO 16140 scheme and ISO 16140 Part 2
Validation of a proprietary method against ISO 16140 Part 2 is the responsibility of the method supplier. This is a given. However, the laboratory should nevertheless study the validation dossier submitted to a certification body closely, in order to independently ascertain whether the method is indeed fit for purpose, that the scope matches the food matrices analysed, and the work to be done in accordance with ISO 16140 Part 3 can be scheduled, before considering implementation in the premises.
While it is important to partner with the right supplier, it is also essential to have the capability to challenge the partnership and ensure the rightfulness of the final decision. Expertise in assessing a method should not only lay with the supplier, but on the side of the end-user as well. This means that continuous training and maintaining expertise is paramount for an analytical laboratory to ensure the efficiency of the global analytical regime and service to the consumer.
Non-targeted testing
Since 2012, the implementation of Next-Generation Sequencing (NGS) techniques has changed the paradigm in culture-based microbiology, unravelling unknown taxa and/or the importance of non-viable cells in a food product (both technological, spoilage and pathogenic microbiota).
ISO Standards and proprietary methods target defined microorganisms; ie, they only find what they’re looking for. NGS techniques offer the opportunity to identify the global microbiota present in samples, detecting the unknown if not expected. In the coming years, one of the biggest challenges in analytical testing will be implementing (and understanding) these new methods and adapting them accordingly to microbiological criteria.
Conclusion
Microbiological testing has its limits, which lie not only in the analytical method itself, but in the statistical representation of the food sample, considering the heterogenous distribution of the microorganisms in a food product. As stated by Habraken, et al.1 in 1986: “The lack of reliability of the mere examination of finished products when evaluating the microbiological wholesomeness of food products has been known to microbiologists for a long time” – not just the microbiological distribution but also the relevance of the method.
ISO TC34 SC9 (Technical Committee Food, Sub Committee Microbiology) has recently challenged and validated the majority of the microbiological standard,2 and will continuously do so according to ISO 17468. The recently published standard ISO 16140 Part 3, and eventually the accreditation schemed ISO 17025, aim to ensure the capability of the analytical laboratory to perform the method rightfully and to provide robust results.
…But this remains the realm of microbiology. Despite all considerations and actions in place, mystery and unexpected results will ensue, and microbiologists will continue to live with this.
References
- Habraken CJM, Mossel DAA, Van Den Reek S. 1986. Management of Salmonella risks in the production of powdered milk products. Netherlands Milk Dairy Journal,
40: 99-116
- Leclercq A, Hardouin G, Lombard B. 2019. European and International validation of 15 main reference methods in the microbiology of the food chain. International Journal of Food Microbiology 288: 1-102.
About the authors
François Bourdichon
François holds a Masters in science, major general microbiology from Pasteur Institute, Paris. He has been a delegate of France at the International Dairy Federation (IDF) since 2010, and is presently chair of the standing committee on microbial hygiene (SCMH), member of the food standards steering group (FSSG), the standing committees on harmonization of microbial method (SCHMM) and analytical methods for dairy micro-organisms (SCAMDM). He represents IDF at ISO, WHO, FAO and Codex Alimentarius on food safety related topics.
Benjamin Diep
Benjamin has spent more than 20 years in food industry focusing on food safety and quality. He is currently working as a scientist at Nestlé Research in Lausanne, Switzerland. His field of expertise comprises methods development and validation (cultural and molecular based methods) for food pathogens and hygiene indicators. He recently published a manuscript on the use of a microarray method and Whole Genome Sequencing data to predict Salmonella serovars (Frontiers in Microbiology) and on the validation of commercial sterility testing methods (Food Control). He also works with operation where he provides technical support to improve the laboratories competency. He is a member of ISO 16140 WG and the co-project leader of the ISO 16140 part 3, as well as a technical reviewer for MicroVal.
Benjamin Junge
Benjamin is the Head of Product Management at BIOTECON Diagnostics. Responsible for most foodproof® real-time PCR pathogen kits in the portfolio such as Salmonella, Listeria, Campylobacter and Cronobacter as well as for external validations such as AOAC or MicroVal. He is a graduate biologist with majors in genetics and biochemistry from the University of Cologne, and Master in Management and Marketing, Willy Scharnow Institute, Free University of Berlin. He has over 18 years of professional experience in the food safety industry as application specialist, sales manager and product manager.
Andy Muirhead
After graduating from Manchester Metropolitan University with a BSc (Hons) in Biological Sciences, Andy joined the Leeds Regional Public Health Laboratory. In 1987, he gained a Fellowship in Biomedical Sciences, and in 1987, founded and ran an independent Food Contract Laboratory based in Leeds, which was later acquired by Eclipse Scientific Limited in 2011. Following the acquisition of Eclipse by ALS, Andy was appointed as the UK Company Microbiologist which involves the provision of technical assistance, training and consultancy services to both ALS colleagues and clients. In 2016, Andy won the Society of Food Hygiene and Technology (SOFHT) Trainer of the Year award.
Returning in 2022…Food Integrity will return 21-25 March 2022 as a virtual conference.
