Common Challenges and Considerations in the Processing of Microbiology Tests in the Food Industry

Microbiological testing plays a critical role in ensuring food safety. Regulatory bodies such as the U.S. Food and Drug Administration (FDA), the UK’s Food Standards Agency (FSA), and the EU’s European Food Safety Authority (EFSA) emphasizes the prevention of adulterated food products from entering the market. Microbiological analyses are a cornerstone of this effort. Here, we will explore the usual challenges met in microbiology testing within the food industry and propose considerations to enhance the application of these methods in food safety programs. 

Reasons for Microbiological Analyses 

Microbiological tests are performed for several reasons; these include: 

• Verifying Hazard Analysis Critical Control Points (HACCP) or food safety plan preventive controls for testing laboratory environmental samples, in-process materials, product contact surfaces, and finished products 

• Detection of patterns and trends to illustrate process control 

• Release of the finished product 

• Regulatory compliance or conformity with customer specifications 

• Troubleshooting microbiological challenges in the product, process, or facility in response to complaints 

• Research associated with product or process development 

Challenges in Microbiological Testing 

Microbiological testing in food safety laboratories is a rigorous, multifaceted process—but even the most validated methods are not without challenges. Numerous issues can compromise results, from inconsistent outcomes and media interference to the complexities of cultural confirmation and operational errors. 

Inconsistent Results 

Validated methods are generally effective, but inconsistencies occasionally occur. Rapid molecular techniques such as PCR enhance pathogen identification and detection rates. Biochemical tests, such as the catalase and oxidase tests, are routinely used to identify bacteria like Salmonella, providing presumptive results confirmed through plated media such as XLD Agar, MacConkey Agar, and Blood Agar. Despite inherent limitations, these are well understood and managed within food labs, ensuring reliable outcomes. 

Discrepant Results 

Estimating the risk of discrepancies is challenging. Proficiency scheme data suggest a false-positive rate of less than 5% [1] [2]; the false-negative rate is variable, depending on the pathogen.  

Factors contributing to discrepancies include matrix interference, the presence of atypical strains, detection of exogenous DNA, high populations of background organisms, equipment failures, cross-contamination, and technician errors. 

Non-uniform Distribution of Pathogens: Microorganisms occur at low concentrations and are non-uniformly distributed in the product, environment, or sample. This makes detection challenging. 

Non-conforming Presumptive Positives: Rapid screening platforms often yield presumptive positive results that require confirmation through cultural isolation methods. Examples of cultural isolation methods include: 

• Plating on Selective and Differential Media: This involves using media that supports the growth of the target organism while inhibiting others, such as Eosin Methylene Blue A agar (EMB), Man, Rogosa and Sharpe agar (MRS) and Sorbitol MacConkey Agar (SMAC).  

• Use of Selective Enrichment Media: Such media enhances the growth of the target organism while suppressing others. Examples include Selenite Broth and Tetrathionate Broth.  

Media Interference: Components of enrichment media can interfere with detection technologies, such as PCR, by inhibiting amplification or recognition of the target nucleic acid sequence. 

Pre-enrichment and Selective Enrichment Media Bias: The combination of a sample's intrinsic microbial flora and the medium's formulation can result in biased selection or repression of a microbial target. Studies show that enrichment medium formulation and the presence of competing organisms can affect the dominant serovar of pathogens such as Salmonella. This can lead to molecular detection without cultural confirmation, despite the presence of viable microorganisms [3]. 

Colonies with Similar Morphology: During cultural isolation, colonies may appear morphologically typical of the target organism, but not be the target organism, extending the confirmation process. For example, Citrobacter braakii can mimic Salmonella on XLD agar. This can be resolved by using Hektoen Enteric agar. 

Background Flora: High levels of background microflora can outcompete the target pathogen for nutrients, making detection difficult. This is particularly challenging for pathogens such as Shiga-toxigenic Escherichia coli (STEC), which are difficult to isolate from other E. coli strains. This can be resolved by CHROMagar STEC agar.  

Viable but Not Culturable (VBNC) Cells: Some bacteria enter a VBNC state in response to adverse conditions, retaining metabolic activity and virulence factors but not growing on standard media. These cells can resuscitate in a host, potentially leading to illness.  

Cross-Contamination: Cross-contamination during sample preparation can lead to false positives. Proper sanitation and handling protocols are essential to prevent this. 

Operational Issues: Laboratory procedural errors, such as using expired materials, improper equipment maintenance, and mislabeling samples, can affect test results. 

Considerations to Enhance Microbiological Testing 

Ensuring food safety relies on accurate, dependable microbiological testing—yet this can be challenging to achieve consistently. From choosing the right methods to understanding the limitations of different techniques, there are many factors that influence the quality and reliability of results. The following points highlight practical steps that can help improve performance across food safety testing programs. Comprehensive Risk Assessment: Conduct thorough risk assessments of problematic results and implement actions to strengthen microbiological methods within food safety programs. 

Use of Validated Methods: Ensure that methods are validated for the target microorganism and detection method. Include quality control measures, such as positive and negative media controls, to identify and correct for media interference. 

Proficiency Testing: Participate in proficiency schemes to benchmark performance and identify areas for improvement. These schemes assess laboratory accuracy by comparing results from test samples with known values. 

Training and Education: Provide ongoing training for laboratory personnel to minimize technician error and ensure proper handling of samples and equipment. 

Enhanced Sampling Strategies: Increase the number of test portions to improve the likelihood of detecting pathogens. However, avoid retesting product lots following a positive pathogen test result to obtain a different result. 

Clear Communication of Results: Understand and communicate the capabilities and limitations of different testing methods. Qualitative methods are designed to detect specific microorganisms, while quantitative methods provide insight into the likelihood of the microorganism's presence. 

Mitigation of Media Bias: Use media validated for the target microorganism and detection method. Employ quality control measures to identify and correct for media bias. 

Addressing VBNC Cells: Use microscopic and molecular techniques to detect VBNC cells if their presence is suspected. 

Laboratory Quality Systems: Implement and maintain laboratory quality systems accredited to the latest version of ISO 17025. These systems ensure consistent and reliable performance, helping to eliminate mistakes and operational errors. 

Validation of Test Methods: Whether you are a manufacturer or user of microbiological test methods, ensuring that your methods are fully tested, validated, and fit for use is crucial.  

ISO 16140 Series: The updated ISO 16140 standard incorporates new insights and experiences from validation studies worldwide. This ensures a reliable common protocol for validating alternative methods, providing performance data for end-users to make informed choices and serving as a basis for method certification by independent organizations. 

By addressing these common challenges and implementing these considerations, food manufacturers can enhance the reliability and effectiveness of microbiological testing, ultimately contributing to safer food products and compliance with regulatory standards. Partnering with a trusted microbiology media manufacturer can help achieve these goals and maintain consumer trust. 

As the manufacturer of Redipor® prepared media, we at AnalytiChem understand the complexities and challenges faced by food industry players in microbiological testing. Our high-quality products include custom Redipor® ️ formulations in contact and settle plates designed to meet the unique requirements of individual customers.   

By partnering with us as a specialist manufacturer of prepared media, food manufacturers and contractual testing labs can enjoy greater confidence thanks to a variety of benefits; these include: 

• Reliable and Consistent Results: All Redipor® prepared media products are rigorously tested to ensure consistency and reliability, reducing the risk of discrepancies and false positive results. 

• Expert Support: Our technical sales specialists, quality teams, and microbiology product specialists are available to guide and support you through the complexities of microbiological testing and advise on best practices to optimize your workflow. 

• Custom Solutions: As well as off-the-shelf stock items, we also offer customized media solutions that can be tailored to meet your specific needs, ensuring optimal performance in your testing processes. 

• Compliance and Quality Assurance: Our products comply with regulatory standards and are manufactured under stringent quality control measures, ensuring the highest level of safety and efficacy. 

For more information, or to discuss your specific prepared media requirements, whether Redipor® formulations, packaging formats or combination, contact our team. 

References 

[1] Edson, D.C., Empson, S. and Massey, L.D., 2009. Pathogen detection in food microbiology laboratories: An analysis of qualitative proficiency test data, 1999–2007. Journal of Food Safety, 29(4), pp.521-530. 

[2] Abdel Massih, M., Planchon, V., Polet, M., Dierick, K. and Mahillon, J., 2016. Analytical performances of food microbiology laboratories – critical analysis of 7 years of proficiency testing results. Journal of Applied Microbiology, 120(2), pp.346-354. 

[3] Legan, J.D., Post, L., Barnes, C., Brookhouser-Sisney, A., Chaney, W.E., Chatterjee, P., Corrigan, N., Demarco, D.R., Hammack, T., Hunt, K.A., Maus, R.D., Pierre, S., Rule, P., Taylor, N., Tudor, A. and Weller, J., 2024. Discrepancies in the Microbiological Analysis of Foods: Causes and Resolutions. Food Protection Trends, 44(4), pp.300-308.