For non-experts who need high quality results in microbial monitoring, the number and variety of testing solutions available can appear daunting and complex. The technique chosen will determine the types of results produced, and so the kinds of knowledge that can be gathered, as well as the decisions available to be made.
Given the significant risks microbial contamination pose to industry, knowing how to select the right microbial testing technique is critical in avoiding useless results and wasted investment, not to mention a lack of protection.
In this article, we will present various methods available, their applications, and the key factors influencing the selection of a microbial monitoring technique, so that even professionals new to this domain can make sense of the available choices.
Applications of Microbial Testing
There is no one-size-fits-all when it comes to microbial test techniques, and the choice often depends on the specific application. Each application requires distinct outcomes for effective monitoring, most often in terms of levels of microbial identification and quantification:
Product Conformity/Final Product Testing
Testing the final product to ensure it meets specifications is non-negotiable, often requiring specific methods approved by regulatory bodies, to ascertain both microbe identity and quantity.
For example: a beverage company sets the criteria for the maximum number of live yeast present per bottle, so that the product will not spoil during prolonged storage or international transport by sea. For that, it will look for a test with the sensitivity appropriate for its chosen acceptance criteria and a specificity sufficient to count/discriminate living yeast.
In-Process Product Testing
Monitoring at various, generally pre-specified stages during production is critical to maintaining quality. The selected method is often chosen to correlate with final product testing techniques to ensure consistency, especially when specified by regulatory compliance requirements.
In the same example as above, the beverage is tested after a final filtration or pasteurisation step and before bottling to ensure its content in living yeast is coherent with the bottled product meeting its release criteria. In-product testing can be applied at multiple steps in the process to ensure that the counts in microorganisms of concern in the product is reduced along the process.
Process Monitoring
Ensuring a stable process is essential for product consistency and so this requires monitoring the parameters and controls of the production process. This typically requires knowing both the quantity and type of microbes present, ensuring the process stays within specifications, often with real-time sensors and control systems, such as temperature, pressure, flow-rates.
In the same example as above, the ability of the pasteurisation or filtration equipment to reduce the yeast concentration is verified, most often by measuring the yeast content prior and after a pasteurisation cycle with parameters set at the minimum so as to represent a worst-case scenario. If the equipment passes the test, this will ensure it effectively accomplishes its job.
Hygiene Monitoring
This application works on the prevention of contamination and maintains production quality by focusing on the cleanliness and sanitary conditions of the production environment and equipment. Knowing both the quantity and type of microbes can be advantageous but is not always essential, depending on the specific industry and process.
If microbial content in a product is important, which it most often is, to ensure the equipment itself does not contribute to increasing those counts, it will be cleaned then sanitized. Hygiene monitoring will consist in swabbing the filling heads of the bottling equipment or testing the rinse water of the filtration equipment after sanitisation to make sure the equipment is clean enough to operate.
Environmental Monitoring
Detecting environmental contaminants on surfaces, in the air or water, or sometimes on personnel, helps in controlling factors that could compromise product quality. Quantitative and qualitative microbial data is valuable but not always critical.
Microbes are easily transported from one location to another through air or contact. Movements of fluids such as water risk re-contaminating previously cleaned equipment. To prevent risks of such recontaminations environmental testing consists in testing the potential sources of recontamination / cross contamination such as the equipment used for maintenance, the cleaning equipment, handles, hands/gloves, floor, etc.
Available Techniques for Microbial Testing
Once the relevant application is clear, it is time to consider appropriate techniques. It is impossible to stress it enough: this choice impacts whether or not a testing plan can gather actionable information and allow the user to make decisions. Here are some of the common methods:
Culture-Based Methods
Techniques based on culture methods involve growing microorganisms from samples on nutrient-rich media to identify and quantify different types of bacteria, fungi, and other microbes. This traditional microbiological technique is the most widely used in clinical diagnostics, food safety, environmental testing, and pharmaceuticals. The process starts with sample collection, followed by inoculation on selective or differential media that supports the growth of specific microorganisms. Incubation at controlled temperatures allows the microbes to grow into colonies but is sometimes used to select certain bacteria while providing others with less than ideal conditions (temperature is then used for selection). These colonies are then examined based on their morphology, colour, and growth patterns, and further biochemical, molecular or microscopic tests may be conducted for precise identification. Culture-based methods provide detailed information about the microbial community in a sample, including biocide resistance. Time to results traditionally range from days to weeks with some recent equipment allowing for detecting in hours, depending on the exact technique and microorganisms being cultured, but culture methods are viewed as a gold standard in microbial analysis.
Molecular (PCR)
Polymerase Chain Reaction (PCR) testing is a molecular technique used to amplify and detect specific DNA sequences, enabling the identification of various microorganisms and genetic materials in samples. This method is known for its specificity, allowing it to differentiate between and identify different types of microorganisms. PCR testing is used in medical diagnostics, research, and environmental monitoring, as well as in food safety and quality control. The process involves extracting DNA from a sample, then using specific primers and enzymes to amplify target DNA sequences in multiple cycles of heating and cooling. The amplified DNA is then detected and analyzed, often through fluorescence-based methods. PCR’s ability to detect even minute quantities of DNA makes it a useful tool for accurate microbial identification and genetic analysis, but it requires specialized equipment and expertise, as well as prior knowledge of the microorganisms being investigated.
The five most common PCR techniques used in the industry are:
- Basic PCR: A method to make many copies of a specific DNA segment. It involves heating and cooling cycles to duplicate DNA.
- Real-Time PCR (qPCR): Similar to basic PCR, but it measures the amount of DNA as it increases during the process. This is useful for quantifying DNA.
- Reverse Transcription PCR (RT-PCR): Converts RNA (like from viruses) into DNA, then amplifies it. This is important for studying gene expression and RNA viruses.
- Multiplex PCR: Amplifies multiple DNA sequences at the same time in one reaction. It’s efficient for analysing multiple genes simultaneously.
- Hot Start PCR: A variation of basic PCR that reduces unwanted non-specific DNA amplification. It uses a special enzyme that activates only at higher temperatures.
These techniques are widely used due to their efficiency, specificity, and versatility in various applications like research, medical diagnostics, and genetic testing.
LAMP (loop‐mediated isothermal amplification) is an alternative molecular method used for pathogens, which does not involve thermal cycling to amplify DNA: the resulting equipment are smaller, and less expensive.
ATP
Adenosine Triphosphate (ATP) microbial testing detects ATP, a molecule found in all living cells, to estimate the presence and quantity of microorganisms in samples. This method is fast but lacks the ability to identify which microorganisms are present. In industries like food, healthcare, and pharmaceuticals, ATP testing is often used for quick assessments of cleanliness and microbial load. The process involves collecting a sample, introducing it to a luciferase-containing reagent (luciferase is an enzyme originally extracted from fireflies, which turns ATP into light), and measuring the light emitted from the reaction, which correlates with ATP levels and poorly with the number of microorganisms. Despite its rapidity and ease of use, ATP testing serves more as a preliminary assessment tool rather than a comprehensive microbial analysis method.
Flow Cytometry
Flow cytometry is a method used in microbial testing to analyze the physical and chemical characteristics of particles in a fluid. The process involves suspending cells or particles from a sample in a stream of fluid and passing them through a laser. As each particle crosses the beam, it scatters light and may also emit fluorescence if fluorescently labeled antibodies or dyes are used. These light signals are collected and analyzed to determine various properties of the particles, such as size, complexity, and the presence of specific markers. Flow cytometry can provide detailed information about the composition of microbial populations in a sample. It allows for the differentiation and quantification of different cell types, including the distinction between live and dead cells. However, flow cytometry requires highly specialized equipment and expertise, and the preparation of samples may influence the results, such as in cases of sample turbidity.
Some recent techniques use impedance instead of light to detect the microorganisms as they pass through the fluid stream.
Enzymatic Methods
These methods involve detecting specific enzymes produced by microorganisms to identify and quantify their presence in samples. The process typically begins with the collection of a sample, followed by the addition of substrates that react with microbial enzymes. The presence and activity of these enzymes are indicated by measurable changes, such as color shifts or the release of fluorescent or luminescent signals. Enzymatic assays are known for their specificity, as different microbes produce unique enzymes, and for their speed. These methods are particularly effective for detecting specific bacteria and pathogens in scenarios where rapid and targeted identification is needed. However, they may not provide a comprehensive overview of the microbial community in a sample and are limited to detecting microorganisms that produce the specific enzymes being tested.
Criteria for Choosing a Technique
After determining the application and looking at which techniques are available, criteria for selection can be set. This involves multiple considerations:
Relevance to Microorganisms of Concern
The method must be effective for the specific types of microorganisms relevant to the industry and product. There is little value in measuring coliform levels if your process is most vulnerable to wild yeast, or the product hostile to coliform survival for example.
Sensitivity and Specificity
The technique should detect contamination at levels above and under those that pose a risk and distinguish relevant microorganisms accurately. If a technique is not sensitive enough, a production batch could be released with quality problems in the making, that will be appear later.
Sampling Method and Site Representation
Choosing the right sampling method and location ensures a true representation of microbial presence and distribution in the batch or equipment to be tested. For example, testing a tank’s CIP rinse water will give an indication of the cleanliness of the entire tank’s inner surface while swabbing the tank ceiling will give a precise indication of the cleanliness of the hardest to clean surface.
Regulatory Compliance and Validation
When applicable, the method must comply with industry regulations and be validated for the specific setting. It should be noted that in most cases, regulations only specify both methods and pass/fail criteria (obligation of means and of results), on the final product, for microbial criteria which represent a safety concern for the consumer.
This is to be distinguished from regulatory imposed auto-control programs, in which case the manufacturer has an obligation of having an appropriate microbial monitoring plan, without imposing the test method or acceptance criteria for those auto-controls. The manufacturer is deemed the most knowledgeable party in the design and implementation of such a plan.
Speed of Detection (Time-to-Result)
While rapid results are desirable, they should never compromise the accuracy and reliability of the data. Speedy outcomes that do not meet requirements are of little value in a contamination event. In fact, detection speed should only be considered after other criteria.
When the uncompromisable criteria are met, the speed in time to result can present a real interest for releasing an intermediate or final product faster, which improves cash-flow.
In addition to these considerations, the cost of the technique, the ease of use, hands on-time, integration into the normal workflow are also important factors.
Conclusion
Selecting a microbial measurement method is a critical decision that must be made with an understanding of the unique demands of the specific industrial environment. This choice should be dictated by criteria that encompass the ability to accurately identify and quantify microorganisms, compliance with industry and regional regulations, and the validation of the method’s effectiveness within the intended setting.
The complexity of this decision is partly due to the necessity for precision—sensitivity to detect potentially hazardous levels of contamination and specificity to pinpoint the exact nature of the microbial presence. Moreover, the sampling process must be robust enough to provide a true reflection of the microbial landscape, capturing the scope of contamination risk.
Speed of detection is another critical factor, especially in time-sensitive industries, but it cannot supersede the accuracy and reliability of the results. While rapid testing offers the advantage of prompt responses, it should not compromise the rigor of the testing process.
Ultimately, the choice of a microbial measurement method must be made with an informed understanding of these considerations and the needs of the setting in question. BioMire does offer nomad as an on-site testing solution that uses Membrane Filter (MF) Technique because it permits our users to establish custom microbial monitoring systems with a high-value balance between sensitivity, detection speed, and cost, but it is critical to select a technique that best fits your situation.
If you have questions about which microbial testing technique can best fit your needs, don’t hesitate to contact us!