MICROBIOLOGY
Traditional cultural techniques versus non targeted molecular detection methods Andy Muirhead, ALS company microbiologist, considers established versus more recent and developing testing and detection techniques. WHAT’S CHANGED? This year marks the bicentenary of the birth of Louis Pasteur (1822-1895), and if the famous microbiologist would have had access to a time machine and managed to travel forward in time by around 170 years, he could walk into one of our laboratories and still recognise many of the techniques which were performed in his laboratory in the middle of the 19th century. We still detect and enumerate bacteria by putting them in broths and agars, we place them in optimal growth conditions and demonstrate their presence by allowing the individual cells to multiply to such an extent that we can observe visible colonies on selective or non-selective agars. Although there has been considerable advances in the agars and cultural techniques which we have at our disposal, it does seem inconceivable that routine diagnostic microbiology (both clinical and food) hasn’t advanced more rapidly than it actually has, and that faster and more sensitive molecular techniques haven’t replaced the more conventional methodologies. Whilst molecular methods have become the mainstream in virology (we are all familiar with the Polymerase Chain Reaction, or PCR test used for Covid), in bacteriology they are still used very rarely in bacteriology. A possible reason is that traditional cultural virology methods using cell culture lines were difficult to perform. They required a considerable amount of expertise, both in the growth and maintenance of the tissue culture lines and in the inoculation, incubation and the microscopic interpretation of the changes to the tissue cell cultures. It could often take several weeks to get a result, so is no surprise therefore that alternative methods such as ELISA and PCR have gained widespread acceptance in virology, but what about bacteriology?
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ALS Laboratories (UK) Ltd (www.als-testing.co.uk) is one of the UK’s leading providers of food and drink testing services. With six accredited laboratories located across the country, they offer a comprehensive range of high quality, analytical testing services, including microbiological, nutritional, vitamins and minerals, pesticides and contaminants, allergens and speciation. They also provide clients with a wide range of consultancy services and technical support on food safety, labelling requirements, allergens management and sensory testing. TRADITIONAL METHODS AUGMENTED The main reason why bacteriology has fallen behind in terms of embracing new molecular methodologies is that by and large, the traditional methods work, and perhaps more importantly can be delivered at a cost which is acceptable to our clients. However, we aren’t completely working in the dark ages as over the last 20 years, traditional methods in food microbiology have been augmented by more rapid methods of isolation and identification such as ELISA and MALDI-TOF MS. Performing our Salmonella and Listeria screening tests by ELISA saves an incredible amount of time and money compared to the traditional cultural ISO methodologies.
When we get a presumptive result by ELISA, we then go back to our broth culture and plate it out and look for typical colonial morphology on the selective agar plates. If the colonies look typical for the target organism we obtain a confirmed result by performing conventional biochemical and serological techniques. This however can take another two to three days, which is why we can make use of a mass spectrometry technique known as MALDITOF MS, which enables us to confirm the presumptive isolate much faster. If MALDI identification leads to a confirmed Salmonella, then we are able to serotype the Salmonella isolates by PCR analysis of the genetic material of the bacteria rather than the antigenic structure of the organism as is used in traditional serological techniques. This gives an accurate and rapid identification of the particular type of Salmonella which can be incredibly useful when attempting to identify a potential cause of contamination of a product. It gives the same result as traditional serological testing, but much quicker and without the requirement to hold expensive banks of antisera. So, despite my assertion that Louis Pasteur would recognise the basic principles of the traditional cultural techniques which are still in common use in our lab, we have actually embraced many newer methodologies such as ELISA, MALDI-TOF MS and in the serotyping of Salmonella by PCR, which enables us to process large volumes of samples rapidly and cost effectively. TIME-SAVING However, even with these techniques we are still relying on initially growing the organisms to levels where they can become detectable, so is it possible to get rid of the requirement to grow the organisms first, as after all that is the time-consuming stage, and instead demonstrate the presence of organisms
