Concentration Techniques for Bacterial Cells
Separation and Concentration of Microorganisms from Food Matrices
- Rapid pathogen tests are not sensitive enough to detect very low numbers of cells in food without an initial separation and concentration procedure
- Traditional enrichment culture methods are effective, but time consuming, often requiring several days to complete
- Development of rapid separation and concentration techniques has been limited to date, but validated methods based on physical and immunomagnetic separation (IMS) are available, some of which have been automated
One of the most important tasks undertaken by food microbiology laboratories is the testing of samples for the presence of foodborne pathogens and potential spoilage organisms. Microbiological standards and criteria applying to food often state that pathogens such as Salmonella, E. coli O157 and Listeria monocytogenes should be absent in a specified weight of sample, usually 25g. This means that methods must be capable of detecting a single viable cell of the target species in a 25g sample of the food being tested. Traditional culture based methods have that capability, but typically take several days to produce a result because of the need to amplify very low numbers of cells to levels that can be detected in culture.
Reducing the time needed to complete pathogen detection tests has been a primary goal for food microbiologists for several decades and much research has been targeted at rapid methodology. The result is that laboratories now have an array of detection methods based on technologies like PCR, and enzyme immunoassay, which can cut test times considerably and be largely automated. Unfortunately, even the most sensitive methods still require at least 103 cells of the target organism to be present before a detection can be accomplished. This means that, where low numbers of pathogens are present, some form of culture-based step is still necessary before the rapid method is employed. It has so far proved very difficult to reduce the total test time for rapid pathogen tests to less than 18-24 hours. This is a crucial limitation, because it means that microbiological testing cannot produce results quickly enough to monitor the critical control points in a HACCP system for managing food safety.
The challenge is magnified still further by the complexity of food matrices. Most foods are composed of many different materials, including large proteins, fats and oils, sugars and complex polysaccharides, often arranged in a complex three-dimensional structure. Many also contain a range of other non-pathogenic microorganisms, sometimes present in high numbers, but posing no health threat to the consumer. The target pathogen cells may be a very small component of the overall microflora and may be attached within the food matrix as single cells or clumps of cells. Before rapid detection methods can be used successfully, it is usually necessary to separate the target cells from the food matrix and from the background microflora. Once the cells have been separated, further time savings are available if they can then be concentrated to allow faster detection.
Compared to the R&D effort focused on detection methods, development of novel separation and concentration techniques for pathogens in food has been quite limited. But in the absence of a detection method that can be applied directly to food samples without any pre-treatment step, separation and concentration remain an essential component of food microbiology test protocols. Traditional methods are still widely used, but a range of physical, chemical and immunological separation methods have been investigated and some have been developed into practical applications and commercial products.
As yet, methods that are able to remove microbial cells directly and quantitatively from non-liquid foods are not available. The sample must therefore be suspended in a relatively large volume of liquid - diluent or growth medium - before separation and concentration can be attempted. Virtually all current pathogen detection methods include either an enrichment culture or suspension step.
The enrichment culture is the traditional method of detecting low numbers of pathogens in food samples. Many standard methods include a two-stage enrichment culture. The first step, or pre-enrichment, involves suspending the required sample weight (10-25g) in a large (100-250ml) volume of a non-selective medium designed to resuscitate damaged cells and promote microbial growth. After incubation, typically at 37oC for 18-24 hours, a small volume of the culture is transferred to a suitable selective enrichment medium designed to suppress the growth of competing organisms while allowing the target pathogen to multiply. In theory, such methods can amplify a single viable cell to detectable levels, even if it remains attached to the food matrix. There is no need to treat the sample to ensure that microbial cells are freely suspended in the medium. Though simple and reliable, traditional enrichment culture-based methods are extremely time-consuming, often taking several days to complete.Food sample suspensions Pathogen detection methods that do not utilise some form of enrichment culture still require the preparation of a sample suspension before separation, concentration and detection techniques can be applied. Ideally the initial suspension will be prepared in such a way that each target cell present is freely suspended in the diluent. This is difficult to achieve, but a variety of techniques have been investigated.Simply shaking a food sample in diluent will break it up to some degree, but is unlikely to result in a homogeneous suspension. Traditional quantitative methods (e.g. for total viable counts) often specify some form of mechanical agitation to release microbial cells from the sample matrix. Some protocols still include rotary bladed blenders as the preferred method of producing a suspension, but the need to clean and sterilise the blender between samples and problems with heat generation in the suspension limit their effectiveness. Paddle type blenders like the Seward Stomacher® overcome some of these problems by processing the sample in a sterile plastic bag so that the paddles do not make contact with the sample and remain clean. They are also able to handle large volumes of liquid. Paddle blenders are now widely used in food microbiology laboratories and have largely replaced bladed blenders. A more recent development is the Pulsifier® from Microgen Bioproducts, which replaces the paddles with a high-speed oscillating ring producing shock waves as well as agitation. The Pulsifier is able to release more microbial cells into suspension with less disruption to the food matrix than other blenders, and without creating large quantities of food debris in the suspension. Other methods for preparing initial suspensions, such as sonication, have been investigated, but are not widely used. Separation and concentration Once microbial cells have been released from the food matrix into suspension, the target pathogen can be separated and the cells concentrated for more rapid detection. A number of different physical, chemical and immunological methods have been devised to accomplish this, including ion exchange columns and biphasic partitioning. But three techniques form the basis of most practical applications: membrane filtration, centrifugation and immunomagnetic separation (IMS). Membrane filtration Filtration has obvious attractions as a means of separating and concentrating microbial cells from sample suspensions. It is a relatively simple and inexpensive operation and has the potential to process large volumes of suspension quickly. The main disadvantage is that many food suspensions are not easy to filter, especially high-fat processed food products, thus severely limiting possible applications. It may be possible to pre-filter some suspensions to remove food debris that may block the membrane, but this can also remove some of the target cells. Some improvement in the filterability of food suspensions can be achieved by techniques such as trypsin digestion or the addition of surfactants, but this may also interfere with pathogen detection and reduce cell viability.For filterable samples, such as beverages and some dairy products, practical separation and concentration methods based on membrane filtration have been developed and combined with a variety of microbial detection technologies as commercially available instruments and test systems. Examples include the AES Chemunex ChemScan®RDI Analyser, which uses a solid-phase cytometry technique to detect viable cells directly on filters, and the ATP-bioluminescence based BeviStat™ Rapid Microbiology Detection System from Millipore. Microscopy-based methods, such as the direct epifluorescent filter technique (DEFT), are also still used in some laboratories to count bacterial cells on membrane filters.Centrifugation Centrifugation has also been used successfully to separate and concentrate microbial cells in food suspensions, although applications are limited by the practical considerations of processing large volumes of liquid. Low speed centrifugation can be used to remove food debris while leaving bacterial cells in suspension, and higher speeds can then be used to concentrate bacteria. Alternatively, methods based on density gradient centrifugation can be used. For example, buoyant density centrifugation (BDC) has been used in rapid detection of foodborne pathogens to separate bacterial cells and food particles with different buoyant densities by flotation and sedimentation and to remove PCR inhibitors. Some commercial applications have also been developed, notably the automated Foss Bactoscan™ FC instrument designed for the dairy industry. This is able to separate bacterial cells from milk with the aid of a density gradient, before detection and enumeration by flow cytometry.Immunomagnetic separation IMS technology was first developed in the 1980s and is now widely used in food microbiology to separate target pathogens from other microorganisms. Magnetised polystyrene beads or other particles are coated with antibodies specific to the target organism and these are then added to a volume of sample directly (where high counts are likely), or more often to a pre-enrichment culture. After a short incubation the target cells are immobilised on the surface of the beads and can be concentrated into a pellet using a powerful magnetic field. The sample can then be discarded and the pellet re-suspended and washed to remove food debris and other material. The immobilised pathogens can be detected by culture, or by rapid methods, including PCR and ELISA. IMS is a very effective separation and concentration technique, although it is not suitable for processing large volumes of sample or culture, partly because high concentrations of beads are needed to bind the target cells.Magnetic beads and particles for food microbiology applications have been available commercially for many years. The best known are Dynabeads®, supplied by Life Technologies and designed for selective capture and concentration of a range of foodborne pathogens from pre-enrichment cultures. Dynabeads specific to Salmonella Listeria and a number of VTEC serotypes are available. Dynabeads can also be used in an automated process using the BeadRetriever™ system.A further development of automated IMS technology is illustrated by the Pathatrix® Auto System, also from Life Technologies. Pathatrix is unique in allowing up to ten samples to be pooled after pre-enrichment, providing considerable cost savings through reduced detection testing. The separation and concentration process can be completed within 15 minutes.Future developments Although methods and instruments designed to speed up the process of separating and concentrating microorganisms from foods are available, there is still a need for new methods to be developed to support PCR- and immunology-based detection tests. At present food microbiologists are still reliant on enrichment cultures to amplify low numbers of pathogens to a detectable level and the goal of instant detection has yet to be achieved.One approach to moving away from dependence on enrichment procedures is to combine some of the technologies described above in multi-step methods. For example, a protocol using a combination of filtration, low and high speed centrifugation and buoyant density gradient centrifugation has been described that is capable of a 250 fold concentration of target pathogens in food samples and detected 10 CFU/g of Salmonella in chicken in 3 hours when combined with a PCR detection technique. Such a method would not be practical for routine application in a food microbiology laboratory, but does indicate the potential for development of multi-step methods, especially if they can be automated.
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