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Author: admin, 03.12.2014. Category: Organic Foods

Good understanding of microbiological aspects of food production as well as awareness of possible microbial contaminations of foodstuffs is crucial for the general food safety. The problem of microbial contaminants in foodstuffs is not a new one and has been getting more and more into the focus of public health. Other organisms present in the environment of food production facilities, such as Staphylococci, Listeria, Bacilli and last but not least yeasts and molds, could pose additional threats if they are not monitored well. Finally, the main parameter a food microbiologist has to look for is the total amount of bacteria, the so called “total viable count”. The most efficient way to monitor the production process on microbial contaminants is the implementation of in-process controls at the production site. Compact Dry plates can be used everywhere at any time since the dry plates are not reconstituted before the sample implementation and are therefore stable at common temperatures for long periods of time. Compact Dry provides alternative solutions to classic agar plates and with the following issues of “Compact Dry: Food Microbiology Made Easier” we intent to bring you step by step to this fine product by sharing a deeper insight into the necessity and advantages of microbiological food controls. The pathogen diagnostic market continues to grow at approximately seven per cent a year with an estimated global market value of $2.4 billion per annum.
Pathogen testing is mainly performed in the water and food industries, including to monitor producers of high risk products such as deli meats. Before HACCP, end product testing was considered a safety net to catch contaminated product.
Apart from cost per test, a common theme in pathogen screening is how to decrease the time of analysis, increase sample throughput and perform screening on site. Real Time-PCR was developed in the 1990s and essentially is based on the increase in fluorescence as the copy number of the target gene increases. A major advance in genetic based diagnostics is the commercialization of isothermal amplification that was first developed in the late 1990s. Figure 1: Schematic of isothermal amplification based on signal transduction using bioluminesence (Image reproduced by permission of 3M). Although there are numerous diagnostic platforms that have been developed based on different transduction methods (for example, optical or electrochemical), very few are commercialized. In traditional methods, pathogen levels are increased through an enrichment step which also functions to differentiate living cells from dead cells, reduce background microflora, dilute interferents, in addition to facilitating cellular repair.
The concept of culture-free, on-site, pathogen screening is on the verge of becoming a reality with advances in sample preparation and diagnostic platforms. In conclusion, we have come a long way since Robert Koch and his team prepared the first culture media and there is strong commercial potential for non-culture techniques for pathogen screening. In order to control food safety and quality from primary production to consumption, solid knowledge of Food Microbiology is necessary. There are two bachelor studies and three master studies in which the Food Microbiology Laboratory offers a major part.
Biology (BO101) at NUI, Galway is a joint course run by the Disciplines of Biochemistry, Botany, Zoology and Microbiology. Reliable tests systems for analytical evaluation of microbial safety shall ensure delivery of safe products to the consumers and decrease the risk of contamination with life-threatening bacteria. By elaborating on the available analytical solutions, Compact Dry Food Microbiology Made Easier guide will therefore serve as a comprehensive know-how base for all practicing food microbiologists and food safety professionals. Even if the consumers do not know much about microorganisms in general they already understood that microbiological testing is necessary to avoid infections caused by foodborne pathogens. Salmonella is, yet, only one of numerous parameters which have to be tested in relevant matrices – even if it is one of the most commonly known pathogens.
All of them are naturally present on raw materials or humans and some of them have a highly pathogenic potential and can lead to serious diseases. These controls mitigate problems at an early stage when set up in the right way: taking samples at critical control points and analyzing them with reliable microbiological methods.
On the other hand, the end product controls are usually done by accredited laboratories but could be also conducted “in-house” with officially validated and easy to handle methods. Chromogenic agents present in all Compact Dry media types enable easy counting of colonies (product example: Compact Dry Total Count) after incubation. The ability to selectively isolate pathogens enabled major advances in clinical, environmental and food microbiology-indeed it was the very foundation. Yet the basic approach of enrichment, plating and confirmation has essentially remained unchanged for over a century. The increase in pathogen screening within the food sector is based on regulatory requirements implemented by federal and international agencies, the need to conform to retailer specifications and to demonstrate due diligence to consumers.

The most extensive testing is end product screening and environmental sampling, although, raw ingredient testing is also performed. However, in this “HACCP” age the emphasis of end product testing is to verify that interventions in place can eliminate or reduce the risk derived from pathogens. Such features would allow for rapid release of product to the market or corrective action plans to be implemented.
Through sustained research efforts there has been a diverse range of diagnostics developed to meet this need. Therefore, one could see in real time if a sample is positive for pathogens and have results within an hour. In traditional RT-PCR the reaction goes through a thermal cycle requiring precise control of the temperature to perform the denaturation, annealing and elongation step. However, as previously mentioned, the enrichment step adds a significant amount of time for analysis and requires containment laboratories. The most promising technology is tangential flow filtration (TFF also referred to hollow fiber filtration) that can non-selectively concentrate samples of any volume by 1,000 fold. The focus on the medical sector has driven the development of miniaturized sensors to facilitate point-of-care testing and also to use ever smaller volumes of samples.
Still the area is progressing with demonstration models of integrated systems based on hollow fiber filtration used in combination with flow through immunoassays or RT-PCR.
It is now possible to screen for multiple pathogens within a sample on-site within a matter of hours.
Specifically, there is a need to differentiate between viable and non-viable cells to minimize false-positive reactions. Although the science has almost delivered the hardware to make integrated sample preparation and diagnostics a reality, the big unanswered question is if there is an actual market for such systems which is yet to be determined.
In Food Microbiology both positive (fermentation) and negative aspects (spoilage, disease) of micro-organisms are studied. Furthermore, the Laboratory is involved in numerous courses that are part of other study programs and there are several BSc and MSc theses available for students. Results appear on this page.Also searches the entire Web site (with results on a new page) if you press enter or click on 'Go'. The next group of interest is the “fecal contaminant bacteria” which are exemplified by coliform bacteria, Enterobacteria or E.
Such a control system allows the producer to react quickly and take the right measures to ensure the food safety for the end consumers. This is the mission of the portfolio of Compact Dry: a robust, easy to handle microbiological test system with a proven reliability by official validations according to ISO 16140 as well as AOAC-RI standards.
Indeed, culture based techniques have formed the very foundation of developing microbiological criteria for testing foods, water and the environment.
With respect to specific testing, the usual targets are Listeria monocytogenes, Shiga Toxin producing Escherichia coli and Salmonella although it can be envisaged that enteric viruses will be added to the list given that the group of microbes account for over 50 per cent of foodborne illness cases. Consequently, there is a continuing focus of moving away from culture-based techniques to the golden-grail of culture-free methods that both speed up analysis and can be performed on-site.
To date the main focus has been immuno (antibodies or receptors) and genetic based techniques.
The hardware to step through the temperature points adds to the expense and complexity of the system. All are relevant but the lack of sensitivity has been the Achilles Heel of most diagnostic devices. The technique has become widely used to concentrate viruses and bacteria although largely confined to research as opposed to routine diagnostics. In this context, miniaturization is possible given that pathogen levels in clinical samples are typically high.
One of the most impressive systems is the remote water testing unit developed by NASA in collaboration with the University of Waterloo and K-State University (Figure 2). The key has been to bring down the cost of analysis and make systems sufficiently robust to withstand the rigours of life outside the laboratory. More critical is how to relate results to currently based microbiological criteria which were established using culture techniques. Yet, there are clear limitations with culture-based techniques such as long analysis times, low sample throughput and inability to grow certain microbes. The best known immune-technique is the lateral flow immune assay that can deliver results within 15 minutes.

However, with advances in technology it is now possible to purchase a RT-PCR for less than $14,000, although, cost-per-test remains relatively high (>$30). However, with isothermal amplification the temperature is kept constant that greatly reduces the cost of hardware, making for a more robust system. A more high tech approach is to apply dielectrophoresis capture that separates cells based on charge, although, to date the method has been applied in microfluidic devices as opposed to large volume sample preparation. The unit contains an auto-sampler that collects 10 litres of water then concentrates using hollow fiber filtration. This is less critical if standard sample sizes of 25g are used but more problematic if larger sample sizes are applied. The latter is relevant to Viable But Non-Culturable (VBNC) bacteria and the parasites (for example, enteric viruses and protozoan).
Biosensor research has developed microfluidic devices with the inner chambers coated with antibodies that can be used to detect pathogen targets in micro litre volumes of samples.
A recent advance on RT-PCR technology is the ability to perform quantitative analysis (qPCR) that is compatible with trend and risk analysis. To increase the sensitivity of sensors the options are to: 1) use larger volumes then concentrate the target, 2) extract the target or as common practice, 3) to enrich the sample. Innovations such as Immuno magnetic separation (IMS) and similar technologies have become routine for concentrating microbial targets although still require an initial enrichment step, albeit shorter than standard culturing.
A related technique has been to use charged filters based on ion exchange resins (electropositive or negative). The retentate is then placed into a chamber where the target pathogens are captured using IMS and then RNA extracted from the cells. There is also the issue of AOAC approval which is a necessity for any pathogen screening system. In addition, culture based techniques are required to be performed in containment laboratories making on-site testing problematic.
Although such systems are a marvel of engineering, none are compatible with the demands of pathogen testing within the food industry. There are several formats for isothermal amplification with current commercial pathogen detection systems being based on Loop-Mediated Isothermal Amplification (LAMP). Consequently, such techniques reduce the analysis time and cannot be considered culture free methods. The main issue with ion exchange filters is the cost ($150 each) and need for regeneration should the filter be re-used. The LAMP method applies the enzyme Bst DNA polymerase derived from the thermophile, Bacillus stearothermophilus. There are flow systems whereby the pathogen target is extracted from sample homogenate (typically 250 ml) is flowed over the surface of a magnet coated with antibody modified paramagenetic beads thereby negating the enrichment step.
Electropositive Glass wool is more cost effective at $0.75 per filter depending on dimensions. The system is a classic example on how current technologies can be combined to produce an integrated, culture free, system that is compatible with on-site testing.
In brief, the Bst polymerase forms single stranded DNA via strand displacement, thereby enabling the primers (four in total) to bind to the target sequence. Studies have shown that ion exchange techniques provide equal performance to tangential flow filtration with the added advantage of partial selectivity. A fully commercial system is still several years down the road and one can envisage the price would be a key factor that determines if it will be a success. Filtration based techniques also requires more hardware (for example, pumps), so they could be difficult to deploy on-site. The issue of clogging of filters or columns due to the constituents within systems is also a problem although this can be addressed by using a combination of techniques (i.e.
There are several approaches to transduce (report) the replication event that includes turbidity or fluorescence. An elegant approach in currently available commercial systems based on LAMP is to phosphorylate ADP via the phosphate groups released during the elongation of DNA to form ATP that then generates bioluminescence via luciferase (Figure 1). The potential of isothermal application is almost limitless and opens the door to produce miniaturized sensor or arrays that can screen for multiple pathogen targets on-site.

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