Isolation and Confirmation

In today’s modern microbiology laboratory, many scientists recognize the benefits of rapid bacterial testing and identification methods for target pathogens. With the onset of technology, many rapid methods have been developed to significantly reduce time to detection, all the while reducing the cost of the testing process and allowing the release of time-sensitive products more quickly. Imagine a food lab with a time sensitive product. However, due to their traditional Listeria detection method, they cannot release product to the supermarket for five or more days after production. This reduces the stability of the final product, as well as its shelf life. The costs involved with this timely process are very high. If a product could be released after only two days of testing, it would be fresher and have a longer shelf life, not to mention significantly reduced labor costs. This quicker product release time can save companies millions of dollars and provide a definitive competitive edge. Each year, an alarming number of food recalls takes place in the United States due to pathogen contamination. Recalls cost food and ingredient manufacturers millions per year and jeopardize the health of many consuming contaminated products.

A Key Pathogen

The food industry needs to be vigilant when it comes to controlling microbiology contamination throughout its production process. A key bacterial pathogen which can find its way into a wide range of food products is Listeria monocytogenes. While a number of Listeria species are not considered potential human threats, Listeria monocytogenes is. It is important that food testing laboratories isolating Listeria species can rapidly confirm which species they are dealing with. The genus comprises six different species and these have distinct characteristics in terms of metabolism and potential to act as human pathogens. The most commonly found species are Listeria monocytogenes, a pathogenic species, and Listeria innocua, considered as non-pathogenic. Recently AOAC-RI approved identification strips have been designed to enable users to generate rapid confirmation of the identity of any Listeria species that they isolate from food or food ingredient samples. The product is normally used on “Listeria-like colonies” that have been isolated on a selective agar plate such as CHROMagar Listeria and ALOA media (both recommended by FDA and BAM). One colony is then taken and suspended in the Listeria suspending broth (supplied in the kit). The bacterial suspension is then added to all 12 wells of the micro-well strips provided. Finally, the haemolysin reagent (containing stabilized red blood cells) is added to well 12. The micro-well strip is then incubated for 18 to 24 hours in a non-fan-assisted incubator at 35 to 37 degrees C. The product then delivers its results within 18 to 24 hours. The unique feature of this product is an in-well haemolysis test based on the ability of a Listeria species isolate to lyse red blood cells. This is one of the key pathogenicity markers and provides a clear discrimination between Listeria monocytogenes (haemolytic) and Listeria innocua (non-haemolytic). The substrate reactions are easily read. Well 1 is esculin and should have turned black for all Listeria species isolates tested. The next 10 wells are sugars and will have either remained purple (negative) or turn yellow (positive). The final haemolysis well will show a button of red blood cells with clear liquid (negative) or a cloudy liquid with no red cell button visible (positive / haemolytic). The results are recorded and used to produce a four digit code which is then entered into the dedicated software program. The program analyzes the four digit code and suggests the most probable Listeria species. The identification strips offer several advantages over the conventional method and other miniaturized biochemical test systems for Listeria species identification and confirmation. The first advantage is that the strips can be used with bacterial colonies taken directly from selective agar plates. Most other systems require the test to be performed on colonies from non-selective plates. Additionally, only one colony is required per test, so there is no problem of multiple species contamination. All distinct colonies can be tested separately. The multi-well strip is a self-contained test system, which requires absolutely no setup and delivers the complete result without the need of additional confirmatory tests such as CAMP or a separate blood plate for haemolysis. Finally, the software program that interprets the results will deliver a most probable species result to confirm the identity of the isolate. The software also incorporates a feature ensuring that in the case of introduction of a non-Listeria species isolate, the program will identify and then prompt the operator to go back to re-confirm that it is indeed a member of the genus Listeria and suggest methods to further confirm.

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