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From: Food Quality & Safety magazine, February/March 2005

Isolation and Confirmation

Strip ID method paves the way to results and specificity

by Gina M. Dunn

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.

Debunking Myths

Myth 1: True rapid systems which provide identifications within four to six hours of inoculation are as reliable as those systems which require overnight incubation. The achievement of identification after such a short incubation period relies on the identification system being inoculated with a very large number of bacteria. To achieve this density of organisms, multiple colonies must be selected and in doing so, the potential always exists to select different organisms and inoculate the system with mixed cultures. Working from a single colony with a system which requires overnight incubation eliminates this problem and ensures no mixed culture. Myth 2: It is not possible to detect Listeria and/or Listeria monocytogenes from an initial sample to a biochemical ID in less than five days. Using the FDA-BAM recommended ALOA media or CHROMagar Listeria media in conjunction with the identification strips, presumptive to final identification confirmation can be achieved in two days, with no additional tests. Myth 3: Biochemistry identification kits are difficult to assemble, inoculate, handle and read. Biochemistry strip kits are extremely easy to use. Unlike other methods on the market, there is absolutely no product assembly, no water addition and no setup time necessary at all. Inoculation is as simple as pipetting 100 µl of inoculum solution into a microwell. The lid then simply pops back on the microwell unit. The convenient and compact size and microwell format (only 5 inches by 1/2 inch total size) ensures that the strips can be stacked in an autoclave without worry of tipping over. Myth 4: I do not need a Haemolysis test for Listeria identification. The two virulent species of Listeria (L. monocytogenes and L. ivanovii ) both produce a Phospholipase C (phos-phatidylinositol specific) enzyme. Mutant strains of L. monocytogenes that lack the Phospholipase C enzyme are avirulent or its virulence is significantly reduced. The detection of haemolysin provides a more practical method of discriminating between virulent and avirulent Listeria species. As the detection of the haemolytic activity is fundamental to the identification of Listeria species (in particular L. monocytogenes and L. ivanovii) and the interpretation of this reaction is sometimes difficult. A haemolysin test is simply performed by adding one drop of the specially stabilized sheep red blood cells to well 12 of the test strip. If the organism being identified produces haemolysin, the red blood cells will rapidly be lysed and the cellular contents will be released into the suspending medium. The well's contents appear as a homogeneous red/brown solution. Alternatively, if the organism being identified does not produce haemolysin, the stabilized red blood cells will remain intact. These cells will settle to the bottom of the microwell forming a distinct red layer with a clear supernatant. The multiwell strip is a self-contained test system which delivers the complete result without recourse to additional confirmatory tests such as CAMP or a separate blood plate for haemolysis. Myth 5: Interpreting the results of a biochemistry ID test is extremely difficult and confusing. The results of the test strip are recorded and used to produce a four digit code which is entered into the dedicated software program. The software is simple to use; yet provides comprehensive data analysis system for the interpretation of results. Organisms are identified using a range of biochemical or growth characteristic tests with the results being compared to separate accumulated results of known cultures of similar organisms. This is straightforward when only a few organisms make up the database being considered and only a small number of tests are required to differentiate them. This task becomes significantly more complicated as the numbers increase. To simplify this, a computer-aided, probability-based approach employed in the software may be used. The software also recommends a range of supplementary tests that may be used to further differentiate the species selected as the most likely identification choices. The program has now incorporated a new feature which ensures that the introduction of non-Listeria species isolated on the test strip will be identified and prompt the operator to go back to reaffirm that the isolate under test is indeed a member of the genus Listeria, using standard pre-tests of gram stain (positive), Oxidase (negative), Catalase (positive) and motility at 25 degrees C but non-motile at 37 degrees C. -FQ

Gina M. Dunn is a marketing and sales associate at Microbiology International. Reach her at 800-396-4276 or Gina.Dunn@800EZMICRO.com.

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