Isolation and Confirmation

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