Identification to genus. The bacterial isolates that test positive for a pathogen are most often identified to genus using a biochemical key as, for instance, described in Schaad et al. (2001). Identification to genus includes performing the following biochemical and physiological determinations for each isolate: Gram staining, glucose fermentation on Hugh-Leifson (HL) medium, pigment production on Yeast extract Dextrose Calcium agar (YDC), preparation on King B medium of a pigment fluorescing under UV light and the ability to grow on D1M medium.

Identification to species

API tests

API tests (BioMerieux, France), in particular API 20E and API 20NE tests, are commonly used for the initial biochemical identification of tested bacterial plant isolates that satisfy Koch's postulates. API 20E tests have been used to test fermenting Gram-negative rods, while API 20NE tests have been used to characterize non-fermenting Gram-negative rods.

The BIOLOG system

The BIOLOG system (from BIOLOG Inc., Hayward, CA) is used to confirm and extend the initial biochemical API test identification. BIOLOG is an off-the-shelf kit containing powdered media for the identification of Gram-positive and Gram-negative bacteria. This system enables the simultaneous determination of 95 biochemical traits of the tested bacterial isolate to create its unique biochemical profile, which, once read and entered into program database, is compared by a specially developed algorithm against nearly 3,000 other biochemical profiles, including those of phytopathogenic bacteria. This algorithm uses differences between particular biochemical profiles to assess affinity of tested isolates, expressed in special units. On this basis, the tested bacteria are identified and the names of the species with the most similar biochemical profile are given.

FATTY ACID ANALYSIS

Another frequently used method of biochemical identification of bacteria is fatty acid methyl analysis (FAME), which has been in use for over half a century. So far, research has revealed the existence of over 300 different fatty acids and their derivatives in the cytoplasmic membrane and outer cell envelope (in Gram-negative bacteria). Research has also found that many bacterial species can be diagnosed on the basis of not only their fatty acid composition, but also their amounts. Therefore, the combination of over 300 different fatty acids and their quantities offers a reliable bacterial diagnostic tool as nearly every species comes with a unique fatty acid profile.