Observe the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1000x magnification under immersions oil. With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the sample are stained fluorescent green. Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of C. m. subsp. sepedonicus appear fluorescent red. Compare cell morphology with that of the positive controls. Cells must be bright fluorescent and completely stained The FISH test (section 9.4) must be repeated if the staining is aberrant. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields.

Observe for bright fluorescing cells with characteristic morphology of C. m. subsp. sepedonicus in the test windows of the test slides (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent or better than that of the positive control strain. Cells with incomplete staining or with weak fluorescence must be disregarded.

If any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the slide coating.

There are several problems inherent to the specificity of the FISH test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to C. m. subsp. sepedonicus may occur, although much less frequent than in the IF test, in potato heel end core and stem segment pellets.

Consider only fluorescing cells with typical size and morphology, see in 5.3.2.

Interpretation of the FISH test result:

1. (i)

   Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of C. m. subsp. sepedonicus are observed using the FITC filter and if bright red fluorescent cells using the rhodamine filter in all positive controls and not in any of the negative controls. If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 4). Samples with at least 5 × 10³ typical cells per ml of resuspended pellet are considered potentially contaminated. Further testing is required. Samples with less than 5 × 10³ typical cells per ml of resuspended pellet are considered negative.

2. (ii)

   The FISH test is negative if bright red fluorescent cells with size and morphology typical of C. m. subsp. sepedonicus are not observed using the rhodamine filter, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter.