S.L. 433.7 Art. 000the perimeter. 40 microscope and The IF test of R. http:// intensity must Cells with be the case of buffer or if of the cells bacteria with to occur in at the are found, field and pellet typical cells potentially × 103 cells per Further

Scan windows across two diameters at right angles and around For samples showing no or low number of cells observe at least fields.

Check the positive control slide first. Cells must be bright fluorescent completely stained at the determined antibody titre or working dilution. (Section VI.A.5.) must be repeated if the staining is aberrant. 5.4.2. Observe for bright fluorescing cells with characteristic morphology in the test windows of the test slides (see website solanacearum forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence be equivalent to the positive control strain at the same antibody dilution. incomplete staining or with weak fluorescence must be disregarded. If any contamination is suspected the test must be repeated. This may when all slides in a batch show positive cells due to the contamination positive cells are found (outside of the slide windows) on the slide coating.

5.4.3. There are several problems inherent to the specificity immunofluorescence test. Background populations of fluorescing with atypical morphology and cross reacting saprophytic size and morphology similar to are likely R. solanacearum potato heel end core and stem segment pellets.

5.4.4. Consider only fluorescing cells with typical size and morphology titre or working dilution of the antibodies as in 5.3.

5.4.5. Interpretation of the IF reading:

(i) If bright fluorescing cells with characteristic morphology estimate the average number of typical cells per microscope calculate the number of typical cells per ml of resuspended (Appendix 5). The IF reading is positive for samples with at least 5 × 103 per ml of resuspended pellet. The sample is considered contaminated and further testing is required.

(ii) The IF reading is negative for samples with less than 5 ml of resuspended pellet and the sample is considered negative. testing is not required.