F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
SECTION VIOPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM
A.DIAGNOSTIC AND DETECTION TESTS
5.IF Test
5.4.Reading the IF test:
5.4.1
Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil or water immersion at a magnification of 500-1 000. 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.
Check the positive control slide first. Cells must be bright fluorescent and completely stained at the determined antibody titre or working dilution. The IF test (Section VI.A.5.) must be repeated if the staining is aberrant.
5.4.2.
Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent to the positive control strain at the same antibody dilution. 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.
5.4.3.
There are several problems inherent to the specificity of the immunofluorescence test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum are likely to occur in potato heel end core and stem segment pellets.
5.4.4.
Consider only fluorescing cells with typical size and morphology at the 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 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 5).
The IF reading is positive for samples with at least 5 × 103 typical cells per ml of resuspended pellet. The sample is considered potentially contaminated and further testing is required.
- (ii)
The IF reading is negative for samples with less than 5 × 103 cells per ml of resuspended pellet and the sample is considered negative. Further testing is not required.
Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al..