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[F1ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
Textual Amendments
SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM
A. DIAGNOSTIC AND DETECTION TESTS U.K.
7. FISH test U.K.
Principle U.K.
When the FISH test is used as the first screening test and found to be positive, Isolation or the IF test must be performed as a second compulsory screening test. When the FISH test is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.
Note: Use validated R. solanacearum -specific oligo-probes (see Appendix 7). Preliminary testing with this method should permit reproducible detection of at least 10 3 to 10 4 cells of R. solanacearum per ml added to sample extracts which previously tested negative. U.K.
The following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at -16 to -24 or -68 to -86 °C.
As negative controls, use aliquots of sample extract that previously tested negative for R. solanacearum .
As positive controls prepare suspensions containing 10 5 to 10 6 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 3) in 0,01M phosphate buffer (PB) from a 3 to 5 day culture). Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum , suspended in potato extract, as specified in Appendix 3 B.
The use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.
Standardized positive and negative control material available for use with this test are listed in Appendix 3A).
Test control material in an identical manner as the sample(s).
7.1. Potato extract fixation U.K.
The following protocol is based upon Wullings et al. (1998): U.K.
7.1.1. Prepare fixative solution (see Appendix 7). U.K.
7.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for 7 minutes at 7 000 g. U.K.
7.1.3. Remove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24 hours previously. Vortex and incubate for one hour in the refrigerator. U.K.
7.1.4. Centrifuge for 7 minutes at 7 000 g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 7). U.K.
7.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 7.1. Applying two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01 M PB). The remaining sample solution (49 µl) can be stored at -20 °C after addition of one volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01 PB (mix by vortexing). U.K.
Fig. 7.1
Layout for FISH slide
| Sample 1 | Blank | Blank | Blank | Sample 2 |
 |  |  |  |  |
| window 1 | window 2 | window 3 | window 4 | window 5 |
| Sample 1 | Blank | Blank | Blank | Sample 2 |
 |  |  |  |  |
| window 6 | window 7 | window 8 | window 9 | window 10 |
| Coverslip 1 | Coverslip 2 | |||
7.1.6. Air-dry the slides (or on slide dryer at 37 °C) and fix them by flaming. U.K.
At this stage the procedure may be interrupted and the hybridisation continued the following day. Slides should be stored dust-free and dry at room temperature.
7.2. Hybridisation U.K.
7.2.1. Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each. Air dry the slides in a slide-holder. U.K.
7.2.2. Prepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-incubate the box in the hybridisation oven at 45 °C for at least 10 minutes. U.K.
7.2.3. Apply 10 μl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty. U.K.
7.2.4. Apply coverslips (24 × 24 mm) to the first and last four windows without trapping air. Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at 45 °C in the dark. U.K.
7.2.5. Prepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water). Pre-incubate each in a waterbath at 45 °C. U.K.
7.2.6. Remove the coverslips from the slides and place the slides in a slide holder. U.K.
7.2.7. Wash away excess probe by incubation for 15 minutes in the beaker with 1x hybmix at 45 °C. U.K.
7.2.8. Transfer the slide holder to 1/8 hybmix washing solution and incubate for a further 15 minutes. U.K.
7.2.9. Dip the slides briefly in Milli Q water and place them on filter paper. Remove excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide. U.K.
7.3. Reading the FISH test U.K.
7.3.1. Observe the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1 000 × magnification under immersion 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 R. solanacearum appear fluorescent red. Compare cell morphology with that of the positive controls. Cells must be bright fluorescent and completely stained The FISH test (Section VI.A.7.) 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. U.K.
7.3.2. Observe for bright fluorescing cells with characteristic morphology of R. solanacearum 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. U.K.
7.3.3. 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 the slide coating. U.K.
7.3.4. 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 R. solanacearum may occur, although much less frequent as in the IF test, in potato heel end core and stem segment pellets. U.K.
7.3.5. Consider only fluorescing cells with typical size and morphology. U.K.
7.3.6. Interpretation of the FISH test result: U.K.
Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of R. solanacearum are observed using the FITC filter and 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 × 10 3 typical cells per ml of resuspended pellet are considered potentially contaminated. Further testing is required. Samples with less than 5 × 10 3 typical cells per ml of resuspended pellet are considered negative.
The FISH test is negative if bright red fluorescent cells with size and morphology typical of R. solanacearum 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.]