ANNEX IU.K.TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith) Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al. SCOPE OF THE TEST SCHEME
5.FISH TESTU.K.
5.1.Potato extract fixationU.K.
The following protocol is based upon Wullings et al., (1998):U.K.
5.1.1.Prepare fixative solution (see Appendix 7).U.K.
5.1.2.Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for eight min at 7 000 g.U.K.
5.1.3.Remove the supernatant and dissolve the pellet in 500 µl of fixative prepared < 24 hours previously. Vortex and incubate overnight at 4 °C.U.K.
An alternative fixative is 96 % ethanol. To use this dissolve the pellet from step 5.1.2 in 50 µl 0,01M PB and 50 µl 96 % ethanol. Vortex mix and incubate at 4 °C for 30 to 60 minutes.
5.1.4.Centrifuge for 8 min. at 7 000 g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 3).U.K.
5.1.5.Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 3. Apply two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01M PB). The remaining sample solution (49 µl) can be stored at -20 °C after addition of 1 volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01M PB (mix by vortexing).U.K.
Figure 3.
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 | |||
5.1.6.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.