[ANNEX I U.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
8. ISOLATION OF C. M. SUBSP. SEPEDONICUS U.K.
8.1. Selective plating U.K.
8.1.1. From a 100 µl aliquot from a resuspended potato pellet sample or eggplant sap make 10-fold dilutions in pellet buffer (Appendix 3). U.K.
8.1.2. Isolation from undiluted potato pellet usually fails due to the fastidious growth habit of Cms and competition by saprophytes. Since the bacterium is usually present in high populations in infected tissues, the saprophytes can usually be diluted out, whilst the pathogen remains. It is therefore recommended to spread 100 µl from each of the samples, 1/100 up to 1/ 10 000 dilutions onto MTNA medium or NCP-88 medium (Appendix 5) (if using 90 mm diameter petri dishes- adjust volume for alternative dish sizes), using spreaders (hockey sticks) and the spread plate technique. U.K.
Note: U.K.
An alternative strategy is to spread out the initial 100 µl potato pellet aliquot onto a first agar plate with a spreader and then remove the spreader to a second agar plate, streaking out any residue left on the spreader; finally repeat this with a third plate, thus giving a dilution plating effect via the spreader.
8.1.3. Incubate plates in the dark at 21 to 23 °C. U.K.
8.1.4. Initial examinations of the plates including, by reference to the control plates, counts of any C. m. subsp. sepedonicus like colonies are made after 3 days, with further counts after 5, 7, eventually 10 days.] U.K.