1DLO-Centre for Plant Breeding and Reproduction Research, PO Box 16, 6700 AA Wageningen, The Netherlands; 2WAU- Agricultural University of Wageningen, Bomenweg 2, Wageningen, The Netherlands

Background and objectives
Conventional direct-count assays of bacterial viability are based on metabolic characteristics and multiplication and involves plating of the organisms on selective or semi-selective media. The isolated organisms have to be identified by a range of biochemical or serological tests. Altogether such protocols can take a long time. New methods to measure the viability of microorganisms rapidly, accurately and reliably are higly desirable. Fluorescent probes can be useful tools which allow measurements of cellular parameters, such as enzyme activity, membrane potential and intracellular pH, for individual cells. In this study, a rapid and reliable method to measure viability of Clavibacter michiganensis subsp. michiganensis was evaluated using the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) for determination of the intracellular pH ( pHin). The fluorescence of this probe is pH dependent and it can be easily taken up by bacteria during incubation with its diacetate ester 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester (cFDASE). Once it is inside the cell, its succinimidyl group forms conjugates with aliphatic amines and its fluorescence can be detected after intracellular esterase activity.

Materials and methods
Clavibacter michiganensis subsp. michiganensis NCPPB 1064 was grown on 1% Glucose-Nutrient-Agar (Oxoid CM3) medium for 48 hours at 25C or in Nutrient Broth (Oxoid CM1) medium, supplemented with 10g/l glucose, on a rotary shaker (180 rpm) to an optical density value of 0.4. Hot water treatments at temperatures of 25 ( control ), 40, 45 or 50C and HCl treatments at concentrations of 0 (control), 0.1, 0.2 or 0.6M were given to the cells for 1 hour. The cells from both the hot water treatment and acid treatment were incubated for 15 minutes at room temperature in the presence of 1.0 M of 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) in CPK buffer containing citric acid ( 50 mM ), Na2HPO42H2O ( 50 mM ) and KCl ( 4M ), pH 5.5 or pH 7.0. Spectrofluorometer measurements were performed during 25 minutes assays and the results were compared with the conventional plate count technique.

Results and conclusions
When the cells were treated at 50C, no colony forming units ( CFU/ml ) and no pH gradient ( difference between intracellular and extracellular pH ) were observed, neither for bacteria grown in solid or liquid medium nor for the bacteria incubated in pH 5.5 or 7.0. For 45C treated cells grown in liquid medium and incubated in buffer at pH 5.5 and 7.0, a small pH gradient ( 0.1 ) was observed, but no colonies were detected by the plate count technique in both cases. For 25 and 40C treatments a good correlation was found between the number of colony forming units determined by plate counting and the maintenance of an intracellular pH different from the external pH of the medium. Bacterial cells grown on solid medium for 48 hours showed to be more tolerant to high temperatures than when grown in liquid medium. Bacterial cells treated with HCl showed to be very sensitive to acid treatment, independently of the culture medium and the pH of the buffer. Although a relatively low number of cells could be recovered in the plate count assays, no pH gradient was observed in any of the treated cells.

In conclusion, the assessment of viability of the plant pathogen bacterium Clavibacter michiganensis subsp.michiganensis can be successfuly achieved in a rapid test by measuring the intracellular pH using the fluorescent probe cFSE.