Ls analyser

Bacterial cultures were grown up to the stationary phase, washed up with PBS and diluted to OD₆₀₀ = 0.2 (10⁷ CFU/ml) by (i) Sauton medium (pH 5.5) with ADC growth supplement and 0.05% Tween for low pH stress; (ii) by the culture supernatant to study inhibitory effects of NO (provided by the DETA/NO donor, 0.5 mM) and H₂O₂ (10 mM). Cell viability after 24 h and 48 h of stresses exposure were measured by incorporation of [³H]-uracil label. Two microliters of 5,6,-[³H]-uracil (2 μCi) were added to 1-ml culture samples and incubated at 37°C with agitation for 20 h. Two hundred microliters of culture were put in 3 ml 7% ice-cold CCl₃COOH, incubated at 0° C for 15 min and filtered through glass microfiber filters (Whatman, USA). Precipitated cells were washed with 3 ml 7% CCl₃COOH and 3 ml 96% ethanol. Filters were put in 10 ml of scintillation mixture; CPM were determined by LS analyser (Beckman Instruments Inc., USA).

One microlitre of 5,6,-³H uracil (1 µCi) was added to 1 ml culture samples and incubated at 37°C with agitation for 20 h. Two hundred microlitres of this culture was placed in 3 ml 7% ice-cold CCl₃COOH and incubated at 0°C for 20 min, followed by filtration through a glass microfibre filter (Whatman). Precipitated cells were washed with 3 ml 7% CCl₃COOH and 6 ml 96% ethanol. Filters were placed in 10 ml scintillation mixture; impulse counts were determined by LS analyser (Beckman Instruments) and expressed as counts per minute (cpm).