Bacterial populations usually include minority sub-populations of cells exhibiting increased tolerance to ultraviolet radiation (UV), whose presence is evidenced by a reduction in the slope of the survival curve at high doses. For this reason, bacterial survival to high UV doses could be considerably higher than the one expected from the analysis of the inactivation kinetics of the susceptible majority sub-population. The ability of some cells to overcome the action of UV is usually ascribed to shielding due to turbidity or clumping, and the presence of persistent cells has also been proposed. The aim of the present study was to obtain experimental evidence supporting the involvement of these mechanisms in UV tolerance, and look for a procedure to eliminate cells able to tolerate high doses of radiation. The model organism was Pseudomonas aeruginosa, and the radiation source was a low pressure mercury lamp. Survival curves were obtained at different bacterial concentrations to test the influence of turbidity, and the effect of clumping was appraised using chemical and physical agents which produce disaggregation. Since the condition of persistence is transient and reversible, its contribution to UV tolerance was evaluated by the ability of survivors to regain the susceptible condition after irradiation. Although P. aeruginosa is prone to aggregation, dilution of the bacterial suspensions, ultrasonic treatment or addition of a surfactant had no detectable effect on their response to radiation, suggesting that shielding is not responsible for UV tolerance. Conversely, most survivors to high UV doses became susceptible when they were incubated in the dark after irradiation, as it would be expected for persistent cells. The results suggest that treatment schemes involving split irradiations could improve the effectiveness of UV disinfection.
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