| Summary: | Ozone is a triatomic allotropic modification of oxygen with very high oxidation potential and strong antimicrobial properties, and can be used as a disinfecting agent. The aim of this work was to investigate the effectiveness of gaseous ozone in reducing the number of bacteria and the total biomass of <i>E. coli</i> biofilm using different methods of quantification and detection. Biofilm of all tested clinical isolates and standard strain was grown on ceramic tiles with dimensions of 1.0 × 1.0 cm over 24 h. These plates were then treated with gaseous ozone for 1 h. After washing, CFU/cm<sup>2</sup> was determined, ATP bioluminescence was measured with a luminometer, and the total biomass reduction was measured after crystal-violet staining. Gaseous ozone proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Treatment caused a reduction in total bacteria number of up to 2.00 log<sub>10</sub>CFU/cm<sup>2</sup>, followed by a reduction in total biomass of up to 61.40%. Inhibition rates varied from 35.80% to 99.41%, depending on the method of quantification used. All methods used in this study are effective in determining the anti-biofilm activity of gaseous ozone, but more research is needed.
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