Modification of a method for quantitative assessment of biofilm-forming capacity of microorganisms using safranin

Biofilms are organized communities of microorganisms. Their existence in this form mediates their increased resistance to antibiotics, disinfectants and immune system. The standard method for quantitatively assessing the ability of bacteria to produce biofilm, which involves determining optical density of biomass in a microtiter plate, is based on staining the biofilm with crystal violet. The use of this dye has several limitations, including extraction variability and safety requirements. The aim of the study was to modify the method for assessing the biofilm-forming capacity of microorganisms by using safranin as an alternative indicator dye and to conduct a comparative analysis of results with the classical method. The study was performed on clinical isolates of Escherichia coli (n=5) and Staphylococcus aureus (n=5), as well as reference control strains. The ability of cultures to form biofilms was determined quantitatively in microtiter plates. The method included a parallel comparison of the parameters obtained using safranin and the classical crystal violet staining method, with each experiment performed in paired independent plates. Staining with safranin provided systematically higher optical density values (the average increase was 0.09; 10.2%), which was statistically significant (t=5.16; p=0.00059). A high positive correlation was found between the methods (r=0.96; p<0.001). In 90% of cases, the adhesion classification remained consistent, with safranin demonstrating better reproducibility for most strains. These results confirm the potential of safranin as an alternative dye for biofilm quantification. The modified method can be implemented into routine laboratory testing after validation and standardization and can also be used in the screening of antimicrobial and antibiofilm drugs.