Determination of biofilm biomass by crystal violet assay

In our study, we employed two distinct assays to evaluate the effects of the tested compounds on biofilm formation and on preformed biofilms. For the assessment of biofilm formation, we used a method that involves the direct exposure of bacterial cultures to the compounds during the initial stages of biofilm development. This approach allows us to observe the impact of the compounds on the early stages of biofilm formation, including bacterial adhesion and microcolony formation. Specifically, bacterial suspensions (200 µL, 10⁶ CFU/mL) were treated with 0.125 mg/L AZT, 4 mg/L NIT, or both, transferred to a 96-well plate, and cultured at 37°C for 36 h. In contrast, the assay for preformed biofilms involves the treatment of established biofilms with the compounds. This method is designed to simulate the conditions where biofilms have already formed and are exposed to treatment, enabling us to assess the compounds’ activity to disrupt or reduce the viability of mature biofilms. Specifically, bacterial suspensions (200 µL, 10⁶ CFU/mL) were transferred to a 96-well plate, cultured at 37°C for 24 h, and the resultant biofilms were treated with 0.125 mg/L AZT, 4 mg/L NIT, or both. The incubation was then continued at 37°C for 12 h. For both methods, the wells were subsequently rinsed with PBS without destroying the established biofilm. After a 10-min methanol fixation, the biofilms were stained with 0.1% crystal violet solution for 10 min, followed by a rinse with water. After drying, the stained biofilms were photographed before adding 33% acetic acid and measuring OD₅₉₀ (38).

Both assays are critical for a comprehensive understanding of the antimicrobial effects of the compounds on biofilms at different stages of development.