4.4.5. Microplate Microtiter Assay

The influence of the compounds on the biofilm formation was determined using the microtiter plate assay. Briefly, after the determination of the MIC values, the microplates were emptied and washed three times with sterile distilled water for the removal of planktonic microbial cells. The biofilms adherent on the plastic wells were fixed with methanol for 5 min and stained with 1% crystal violet solution for a quarter of an hour. The excess dye was removed and thereafter the fixed dye was resuspended in 33% acetic acid. Ciprofloxacin (Oxoid) was used as a positive control for antibacterial tests and fluconazole (Oxoid) for antifungal testing. The absorbance of the colored solutions was recorded with an ELISA Reader (Apollo LB 911). The minimal biofilm inhibitory concentration (MBIC), defined as the lowest compound concentration required to inhibit biofilm formation, was determined. The results from three separate biological replicates were mediated [83].
