A rapid and simple measurement method for biofilm formation inhibitory activity using 96-pin microtiter plate lids

The purpose of this study was to develop a rapid and simple measurement method for biofilm formation inhibitory activity, and to screen food additives and foodstuffs that inhibit biofilm formation. The measurement method for biofilm formation inhibitory activity was developed by combining biofilm formation on pins of microtiter plate lids and staining using crystal violet. The optimum conditions for biofilm formation on the pins were established for seven Gram-positive and six Gram-negative bacteria by investigations of media, incubation time, and pin materials. Minimum concentrations of food additives required to inhibit biofilm formation were determined using the proposed method. The values obtained by the proposed and conventional methods agreed well. In addition, by sequential measurements of minimum inhibitory concentrations and minimum bactericidal concentrations using the proposed method, mechanisms of inhibition of biofilm formation were assessed. Furthermore, inhibitory activities of the water extracts of 498 different plant foodstuffs on biofilm formation by Streptococcus mutans were measured; five of the extracts showed potent inhibitory activities. The method proposed here circumvents the tedious and time-consuming conventional method in which biofilms are cultivated on the bottom of wells of microtiter plates.