Antibiofilm Activity Quantification Protocol

The quantification of antibiofilm activity was based on the research by Waturangi et al. [4 (link)] with some modifications. The activities of antibiofilm were divided into inhibition and destruction activity. For inhibition activity, B. cereus and B. subtilis inoculated into Brain Heart Infusion Broth (BHIB) and incubated at 37 °C, 120 rpm 24 h, whereas S. putrefaciens inoculated into BHIB and incubated at 28 °C, 120 rpm 24 h. A total of 100 μL cultures (OD₆₀₀ = 0.132) and 100 μL crude extract were transferred into a 96-wells microplate and incubated at 37 °C 24 h for B. cereus and B. subtilis and 28 °C for S. putrefaciens. For destruction activity, a total of 100 μL cultures (OD₆₀₀ = 0.132) were transferred into a 96-wells microplate and incubated to form biofilms. After 24 h, 100 μL crude extract was added to each well then reincubated. Overnight culture of food spoilage bacteria without any treatment were used as a positive control, while sterile BHIB was used as a negative control.
After incubation, the planktonic cells and media were discarded and the adherent cells were rinsed twice with deionized water and allowed to air dry. A total of 200 μL of 0.4% crystal violet was used to stain the adherent cells for 30 min. Thereafter, the dye was discarded and the wells were rinsed five times with deionized water and allowed to air dry. Afterward, 200 μL of ethanol was added to solubilize the crystal violet. The optical density was determined at 595 nm with a microplate reader (TECAN M200 PRO). The percentage of inhibition or destruction activity calculated using the following equation:
$$\%\text{inhibition or destruction}=\frac{\mathrm{OD}\text{positive control}-\mathrm{OD}\text{sample}}{\mathrm{OD}\text{positive control}}\mathrm{x}100\%$$