Synergy htx elisa multi mode reader

The evaluation of the inert substrate adhesion potential of the tested strains was carried out by the crystal violet staining method. After 24 h of incubation, 96-well plates are washed thrice with phosphate buffered saline solution and fixed with cold CH₃OH (5 min). After its removal, the dried plates are stained with 1% crystal violet solution (15 min). The excess dye is removed by washing, and in order to determine the MAIC values (minimum adhesion inhibition concentration), the dye included in the cells adhered to the walls of the well is dissolved with 33% acetic acid. Spectrophotometer readings will be performed at 490 nm with the BioTek Synergy™ HTX ELISA Multi-Mode Reader (BioTek, Winooski, VT, USA) [52 ,53 (link),54 (link)].

Determination of minimum inhibitory concentration (MIC) was performed by an adapted binary serial microdilution standard method [46 (link),47 (link)] in liquid medium (NB for bacteria and YPG for yeasts), using 96-well microtiter plates. From each BPE sample, serial two-fold microdilutions were achieved in 150 μL of corresponding broth medium seeded with the standard inoculum of 1.5 × 10⁸ CFU/mL (for bacterial strains) and 3 × 10⁸ CFU/mL (for yeast strains). Due to the BPE samples containing ethanol, a control (C_Et) was performed. The negative control was considered the sterile liquid medium, and the positive control (C+) was the broth medium inoculated with microbial suspensions, following the same conditions described before. The dishes were incubated at 37 °C for 24 h for bacterial strains and 48 h for yeast strains. The MIC values were established by visual analysis and spectrophotometric measuring of absorbance at 620 nm using a BIOTEK SYNERGY-HTX ELISA multi-mode reader (Winooski, VT, USA).