Anti-biofilm Effects of Oleuropein

One millilitre of buffered RPMI-1640 with 0.1% DMSO containing an MIC/2 concentration of oleuropein was added to each well of the sterile 24-well plates, along with 1 ml of prepared microbial inoculum. In the next well, a mixture of culture medium and yeast or bacteria suspension was used instead of the oleuropein solution. Also, a suspension of C. albicans and E. coli (1: 1 ratio) was used to observe the effect of oleuropein in the biofilm of the microbial mixture according to the above method. The biofilm formed on very thin PVC slides measuring 7 mm which were placed inside the wells and incubated for 48 h at 37 °C.
After incubation, each slide was removed and washed with PBS, followed by fixation with 2.5% glutaraldehyde for 2 h at 4 °C. The samples were then dehydrated using alcohol concentrations of 30, 70, 80, 90, 95 and 99%. After coating the samples with a layer of gold, the three-dimensional structure of the samples was imaged using a JEOL JSM-840 scanning electron microscope [41 (link), 44 (link)].