In Vitro Biofilm Synthesis by Staphylococcus aureus

Begin by obtaining isolated colonies of Staphylococcus aureus from a cryopreserved stock using a streak plate technique on a nutrient-rich agar plate such as tryptic soy agar. Coat individual wells of a 96-well plate with 100 microliters of PLL diluted in sterile water, and incubate at room temperature for 30 minutes. Aspirate the PLL solution aseptically, using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature.

Prepare an overnight culture by inoculating a colony of S. aureus in MEM-alpha supplemented with 2% glucose, and incubate it at 37 degrees Celsius for 16 to 18 hours at 200 rotations per minute. Dilute the overnight culture by transferring 50 microliters to 5 milliliters of fresh MEM-alpha supplemented with 2% glucose. Then, incubate it at 37 degrees Celsius at 200 rotations per minute until mid-logarithmic phase is achieved. Use MEM-alpha to normalize mid-logarithmic culture to an OD of 0.1.

Transfer 150 microliters of normalized culture to each well of the PLL-treated 96-well plate. Incubate the plate in a humidified chamber at 37 degrees Celsius for 18 to 20 hours. Aspirate the supernatant to remove the planktonic cells. Gently, wash the remaining biomass with 150 microliters of HBSS to remove the unattached cells. Repeat at least twice to remove all the planktonic cells.