In Vitro Biofilm Synthesis by Staphylococcus aureus

1. Preparation of in vitro biofilm

1. Obtain isolated colonies of S. aureus from a cryopreserved stock using a streak-plate technique on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials).
2. Coat individual wells of a 96-well plate with 100 µL of 0.001% (v/v) poly-L-Lysine (PLL) diluted in sterile H₂O and incubate at room temperature for 30 min. Aseptically, aspirate the PLL solution using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature.
   NOTE: All aspiration steps in the protocol are performed using a vacuum-assisted aspiration trap unless otherwise stated.
3. Prepare an overnight culture by inoculating a colony of S. aureus in minimal essential media alpha (MEMα) supplemented with 2% glucose and incubate at 37 °C, shaking at 200 rpm for 16-18 h.
4. Dilute the overnight culture by transferring 50 µL to 5 mL of fresh MEMα supplemented with 2% glucose and incubate at 37 °C, shaking at 200 rpm, until mid-logarithmic phase, generally between optical density 600 (OD_600nm) of 0.5-0.8. Use MEMα to normalize mid-logarithmic culture to an OD_600nm of 0.1.
5. Transfer 150 µL of normalized culture to each well of the PLL-treated 96-well plate. Incubate statically for 18-20 h in a humidified chamber at 37 °C.
   NOTE: The biofilms can also be grown in other formats such as µ-channel slides (see Table of Materials).
6. Aspirate the supernatant to remove the planktonic cells. Gently wash the remaining biomass with 150 µL of Hanks' Balanced Salt Solution (HBSS) to remove the unattached cells. Add HBSS dropwise to avoid disrupting the biofilm.
   NOTE: While aspirating the supernatant and HBSS during washes, leave just enough liquid (supernatant or HBSS) in the wells containing biofilm such that the biofilm is still immersed. This prevents the disruption of the biofilm structure when HBSS is added dropwise to wash the biofilm.
7. Repeat step 1.6 at least two more times to remove all the planktonic cells. At this point, biofilms are ready for immediate downstream experiments.
   NOTE: If the biofilms are not used for neutrophil experiments, HBSS can be substituted with phosphate-buffered saline (PBS). HBSS is preferred over PBS as HBSS contains components, including glucose, that provide optimum conditions for neutrophil activation.