50 ml reaction tube

Plasma preserves and buffy coats from anonymous donors were obtained from the DRK-Blutspendedienst West (Hagen, Germany). First, the buffy coat was transferred to a 50 ml reaction tube (Sarstedt AG & Co., Nürnbrecht, Germany) and centrifuged at 3000 rpm for 30 min at room temperature (RT) to separate remaining erythrocytes. The resulting supernatant of blood plasma and layer of immunocompetent cells was collected and added to the plasma preserve of the same donor in a sterile glass bottle. The resulting mixture of plasma and buffy coat was gently mixed and continuously shaken at 100 rpm at 22 °C. Subsequently, a bacterial solution of the individual test pathogen was prepared by colony-picking and the solution adjusted to a 0.1 McFarland standard. For final preparation a corresponding amount of prepared bacterial solution was added to the plasma/buffy coat mixture to result in initial pathogen concentration of 2*10⁶ cfu per 3 ml plasma solution, creating a master mixture of plasma, buffy coat and pathogen. To induce fibrin polymerization (for disc-formation), 18.3 µl CaCl₂ (500 mM) per ml plasma was added to the master mixture. Finally, to prepare individual hpBIOMs, 3 ml master mixture per well were immediately transferred into 6-well culture plates (Sarstedt AG & Co., Nürnbrecht, Germany). The plates were incubated for at least 12 h on a rotation shaker at 50 rpm at 37 °C.

Biofilms were collected by swab method (Copan Diagnostics Inc., Murrieta, CA, USA) from standard household siphons (bathroom sink, kitchen sink, shower 1, and shower 2) in Kleve, Germany. The biofilm was transferred into a 50 mL reaction tube (Sarstedt AG & Co. KG., Nümbrecht, Germany) containing 25 mL of PBS solution (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). After vigorous vortexing for 2 min, the suspension was mixed 1/1 with 80% glycerol. The biofilm suspension was homogenized by end-over-end rotation (IKA-Werke GmbH & Co. KG, Staufen, Germany) for 5 min. Additionally, 1 mL aliquots were prepared and stored at −80 °C.