Cultivation and Biofilm Formation of Bacterial Strains

The aerobic bacterial strains (S. aureus, S. epidermidis, E. coli, and P. aeruginosa) obtained from the DSMZ were starved as equivalents of 0.5 mL by freezing at −80 °C and re-cultivated on the LB-medium for further investigations at 37 °C in Erlenmeyer flasks by continuously shaking in a laboratory shaker for an oxygen supply for 12 to 24 h depending on the microorganism. The pH value was 7.0 ± 0.2. The anaerobic strain A. denticolens obtained from the DSMZ was starved as equivalent of 0.5 mL by freezing at −80 °C and re-cultivated on a modified PYG-Medium at pH 7.2 (see DSMZ medium No. 104, where the soluble oxygen was displaced by gassing with a mixture of 80% H₂ and 20% CO₂ for 30 min) for further investigations at 37 °C using an anaerobic jar with an atmosphere of 80% H₂ and 20% CO₂ for 24 h.
The bacterial growth was monitored by measuring the optical density (OD) at 600 nm as well as counting the colony forming units (cfu) on appropriate agar plates. The microorganisms were harvested using centrifugation and suspended in a fresh growth medium in micro-titer plates with sterilized apatite samples for biofilm formation, with an incubation temperature of 37 °C.