R2a agar

Washed biofilm (10 mg) was added to 1 ml of pharmaceutical grade distilled water in an eppendorf tube. Thereafter, 1 g of acid washed, sterile 2 mm glass beads were added to the tube which was then vortexed at maximum speed for 2 × 5 min with intermittent cooling (2 min) on ice. This approach seemed to completely disrupt the film and free bacteria were visible in the microscope. Aliquots (0.1 ml) were spread on R₂A-agar (Oxoid, Thermo Scientific, UK) to obtain the total mesophilic heterotrophic bacterial plate count and Rose Bengal Chloramphenicol agar (Oxoid) to obtain the total mesophilic heterotrophic fungal plate count. Plates of each agar type were incubated at 22 ± 2 °C (7 days). An additional R₂A plate was incubated at 55 ± 1 °C for 48 h to detect thermophiles. Samples were also spread on sheep blood agar plates (Oxoid) for the detection of rapidly growing strains of potential clinical interest. Plates were examined for colony counts and types after aerobic and anaerobic incubation at 37 ± 1 °C (48 h).
Free-living protozoa were detected as previously described.⁷ (link) In brief, washed biofilm pieces (10 mg) were added to non-nutrient agar seeded with heat-killed Escherichia coli. Plates were incubated at two different temperatures, 22 ± 2 °C and 37 ± 1 °C, and examined for the presence of protozoa and protozoal cysts over a period of 7 days.

Culturability was assessed for 24 and 48 h old biofilms. Colonized coupons were washed in 1 mL of STW in new 24 wells microtiter plates in order to remove weakly or non-adhered bacteria. Each coupon was inserted in a 50 mL centrifuge tube containing 3.5 mL of saline water (8.5 g L⁻¹ of NaCl) after being scrapped with a micropipette tip while 1 mL of saline water was dispensed on the coupon to help the removal of the scrapped cells.^{32 (link)} Then, 0.5 mL of thiosulphate (0.5% w/v) was added to the tube containing the scrapped suspension in order to neutralize the effects from the copper ions released.³⁶ Tubes containing coupons were then vortexed for 2 min to complete the removal of adhered bacteria and to dissociate possible bacterial aggregates.^{32 (link)} Serial dilutions from the obtained suspension were then performed and plated on R2A agar (Oxoid, Hampshire, UK) plates. Colony forming units (CFU) were enumerated after incubation for 48 h at 25 °C. For dual species biofilms, the number of CFU per cm² was also assessed for each bacterium. This was possible because the selected bacteria had clearly distinct colony morphologies when grown on R2A agar. The detection limit for CFU enumeration was 2 log CFU per cm².