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.
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.