2800 ultrasonic cleaner

Particle size, zeta potential, and polydispersity index (PDI) were obtained using a Malvern Zetasizer NanoZS (Malvern Instruments Inc., Worcestershire, UK) at 25°C with a 633-nm laser and measurement angle of 173° for 120 continuous accumulation times. Chitosan nanoparticles (200 μg) were suspended in 2 mL of 0.2 μm-filtered deionized water and sonicated (Branson 2800 ultrasonic cleaner, 60 W, Danbury, CT, USA) for 15 min before analysis.
The morphology of the nanoparticles was analyzed using a Scanning Electron Microscopy (SEM, JEOL JCM-5700, Peabody, MA, USA) at 5 kV and magnifications ranging from 30 to 10,000x. Dried nanoparticle samples were adhered to a conductive carbon tape with a thin aluminum foil core (Nisshin EM Co., Ltd, Japan). Subsequently, samples were mounted on specimen stubs and coated with a thin film (< 20 mm) of gold and platinum layer using a sputter coater (Denton Desk V, Denton Vacuum, Moorestown, NJ, USA).

Six pieces of 5.5 × 6 cm and average weight of 0.13 ± 0.03 g of PCL membranes were placed in 50 mL flasks with 30 mL of a L. sphaericus CBAM5 suspension with a concentration of 1.02 × 10⁹ CFU/mL in saline solution (0.85%). The flasks were incubated at 30 °C, 150 rpm for 5 h. The membranes were then washed twice with sterile saline solution (0.85%) to remove non-immobilized cells.
Bacterial immobilization was confirmed by scanning electron microscopy (SEM) analysis and a detachment protocol was used to estimate the bacterial concentration immobilized in the membranes.
The detachment protocol was made as previously reported [11 (link)]. Firstly, three membranes were transferred to flasks containing 15 mL of sterile saline solution (0.85%) and these were vortexed for 30 s. Then, sonication was performed at 40 kHz and 4 °C in a Branson 2800 Ultrasonic cleaner during 2 cycles of 1 min sonication and 30 s rest for a total of 10 min of sonication. After sonication, the membranes were vortexed for 1 min and between each cycle, fresh saline solution (0.85%) was used. Standard serial 10-fold dilutions were inoculated (10 µL) in nutrient agar and incubated for 24 h at 30 °C for the drop plate count method [19 ].