0.45 μm filter

Effluents from both WWTPs were sampled monthly for one-year period. Effluent water was refrigerated until further processed in the laboratory, usually within 6 hours of collection.
For selective isolation of carbapenem-resistant P. aeruginosa 100 ml of effluent water was filtered through 0.45 μm filter (Whatman). Filters were plated onto in-house selective medium (cetrimide agar (Merck)) supplemented with imipenem with a final concentration of 4 μg/mL) and incubated at 42°C for 48-hours.
After incubation up to ten suspected P. aeruginosa colonies were subcultured and identified by MALDI-TOF. All isolates confirmed as P. aeruginosa were submitted for identical antibiotic susceptibility testing as described above for clinical strains.
Isolates of P. aeruginosa resistant to either imipenem or meropenem or both were stored at -70°C for further processing.

The dissolution profile of LCM and prepared LCM-E100 MPs was evaluated using a USP dissolution test apparatus II (Hanson elite 8, Chatsworth, CA, USA). The number of prepared MPs corresponded to 50 mg of LCM. The formulations were suspended in 900 mL of distilled water and in a buffer solution of pH 1.2, separately [16 (link),17 ]. A paddle speed of 50 rpm and temperature of 37 ± 0.5 °C were maintained during the experiment. At predetermined time intervals (0, 5, 10, 15, 30, 45, 60, and 120 min), 3 mL of sample was collected and passed through a 0.45-μm filter (Whatman, Maidstone, UK). The aliquots were replaced with fresh dissolution medium. The concentration of LCM was analyzed by HPLC. All experiments were performed in triplicate.

Qualitative and quantitative analyses of the most promising B. hispida extract were performed using an HPLC system (Prominence LC2030C, Shimazu, Japan). A Knauer^® vertex III reversed-phase HPLC column C18 (250 mm × 20 mm) (KNAUER Wissenschaftliche Geräte GmbH, Berlin, Germany) was used as the stationary phase. A gradient system was used as an analytical method with a detector at a wavelength of 360 nm. The mobile phase consisted of A (0.1% acetic acid in water) and B (acetonitrile) with a flow rate of 1.0 mL/min for 33 min. Gradient elution of the mobile phase was carried out as follows: 0.01 min, 95% A; 2 min, 95% A; 7 min, 80% A; 22 min, 70% A; 23 min, 95% A; and 33 min, 95% A. The analytical samples were dissolved in absolute ethanol and filtered through a 0.45 μm filter (Whatman, Marlborough, MA, USA) before analysis with an injection volume of 10 μL. B. hispida extract at 1.0 mg/mL was prepared for analysis. Rutin was used as a reference compound to analyze the active compounds in B. hispida extract. The retention time of rutin was approximately 17 min. The concentration of rutin in B. hispida extract was calculated from the peak area using a linear equation constructed from a standard curve of rutin (0.0 to 50 µg/mL).

Fry mortalities and survivors from the challenged tanks and control tanks were tested for the presence of IPNV. Fry were weighed, homogenised using sterile pestle, mortar and sand then diluted 1:10 in cell culture medium. The homogenate was centrifuged at 2500 ×g for 15 min. at 4 °C then the supernatant removed and filtered through 0.45 μm filter (Whatman) before inoculation onto 24 h old confluent monolayers of CHSE-214 cells in 96-well cell culture trays for titration. Culture trays were incubated at 15 °C and titres read after 7 days. Wells showing positive cytopathic effect (CPE) for each sample were further tested by ELISA (Test-Line) to confirm the presence of IPNV. Subsequently, for the determination of viral load in the samples used for the microarray experiment, an RT-QPCR assay applied in an accredited commercial laboratory (Integrin Advanced Biosystems, UK) was used.