Micro centrifuge tube

An overnight (18 h) culture of P. aeruginosa prepared as described above was diluted 1:100 in fresh LB broth and 100 μl of the dilution was added to each well of a 96-well polystyrene microtiter plate with a flat bottom (pureGrade^TM S, Brand, Germany). Uninoculated LB broth was used as a sterile control.
After 24 h, the planktonic bacteria (90 μl) were removed by pipetting and were transferred to a microcentrifuge tube (Sarstedt, 1.5 ml). An aliquot of 65 μl was removed and transferred to a new microcentrifuge tube. Planktonic bacteria were harvested by centrifugation (10 min, 10477 × g, 18°C), washed twice in 200 μL deionized water, and finally resuspended in the equivalent test substance. In parallel the biofilms attached to the walls of the well were washed twice with 200 μl of deionized water. From a second microtiter plate the washed biofilms were suspended by scraping the biofilm off the well surface using the equivalent test substance. Planktonic bacteria, attached biofilms, and suspended biofilms were exposed to AgNO₃ (100 μg/ml Ag), AgNPs (500 μg/ml), and deionized water as a control. The microtiter plates were statically incubated at 36°C for 24 h. Silver was neutralized using sodium thioglycolate (0.1%) and sodium thiosulfate (0.14%).

Genomic bacterial DNA was extracted using a GenoLyse^® kit (Hain Lifescience GmbH, Nehren, Germany) from mycobacteria growth indicator tube liquid media by transferring 1.0 mL to a Sarstedt micro-centrifuge tube. The tube was centrifuged for 15 minutes at 14 000 rpm. The supernatant was carefully removed and the pellet was re-suspended in 100 µL lysis buffer, vortexed thoroughly and incubated for five minutes at 95°C in a heating block. At the end of the five-minute incubation, tubes were briefly centrifuged to remove condensation. Neutralization buffer (100 µL) was added to each tube, vortexed for five seconds and then centrifuged for five minutes at 14 000 rpm. Supernatant was transferred to a new tube and the pellet discarded. Samples were stored at -80°C for use as DNA template.

Genomic bacterial DNA was extracted from cultures at the UNC laboratory using the HAIN GenoLyse kit (Hain Lifescience GmbH, Nehren, Germany). From MGIT liquid media, 1.0 mL was transferred to a Sarstedt micro-centrifuge tube. The tube was centrifuged for 15 minutes at 14 000 rpm. The supernatant was carefully discarded and the pellet was re-suspended in 100 µL Lysis Buffer from the kit, vortexed thoroughly and incubated at 95°C in a heating block for five minutes. Tubes were removed and briefly centrifuged to remove condensation. To each tube, 100 µL of Neutralization Buffer was added, vortexed for five seconds and then centrifuged for five minutes at 14 000 rpm. Supernatant was transferred to a new tube and the pellet was discarded. The extracted DNA was stored at -80°C for use as DNA template.