L13152 live dead baclight bacterial viability kit

Following incubation of the scaffolds with bacteria overnight, scaffolds were washed with saline, incubated for 15 min in a solution containing propidium iodide and Syto9 (L13152 LIVE/DEAD^® BacLight™ Bacterial Viability Kit, Molecular Probes, OR, USA) and washed with saline again. Fluorescence emission was detected using an ECLIPSE E600 fluorescent microscope (Nikon, Japan). Results displayed are representative of three independent experiments conducted.

E. coli bacteria (ATCC 25922) were grown overnight in M9 minimal media and diluted 1000-fold in M9 and grown for 5 h at 37 °C. Following nanostructure formation treatment 500 µl of each test compound diluted to 0.5 mg/ml in M9 minimal media was added to 500 µl of growth medium containing the bacteria (5 × 10⁶ CFU/ml). At each time point (initial incubation, 1 and 24 h) samples were taken and washed with saline, incubated for 15 min in a solution containing propidium iodide and Syto9 (L13152 LIVE/DEAD^® BacLight™ Bacterial Viability Kit, Molecular Probes, OR, USA) and washed with saline again. Fluorescence emission was detected using an ECLIPSE E600 fluorescent microscope (Nikon, Japan).

Medical grade silicone sheets (thickness:
1 mm) were purchased from Goodfellow, Ltd. (Cambridge, UK), and cut
into disks with a diameter of 1 cm. All-silicone urinary catheters
were purchased from Bard Ltd. (West Sussex, U.K.) and cut into segments
1 cm long. LIVE/DEAD BacLight Bacterial Viability Kit L13152, Alexa
Fluor 488 Phalloidin, and 4′,6-diamidino-2-phenylindole, dihydrochloride
(DAPI) were purchased from Thermo Fisher Scientific (Paisley, U.K.). Escherichia coli ATCC 25922 (E. coli) and Proteus mirabilis ATCC 51286 (P. mirabilis) were obtained from the American Type Culture Collection (ATCC, Buckinghamshire,
U.K.). Low swarm agar (LSW) and Mueller Hinton Agar (MHA) were purchased
from Oxoid Ltd. (Hampshire, U.K.). Other chemicals used in this study
were purchased from Merck Life Science UK, Ltd. (Dorset, U.K.) and
used without further purification.

Medical-grade unplasticized PVC sheets were
purchased from Goodfellow Cambridge Ltd. (Huntingdon, UK). Acetic
acid glacial was purchased from VWR Chemicals (Lutterworth, UK). The
SYLGARD 184 elastomer kit was purchased from Dow Corning Corporation
(Midland, UK). Kollidon 90F was purchased from BASF (Ludwigshafen,
Germany). Escherichia coli (E. coli, ATCC 25922) and Staphylococcus
aureus (ATCC 29213) were obtained from the American
Type Culture Collection (ATCC, Buckinghamshire, UK). The LIVE/DEAD
BacLight Bacterial Viability Kit L13152 was purchased from Thermo
Fisher Scientific (Paisley, UK). Other chemicals used in this study
were purchased from Merck Life Science UK Ltd. (Dorset, UK) without
further purification.

Biofilm formation of S. suis HA9801 under sub-concentration of Amoxicillin (0.078125 μg/mL) and Tylosin (0.3125 μg/mL) was evaluated using live/dead bacterial viability kits (LIVE/DEAD BacLight Bacterial Viability Kit L13152, Thermo Fisher Scientific, Beijing, China). Biofilms of S. suis HA9801 were cultured according to method 2.3, and cell slides were placed in 6-well plates and incubated at 37 °C for 24 h. The biofilm attached to the cell slides was gently washed with sterile PBS, then stained with a backlight live/dead viability kit consisting of Syto9 and propidium iodide (PI). Following staining, they were washed with distilled water and dried at room temperature. The samples were observed by laser confocal scanning microscope (Zeiss LSM CLSM); 488 nm laser excitation shows a green fluorescence emission of live bacteria, while 543 nm laser excitation excites a red fluorescence emission of bacteria whose membrane was damaged.