LIVE/DEAD™ BacLight™ Bacterial Viability Kit, for microscopy

Protocol tips

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	Add 3 µL of the dye mixture for each mL of the bacterial suspension. Mix thoroughly and incubate at room temperature in the dark for 15 minutes

Protocol tips
Add 3 µL of the dye mixture for each mL of the bacterial suspension. Mix thoroughly and incubate at room temperature in the dark for 15 minutes

Publication protocol

The bactericidal effect of photocatalytic treatment as a function of UV-A dose was evaluated with CFU counting, metabolic activity assays and Live/Dead staining combined with fluorescent intensity measurements. For antibacterial tests with both planktonic S. epidermidis and S. mutans, 10 μL of bacterial suspension (bacterial population ~107) was spread on each NP adhesive disk using a pipette tip. The disks with bacteria were irradiated with a high-power UV-A diode (λ = 365 nm, NSCU033B(T), Nichia, Japan). A collimating lens ensured an even UV-A light intensity of 15 mW/cm2 over the irradiated area (UV light meter, UV-340, Lutron), and the treatment times were varied to provide UV-A doses ranging from 0 to 13.6 J/cm2. The 0 J/cm2 dose refers to control disks that were not exposed to UV-A light and were included to provide a reference level for determining the log reduction in viability of the samples subjected to UV-A irradiation. Four disks at each UV-A dose and for each bacteria strain were irradiated. The disks were inspected for moisture loss on the surface so that any bactericidal effect due to desiccation would be minimized. After photocatalytic treatment, each disk was immediately put into a well in a 48-well plate containing 100 μL of sterile water. The 48-well plate was then fixed to an incubating orbital shaker (Talboys, Troemner, USA) and shaken at 500 rpm for 2 min to re-suspend the bacteria from the disk surfaces. The sample disks were removed from the wells and bacterial viability was subsequently analyzed.

From the 100 μL of bacteria suspension of S. epidermidis after each test, 10 μL was taken for CFU counting, 10 μL for the metabolic assay incorporating resazurin and 50 μL for fluorescence intensity measurements following Live/Dead staining. From the 100 μL of bacteria suspension of S. mutans after each test, 10 μL was taken for the metabolic assay incorporating resazurin, 10 μL for the metabolic assay incorporating phenol red and 50 μL for fluorescence intensity measurements following Live/Dead staining.

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Manufacturer protocol

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