Live dead bacterial viability kit

The Live/Dead Bacterial Viability Kit (Molecular Probes, Invitrogen, USA) was used to stain biofilm-coated specimen for 15 min (n = 1 for each group). Specifically, the live bacteria could be penetrated by SYTO-9 to emit green fluorescence at 488 nm wavelengths, whereas the dead bacteria with damaged membrane could be stained by propidium iodide (PI) to emit red fluorescence at 568^{23 (link)}. The specimens were rinsed gently and observed via a confocal laser scanning microscope (CLSM; Fluoview FV1200, Olympus, Tokyo, Japan) at 40 × magnification. A series scanning at a Z-stack of 2 μm was performed to produce ten images from the adhesive surface to the top of the biofilm. BioImageL software (Faculty of Odontology, Malmö University, Malmö, Sweden) was used to produce 2D overlay images of CLSM along the Z-stack, the total biomass and live/dead bacterial distributions at each scanning layer were also analyzed.

Fluorescence microscopy via live/dead assay was used to directly visualize the early bacterial attachment (4 h after inoculum) over the studied materials. A dental plaque microcosm biofilm model using human saliva was used to promote the biofilm grown over the composites, according to a previous report [33 (link)]. The biofilms on the disks were gently washed three times with phosphate-buffered saline (PBS), and then stained using a live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA). Live bacteria were stained with Syto 9 to produce a green fluorescence, and bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence [19 (link)]. The corresponding images were acquired using appropriate selective filters in the epifluorescence microscope (TE2000-S, Nikon, Melville, NY, USA). The area of green staining (live bacteria) was computed with NIS-Elements imaging software (Nikon, Melville, NY, USA). The area fraction of live bacteria was calculated based on green staining area/total area of the image.

Methicillin-sensitive Staphylococcus aureus (MSSA; strain ATCC 29213) and methicillin-resistant Staphylococcus aureus (MRSA; strain ATCC 43300) were obtained from the American Type Culture Collection. LB broth and LB agar were purchased from Sangon Biotech (Shanghai, China). Distilled deionized water was obtained from a Millipore purification system (MilliporeSigma, USA). EM was acquired from Meilun Biotech (Dalian, China). Crystal violet was bought from Beyotime (Shanghai, China). The LIVE/DEAD bacterial viability kit was procured from Invitrogen Molecular Probes (Eugene, OR, USA).
The LFLIU instrument (Nu-Tek Soni-Stim pro UT1021), with adjustable dual frequencies (1 MHz and 3 MHz), effective radiation area of 5.0 cm², and adjustable output power of 0.1 to 3 W/cm², was purchased from Kang Zan Medical Device Technology Co., Ltd. The OD values were measured using an M5 multifunctional microplate reader (SpectraMax M5; Molecular Devices, USA), fluorescence photos were taken using an SP8 Lightning confocal microscope (Leica, Germany), and the microstructure of bacteria was photographed using a transmission electron microscope (TEM) (Hitachi H-7650).

The viability of the adhered bacteria was determined by staining using a LIVE/DEAD bacterial viability kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s protocols [10 (link),11 (link)]. Equal volumes of SYTO 9 dye and propidium iodide (which stain live and dead bacteria, respectively) from the kit were mixed thoroughly. Subsequently, 3 µL of the mixture was added to 1 mL of the bacterial suspensions prepared as described above. After 15 min of incubation at room temperature in the dark, the stained samples were examined by confocal laser microscopy (CLSM, LSM880; Carl Zeiss, Thornwood, NY, USA). Live bacteria appeared green, while dead bacteria appeared red.

After performing the process described in sub-section “Bacterial attachment to the specimen”, three specimens from each group were stained using a live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA). Equal volumes of propidium iodide and Syto 9 dye, which stain live and dead bacteria, respectively, were thoroughly mixed. Then, 1 mL of PBS was added to 3 µL of the propidium iodide and Syto 9 dye mixture in which each specimen attached with S. mutans was immersed for 20 min at 37 ± 1 °C in the dark environment. The stained S. mutans on the experimental resin surface was observed using a confocal laser microscope (LSM700, Carl Zeiss, Thornwood, NY, USA). Live bacteria appear green, whereas dead bacteria appear red.