Live deadtm baclighttm bacterial viability kit

Poly-L-lysine (PLL) (average molecular weight: 4700) was kindly provided by JNC corporation (Tokyo, Japan) as a gift. Sodium chloride and L-cysteine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bacto^TM Lactobacilli MRS Broth (MRS) and agar were purchased from BD biosciences (San Jose, CA, USA). Bromocresol purple, ciprofloxacin and LIVE/DEAD^TM Baclight^TM Bacterial Viability Kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Flamma^® 648 NHS ester was purchased from BioActs (Incheon, Korea). All other materials and solvents used were of the highest analytical grade.

Carvedilol (PHR1265) and ciprofloxacin (17850) were purchased from Sigma-Aldrich (Poznań, Poland). Cation-adjusted Mueller Hinton II Broth (MHB) was obtained from Becton Dickinson (Warsaw, Poland). Fluo 3-AM and a LIVE/DEADTM BacLightTM Bacterial Viability Kit were purchased from Thermo Fisher Scientific (Warsaw, Poland). Glutaraldehyde and osmium tetroxide were obtained from Agar Scientific (Stansted, UK). All of the other reagents with a high analytical purity grade were obtained from Sigma-Aldrich (Poznań, Poland).

Chamber slides were inoculated with bacterial suspension (S. suis alone, A. pleuropneumoniae alone and combination in 1:1 ratio) for 24 h at 37°C. The slides were washed twice with PBS to remove the medium and unattached bacteria. Samples was stained with SYTO 9 solution following the manufacturer’s instructions from LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit (Thermo Fischer Scientific, Inc., Waltham, MA, United States), and then washed with PBS (Tawakoli et al., 2013 (link)). Biofilms were observed using a Zeiss LSM800 CLSM (Carl Zeiss, Jena, Germany).

S. aureus strain NCTC 8325 was obtained from Public Health England. Tryptone soya broth (TSB), tryptone soya agar (TSA), Ciprofloxacin 98%, D(-)-aspartic acid 99 + %, D(-)-glutamic acid 99 + % were all obtained from Fisher Scientific whereas L-aspartic acid 98 + % and L-glutamic acid 99% were obtained from Sigma Aldrich. Crystal violet (CV) was obtained from Sigma Aldrich. Phosphate buffered saline (PBS) tablets and LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit were obtained from Thermofisher Scientific.

Live-dead staining was performed on 3 samples per group and time point. For fluorescent sample staining, LIVE/DEADTM BacLight^TM Bacterial Viability Kit (Thermo Fisher Scientific, Wesel, Germany) was utilized, and photographs were then taken (ColourView III, Olympus Europa GmbH, Hamburg, Germany) using a stereomicroscope (SZ61, Olympus Europa GmbH, Hamburg, Germany).

Cell viability analysis of C. albicans was performed using confocal laser scanning microscopy (Lsm 800 with Airyscan with GaAsp detector, Carls Zeiss, Germany). After the 48-hour adhesion and biofilm phases, samples randomly assigned to this assay were washed once in PBS and stained with the Live/Dead^TM BacLight^TM Bacterial Viability Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing SYTO-9 and propidium iodide (excitation/emission at 488/400–540 nm and 488/600–700 nm, respectively). For all images, 3% power and pinhole opening of 60μm were used. The resin discs were immersed in 1.5 mL of the dye solution in sterile 12-well plates and incubated in the dark for 45 minutes, as instructed by the manufacturer. After the incubation period, the specimens were washed once in PBS to be observed under confocal laser scanning microscopy. Fluorescence measurement was performed by capturing five images in random fields per disc, totaling ten images per group. Biofilm thickness was determined with z-stack readings at 1 μm [37 (link)].

S. pneumoniae was treated with different concentrations of shionone (0, 4, and 8 μg/mL) for 6 h, and then 500 μL of the bacterial suspension was collected at 10,000× g for 2 min. After washing with sterile PBS 3 times, the pellet was suspended in 500 μL PBS. According to the instructions of the LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit (Invitrogen, Waltham, MA, USA), the samples were mixed with 1.5 μL working regent and then incubated in the dark for 15 min. Then, 5 μL bacterial suspension samples were dropped on a slide for imaging with a fluorescence microscope (Fv1000, Olympus).