Protocol detail

Biofilm Imaging and Analysis

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Biofilms were grown in μ-Slide slides (Ibidi, Gräfelfing, Germany) inoculated with ∼1 × 10⁵ cells in 500 μL of fresh BHI medium and incubated for 48 h at 37°C. The culture medium was changed after 24 h of biofilm growth. Biofilms were stained using the Live/Dead BacLight bacterial viability kit (Life Technologies, New York, NY, USA) and/or TOTO-1 iodide staining (Thermo Fisher Scientific, catalog [cat.] no. T3600; dilution, 1:1,000) for detection of free eDNA surrounding living and dead cells (85 (link), 86 (link)) and examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Data were analyzed with the ZEN 3.1 (blue edition) software (Zeiss).

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Sivori F., Cavallo I., Kovacs D., Guembe M., Sperduti I., Truglio M., Pasqua M., Prignano G., Mastrofrancesco A., Toma L., Pimpinelli F., Morrone A., Ensoli F, & Di Domenico E.G. (2022). Role of Extracellular DNA in Dalbavancin Activity against Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms in Patients with Skin and Soft Tissue Infections. Microbiology Spectrum, 10(2), e00351-22.

Publication 2022

μ-Slide slides Live/Dead BacLight bacterial viability kit TOTO-1 iodide staining Apotome system Axio Observer inverted fluorescence microscope ZEN 3.1 (blue edition) software

Bacterial viability Biofilms Cells Culture medium Dilution Edna Fluorescence microscope Iodide Toto 1

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Variable analysis

independent variables

Inoculation of μ-Slide slides with ∼1 × 10^5 cells
Incubation time (48 h)
Incubation temperature (37°C)
Change of culture medium after 24 h of biofilm growth

dependent variables

Biofilm formation
Bacterial viability (live/dead cells)
Extracellular DNA (eDNA) surrounding living and dead cells

control variables

Biofilm growth medium (BHI medium)
Slide type (μ-Slide slides from Ibidi)
Staining methods (Live/Dead BacLight bacterial viability kit and TOTO-1 iodide staining)
Microscopy system (Apotome system connected to Axio Observer inverted fluorescence microscope)
Image analysis software (ZEN 3.1 (blue edition))

controls

No explicit mention of positive or negative controls

Annotations

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