Protocol I - Labelling of surface biofilm using EbbaBiolight

This protocol describes how to grow biofilm at the air-liquid interface using inclined glass coverslips and how to detect & visualize it using EbbaBiolight. When used in recommended concentrations in Salmonella biofilms, EbbaBiolight does not influence biofilm formation [1]. Therefore we advise adding EbbaBiolight while the biofilm is growing. EbbaBiolight can also be added after the biofilm has assembled (see EbbaBiolight - Protocol II). The method described here is adapted from Choong et al. where the extracellular matrix components curli and cellulose were identified as the targets for optotracer binding. Direct labeling of Salmonella bacteria has not been observed.

Disclaimer: When adapting this technique, please make sure to include relevant controls to verify that EbbaBiolight does not affect biofilm formation, to confirm curli as EbbaBiolight binding target and to exclude pH effects. Please be aware that EbbaBiolight is suited for labelling live bacteria only and is not compatible with fixation.


  • EbbaBiolight
  • LB broth (w/o salt)
  • Bacteria on standard culture plate
  • Sterile glass coverslips (24x24 mm)
  • 6-well plate with cover or adhesive seal
  • Mounting medium
  • Nail polish
  • EtOH 70%


  • Incubator (28°C)
  • Shaking Incubator (37°C)
  • Fluorescence microscope

Assay Procedure:

Plate preparation:

  • Place two sterile glass coverslips opposite to each other and inclined towards the walls of the wells in a 6-well plate

Prepare bacterial inoculum:

  • Pick a colony from a standard culture plate
  • Transfer colony to LB broth
  • Prepare an overnight or exponential culture under continuous shaking at 37°C
  • Dilute bacterial culture 1:100 in fresh LB broth
  • Add EbbaBiolight (1:1000) and mix gently
  • Pipett 6 ml of EbbaBiolight supplemented bacterial culture into each well fitted with glass coverslips


  • Seal the plate with cover or adhesive seal & incubate at a suitable temperature for 24-48 h

Mounting & Sealing:

  • Remove the glass coverslips from each well & wipe the backside
  • Add a drop (ca 30 µl) of mounting medium to the sample and flip the coverslip on a microscope slide
  • Remove excess liquid, seal the coverslip with nail polish & clean the slide with 70% EtOH

Endpoint analysis:

  • Visualize biofilm using fluorescence microscopy. Use filter-sets or excitation- and emission parameters as indicated in the table below

EbbaBiolight Fluorescence Detection:

EbbaBiolight fluorescent tracer molecules are optotracers. Unlike conventional fluorescent dyes, optotracers bind promiscuously to a range of targets with repetitive motifs. EbbaBiolight has been shown to bind to curli and cellulose in Salmonella extracellular matrix [1,2], peptidoglycan and lipoteichoic acids in the cell envelope of Staphylococci [2], β-glucans from S. cerevisiae and Chitin in C. albicans [3]. Upon binding, the fluorescence intensity of the optotracer increases. This property makes it possible to use EbbaBiolight for live fluorescent tracking of microorganisms, without the need to wash away unbound molecules. It is possible to read out fluorescence intensity at the emission maximum (Emmax) when excited at or close to the excitation maximum (Exmax). This is useful for microscopy or fluorescence spectroscopy when straight-forward data analysis is required. Yet, due to the unique properties of the optotracers, a unique optical fingerprint is produced reflecting the specific nature of the target (sample composition) and environment (pH, osmolarity, polarity of the medium). This means that depending on the specific properties of the sample, Exmax or Emmax can shift, or the appearance of double peaks or shoulders might indicate binding to multiple targets. We therefore recommend acquiring fluorescence excitation and emission spectra whenever possible within experimental limitations.

Table: EbbaBiolight spectral properties with maximum excitation (Exmax) and emission (Emmax) when bound and recommended range for acquisition of excitation- and emission spectra as well as recommended filter sets for microscopy.
Exmax Emmax Excitation spectrum (detect at Emmax) Emission spectrum (excite at Exmax) Recommended
EbbaBiolight 480 420 nm 480 nm 300 - 450 nm 450 - 800 nm DAPI
EbbaBiolight 520 460 nm 520 nm 300 - 490 nm 490 - 800 nm FITC, GFP
EbbaBiolight 540 480 nm 540 nm 300 - 510 nm 510 - 800 nm FITC, GFP, YFP
EbbaBiolight 630 520 nm 630 nm 300 - 600 nm 550 - 800 nm PI, Cy3, TxRed, mCherry, Cy3.5
EbbaBiolight 680 630 nm 680 nm 300 - 650 nm 660 - 800 nm PI, mCherry, Cy3.5

Access EbbaBiolight spectra


  1. Choong FX et al. (2016) Real-Time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes. npj Biofilms and Microbiomes, 2, 16024
  2. Choong FX et al. (2021) A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces. Biofilm, 3, 100060
  3. Butina K. et al. (2020) Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time. npj Biofilms and Microbiomes, 6(1), 35
  4. Kärkkäinen, E. et al. (2022) Optotracing for live selective fluorescence-based detection of Candida albicans biofilms. Frontiers in Cellular and Infection Microbiology, 12, 2235-2988

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