Labeling of surface biofilm using EbbaBiolight

Labeling of surface biofilm using EbbaBiolight

This protocol describes how to grow Salmonella biofilm at an air-liquid interface using inclined glass coverslips and how to visualize Salmonella extracellular matrix component curli using EbbaBiolight. The method described here is based on Choong et al. (2016) npj Biofilms and Microbiomes, 2, 16024 where isogenic mutants of S. Enteritidis were used to identify the extracellular matrix components curli and cellulose as targets for optotracer binding. When used as recommended, EbbaBiolight does not label Salmonella cell wall and does not influence biofilm formation. If adding EbbaBiolight during biofilm growth is not feasible, it can also be applied after the biofilm has assembled and incubated for 30-60 min.

Materials:

  • 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%

Equipment:

  • 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.
  • Incubation:
    • 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.
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