Monitoring curli in bacterial biofilms forming on semi-solid agar

Monitoring curli in bacterial biofilms forming on semi-solid agar

This protocol describes how to use EbbaBiolight to visualise curli in biofilm forming on semi-solid agar in real-time. Curli is a functional amyloid produced by many Enterobactericeae involved in adhesion to surfaces, cell aggregation, and biofilm formation. EbbaBiolight are versatile molecules that have been reported to target various structures in the cell wall of gram-positive bacteria and the extracellular matrix of gram-negative bacteria. Curli has been identified as one of the major targets for EbbaBiolight in Salmonella biofilms using wildtype bacteria as well as curli deficient (ΔcsgA) strains. For reference, see Choong et al. (2021) Biofilms, 3, 100060. We recommend using fluorescent protein tagged bacteria to be able to distinguish between bacterial cells and curli in the bacterial extracellular matrix. The method described here is a semi-high throughput approach which allows for analysis at a faster pace and larger volume compared to conventional methods.

Materials:

  • EbbaBiolight
  • LB broth (w/o salt)
  • LB agar (w/o salt)
  • 6-well plate with cover
  • Curli-producing bacteria on standard culture plate

Equipment:

  • Incubator (28°C)
  • Shaking Incubator (37°C)
  • Microwave
  • Fluorescence microscope OR Fluorescence Plate Reader

Assay Procedure:

  • Prepare plates with EbbaBiolight supplemented LB agar:
    • Microwave LB agar until it is melted.
    • Let the microwaved agar rest at RT for 5-10 min.
    • Add 2 µL/mL EbbaBiolight.
    • Pour 2 mL of supplemented LB agar per well in a 6-well plate.
    • Cover plates and let cool down at RT.
    • Optional: Store at 4°C.
  • 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.
    • Transfer 10 µl of bacterial inoculum to the centre of a dedicated well on the previously prepared 6-well plate.
  • Incubation:
    • incubate the plate in an incubator at 28°C for as long as required for the biofilm to form.
  • Endpoint analysis:
    • Analyse in fluorescent microscope for optotracer based morphotyping OR in Fluorescence Microplate Reader for curli quantification.
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