Monitoring curli production in liquid culture using EbbaBiolight

This protocol describes how to monitor kinetics of Salmonella extracellular matrix (curli) production in liquid culture. 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 described, EbbaBiolight does not label Salmonella cell wall and does not influence biofilm formation.

Materials:

• EbbaBiolight
• Growth medium
• Bacteria on standard culture plate
• 96-well plate (round bottom) with cover
• Deionized water

Equipment:

• Incubator (28°C)
• Shaking Incubator (37°C)
• Fluorescence plate reader

Assay Procedure:

• Prepare bacterial inoculum:
• Pick a colony from a standard culture plate.
• Transfer colony to growth medium.
• Prepare an overnight or exponential culture under continuous shaking at 37°C.
• Dilute bacterial culture 1:100 in fresh growth medium.
• Add EbbaBiolight (1:1000) and mix gently.
• Prepare 96-well plate:
• Use 100-200 µl liquid culture supplemented with EbbaBiolight per well.
• Fill unused wells with sterile water to avoid drying during incubation.
• Seal the plate with a cover.
• Incubation & Readout:
• incubate the 96-well plate directly in the plate reader under static conditions.
• Record excitation- and emission at regular time points during biofilm growth.