Protocol I: Staining of surface biofilm using EbbaBiolight 680

This protocol describes how to grow biofilm at the air-liquid interface using the inclined-coverslip method and how to detect the air-surface biofilm using EbbaBiolight 680. As the optotracer does not influence biofilm formation when used in recommended concentrations, it can be present in growing cultures. It is also possible to grow the biofilm using the described method without EbbaBiolight 680, and then use EbbaBiolight 680 to visualize the grown biofilm as described in Protocol II. If necessary for imaging, we recommend light fixation in ice-cold ethanol, but fixation in 4% paraformaldehyde works as well. We have tested this procedure with Salmonella Enteritidis and Salmonella Typhimurium strains. For these strains, we have not observed staining of intracellular or membrane components.

Solutions and Reagents:

EbbaBiolight 680 is provided as 1000-fold concentrated solution. The following common reagents are required (not supplied):

  • Sterile glass coverslips (24x24 mm)
  • 6-well plate with cover or adhesive seal
  • Growth medium
  • Phosphate buffered saline (PBS) pH 7.4
  • Ethanol, 95% (-20°C) or 4% PFA
  • Mounting medium

Assay Procedure:

  • Place two sterile glass coverslips opposite to each other and inclined towards the walls of the wells in a 6-well plate.
  • Prepare your bacterial culture at appropriate bacterial density. Then, add EbbaBiolight 680 (1:1000) and mix gently before pipetting 5-7 ml of the solution into each well fitted with glass coverslips.
  • Seal the plate with a cover or adhesive seal and incubate at a temperature of choice for 24-48 h.
  • Remove the glass coverslips from each well. Wipe the backside clean and fix biofilm formed at the air-liquid interface on the glass coverslip by immersing the coverslip in cold ethanol for 4 min or PFA for 30 min.
  • Wash 3 x 1 min in PBS (Optional).
  • Mount the samples using mounting medium and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • EbbaBiolight 680 is excited at 561 nm (standard laser line) and emission is detected using a standard PI (Propodium Iodide), mCherry or Cy3.5 filter set. Optional: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets.

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