EbbaBiolight Protocols
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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Protocol II: Optotracing of biofilm in tissue sections using EbbaBiolight 680
This protocol describes how to stain for the biofilm markers curli and cellulose in tissue sections. EbbaBiolight can be used to stain tissue sections prepared by the most common techniques like paraffin embedding and freezing. We recommend 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. As EbbaBiolight is only fluorescent when bound, you can consider to omit washing steps when working with sensitve tissues.
Solutions and Reagents:
EbbaBiolight is provided as 1000-fold concentrated solution. The following common reagents are required (not supplied):
- Ethanol, 95% (-20°C) or 4% PFA
- Phosphate buffered saline (PBS), pH 7.4
- Deionized water
- Glass coverslips
- Mounting medium
Assay Procedure:
- Fix infected cells or tissue sections with method of choice. We recommend fixation with ice-cold ethanol (5 min) at room temperature.
- Rehydrate tissue sections in a mix of ethanol and deionized water (1:1) for 5 min. The rehydration step may need to be repeated with lower ethanol ratio depending on the tissue.
- Equilibrate sections in PBS for 5 min.
- Dilute EbbaBiolight in PBS 1:1000.
- Apply diluted EbbaBiolight generously. Use enough liquid (ca 0.5 ml) to prevent the sections from drying out during incubation. Incubate for 30 min.
- Wash 2 x 5 min in PBS (Optional).
- Mount tissue sections 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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Protocol III: Biofilm tracking in live cultures using EbbaBiolight 680
This protocol describes how to trace the formation of biofilm in bacterial cultures using EbbaBiolight 680 which binds to the biofilm markers curli and cellulose. As EbbaBiolight 680 does not influence biofilm formation when used in recommended concentrations, it can be present in growing cultures. We have tested EbbaBiolight 680 for tracing biofilm produced by Salmonella Enteritidis and Salmonella Typhimurium during growth. For these strains we have not observed staining of intracellular or membrane components. Please make sure that the pH remains constant during your growth experiment. EbbaBiolight 680 works best at pH 7.4
Solutions and Reagents:
EbbaBiolight 680 is provided as 1000-fold concentrated solution. The following common reagents are required (not supplied):
- Growth medium
- Phosphate buffered saline (PBS), pH 7.4
- 96-well plate (round bottom) with cover
- Deionized water
- Spectrophotometer
Assay Procedure:
- Dilute EbbaBiolight 680 in growth medium 1:1000
- Inoculate supplemented growth medium with bacterial culture.
- Fill the wells of the 96-well plate with 100 μl inoculated medium.
- Fill unused wells with sterile water to avoid drying during incubation.
- Seal the plate with a cover or adhesive seal.
- To maximize the temporal resolution when monitoring the biofilm formation, we recommend to incubate the 96-well plate directly in the spectrophotometer. Alternatively, position the 96-well plate in a standard incubator and move it to the spectrophotometer at regular time intervals for recording.
Spectrophotometer Settings:
- EbbaBiolight 680: Excite at 540 nm and collect emission at 680 nm. Optional: Record an emission spectrum (560 - 800 nm) with 540 nm excitation.
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Protocol IV: Biofilm quantification in colony re-suspensions using EbbaBiolight 680
This protocol describes how to quantify the biofilm markers curli and cellulose in colony re-suspensions using EbbaBiolight 680. 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):
- Agar plates
- Phosphate buffered saline (PBS), pH 7.4
- 96-well plate (round bottom)
- Spectrophotometer
Assay Procedure:
- Grow bacterial colonies on an agar plates under biofilm forming conditions. Notice: no morphotyping is required for this procedure.
- Dilute EbbaBiolight 680™ in PBS 1:1000
- Add 100 μl into each well of a 96-well plate.
- Pick bacterial colonies from the agar plate and resuspend thoroughly into each of the pre-filled wells.
- Place the plate in a spectrophotometer to quantify biofilm in the colony resuspensions.
Spectrophotometer Settings:
- EbbaBiolight 680: Excite at 540 nm and collect emission at 680 nm. Optional: Record an emission spectrum (560 - 800 nm) with 540 nm excitation.