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ECtracer™

General Information

ECtracer™ are fluorescent molecular tracers for detection of bacterial biofilm components. ECtracer™ allow direct visualisation of biofilm, without using antibodies or any toxic chemicals. ECtracer™ are highly fluorescent only when they are bound to their target, non-toxic, and do not interfere with bacterial growth or biofilm formation at recommended concentrations. This allows for a variety of applications: ECtracer™ can be used to stain biofilm at an air-liquid interface or used to detect biofilm in sections of infected tissues. Further, ECtracer™ can be used to quantify colony biofilm components by spectrophotometric detection of biofilm components in colony resuspensions. Strikingly, ECtracer™ can be added to a live culture to track biofilm formation in real-time using spectrophotometric analysis. Further, ECtracer™ are photo- and thermostable and allow for easy handling in any application. We offer ECtracer ex vivo products for staining of biofilm at surfaces or in tissue sections, biofilm tracking in live cultures and biofilm quantification from colony resuspensions. ECtracer™ ex vivo products are available in aliquots of 50 µl or 100 µl.

ECtracer™ Mix&Try Kit is our Test Kit for Getting Started

ECtracer™ Mix&Try Kit contains 10 µl each: the blue ECtracer™480, the yellow ECtracer™520, the orange ECtracer™630 and the red ECtracer™680. ECtracer™480 is excited between 405-458 nm and fluorescence emission occurs between 470–550 nm. ECtracer™520 is excited between 405-488 nm and emission is detected between 500-600 nm. ECtracer™630 is excited between 458-514 nm and emission is detected between 600-650 nm. ECtracer™680 is excited between 530-565 nm emission is detected between 600-800 nm. Using all these different options will allow you to select the best ECtracer™ for your experiment.

ECtracer™ Mix&Try Kit is available as ex vivo variant. Please contact us for customs options.

contact us for custom options.

ECtracer™680 is our red fluorescent tracer molecule for staining of curli and cellulose in the bacterial extracellular matrix.

ECtracer™680 fluorescence is readily visualized using standard microscopy equipment. Excitation is achieved using the 561 nm laser line, and emission can be detected at 680 nm using the standard Cy5 filter set. The optical spectrum of ECtracer™680 allows custom settings to be applied, using an excitation range of 530-565 nm and a detection range of 600-800 nm.

With exceptionally high signal-to-noise ratio and spectral properties that are clearly distinguishable from biological autofluorescence, we especially recommend ECtracer™680 for tracking of biofilm in live cultures.

ECtracer™680 is available as ex vivo variant in 50 µl and 100 µl aliquots. Please contact us for customs options.

contact us for custom options.

ECtracer™

  • are provided as ex vivo variant in volumes of 50 µl and 100 µl
  • are diluted 1000-fold
  • are non-toxic
  • are photo- and thermostable

Applications

Storage

  • Store ECtracer™ at 4°C
  • Use the opened vial within 12 month

Note

  • ECtracer™ is for research use only.
  • ECtracer™ is not for diagnostic use or use in humans.
  • ECtracer™ is not for resale.
  • ECtracer™ is a trademark belonging to Ebba Biotech AB

Download Info

Protocol I: Staining of surface biofilm at an air-liquid interface

This protocol describes how to grow biofilm at the air-liquid interface using the inclined-coverslip method. As ECtracer™ doesn't influence biofilm formation when used in recommended concentrations, it can be present in growing cultures. 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:

ECtracer™ 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 ECtracer™ (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.
  • Mount the samples using mounting medium and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • ECtracer™480 (part of ECtracer™ Mix&Try Kit): Excite at 405 nm (standard laser line) and detect emission using the DAPI or FITC filter sets. Optional: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™520 (part of ECtracer™ Mix&Try Kit): Excite at 458 or 488 nm (standard laser lines) and detect emission using a standard FITC filter set. Optional: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™630 (part of ECtracer™ Mix&Try Kit): Excite at 488 or 514 nm (standard laser lines) and detect emission using standard TRITC or TxRed filter set. Optional: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™680: Excite at 561 nm (standard laser line) and detect emission using a standard Cy5 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.

Download Protocol

Protocol II: Staining of biofilm in infected cells or tissues

This protocol describes how to stain for the biofilm markers curli and cellulose in tissue sections. ECtracer™ 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 ECtracer™ is only fluorescent when bound, you can consider to omit washing steps when working with sensitve tissues.

Solutions and Reagents:

ECtracer™ 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 ECtracer™ in PBS 1:1000.
  • Apply diluted ECtracer™ 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.
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • ECtracer™480 (part of ECtracer™ Mix&Try Kit): Excite at 405 nm (standard laser line) and detect emission using the DAPI or FITC filter sets. Optional: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™520 (part of ECtracer™ Mix&Try Kit): Excite at 458 or 488 nm (standard laser lines) and detect emission using a standard FITC filter set. Optional: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™630 (part of ECtracer™ Mix&Try Kit): Excite at 488 or 514 nm (standard laser lines) and detect emission using standard TRITC or TxRed filter set. Optional: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets.
  • ECtracer™680: Excite at 561 nm (standard laser line) and detect emission using a standard Cy5 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.

Download Protocol

Protocol III: Spectrophotometric monitoring of biofilm formation in bacterial cultures

This protocol describes how to trace the formation of biofilm in bacterial cultures using the bacterial amyloid curli and the carbohydrate cellulose as biofilm markers. As ECtracer™ doesn't influence biofilm formation when used in recommended concentrations, it can be present in growing cultures. We have tested ECtracer™ 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.

Solutions and Reagents:

ECtracer™ 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 ECtracer™ 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:

  • ECtracer™480 (part of ECtracer™ Mix&Try Kit): Excite at 430 nm and collect emission at 480 nm. Optional: Record an emission spectrum (450 - 700 nm) with 430 nm excitation.
  • ECtracer™520 (part of ECtracer™ Mix&Try Kit): Excite at 470 nm and collect emission at 530 nm. Optional: Record an emission spectrum (490 - 700 nm) with 470 nm excitation.
  • ECtracer™630 (part of ECtracer™ Mix&Try Kit): Excite at 510 nm and collect emission at 635 nm. Optional: Record an emission spectrum (530 - 800 nm) with 510 nm excitation.
  • ECtracer™680: Excite at 540 nm and collect emission at 680 nm. Optional: Record an emission spectrum (560 - 800 nm) with 540 nm excitation.

Download Protocol

Protocol IV: Spectrophotometric quantification of biofilm in colony resuspensions

This protocol describes how to quantify the biofilm markers curli and cellulose in colony re-suspensions. 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:

ECtracer™ 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 ECtracer™ 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:

  • ECtracer™480 (part of ECtracer™ Mix&Try Kit): Excite at 430 nm and collect emission at 480 nm. Optional: Record an emission spectrum (450 - 700 nm) with 430 nm excitation.
  • ECtracer™520 (part of ECtracer™ Mix&Try Kit): Excite at 470 nm and collect emission at 530 nm. Optional: Record an emission spectrum (490 - 700 nm) with 470 nm excitation.
  • ECtracer™630 (part of ECtracer™ Mix&Try Kit): Excite at 510 nm and collect emission at 635 nm. Optional: Record an emission spectrum (530 - 800 nm) with 510 nm excitation.
  • ECtracer™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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