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EbbaBiolight Mix&Try

EbbaBiolight Mix&Try Kit is our recommended option for starting out with using EbbaBiolight. It contains a sample volume (10 µL) of each variant. Testing each variant in the EbbaBiolight Mix&Try Kit will allow you to determine which one of our optotracers is best suited for your experiment.

All EbbaBiolight variants label extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Specifically, the optotracers have been used as follows: EbbaBiolight 480 labeling extracellular matrix produced by P. aeruginosa but not C. albicans in a mircofluidic device during mono- and co-culture. EbbaBiolight 630 labeling peptidoglycan and lipoteichoic acids in the cell wall of certain gram positives like Staphylococci and E. faecalis. EbbaBiolight 680 labeling curli in the extracellular matrix produced by Salmonella as well as β-glucans, chitin and intracellular amyloid aggregates in yeast- and hypal forms of C. albicans. Furthermore, it has been used to track pellicle biofilm formation in B. cenocepacia. Contact us to learn more about EbbaBiolight applications.

All EbbaBiolight variants are exceptionally photostable and fluorogenic. When bound to a target, the optotracers can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Excitation and emission wavelengths for each molecule are shown in Table 1. EbbaBiolight are intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 480 420 nm 480 nm DAPI
EbbaBiolight 520 460 nm 520 nm FITC, GFP
EbbaBiolight 540 480 nm 540 nm FITC, GFP, YFP
EbbaBiolight 630 520 nm 630 nm PI, Cy3, TxRed, mCherry, Cy3.5
EbbaBiolight 680 530 nm 680 nm PI, mCherry, Cy3.5
contact us for custom options.
EbbaBiolight 680

EbbaBiolight 680 is our red fluorescent optotracer for labeling extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Specifically, EbbaBiolight 680 labels curli in the extracellular matrix produced by Salmonella as well as β-glucans and chitins in yeast- and hyphal forms of C. albicans. Furthermore, it has been used to track pellicle biofilm formation by B. cenocepacia. Contact us to learn more about EbbaBiolight applications.

As all our optotracers, EbbaBiolight 680 is exceptionally photostable and fluorogenic. When bound to a target, EbbaBiolight 680 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Use recommended filter sets as well as excitation- and emission wavelengths according to the table below. EbbaBiolight are intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 680 530 nm 680 nm PI, mCherry, Cy3.5

EbbaBiolight 680 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). AAs our standard solvent, we use ultrapure water (MilliQ). We also provide EbbaBiolight 680 dissolved in DMSO Please contact us for custom options.

contact us for custom options.
EbbaBiolight 630

EbbaBiolight 630 is our orange fluorescent optotracer for labeling extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Specifically, EbbaBiolight 630 labels peptidoglycan and lipoteichoic acids in the cell wall of certain gram positives like Staphylococci and E. faecalis. Contact us to learn more about EbbaBiolight applications.

As all our optotracers, EbbaBiolight 630 is exceptionally photostable and fluorogenic. When bound to a target, EbbaBiolight 630 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Use recommended filter sets as well as excitation- and emission wavelengths according to the table below. EbbaBiolight are intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 630 520 nm 630 nm PI, Cy3, TxRed, mCherry, Cy3.5

EbbaBiolight 630 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

contact us for custom options.
EbbaBiolight 540

EbbaBiolight 540 is our yellow fluorescent optotracer for labeling extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Contact us to learn more about EbbaBiolight applications.

As all our optotracers, EbbaBiolight 540 is exceptionally photostable and fluorogenic. When bound to a target, EbbaBiolight 540 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Use recommended filter sets as well as excitation- and emission wavelengths according to the table below. EbbaBiolight are intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 540 480 nm 540 nm FITC, GFP, YFP

EbbaBiolight 540 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

contact us for custom options.
EbbaBiolight 520

EbbaBiolight 520 is our green fluorescent optotracer for labeling extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Contact us to learn more about EbbaBiolight applications.

As all our optotracers, EbbaBiolight 520 is exceptionally photostable and fluorogenic. When bound to a target, EbbaBiolight 520 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Use recommended filter sets as well as excitation- and emission wavelengths according to the table below. EbbaBiolight are intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 520 460 nm 520 nm FITC, GFP

EbbaBiolight 520 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

contact us for custom options.
EbbaBiolight 480

EbbaBiolight 480 is our blue fluorescent optotracer for labeling extracellular and intracellular bacterial amyloids as well as certain glucans produced as part of the extracellular matrix. Specifically, EbbaBiolight 480 has been used to label extracellular matrix produced by P. aeruginosa but not C. albicans in a mircofluidic device during mono- and co-culture. Contact us to learn more about EbbaBiolight applications.

As all our optotracers, EbbaBiolight 480 is exceptionally photostable and fluorogenic. When bound to a target, EbbaBiolight 480 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. As spectral information can give hints of the nature of the target, we recommend to acquire excitation and emission spectra whenever possible. Use recommended filter sets as well as excitation- and emission wavelengths according to the table below. EbbaBiolight is intended for use in live-cultures and are not compatible with fixation. If fixation needs to be performed, we recommend to apply fixation after labelling.

Table: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
EbbaBiolight 480 420 nm 480 nm DAPI

EbbaBiolight 480 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

contact us for custom options.

EbbaBiolight are optotracers for detection of bacteria & components of bacterial biofilm.

EbbaBiolight molecules allow direct visualisation of bacteria or bacterial biofilm, without using antibodies or any toxic chemicals. The molecules become highly fluorescent when bound to a target, are non-toxic, and do not interfere with bacterial growth or biofilm formation at recommended concentrations so EbbaBiolight can be added to live cultures to track bacterial growth or biofilm formation in real-time using spectrophotometric analysis. Further, EbbaBiolight are photo- and thermostable and allow for easy handling in any application.

We offer the EbbaBiolight optotracers in five different variants that might be screened for applications in various bacterial species and strains. All molecules are available in aliquots of 50 µl, 100 µl, 150 µl or 200 µl (all delivered in 50 µl vials). We also offer an EbbaBiolight Mix&Try kit, that contains 10 µl of each of the five optical tracer molecules from the EbbaBiolight series.

Optotracing

Optotracing is an innovative visualization method developed by scientists for scientists. With optotracing – using fluorescent tracer molecules – Ebba Biotech provides an innovative method for expanding areas of research. The optotracing technology has been developed by a scientific expert team of researchers at Karolinska Institutet, Linköping University and the Royal Institute of Technology (KTH) in Sweden and is a result of many years of cutting-edge nanoscience and organic chemistry research.

We supply and deliver the fluorescent tracer (optotracer) molecules for detection of bacteria and bacterial biofilm in the EbbaBiolight product series. The EbbaBiolight optotracers are a tool to search and detect bacteria & biofilm in hospitals and microbiology laboratories, as well as in industry with high demands to detect bacterial contamination and biofilm formation.

Quick Facts

EbbaBiolight optotracer molecules are:

  • Provided as 1 mg/ml solution in volumes of 50 µl, 100 µl, 150 µl and 200 µl and as a Mix&Try Kit with 5x10 µl
  • Should be diluted 1000-fold before use
  • Non-toxic
  • Photo- and thermostable

Quickstart Guide

Depending on the bacterial species or strain, EbbaBiolight optotracer molecules will bind to components in the bacterial biofilm. We recommend to follow this Quickstart Guide when using any of the EbbaBiolight molecules for the first time.

  • Dilute EbbaBiolight 1:1000 in growth medium (pH 7.4).
  • Dilute a bacterial over-night culture 1:100 with growth medium containing EbbaBiolight.
  • Fill (at least) two wells of a 96-well microtiter plate with 100 μl of the diluted over-night culture containing EbbaBiolight.
  • Prevent drying by filling unused wells with sterile water and seal the plate with a cover or adhesive.
  • Incubate the plate at optimal growth temperature and measure absorbance as well as fluorescence at regular intervals during a period of at least 48h.
  • Take samples (10 µl) during exponential- and stationary phase and pipet onto a microscope slide for imaging. Cover with a glass coverslip and seal the edges to prevent drying. Image at recommended excitation/emission to detect fluorescent bacteria or biofilm.

Applications for EbbaBiolight 680

EbbaBiolight 680 is our optotracer for biofilm visualization. EbbaBiolight 680 binds to cellulose and amyloid components in the biofilm of Salmonella enterica, serovars S. Enteritidis and Salmonella Typhimurium.

Storage

  • Store EbbaBiolight at 4°C.
  • Use the opened vial within 12 month.

Note

  • EbbaBiolight is for research use only.
  • EbbaBiolight is not for diagnostic use or use in humans.
  • EbbaBiolight is not for resale.

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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.

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Protocol V - Real-time monitoring of curli production in bacterial biofilms

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. We recommend using fluorescent protein tagged bacteria to be able to distinguish between bacterial cells and curli in the bacterial extracellular matrix.

Disclaimer: When adapting this technique, please make sure to include relevant controls to verify that EbbaBiolight does not affect biofilm formation, to confirm curli as EbbaBiolight binding target and to exclude pH effects. Please be aware that EbbaBiolight is suited for labelling live bacteria only and is not compatible with fixation.

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
  • Automated temperature-controlled fluorescence microscope OR Automated temperature-controlled fluorescence microwell 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

Real-time recording:

  • Incubate the plate in an automated microscope or fluorescence reader at 28°C
  • Program the reader or microscope to record spectra/images at defined time intervals for ca. 70 h. Use filter-sets or excitation- and emission parameters as indicated in the table below.

EbbaBiolight Fluorescence Detection:

EbbaBiolight fluorescent tracer molecules are optotracers. Unlike conventional fluorescent dyes, optotracers bind promiscuously to a range of targets with repetitive motifs. EbbaBiolight has been shown to bind to curli and cellulose in Salmonella extracellular matrix [1], peptidoglycan and lipoteichoic acids in the cell envelope of Staphylococci [2], β-glucans from S. cerevisiae and Chitin in C. albicans [3]. Upon binding, the fluorescence intensity of the optotracer increases. This property makes it possible to use EbbaBiolight for live fluorescent tracking of microorganisms, without the need to wash away unbound molecules. It is possible to read out fluorescence intensity at the emission maximum (Emmax) when excited at or close to the excitation maximum (Exmax). This is useful for microscopy or fluorescence spectroscopy when straight-forward data analysis is required. Yet, due to the unique properties of the optotracers, a unique optical fingerprint is produced reflecting the specific nature of the target (sample composition) and environment (pH, osmolarity, polarity of the medium). This means that depending on the specific properties of the sample, Exmax or Emmax can shift, or the appearance of double peaks or shoulders might indicate binding to multiple targets. We therefore recommend acquiring fluorescence excitation and emission spectra whenever possible within experimental limitations.

Table: EbbaBiolight spectral properties with maximum excitation (Exmax) and emission (Emmax) when bound and recommended range for acquisition of excitation- and emission spectra as well as recommended filter sets for microscopy.
Exmax Emmax Excitation spectrum (detect at Emmax) Emission spectrum (excite at Exmax) Recommended
filter-sets
EbbaBiolight 480 420 nm 480 nm 300 - 450 nm 450 - 800 nm DAPI
EbbaBiolight 520 460 nm 520 nm 300 - 490 nm 490 - 800 nm FITC, GFP
EbbaBiolight 540 480 nm 540 nm 300 - 510 nm 510 - 800 nm FITC, GFP, YFP
EbbaBiolight 630 520 nm 630 nm 300 - 600 nm 550 - 800 nm PI, Cy3, TxRed, mCherry, Cy3.5
EbbaBiolight 680 630 nm 680 nm 300 - 650 nm 660 - 800 nm PI, mCherry, Cy3.5

Access EbbaBiolight spectra

References

  1. Choong FX et al. (2021) A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces. Biofilm, 3, 100060
  2. Butina K. et al. (2020) Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time. npj Biofilms and Microbiomes, 6(1), 35
  3. Kärkkäinen, E. et al. (2022) Optotracing for live selective fluorescence-based detection of Candida albicans biofilms. Frontiers in Cellular and Infection Microbiology, 12, 2235-2988

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