Staining Proteinaceous Matrix In Bacterial Biofilms

December 02, 2016

Idival Research Institute
Microbiology
Principal Investigator

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Company:

Thermo Fisher Scientific

Product Name:

FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain

Catalog Number:

F10318

We used FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain to stain the proteinaceous matix in bacterial biofilms to see protein distribution in these matrices using confocal laser scanning microscopy. Bacterial strains which produce dense and compact biofilms with abundant extracellular matrix were strongly stained with the FilmTracer. Similar extracellular matrix was not observed in other strains with lower biofilm production. In positive strains, we observed an excellent matrix staining with very good signal and low background.

Experimental Design and Results Summary

Application

Detection of proteinaceous matrix in bacterial biofilms

Starting Material

Live unfixed bacterial biofilms.

Protocol Overview

Bacterial biofilms were grown in 4-well µ-chamber uncoated slides (Ibidi, Martinsried) without shaking. The slides were incubated at 37°C for 48h. After 48h, planktonic bacteria were removed by rinsing with distilled water. For matrix visualization, unfixed biofilms were stained with 200 µl of FilmTracerTM SYPRO Ruby Biofilm Matrix (Invitrogen) per well, incubated in the dark for 30 min at RT, and rinsed with distilled water. A series of optical sections were obtained with a Nikon A1R confocal scanning laser microscope. Images were captured at random with a ×20 Plan Apo 0.75 NA objective. Reconstructions of confocal sections were assembled using the NIS-Elements 3.2 software. Z-stacks of confocal images were rendered into 3D mode using the ImageJ software.

Tips

None

Results Summary

We used FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain to stain the proteinaceous matix in bacterial biofilms to see protein distribution in these matrices using confocal laser scanning microscopy. Bacterial strains which produce dense and compact biofilms with abundant extracellular matrix were strongly stained with the FilmTracer. Similar extracellular matrix was not observed in other strains with lower biofilm production. In positive strains, we observed an excellent matrix staining with very good signal and low background. Image: Bacteria were stained with DAPI (blue) and biofilm matrix was stained with FilmTracer (pink). Left, strain with low proteinaceous matrix (red); Right, strain overproducing a biofilm matrix. Scale bars, 40 um. Original Magnification x200.

DOI or PMID #

N/A

Additional Notes

For FilmTracerTM a 405 nm excitation, 662–737 nm emission filter was used.

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Summary

The Good

Easy to use. Ready to use. Excellent results with confocal laser scanning microscopy.

The Bad

Requires some optimization when used with other colors/antibodies in the same preparation. Requires good skills with confocal laser microscopy.

The Bottom Line

Easy differentiation between proteinaceous and non-proteinaceous bacterial biofilms. Great potential for rapid screening of biofilms matrices. Easy to use.

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