Protocol for FilmTracer™ SYPRO® Ruby biofilm matrix stain

Protocol for FilmTracer™ SYPRO® Ruby biofilm matrix stain: 

1. Wash bacterial biofilms twice with PBS.
2. Cover the biofilms with  FilmTracer™ SYPRO® Ruby biofilm matrix stain (Invitrogen, Molecular Probes, Eugene, OR) (e.g. 100 μl for 96-well plates) for 30 min at room temperature in the dark.  
3. Wash the stained biofilms several times with double distilled water. 

4. Measure the fluorescence of the biofilms in a M200 Tecan microplate reader (or another fluorescent plate reader) with excitation at 450 nm and emission at 610 nm. 

    

Comments: 

1. It is recommended to use black microclear 96-flat-bottomed tissue culture grade plates (Greiner).
2. The assay is easy to perform. 
3. FilmTracer™ SYPRO® Ruby biofilm matrix stain should be complemented with crystal violet staining and MTT metabolic assay for comparison. 
4. So far, we have only used the reagent for Streptococcus mutans biofilms. But, it is expected to be useful for quantifying biofilms of other bacteria too.