I understood that the congo red agar is used for detection of biofilms. I wonder if the method can be used also for detection of antibiofilm activity. Could this be a sensible solution:
1. I test a control group of pathogenic bacteria using congo red agar in order to prove that they are biofilm producers;
2. In experimental group I cover the same pathogenic bacteria with plant extract and I check the mix again with congo red agar in order to see if the biofilm still exists or not.
Dear Philip,
Congo red has been used in the past as a non-specific indicator of certain types of EPS expressed by bacteria and often used in biofilms - we have used it to determine whether mutants of Pseudomonas fluorescens SBW25 are expressing cellulose, and we have used other, related direct-red dyes to see if there are differences in expression associated with cellulose-modification.
Congo red is not that easy to use in plates and in liquid assays - it's colour is not particularly strong, and is easily influenced by colony age etc, as well as plate depth (our eyes can see many shades of red but cameras cannot always capture these!).
However, I think that you could use Congo red to survey a collection of bacterial cells for their ability to express cellulose and related EPS. I'd suggest dotting out 10 µl samples of over-night cultures onto Congo red plates (always pipette out the same volume as the depth o the plate influences the 'redness'; don't use high NaCl concentrations in the media as this caused the dye to aggregate (e.g. LB without NaCl); don';t use old dye mixes as this sometimes percipitates out; photograph/determine results after a specific incubation period as colony age changes the colour). You could compare Congo red staining (qualitatively, or quantitatively using absorbance measurements) with a biofilm-forming assay (e.g. 3 days incubation in liquid broth with no shaking), and you could use other dyes (e.g. Calcofluor for cellulose) to see if you can confirm EPS expression in strains that produce colonies with high-Congo red staining.
Andrew.
Because of the great diversity of the chemical composition of biofilms, it is impossible to predict the dye that may or may not be appropriate for detection of biofilms without experimentation. Therefore, I suggest that you give it a try and report your findings to us later.
Dear Philip,
as Andrew already told, Congo red only stains specific polymers that are present in the biofilms (i.e. cellulose, or amyloid fibers of E. coli, etc etc.). So if you're particularly interested in the production of these, you can give it a try.
However, the classic method for biofilm detection and quantification is crystal violet assay. People routinely use this dye to detect the antibiofilm activity of various chemicals as well.
The method is described here, please take a look.
Best regards,
Nataliya
Article Microtiter Dish Biofilm Formation Assay
Hello,
I am working on production and purification of functional amyloids, that are also involved in biofilm formation. The thing is that to identify amyloid producing strains I would like to have a quick preliminary method. My lab has worked with Salmonella and E. coli and they use T agar supplemented with Congo Red and red colonies indicate amyloid production (in this case curli). I will be working with other species, such as, Pseudomonas, Streptococcus and Staphylococcus and I am not sure if this method will work for these bacteria. I saw that it has been done for Pseudomonas, but I got confused with Staphylococcus and Streptococcus since for Staph I have seen that if you do Congo Red for biofilm formation due to exopolysaccharides, colonies will be black so I wonder if I should look only for red colonies or if this does not apply at all
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