Semiquantitative Analysis of the Red, Dry, and Rough Colony Morphology of Salmonella enterica Serovar Typhimurium and Escherichia coli Using Congo Red

The Congo Red (CR) assay is a standard biofilm test assessing the colony morphology of bacteria growing on agar plates supplemented with the diazo dye Congo Red. Biofilm forming Salmonella enterica serovar Typhimurium and Escherichia coli produce a red, dry, and rough (rdar) morphotype on CR-plates. The phenotype is characterized by staining of the extracellular matrix components curli (brown color) and cellulose (pink color) by CR. This method allows semiquantitative determination of the expression level of the individual matrix components and dissection of the regulatory networks controlling their production in response to c-di-GMP levels. Here, we describe the CR-assay and its variations and discuss the effect of deletion or overexpression of c-di-GMP turnover proteins on colony morphology.