Figure 4. EPS analysis and agglutination assay.

(A) Congo red dye was added to mid-exponential phase cultures of the given strains, cells were centrifuged and the resulting supernatants analyzed in a spectrophotometer at 490 nm. Values were normalized to wildtype binding. An increase is indicative of more Congo red bound by the cells and increased EPS production, such as observed in pilT. pilA and frzS cells show reduced EPS production. Data is from three independent trials. (B) Cells were also analyzed with agglutination assays performed on wildtype (black ♦), frzS (green ▴), pilA (orange ▪), and pilT (red •), and a 1∶1 mixture of wildtype with the mutant (open symbols). In wildtype, absorbance at OD₆₀₀ at the top of a cuvette decreases over time as cells agglutinate and settle to the bottom of the cuvette. pilA and frzS inhibit agglutination by wildtype, whereas pilT was observed to behave similar to wildtype. Data shown is representative of three trials.