Biofilm formation by wild type and scl1 -inactivated isogenic mutants. Crystal violet staining and confocal laser scanning microscopy (CLSM) of the GFP-expressing GAS were used to compare biofilm formation by GAS strains. Wild type (WT) M41-, M28-, and M1-type strains, scl1-inactivated mutants (scl1), and M41 mutant complemented for Scl1.41 expression (M41 C) were used. (a-c) Isogenic GAS strains were grown under static conditions for 24 h and bacterial biomass was detected spectrophotometrically at indicated time points following crystal violet staining. Absorbance values at OD600 are representative of at least three experiments performed in quadruplicate. Statistical significance is denoted as *P ≤ 0.05 and **P ≤ 0.001. (d-f) CLSM analysis of corresponding 24 h biofilms from same experiment. Images are X-Y orthogonal Z-stack views of WT (top) and mutant (bottom) GAS strains. Views are representative of ten images within a single experiment. Average vertical biofilm thickness is indicated in micrometers (top right).