Fig. 3. ΔcprS exhibits increased surface growth characteristics compared with WT.

A. ΔcprS adheres to shaking broth culture tubes. WT and ΔcprS were grown overnight with shaking at 200 r.p.m. in MH broth, and adherent bacteria were visualized by staining cultures directly with 0.2% crystal violet.

B. ΔcprS shows enhanced biofilm formation. Overnight liquid cultures of WT, ΔcprS, and ΔcprS ^C were diluted to an optical density of 0.0002 in MH broth, added to borosilicate glass tubes and incubated without shaking for 2 days. Biofilms were stained by the addition of crystal violet to a final concentration of 0.2% and tubes were photographed (left). Biofilms formed by WT (black bar), ΔcprS (grey bar) and ΔcprS ^C (white bar) were quantified (right) by dissolving adhered crystal violet with 30% methanol/10% acetic acid and measuring the A570 of the resulting solution. Quantifications were performed in triplicate.

C. ΔcprS biofilms are enhanced and accelerated compared with WT and are comprised of spiral-shaped bacteria. Biofilms were grown on glass coverslips in tubes prepared as in Fig. 4, and the region at the air-liquid interface was either stained with crystal violet for bright field microscopy (10×, 40×, 100×; first three panels) or prepared for scanning electron microscopy (SEM) (5000×; far right panels). Pictures are representative of a large region of each slide. From left to right, the bars represent ~400 μm, 100 μm, 40 μm, and 10 μm.