Biofilm formation on coupons in the continuous culture biofilm model. Continuous culture biofilm was analysed for TIGR4 (closed square), its rough mutant FP23 (open square) and the comD mutant FP184 (closed triangle). Bacterial counts in flow through (panel A) and on the coupon (panel B) are from a single experiment while data on biomass (panel C) and the surface area of the biofilm (panle D) are from 15 measurements at each timepoint. Biofilm samples grown on polycarbonate disks were collected at 12, 24, 36, and 48 hours and fixed in formaldehyde. Biofilm was stained with Sybr Green I, a general double stranded DNA stain, and examined with a Zeiss epifluorescence microscope with an ApoTome attachment. Image stacks through the depth of the biofilm were obtained (Z-stacks) from each disk, with 5 stacks per disk and 3 disks per time point and per strain. Biomass in each image stack was enumerated in the COMSTAT image analysis program. Data was transformed by multiplying each point by 10,000 and obtaining the log (base 10) value.