Fig 1. P. aeruginosa cells form vertical striations across depth in colony and pellicle biofilms.

(A) Left: Top view of a P. aeruginosa colony biofilm grown for 3 days on 1% tryptone + 1% agar, and schematic showing orientation of the sample used for SEM imaging. Right: SEM images of a full colony biofilm cross-section. Insets of higher magnification show cellular arrangement and morphology for the indicated locations in the biofilm. (B) Top: Schematic of mixing assay method. Left: Fluorescence micrograph of a thin section prepared from a colony biofilm grown in the mixing assay. Center: Orientation across depth for fluorescent cells detected in biofilm thin section micrographs. The “spread of orientation” is the standard deviation of orientation values for each pixel across biofilm depth; the values shown in the plot are the average “spread of orientation” at each depth for thin section images taken from 6 biological-replicate biofilms. Shading represents the standard deviation for this average. Right: Schematic of cellular arrangement across depth in mature biofilms. (C) Micrographs of biofilms prepared as described in (B), but sacrificed at the indicated time points. Scale bar applies to all images. The data underlying Fig 1B and 1C can be found in S1 Data. (D) Top: Setup used to grow pellicle biofilms for microscopy. Bottom: Fluorescence micrograph of a thin section prepared from a pellicle biofilm. The inoculum contained 2.5% cells that constitutively express mScarlet. Images shown in this figure are representative of at least 2 independent experiments. mScarlet fluorescence is colored yellow. Quantification of colony-forming units confirmed that expression of mScarlet did not affect fitness during growth in mixing assay biofilms (S1 Fig).