Figure 2. Cell volume increases continuously throughout the cell cycle.

(A) Time-lapse images of S. aureus cells stained with FM 4-64 (left) and outlined by fitting with ellipses (right). (B) Average aspect ratio of S. aureus cells throughout the cell cycle (from immediately after previous popping to ready-to-pop) and overlay of the cell outlines (inset) from a typical cell at different points of the cell cycle colored from blue (early) to red (late). Error bars denote standard errors (n=27). Red bars on top indicate the time fraction into the cell cycle when septation starts (left, 0.35 ± 0.03 SEM) and completes (right, 0.77 ± 0.02 SEM), respectively (n=26). (C) Representative traces of cell volume as a function of time following a microcolony starting from a single cell; solid blue traces indicate cell volumes of individual cells before popping and the dashed black line denotes the total cell volume of all the cells present at a given time. Cell volume and surface area were estimated from the 2D cell outlines by fitting to ellipses and assuming prolate cell shapes (i.e., that each cell was rotationally symmetric around the long axis). (D, E) Distribution of relative changes in volume (D) and surface area (E) during popping, after correcting for baseline growth rate. Black solid line represents kernel density estimate of the distribution and red dashed line denotes the average (2% ± 10% SD for volume, −11% ± 6.5% SD for surface area, n=69). (F) (top) 3D SIM images and corresponding extracted data (see also Fig. S5); (bottom) fraction of old surface before (0.71 ± 0.01 SD, n=15) and after (0.73 ± 0.03 SD, n=36) popping. Cells were modeled as ellipsoids and the contribution of the old, WGA-labeled wall to the daughter cells’ total surface area was measured by fitting a plane to the old/new boundary (Fig. S5A). Scale bars: 1 μm.