Fig. 1. S. aureus cells divide in two, but not necessarily in three, alternating orthogonal planes.

S. aureus strain COL cells were initially stained with cell wall dye WGA-488 (green) and membrane dye Nile Red (magenta). Excess of non-bound WGA-488 was removed by washing and Nile Red was added again before cells were placed on a medium-containing agarose pad and imaged every 20 min by structured illumination microscopy. a The border of the WGA-488-labelled region provides information on the orientation of the previous division plane (solid line), while the Nile Red-labelled septum indicates the orientation of the current division plane (dotted line). The angle (α) between the solid and dotted lines corresponds to the angle between the two consecutive planes of division. b Histogram of the angle α formed between two consecutive planes of division shows that these planes are orthogonal (with an average deviation from 90° of 6.7°, n = 183 cells examined over three biological replicates). Source data are provided as a Source data file. c First, second and third division planes are indicated by solid, dashed and dotted lines, respectively, showing that in the cell indicated by the arrow the third division plane is parallel, not perpendicular, to the first plane of division. Scale bars, 1 μm. A full field of view can be accessed in https://figshare.com/s/4808495d92a138aae36c.