Figure 7

Different sampling strategies of biofilm development for downstream omics analysis. (A) We depicted some of the possible sampling strategies when a time-lapse recording of biofilm development is not available. Dense sampling is the best approach, but incurs high costs in downstream experiments, e.g. transcriptome sequencing and protein quantification, especially if the sampling includes replicates and covers long biofilm ontogenies. If the number of sampling timepoints is limited for any reason, e.g. to seven samples, one could choose an equidistant or eventually random sampling approach. In both cases, it is uncertain how these samples will reflect underlying expression dynamics. (B) In the case of limited number of samples, we suggest that a better option is to follow morphological change of biofilm development and to adjust sampling timepoints accordingly. By browsing time-lapse videos, timepoints relevant for biofilm development could be determined. For instance, we analyzed here the time-lapse video that covers biofilm development of the B. subtilis strain 108 during seven days (Supplementary Video 6). We selected seven time-points that we consider relevant for sampling: 12 h—early biofilm development, the inoculation droplet circle is visibly filled with cells, the first regions with ticker cell layers already start to appear; 1d—the cells start to spread outside of the inoculation droplet circle to from a ring, at the edge of inoculation droplet circle an irregular ridge made of ticker cell layers is formed; 1d 12 h—the ring continues to enlarge, the outer edge of the ring starts to thicken, inner circle shows additional ridges and wrinkles; 2d—the ring further continues to enlarge, first radial wrinkles across the ring start to appear, the outer belt of the ring becomes the thickest structure; 2d 12 h—the ring further continues to enlarge, radial wrinkles become prominent, inner part of the biofilm becomes obviously thinner compared to the outer belt; 3d—the biofilm continues to grow, has lace appearance, shows strong radial wrinkles and a curvy edge; 5d—at the upper-right sector of the biofilm edge a secondary growth becomes visible.