Fig. 2: Change in confinement alters bacterial population growth.

a We use absorbance-based optical density measurements as an indicator of an increase in biomass over time to quantify bacterial growth in the 3D microgel media. b Representative absorbance-based growth curves over 16 h indicating confinement-dependent growth dynamics for all bacterial strains (mean ± s.d., n = 3 technical replicates); G’ ~ 10 Pa represents a low confinement matrix and G’ ~ 110 Pa represents a high confinement matrix. c Relative biomass ratio values for all bacterial strains across all time points, showing that higher confinement demarcates the different microbial species into two distinct sub-groups, indicated by bar graphs representing the plateau value of the relative biomass ratio at 16 h (mean ± propagated error, which is calculated as described in the statistical analysis, with biological replicates n = 7 for S. gallinarum, n = 5 for A. radioresistens, n = 6 for E. faecium, n = 6 for E. faecalis, n = 6 for P. mosselli, and n = 8 for E. hormaechei). d Absorbance measurements from 3D growth media mirror the actual microbial load, determined by measuring volumetric bacterial density via direct counting of cells using 3D confocal microscopy (also see Supplementary Fig. 2e). Both of these measurements yield comparable relative biomass ratio after 16 h of growth (mean ± propagated error, which is calculated as described in the statistical analysis; for A. radioresistens n = 3 technical replicates for absorbance measurements; while for the manual counting 317 cells were pooled from three unique micrographs for the low confinement gel, and 198 cells were pooled from four unique micrographs for the high confinement gel—all sourced from the same biological replicate; and for the non-motile E. coli, n = 4 technical replicates for absorbance measurements; while for the manual counting 1163 cells and 828 cells were pooled from five unique micrographs each for the low confinement gel and high confinement gel, respectively—all sourced from the same biological replicate). e–g Motility does not confer significantly greater fitness benefits under confinement, as tested using a motile and non-motile strain of E. coli (the latter carries a deletion in the flagellar protein-encoding gene flhDC), which show no significant differences between the growth curves (mean ± s.d., n = 3 technical replicates) or the relative biomass ratio (mean ± propagated error, n = 5 biological replicates).