Fig. 2.

Phage efficacy in colonies. a All colonies (PAO1, PA14 or PAO1 + PA14) were first grown by inoculating cells onto a membrane filter placed on 0.1x LB agar in 9 replicates. After ~12 h, three filters were destructively sampled to quantify CFUs, three were transferred to fresh 0.1x LB agar containing a dried 50 μl drop of phage containing ~$10^6$ PFU/ml, and three to fresh 0.1x LB agar containing no phage. After ~36 h the remaining 6 filters were harvested to quantify CFUs and/or PFUs (see Methods). b Fluorescence microscopy images of colonies at 12 and 48 h. PA14 are tagged with mCherry (purple) and PAO1 with GFP (green). Sectors that formed in untreated mixed colonies (white arrowheads, B6) were absent in the phage treatment (B9) suggesting that phage kill cells at the actively growing colony edges, while cells in the center survive (Supplementary Fig. 3 shows a similar experiment). The scalebar in B1 represents 2 mm. c–f Data coming from triplicate colonies using unlabelled PA14. These data do not correspond to the images in b, whose quantification was less precise (see Supplementary Fig. 5) because PA14-mCherry were difficult to distinguish from PAO1-GFP (identical drug resistances). c The ratio of population sizes at 48 and 12 h (see Supplementary Fig. 1 for growth curves). The mean at 12 h was used due to destructive sampling. Phage decrease PAO1 (green), insensitive PA14 grow similarly across conditions (purple), and phage decrease in the absence of PAO1 and increase in its presence. d Phage-resistant PAO1 were found in whole colonies of PAO1, but not in the centers of PAO1 colonies, nor in colonies mixed with PA14. e The ratio of PFUs to CFUs was lower in the colony centers, and in whole mixed colonies compared to whole colonies of PAO1 alone. f Phage abundance at 48h is inversely proportional to the initial abundance of PA14 in the colony inoculum. PAO1 was constant in all inocula at $10^2$ CFU/col. Note that $10^6$ PFU were added after 12 hours of growth and the plot shows the final PFU values