SUMMARY The Mother Machine is a microfluidics-based high throughput imaging and analysis system for measuring the properties and behaviors of single cells in a large population (thousands to millions of cells). It is capable of identifying rare stochastic variants in a population, such as spontaneous persister cells, and tracking the properties of those rare cells over time, or in response to various treatments. It is equally capable of identifying phenotypically-altered genetic variants in a large pool of mutants, such as a transposon insertion library typically used for high throughput genetic screens such as Tn-seq. However, rather than relying on indirect inferences based on counts of sequences of particular transposon insertion junctions following several cycles of PCR amplification, the Mother Machine allows direct observation of the mutant over time. Moreover, it allows comparison to about 10 other cells of that genetic lineage in the same trench, providing extremely rigorous and precise measurements. Additional advantages over conventional Tn-seq approaches include the ability to identify phenotypes that do not necessary translate to changes in chromosome abundance (and hence sequence counts), such as the formation of multinucleate filamentous cells, or other irregularities in cell growth or division. Finally, direct imaging of single clonal lineages allows direct assessment of the phenotypic consequence for that particular transposon insertion in that particular orientation, rather than summing the effects of all insertions across a gene. This allows phenotypic differences that depend on the precise insertion location of a transposon, and its orientation to be assessed, for individual comparison to other different insertions within the same gene. The Core Director is a pioneer in this field who has made advancements in the technology not available anywhere else, including the ability to image more than 1 million trenches, and the ability to retrieve cells from trenches of interest. This specialized advanced technology cannot now be recreated in individual laboratories, and is not available commercially, providing a tremendous, unique resource for the important investigation of antibiotic resistance and susceptibility of staphylococci and enterococci. Additionally, the Core Director’s field-leading expertise in stochastic variation within bacterial populations provides an important intellectual resource for subprojects investigating the properties of antibiotic persister subpopulations – the effects of antibiotics on them as well as the contribution of variation in efflux expression to their genesis. These capabilities are highly complementary to those of the research subprojects, and significantly enhances both their throughput and the rigor and richness of the data that cannot otherwise be obtained.

NARRATIVE The Mother Machine Technical Core provides access for each of the subprojects to leading-edge technology for continuous single cell imaging of populations of thousands to millions of bacteria per experiment. This capability, which cannot readily be replicated in other labs and is not commercially available, allows rare cells in an altered physiological state, such as those that persist despite the presence of otherwise lethal levels of antibiotics, to be examined for susceptibility to new antibiotics being designed in this P01. It also provides a deep, rich and precise description of every mutant in a large mixed population, such as mariner transposon insertions in a Tn-seq mutant pool, along with the ability to recover and further study mutants of interest, for studies identifying new antibiotic targets.