Throughout the history of science, new tools and techniques have advanced our understanding of nature. In microbiology, the demand for new tools has increased to keep pace with new discoveries, which include the prevalence of bacterial communities. In these microbial communities, heterogeneity and chemical signaling is essential to adaptation and fitness. These discoveries have stimulated research projects that aim to control heterogeneity in bacterial populations, understand variations at the single cell level, and introduce chemicals that modulate biological processes. New tools can have a transformative impact on these areas of microbiology. Each chapter in this thesis introduces a new physical or chemical tool for studying microbial physiology. Chapter 2 describes a biofilm stencil fabricated in a biocompatible polymer. This material serves as a physical scaffold for patterning biofilms on surfaces, and growing them reproducibly in a high-throughput manner. Chapter 3 introduces a microfluidic device for encapsulating microbial cells in agarose microparticles. Encapsulation enables isolation and manipulation cells with rare phenotypes (e.g. drug resistance) in a large population. Chapter 5 describes DCAP, a small molecule that targets bacterial membranes, reduces their transmembrane potential, and increases membrane permeability. The biological activity of DCAP makes it a potent antibiotic against slow-growing and biofilm-associated cells, which are frequently associated with persistent bacterial infections. Chapter 6 introduces the small molecule divin, which targets the assembly and maturation of divisome, a multi-protein complex that drive bacterial cytokinesis. This mechanism of divin makes it a valuable tool for studying the dynamics of the divisome and the function of its protein components. These chemical and physical tools present new capabilities for isolating, manipulating and studying bacteria, and are poised to transform microbiology.