This presentation will discuss fundamental physical, chemical, and biological concepts important to understanding control of detrimental biofilms. Four phenomena that are important in the action of antimicrobial agents against a biofilm will be examined: diffusion, hydrodynamics, physiology, and the genetic basis of biofilm tolerance. Direct microscopic observation of facile diffusion of a fluorophore-tagged antibiotic into biofilm is contrasted with failure of hydrogen peroxide, a much smaller molecule, to penetrate. These seemingly contradictory observations can be reconciled by recognizing that penetration of antimicrobials into biofilms is governed by the balance of reaction and diffusion. Time-lapse imaging of biofilms subjected to antimicrobial treatments reveals that in many cases these treatments do not remove the biofilm. In instances where removal is observed it is clear that forces applied by the flowing fluid are an important component of the removal process. Oxygen and nutrient concentration gradients within biofilms lead to stratified patterns of anabolic activity. For example, microelectrode technology demonstrates the presence of anoxic niches in biofilms exposed to aerated medium. Staining techniques reveal that the same biofilm can harbor, in distinct spatial niches, growing and dormant cells. Variation in the physiological activity is accompanied by alterations in susceptibility to antimicrobials. More specifically, patterns of gene expression within biofilms in response to local environmental chemistry are hypothesized to contribute to protection from antimicrobial agents. As an illustrative example, a transcriptomic and mutant susceptibility analysis is presented. The biofilm transcriptome is found to be enriched for genes associated with oxygen limitation and stationary phase growth. Mutants in regulatory genes associated with the response to growth arrest or hypoxia exhibit diminished tolerance to an antibiotic when grown as biofilms. These analyses suggest a model in which overlapping starvation and stress responses control the expression of multiple genes that are activated in mature biofilms and contribute to biofilm antimicrobial tolerance. The biofilm defense against biocides and antibiotic is multifactorial and so requires integrated and interdisciplinary science. Learning Objectives - Explain two general mechanisms of biofilm protection from antimicrobial agents - Contrast killing and removal processes in biofilms - Discuss fundamental phenomena important in the interaction of an antimicrobial agent with biofilm