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Historically, the focus of diagnostic microbiology has been to detect and accurately identify disease-causing microorganisms in clinical specimens by culture methods. However, over the past few decades, significant technological advances have changed the landscape of diagnostic tools available to the microbiology laboratory. The physical structure of laboratories, staffing patterns, work flow, and turnaround time have all been profoundly impacted by these technical advances. Specifically, nucleic acid amplification-based molecular techniques now provide rapid, on-demand diagnostic results that have meaningful impact on patient management. The laboratory will continue to evolve and with it, new challenges are likely to arise. The field of diagnostic microbiology has two goals: (1) determine the presence of suspected pathogenic microorganisms present in specimens collected from human beings, animals, and environment, and (2) if present, identify and characterize the organism. In medical practice, a fundamental principle of diagnostic microbiology is to define infectious processes and elucidate treatment options through rapid detection and characterization of specific pathogens. Thus, beyond detection, quantification, and identification of microorganisms in clinical specimens, diagnostic microbiology also provides physicians with antimicrobial susceptibility profiles of the identified microorganism. Advanced diagnostic techniques also provide tools in basic microbial pathogenesis studies. This article provides a brief review of the fundamental principles of diagnostic microbiology. For greater detail, the reader is referred to several excellent textbooks listed under the recommended reading section.