This chapter, authored by Judith H. Merritt, Daniel E. Kadouri, and George A. O'Toole, focuses on the study of static biofilms, particularly their early stages of formation, including initial adherence and microcolony development. The authors present several methods for examining these communities, emphasizing the simplicity and adaptability of static biofilm assays compared to continuous-flow systems. These assays require minimal specialized equipment and can be executed with common laboratory tools, making them suitable for both small- and large-scale studies. The chapter highlights four basic protocols for growing and analyzing biofilms in static systems: 1. **Microtiter Plate Biofilm Assay**: This high-throughput method assesses bacterial attachment by measuring the staining of adherent biomass using crystal violet. It is useful for screening a large number of bacterial strains or species. 2. **Air-Liquid Interface (ALI) Assay**: This assay provides a simple system for microscopic analysis of biofilm formation over a time range of 4 to 48 hours. It is particularly useful for observing early stages of biofilm formation, including attachment and microcolony development. 3. **Colony Biofilm Assay**: This method involves growing bacteria on semipermeable membranes placed on agar plates, allowing easy nutrient replenishment and observation of antibiotic resistance properties. 4. **Kadouri Drip-Fed Biofilm Assay**: This system overcomes the limitations of other static systems by constantly refreshing the bacterial growth medium, allowing for the formation of mature biofilms. It is a "low-flow" system that bridges the gap between static and continuous-flow systems. The chapter also discusses the advantages and drawbacks of each assay, provides detailed protocols, and offers troubleshooting tips. It emphasizes the importance of critical parameters such as inoculation conditions, washing steps, and experimental setup to ensure accurate results. The assays can be used individually or in combination to study various aspects of biofilm formation, including antibiotic resistance and the effects of different conditions on biofilm development.This chapter, authored by Judith H. Merritt, Daniel E. Kadouri, and George A. O'Toole, focuses on the study of static biofilms, particularly their early stages of formation, including initial adherence and microcolony development. The authors present several methods for examining these communities, emphasizing the simplicity and adaptability of static biofilm assays compared to continuous-flow systems. These assays require minimal specialized equipment and can be executed with common laboratory tools, making them suitable for both small- and large-scale studies. The chapter highlights four basic protocols for growing and analyzing biofilms in static systems: 1. **Microtiter Plate Biofilm Assay**: This high-throughput method assesses bacterial attachment by measuring the staining of adherent biomass using crystal violet. It is useful for screening a large number of bacterial strains or species. 2. **Air-Liquid Interface (ALI) Assay**: This assay provides a simple system for microscopic analysis of biofilm formation over a time range of 4 to 48 hours. It is particularly useful for observing early stages of biofilm formation, including attachment and microcolony development. 3. **Colony Biofilm Assay**: This method involves growing bacteria on semipermeable membranes placed on agar plates, allowing easy nutrient replenishment and observation of antibiotic resistance properties. 4. **Kadouri Drip-Fed Biofilm Assay**: This system overcomes the limitations of other static systems by constantly refreshing the bacterial growth medium, allowing for the formation of mature biofilms. It is a "low-flow" system that bridges the gap between static and continuous-flow systems. The chapter also discusses the advantages and drawbacks of each assay, provides detailed protocols, and offers troubleshooting tips. It emphasizes the importance of critical parameters such as inoculation conditions, washing steps, and experimental setup to ensure accurate results. The assays can be used individually or in combination to study various aspects of biofilm formation, including antibiotic resistance and the effects of different conditions on biofilm development.