Abstract
Acinetobacter baumannii is an opportunistic pathogen that has a propensity to cause infections in immunocompromised patients. With the increasing incidence of antibiotic-resistant isolates, Acinetobacter baumannii has emerged as one of the most problematic pathogens in recent decades with the World Health Organization urging prioritization for additional research focusing on this nosocomial pathogen. The following protocol outlines the process of culturing and polarizing A549 human alveolar epithelial cells for infection with Acinetobacter baumannii to study biofilm formation. The protocol additionally outlines the process of fixing the infected A549 cells for downstream applications.