Standardized In-house Protocol for Determining the Minimum Inhibitory Concentration (MIC) via Broth Microdilution for Natural Products Against Obligate Anaerobic Bacteria.

Obligate anaerobic bacteria are important constituents of the human microbiota and are frequently involved in opportunistic infections. However, due to their fastidious nature and oxygen sensitivity, antimicrobial susceptibility testing (AST) is often neglected, resulting in empirical treatments and an increased risk of therapeutic failure. The broth microdilution method offers a cost-effective and scalable alternative for antibacterial activity evaluation but is typically restricted to certain genera due to concerns over oxygen exposure. In this study, we present a complete standardized in-house protocol for determining the minimum inhibitory concentration (MIC) of natural and synthetic antimicrobial agents against obligate anaerobes using the broth microdilution technique under strictly anoxic conditions. Our method is optimized for implementation in laboratories equipped with anaerobic chambers and allows high-throughput screening using minimal sample volumes, making it ideal for natural product-based antibacterial discovery. The protocol is based on other guidelines with critical adaptations that ensure bacterial viability throughout the procedure. It provides reproducible results without oxygen interference, thus representing a valuable tool for researchers investigating novel antimicrobials targeting anaerobic pathogens, within a period of approximately 6 days. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of working solutions Basic Protocol 2: Preparation of the 96-well microplate Basic Protocol 3: Inoculum standardization Basic Protocol 4: Reading and interpreting the results.